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Sample records for 3-methylcholanthrene increases ifn

  1. Hyper- and Hypo- Induction of Cytochrome P450 activities with Aroclor 1254 and 3-Methylcholanthrene in Cyp1a2(−/−) mice

    PubMed Central

    Barker, Melissa L.; Hathaway, Laura B.; Arch, Dorinda D.; Westbroek, Mark L.; Kushner, James P.; Phillips, John D.; Franklin, Michael R.

    2009-01-01

    The response of hepatic mono-oxygenase activities to Aroclor 1254 or 3-methylcholanthrene was investigated in wild-type and Cyp1a2(−/−) mice. Cytochrome P450 concentrations were similar in naïve Cyp1a2(−/−) and wild-type mice. There was no difference between naïve wild-type and Cyp1a2(−/−) animals in 7-ethoxyresorufin and 7-ethoxy-4-trifluoromethylcoumarin dealkylase activities, nor was the induction response after 3-methylcholanthrene any different between the two genotypes. However, both activities were induced to a higher extent in Cyp1a2(−/−) mice after Aroclor 1254. In contrast, 7-pentoxyresorufin dealkylation activity was lower in Cyp1a2(−/−) mice and this differential was maintained during induction by both agents. 7-Methoxy- and 7-benzoxyresorufin dealkylation activities were also lower than wild-type in naïve Cyp1a2(−/−) animals and during 3-methylcholanthrene induction, but showed accelerated induction in Cyp1a2(−/−) mice with Aroclor 1254. Bufuralol 1′- and testosterone 6β-hydroxylation activities, and P450 characteristics were evaluated 48 hours after inducer administration. Bufuralol 1′-hydroxylation, a sexual dimorphic activity (female > male) showed no genotype differences in naïve animals. Activity changes varied across gender and genotype, with 3-methylcholanthrene and Aroclor 1254 inducing in male Cyp1a2(−/−), and Aroclor 1254 inducing in female wild-type. Testosterone 6β-hydroxylation activity was 16% higher in Cyp1a2(−/−) mice and neither 3-methylcholanthrene nor Aroclor 1254 elicited induction. After Aroclor 1254, a 24% increase in P450 concentration with a hypsochromic shift in the ferrous-CO maximum characteristic of CYP1A enzymes occurred in wild-type, compared to no change in either parameter in Cyp1a2(−/−) mice. Induction changes with 3-methylcholanthrene were greater in wild-type mice, a 60% increase in concentration and ~2 nm hypsochromic shift versus a 10% increase and ~1 nm hypsochromic

  2. 9,10-Dihydroxy-9,10-dihydro-3-methylcholanthrene-2-one: a principal metabolite of the potent carcinogen 3-methylcholanthrene-2-one by rat liver microsomes.

    PubMed

    Shou, M; Yang, S K

    1990-04-01

    A principal metabolite, formed in the metabolism of the potent carcinogen 3-methylcholanthrene-2-one (3MC-2-one) by liver microsomes from either untreated, or phenobarbital-treated or 3-methylcholanthrene (3MC)-treated rats, was isolated by reversed-phase HPLC. This metabolite has been identified as a 9,10-dihydrodiol with a (9R,10R):(9S,10S) enantiomer ratio of approximately 84:16 by all three rat liver microsomal preparations. The 9,10-dihydrodiol metabolite and its NaBH4 reduction products [a pair of diastereomeric 9,10-dihydroxy-9, 10-dihydro-2-OH-3MC (2-OH-3MC 9,10-dihydrodiols)] were characterized by UV-visible absorption, mass, and circular dichroic spectral, and chiral stationary phase HPLC analyses. Identification of 9,10-dihydroxy-9,10-dihydro-3MC-2-one (3MC-2-one 9,10-dihydrodiol) as the predominant metabolite of the potent carcinogen 3MC-2-one suggests that 3MC-2-one may be metabolically activated to a bay region 9,10-diol-7,8-epoxide, similar to the previously established metabolic activation pathways of 3MC and 1-hydroxy-3-methylcholanthrene (1-OH-3MC).

  3. 3-Methylcholanthrene and other aryl hydrocarbon receptor agonists directly activate estrogen receptor alpha.

    PubMed

    Abdelrahim, Maen; Ariazi, Eric; Kim, Kyounghyun; Khan, Shaheen; Barhoumi, Rola; Burghardt, Robert; Liu, Shengxi; Hill, Denise; Finnell, Richard; Wlodarczyk, Bogdan; Jordan, V Craig; Safe, Stephen

    2006-02-15

    3-Methylcholanthrene (3MC) is an aryl hydrocarbon receptor (AhR) agonist, and it has been reported that 3MC induces estrogenic activity through AhR-estrogen receptor alpha (ER alpha) interactions. In this study, we used 3MC and 3,3',4,4',5-pentachlorobiphenyl (PCB) as prototypical AhR ligands, and both compounds activated estrogen-responsive reporter genes/gene products (cathepsin D) in MCF-7 breast cancer cells. The estrogenic responses induced by these AhR ligands were inhibited by the antiestrogen ICI 182780 and by the transfection of a small inhibitory RNA for ER alpha but were not affected by the small inhibitory RNA for AhR. These results suggest that 3MC and PCB directly activate ER alpha, and this was confirmed in a competitive ER alpha binding assay and in a fluorescence resonance energy transfer experiment in which PCB and 3MC induced CFP-ER alpha/YFP-ER alpha interactions. In a chromatin immunoprecipitation assay, PCB and 3MC enhanced ER alpha (but not AhR) association with the estrogen-responsive region of the pS2 gene promoter. Moreover, in AhR knockout mice, 3MC increased uterine weights and induced expression of cyclin D1 mRNA levels. These results show that PCB and 3MC directly activate ER alpha-dependent transactivation and extend the number of ligands that activate both AhR and ER alpha.

  4. Response of rainbow trout (Oncorhynchus mykiss) D-11 cell line to 3-methylcholanthrene (3MC) exposure.

    PubMed

    Ferraris, M; Flora, A; Fornasari, D; Radice, S; Marabini, L; Frigerio, S; Chiesara, E

    2002-08-01

    The rainbow trout cytochrome P4501A gene subfamily consists of two members, CYP1A1 and CYP1A3, which are induced by polycyclic aromatic hydrocarbons (PAHs). In this study, we investigated the induction of cytochrome P4501A3 in the rainbow trout (Onchorhynchus mykiss) D-11 cell line after 3-methylcholanthrene (3MC) exposure by generating chimeric constructs in which a 2.3 kb fragment or portion of the 5'-flanking region of the trout cytochrome CYP1A3 gene was fused to the firefly luciferase (Luc) gene. The constructs were then transiently transfected into the trout D-11 cells and their transcriptional activity measured by luciferase assay after treatment with different 3MC concentrations. Maximal induction following exposure to 2 microM 3MC was 2.2-fold after 72 h. Deletion of the region specifying the 5' untranslated region (5'UTR) of the mRNA encoding the CYP1A3 gene increased unstimulated luciferase activity but also led to a loss of response to 3MC treatment. This finding suggests that the region specifying the 5'UTR contains a negative element that is also involved in the transcriptional response to 3MC.

  5. Genotoxicity of 3-methylcholanthrene in liver of transgenic big Blue mice.

    PubMed

    Rihn, B H; Bottin, M C; Coulais, C; Rouget, R; Monhoven, N; Baranowski, W; Edorh, A; Keith, G

    2000-01-01

    Transgenic mice provide a unique tool for studying the tissue specificity and mutagenic potential of chemicals. Because 3-methylcholanthrene (3MC) was found mutagenic in bacteria, clastogenic in bone marrow, and induces DNA adducts in animals, we were interested to determinine whether this xenobiotic provokes (1) cell proliferation, (2) transcriptional activity changes, (3) DNA adducts, and (4) hepatic mutations in transgenic Big Blue mice carrying the lambdaLIZ phage shuttle vector. Big Blue C57/Bl male mice were treated with a single intraperitoneal dose of 80 mg/kg 3MC for 1, 3, 6, 14, or 30 days. Cell proliferation was checked by 5-bromo-2-deoxyuridine labeling and immunohistochemical detection. The maximal increase of the mitotic index was evidenced after 3 days (2.9 times the control value; P < 0.01). The relative nucleus area, reflecting the transcriptional activity, was also the highest in the treated group after 3 days: 1.86 times the control value, on average (P < 0.01). Four major DNA adducts, determined according to the [(32)P]-postlabeling method, were evidenced in liver DNA of treated mice, 6 days after the treatment: the spot intensities increased in a time-dependent manner. The mutant frequency of liver DNA was the highest after 14 days: 20.3 +/- 2.9 x 10(-5) in the treated vs. 7.6 +/- 2.7 x 10(-5) in the control mice (P < 0.01). Sequencing of the lambda lacI mutant plaques showed mainly G:C --> T:A and C:G --> A:T transversions. In conclusion, 3MC at first induced nuclear enlargement and a slight increase of cell proliferation in liver, followed by parallel formation of DNA adducts and mutations. This study shows how transgenic models allow in vivo evaluation of mechanistically simultaneous endpoints.

  6. 3-Methylcholanthrene/Aryl-Hydrocarbon Receptor-Mediated Hypertension Through eNOS Inactivation.

    PubMed

    Chang, Chih-Cheng; Hsu, Yung-Ho; Chou, Hsiu-Chu; Lee, Yuan-Chii G; Juan, Shu-Hui

    2017-05-01

    Endothelial nitric oxide synthase (eNOS) modulates vascular blood pressure and is predominantly expressed in endothelial cells and activated through the protein kinase B (Akt/PKB)-dependent pathway. We previously reported that 3-methylcholanthrene (3MC) activates the aryl hydrocarbon receptor (AhR) and reduces PI3K/Akt phosphorylation. This study investigated the mechanism underlying the downregulatory effects of 3-MC on nitric oxide (NO) production occurring through the AhR/RhoA/Akt-mediated mechanism. The mechanism underlying the effects of 3-MC on eNOS activity and blood pressure was examined in vitro and in vivo through genetic and pharmacological approaches. Results indicated that 3-MC modified heat shock protein 90 (HSP90), caveolin-1, dynein, and eNOS mRNA and protein expression through the AhR/RhoA-dependent mechanism in mouse cerebral vascular endothelial cells (MCVECs) and that 3-MC reduced eNOS phosphorylation through the AhR/RhoA-mediated inactivation of Akt1. The upregulation of dynein expression was associated with decreased eNOS dimer formation (eNOS dimer; an activated form of the enzyme). Coimmunoprecipitation assay results indicated that 3-MC significantly reduced the interaction between eNOS and its regulatory proteins, including Akt1 and HSP90, but increased the interaction between eNOS and caveolin-1. Immunofluorescence and Western blot analysis revealed that 3-MC reduced the amount of membrane-bound activated eNOS, and a modified Griess assay revealed that 3-MC concomitantly reduced NO production. However, simvastatin reduced 3-MC-mediated murine hypertension. Our study results indicate that AhR, RhoA, and eNOS have major roles in blood pressure regulation. Statin intervention might provide a potential therapeutic approach for reducing hypertension caused by 3-MC. J. Cell. Physiol. 232: 1020-1029, 2017. © 2016 Wiley Periodicals, Inc.

  7. 3-Methylcholanthrene (3-MC) and 4-chlorobiphenyl (PCB3) genotoxicity is gender-related in Fischer 344 transgenic rats.

    PubMed

    Jacobus, J A; Wang, B; Maddox, C; Esch, H; Lehmann, L; Robertson, L W; Wang, K; Kirby, P; Ludewig, G

    2010-11-01

    Polychlorinated biphenyls (PCBs) are a class of persistent organic pollutants with myriad biological effects, including carcinogenicity. We present data showing gender-specific genotoxicity in Fischer 344 transgenic BigBlue rodents exposed to 4-chlorobiphenyl (PCB3), a hydroxylated metabolite, and the positive control 3-methylcholanthrene (3-MC) where female rats are more resistant to the genotoxic effects of the test compounds compared to their male counterparts. This difference is further highlighted through our examination of gene expression, organ-specific weight changes, and tissue morphology. The purpose of the present study was to explore the complex and multifaceted issues of lower molecular weight PCBs as initiators of carcinogenesis, by examining the mutagenicity of PCB3, a hydroxylated metabolite (4'-OH-PCB3), and 3-methylcholanthrene (3-MC, positive control) in a transgenic rodent model. Previous findings indicated that PCB3 is mutagenic in the liver of male BigBlue transgenic rats under identical exposure conditions. We expected that female rats would be equally, if not more sensitive than male rats, since a 2-year carcinogenesis bioassay with Sprague-Dawley rats and commercial PCB mixtures reported much higher liver cancer rates in female than in male rats. The current study, however, revealed a similar trend in the mutation frequencies across all four treatment groups in females as reported previously in males, but increased variability among animals within each group and a lower overall effect, led to non significant differences in mutation frequencies. A closer analysis of the possible reasons for this negative result using microarray, organ weight and histology data comparisons shows that female Fischer 344 rats 1) had a higher baseline mutation frequency in the corn oil control group and greater variability than male rats; 2) responded with robust gene expression changes, which may also play a role in our observation of 3) highly increased liver

  8. 3-Methylcholanthrene (3-MC) and 4-Chlorobiphenyl (PCB3) genotoxicity is gender-related in Fischer 344 transgenic rats

    PubMed Central

    Jacobus, J.A.; Wang, B.; Maddox, C.; Esch, H.; Lehmann, L.; Robertson, L.W.; Wang, K.; Kirby, P.; Ludewig, G.

    2010-01-01

    Polychlorinated biphenyls (PCBs) are a class of persistent organic pollutants with myriad biological effects, including carcinogenicity. We present data showing gender-specific genotoxicity in Fischer 344 transgenic BigBlue rodents exposed to 4-chlorobiphenyl (PCB3), a hydroxylated metabolite, and the positive control 3-methylcholanthrene (3-MC) where female rats are more resistant to the genotoxic effects of the test compounds compared to their male counterparts. This difference is further highlighted through our examination of gene expression, organ-specific weight changes, and tissue morphology. The purpose of the present study was to explores the complex and multifaceted issues of lower molecular weight PCBs as initiators of carcinogenesis, by examining the mutagenicity of PCB3, a hydroxylated metabolite (4′-OH-PCB3), and 3-methylcholanthrene (3-MC, positive control) in a transgenic rodent model. Previous findings indicated that PCB3 is mutagenic in the liver of male BigBlue transgenic rats under identical exposure conditions. We expected that female rats would be equally, if not more sensitive than male rats, since a 2-year carcinogenesis bioassay with Sprague-Dawley rats and commercial PCB mixtures reported much higher liver cancer rates in female than in male rats. The current study, however, revealed a similar trend in the mutation frequencies across all four treatment groups in females as reported previously in males, but increased variability among animals within each group and a lower overall effect, led to non significant differences in mutation frequencies. A closer analysis of the possible reasons for this negative result using microarray, organ weight and histology data comparisons shows that female Fischer 344 rats 1) had a higher baseline mutation frequency in the corn oil control group and greater variability than male rats; 2) responded with robust gene expression changes, which may also play a role in our observation of 3) highly increased

  9. Simultaneous 3-methylcholanthrene and antioxidant induction enhance the expression of rat liver p-nitrophenol UDP-glucuronyltransferase

    SciTech Connect

    Ananaba, G.A.; Stewart, J.; Boston, C.; McCrary, J.

    1987-05-01

    To elucidate the gene source of the UDP-glucuronyltransferase (UDP-GT) family of enzymes, the authors have investigated the effects on the induction of p-nitrophenol UDP-GT by a combined treatment of rats with the carcinogen, 3-methylcholanthrene (3-MC) and the phenolic antioxidant, butylated hydroxyanisole (BHA). In this study, the elution profile of the enzyme was determined by ion exchange and affinity chromatography. P-nitrophenol UDP-GT activity was found to elute in the salt fractions of a 0-500 mM KCl gradient and required at least 5 mM UDP-glucuronic acid (UDPGA) to elute from the affinity column and has a M/sub r/ of 56,000. The results indicate that BHA + 3-MC is a more potent inducer of UDP-GT compared to either of the inducers used alone. For further investigation of the molecular basis of the enhanced induction, a cDNA library was generated from the BHA + 3-MC treated rat liver mRNA, and some mRNA from the same treatment was in vitro translated to establish the identities of the products compared to the in vivo translated proteins from the different treatments. The authors results suggest that the BHA and 3-MC induce the expression of different UDP-GT genes and the increased induction is due to these respective genes.

  10. Activation of H-ras oncogene in 3-methylcholanthrene-transformed human cell line.

    PubMed

    Rhim, J S; Fujita, J; Park, J B

    1987-08-01

    DNA prepared from the 3-methylcholanthrene (3MC)-transformed human 312H cell line induced foci on NIH/3T3 cells, whereas DNAs prepared from 7,12-dimethylbenz[a]-anthracene-transformed and the dimethylsulfoxide control 312H cell lines failed to induce foci. The transformed gene from the 3MC-transformed 312H cells was identified as an activated form of the human cellular transforming H-ras oncogene. Analysis of the ras oncogene p21 product in this transformant by immunoprecipitation and gel electrophoresis suggested that this gene was activated by mutation in the 61st codon. These findings demonstrate that activation of a member of the ras gene family can occur in a chemically transformed human cell line.

  11. Regulation of nonmuscle myosin II during 3-methylcholanthrene induced dedifferentiation of C2C12 myotubes

    SciTech Connect

    Dey, Sumit K.; Saha, Shekhar; Das, Provas; Das, Mahua R.; Jana, Siddhartha S.

    2014-08-01

    3-Methylcholanthrene (3MC) induces tumor formation at the site of injection in the hind leg of mice within 110 days. Recent reports reveal that the transformation of normal muscle cells to atypical cells is one of the causes for tumor formation, however the molecular mechanism behind this process is not well understood. Here, we show in an in vitro study that 3MC induces fragmentation of multinucleate myotubes into viable mononucleates. These mononucleates form colonies when they are seeded into soft agar, indicative of cellular transformation. Immunoblot analysis reveals that phosphorylation of myosin regulatory light chain (RLC{sub 20}) is 5.6±0.5 fold reduced in 3MC treated myotubes in comparison to vehicle treated myotubes during the fragmentation of myotubes. In contrast, levels of myogenic factors such as MyoD, Myogenin and cell cycle regulators such as Cyclin D, Cyclin E1 remain unchanged as assessed by real-time PCR array and reverse transcriptase PCR analysis, respectively. Interestingly, addition of the myosin light chain kinase inhibitor, ML-7, enhances the fragmentation, whereas phosphatase inhibitor perturbs the 3MC induced fragmentation of myotubes. These results suggest that decrease in RLC{sub 20} phosphorylation may be associated with the fragmentation step of dedifferentiation. - Highlights: • 3-Methylcholanthrene induces fragmentation of C2C12-myotubes. • Dedifferentiation can be divided into two steps – fragmentation and proliferation. • Fragmentation is associated with rearrangement of nonmuscle myosin II. • Genes associated with differentiation and proliferation are not altered during fragmentation. • Phosphorylation of myosin regulatory light chain is reduced during fragmentation.

  12. Enantioselective aliphatic hydroxylations of racemic 1-hydroxy-3-methylcholanthrene by rat liver microsomes.

    PubMed

    Shou, M G; Yang, S K

    1990-01-01

    Enantiomeric pairs of 1-hydroxy-3-hydroxymethylcholanthrene (1-OH-3-OHMC), 3-methylcholanthrene (3MC) trans- and cis-1,2-diols, and 1-hydroxy-3-methylcholanthrene (1-OH-3MC) were resolved by HPLC using a covalently bonded (R)-N-(3,5-dinitrobenzoyl)phenylglycine chiral stationary phase (Pirkle type 1A) column. The absolute configuration of an enantiomeric 3MC trans-1,2-diol was established by the exciton chirality CD method following conversion to a bis-p-N,N-dimethylaminobenzoate. Incubation of an enantiomeric 1-OH-3MC with rat liver microsomes resulted in the formation of enantiomeric 3MC trans- and cis-1,2-diols; the absolute configurations of the enantiomeric 1-OH-3MC and 3MC cis-1,2-diol were established on the basis of the absolute configuration of an enantiomeric 3MC trans-1,2-diol. Absolute configurations of enantiomeric 1-OH-3-OHMC were determined by comparing their CD spectra with those of enantiomeric 1-OH-3MC. The relative amount of three aliphatic hydroxylation products formed by rat liver microsomal metabolism of racemic 1-OH-3MC was 1-OH-3-OHMC greater than 3MC cis-1,2-diol greater than 3MC trans-1,2-diol. Enzymatic hydroxylation at C2 of racemic 1-OH-3MC was enantioselective toward the 1S-enantiomer over the 1R-enantiomer (approximately 3/1); hydroxylation at the C3-methyl group was enantioselective toward the 1R-enantiomer over the 1S-enantiomer (approximately 58/42). Rat liver microsomal C2-hydroxylation of racemic 1-OH-3MC resulted in a 3MC trans-1,2-diol with a (1S,2S)/(1R,2R) ratio of 63/37 and a 3MC cis-1,2-diol with a (1S,2R)/(1R,2S) ratio of 12/88, respectively.

  13. STRAIN-SPECIFIC SENSITIVITY TO INDUCTION OF MURINE LUNG TUMORS FOLLOWING IN UTERO EXPOSURE TO 3-METHYLCHOLANTHRENE

    EPA Science Inventory

    We previously demonstrated that different strains of fetal mice were more sensitive to lung tumor induction by 3-methylcholanthrene (MC) than were adults. Offspring from either a D2 x B6D2F1 backcross or from parental Balb/c mice exhibited a similar high incidence of lung tumors ...

  14. STRAIN-DEPENDENT SUSCEPTIBILITY TO TRANSPLACENTALLY-INDUCED MURINE LUNG TUMORS AND DNA ADDUCTS OF 3-METHYLCHOLANTHRENE

    EPA Science Inventory

    Strain-dependent susceptibility to transplacentally-induced murine lung tumors and DNA adducts of 3methylcholanthrene G B Nelson, J A Ross, J E Moore, M Xu, N D Kock, M S Miller Wake Forest University, Winston-Salem, NC and USEPA, Research Triangle Park, NC.

    It has been de...

  15. 1-Hydroxy- and 2-hydroxy-3-methylcholanthrene: regioselective and stereoselective formations in the metabolism of 3-methylcholanthrene and enantioselective disposition in rat liver microsomes.

    PubMed

    Shou, M; Yang, S K

    1990-06-01

    Absolute configurations of enantiomeric 1-hydroxy-3-methylcholanthrene (1-OH-3MC) and 2-hydroxy-3-methyl-cholanthrene (2-OH-3MC) were determined by the exciton chirality circular dichroism (CD) method as their p-nitrobenzoate derivatives. Enantiomers of 1-OH-3MC were resolved by HPLC using a column packed with chiral stationary phase (CSP) (R)-N-(3,5-dinitrobenzoyl)phenylglycine covalently bonded to gamma-aminopropylsilanized silica. Enantiomers of 2-OH-3MC were resolved as diastereomeric (-)-methoxyacetates by normal-phase HPLC. 1-OH-3MC and 2-OH-3MC, formed in the metabolism of 3MC by liver microsomes from untreated, phenobarbital (PB)-treated and 3MC-treated male Sprague-Dawley rats, were first isolated as a mixture by reversed-phase HPLC and subsequently separated by normal-phase HPLC. Concentration ratios of [1-OH-3MC]:[2-OH-3MC] formed in the metabolism of 3MC by three rat liver microsomal preparations (at 0.5 mg protein per ml of incubation mixture and an incubation time of 10 min) were found to be: 30:70 (control), 21:79 (PB treated) and 10:90 (3MC treated) respectively. R/S enantiomer ratios of 1-OH-3MC formed in the metabolism of 3MC by three rat liver microsomal preparations were determined by CSP HPLC: 35:65 (control), 39:61 (PB treated) and 46:54 (3MC treated) respectively. R/S enantiomer ratios of 2-OH-3MC formed in the metabolism of 3MC by three rat liver microsomal preparations were determined by CD spectral data: 14:86 (control), 6:94 (PB treated) and 6:94 (3MC treated) respectively. Metabolism of racemic 1-OH-3MC and 2-OH-3MC by all three rat liver microsomal preparations was found to be substrate enantioselective; the rate of 1S-OH-3MC metabolism was faster than that of 1R-OH-3MC, whereas the rate of 2R-OH-3MC metabolism was faster than that of 2S-OH-3MC.

  16. Acute exposure to 3-methylcholanthrene induces hepatic oxidative stress via activation of the Nrf2/ARE signaling pathway in mice.

    PubMed

    Jin, Yuanxiang; Miao, Wenyu; Lin, Xiaojian; Pan, Xiuhong; Ye, Yang; Xu, Minjie; Fu, Zhengwei

    2014-12-01

    Polycyclic aromatic hydrocarbons (PAHs) are the most common contaminants in the environment. The primary focus on the toxicity of PAHs is their ability to activate the aryl hydrocarbon receptor (AhR)-mediated pathway and lead to carcinogenesis in different organisms. However, the influence of PAHs on the antioxidant system in mammalian systems has received only limited attention. In the present study, we observed that the intraperitoneal injection of 100 mg/kg 3-methylcholanthrene (3MC) into mice significantly increased reactive oxygen species (ROS) levels and malondialdehyde (MDA) contents and decreased glutathione (GSH) contents and the activity of total antioxidant capacity (T-AOC), indicating that serious oxidative stress had been induced in the liver of mice. Then, the oxidative stress signal activated the nuclear factor erythroid 2-related factor 2/antioxidant response element (Nrf2/ARE) pathway by enhancing the mRNA levels of Nrf2, p38, and Erk2. Moreover, the mRNA levels of Nrf2/ARE target genes, including glutathione peroxidase (Gpx), glutathione reductase (GR), glutathione synthetase (GS), NAD(P)H: quinone oxidoreductase 1 (Nqo1), superoxide dismutase 1 (Sod1), and Sod2, increased significantly after treatment with 3MC for 24 hours. The hepatic levels of NQO1 and the activities of GR and GS were also significantly enhanced at 24 hours after 3MC treatment. Because the expression of NQO1 is co-regulated by Nrf2/ARE and AhR/XRE in mammalian tissues, NQO1 may play an important role in protecting against the oxidative stress induced by 3MC. Taken together, our findings suggested that acute exposure to 3MC altered the cellular redox balance in hepatocytes to trigger Nrf2-regulated antioxidant responses, which may represent an adaptive cell defense mechanism against the oxidative stress induced by PAHs.

  17. The systemic and gonadal toxicity of 3-methylcholanthrene is prevented by daily administration of α-naphthoflavone.

    PubMed

    Rhon-Calderón, Eric Alejandro; Galarza, Rocío Alejandra; Lomniczi, Alejandro; Faletti, Alicia Graciela

    2016-04-15

    In the present study, we investigated the effect of 3-methylcholanthrene (3MC) on sexual maturity and the ability of α-naphthoflavone (αNF) to prevent this action. To this end, immature rats were daily injected intraperitoneally with 3MC (0.1 or 1mg/kg) and/or αNF (80mg/kg). Body weight, vaginal opening and estrous cycle were recorded and ovaries were obtained on the day of estrus. Ovarian weight, ovulation rate (measured by the number of oocytes within oviducts), and follicular development (determined by histology) were studied. No differences were found in body weight, ovarian weight, day of vaginal opening, or the establishment of the estrous cycle among the different groups of rats. However, animals treated with 3MC, at both doses, exhibited a lower number of primordial, primary, preantral and antral follicles than controls. Also, 3MC inhibited the ovulation rate and induced an overexpression of both the Cyp1a1 and Cyp1b1 genes, measured by chromatin immunoprecipitation assay. The daily treatment with αNF alone increased the number of follicles in most of the stages analyzed when compared with controls. Moreover, the αNF treatment prevented completely not only the 3MC-induced decrease in all types of follicles but also the 3MC-induced overexpression of Cyp enzymes and the genetic damage in bone marrow cells and oocytes. These results suggest that (i) daily exposure to 3MC during the pubertal period destroys the follicle reserve and alters the ovulation rate; (ii) the 3MC action seems to be mediated by an aryl hydrocarbon receptor-dependent mechanism; (iii) daily administration of αNF has a clear stimulatory action on the ovarian function; and (iv) αNF may prevent both the systemic and gonadal 3MC-induced toxicity.

  18. Suppressive effects of 3-methylcholanthrene on the in vitro antitumor activity of naturally cytotoxic cells

    SciTech Connect

    Lill, P.H.; Gangemi, D.

    1986-01-01

    Transient suppression of splenic natural killer (NK), natural cytotoxic (NC) and peritoneal macrophage cytotoxicity was observed following a single injection of 3-methylcholanthrene (3-MC) into C3H/HeN mice. Natural killer cell activity was depressed by 30-60% 4-6 d after injection of 1.0 mg 3-MC. Levels of NK reactivity returned to normal 8 d post 3-MC injection, and no suppression of natural killing was seen when tested 6 wk after 3-MC treatment. 3-MC did not affect propionibacterium acnes augmentation of NK cell activity when tested both 6 d and 6 wk after carcinogen injection. The results indicate that the observed suppression of naturally cytotoxic cells may not be important in allowing 3-MC-induced tumors to grow, since suppression is not long-lasting. Therefore, any effect on tumor growth mediated by a suppression of naturally cytotoxic cells would have to be exerted at the earliest stages of tumor development.

  19. Regulation of nonmuscle myosin II during 3-methylcholanthrene induced dedifferentiation of C2C12 myotubes.

    PubMed

    Dey, Sumit K; Saha, Shekhar; Das, Provas; Das, Mahua R; Jana, Siddhartha S

    2014-08-01

    3-Methylcholanthrene (3MC) induces tumor formation at the site of injection in the hind leg of mice within 110 days. Recent reports reveal that the transformation of normal muscle cells to atypical cells is one of the causes for tumor formation, however the molecular mechanism behind this process is not well understood. Here, we show in an in vitro study that 3MC induces fragmentation of multinucleate myotubes into viable mononucleates. These mononucleates form colonies when they are seeded into soft agar, indicative of cellular transformation. Immunoblot analysis reveals that phosphorylation of myosin regulatory light chain (RLC20) is 5.6±0.5 fold reduced in 3MC treated myotubes in comparison to vehicle treated myotubes during the fragmentation of myotubes. In contrast, levels of myogenic factors such as MyoD, Myogenin and cell cycle regulators such as Cyclin D, Cyclin E1 remain unchanged as assessed by real-time PCR array and reverse transcriptase PCR analysis, respectively. Interestingly, addition of the myosin light chain kinase inhibitor, ML-7, enhances the fragmentation, whereas phosphatase inhibitor perturbs the 3MC induced fragmentation of myotubes. These results suggest that decrease in RLC20 phosphorylation may be associated with the fragmentation step of dedifferentiation.

  20. 3-methylcholanthrene induces differential recruitment of aryl hydrocarbon receptor to human promoters.

    PubMed

    Safe, Stephen

    2010-09-01

    The paper by Pansoy and coworkers investigates the effects of the aryl hydrocarbon receptor (AHR) ligand 3-methylcholanthrene (3MC) on recruitment of the AHR complex to human promoters in T47D breast cancer cells. The results are particularly important because they can be compared with a prior study using the potent AHR ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the same cell line. The chromatin immunoprecipitation and promoter-focused microarrays (ChIP-chip) demonstrated that after treatment of T47D cells with 1microM 3MC, there were 241 AHR-3MC bound regions and many of these contained AHR-responsive elements. However, they also observed interactions with regions that do not contain these responsive elements, and subsequent analysis of selected target genes show that 3MC-dependent AHR binding did not necessarily predict Ah-responsiveness because induction, repression, and no effects were observed. A prior study with TCDD demonstrated that both 3MC and TCDD induced AHR binding to 127 common regions; however, there were significant differences in ligand (3MC vs. TCDD)-dependent AHR bound regions. The results illustrate the complexity of AHR signaling and also demonstrate that compared with TCDD as a reference ligand, 3MC is a selective AHR modulator.

  1. Spontaneous appearance of uterine tumors in vehicle and 3-methylcholanthrene-treated Wistar rats.

    PubMed

    Anger, Dana L; Crankshaw, Denis J; Foster, Warren G

    2006-11-01

    During the conduct of a study designed to determine the effect of 3-methylcholanthrene (3-MC), a synthetic polycyclic aromatic hydrocarbon (PAH) that acts through the aryl hydrocarbon receptor (AhR), on uterine contractility in Wistar rats, uterine tumors were identified in both vehicle and 3-MC-treated animals. The objective of the current study was to describe the histological characteristics of these tumors. Sexually mature female rats (110 days old) were treated with 70 micro mol/kg 3-MC or vehicle (olive oil) for 4 days and euthanized by exsanguination. At necropsy uterine tumors were unexpected findings in two vehicle and four 3-MC-treated rats. The tumors appeared as multiple unilateral or bilateral subserosal nodes. No tumors were found in other tissues on gross inspection. Prior to necropsy, tumor-presenting animals were acyclic and arrested in a state of persistent proestrus. Haematoxylin and eosin staining of tumor sections revealed nests of acidophilic granule-containing cells within a highly vascular stroma of the uterine wall below the muscularis. Positive periodic acid Schiff (PAS) staining suggested the presence of glycogen or glycophospholipids within these granules, however, negative PAS diastase staining indicated that the acidophilic bodies were not composed of glycogen. The tumors are histologically similar to human dysgerminomas. We conclude that these tumors are unrelated to treatment and are of a granular type not previously documented in Wistar rats.

  2. Proteomic Analysis of Serum in Lung Cancer Induced by 3-Methylcholanthrene

    PubMed Central

    Li, Minhua; Ye, Bo; Zhang, Yuxia; Chen, Honglei; Xia, Dong; Liu, Mingqiu; Yang, Fei

    2009-01-01

    Lung cancer remains the leading cause of cancer-related mortality worldwide. Early detection of lung cancer is problematic due to the lack of a marker with high diagnosis sensitivity and specificity. To determine the differently expressed proteins in the serum of lung cancer and figure out the function of the proteins, two-dimensional electrophoresis (2DE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) were used to screen the serum proteins of lung cancer model induced by 3-methylcholanthrene (MCA). From optimized 2DE image, 455 spots in the normal sera and 716 spots in the lung cancers sera were detected. Among them, 141 protein spots were differentially expressed when comparing the serum from normal rat and serum from lung cancer model, including 82 overexpressed proteins and 59 underexpressed proteins. Changes of haptoglobin, transthyretin, and TNF superfamily member 8 (TNFRS8) were confirmed in sera from lung cancer by MALDI-TOF-MS. Proteomics technology leads to identify changes of haptoglobin, transthyretin, and TNFRS8 in serum of rat lung cancer model and represents a powerful tool in searching for candidate proteins as biomarkers. PMID:19794824

  3. Metabolism of 2S-hydroxy-3-methylcholanthrene by rat liver microsomes.

    PubMed

    Shou, M G; Yang, S K

    1990-11-01

    Products formed in the metabolism of 2S-hydroxy-3-methylcholanthrene (2S-OH-3MC) by liver microsomes prepared from phenobarbital-treated rats were isolated by sequential use of reversed-phase and normal-phase HPLC. Metabolites of 2S-OH-3MC were characterized by UV-visible absorption, mass and circular dichroic spectra, and chiral stationary phase HPLC analyses. The metabolites that had been identified were 2S-hydroxy-3-hydroxymethylcholanthrene (2S-OH-3-OHMC), 3MC-2-one, 3MC-2-one 9,10-dihydrodiol, 8-hydroxy-2S-OH-3MC, a pair of stereoisomers 3MC (trans)-1R,2R-diol and (cis)-1S,2R-diol in a ratio of approximately 11:89, a pair of diastereomers 2S-OH-3MC 9R,10R-dihydrodiol and 2S-OH-3MC 9S,10S-dihydrodiol in a ratio of approximately 9:1, and a pair of diastereomers 2S-OH-3MC 11R,12R-dihydrodiol and 2S-OH-3MC 11S,12S-dihydrodiol in a ratio of approximately 77:23. A few tentatively identified minor metabolites were 3-OHMC trans-1R,2R-diol, 10-hydroxy-2S-OH-3MC, a 9,10-dihydrodiol derived from 3MC cis-1S,2R-diol, and a 11,12-dihydrodiol and two diastereomeric 9,10-dihydrodiols derived from 2S-OH-3-OHMC. Since the racemic 2-OH-3MC is a known potent carcinogen and 2S-OH-3MC is the most abundant metabolite of 3MC, some of the 2S-OH-3MC metabolites identified in this study may be further converted to proximate and ultimate carcinogens which may contribute to the overall carcinogenicity exhibited by 3MC.

  4. Aryl hydrocarbon receptor-independent activation of estrogen receptor-dependent transcription by 3-methylcholanthrene.

    PubMed

    Shipley, Jonathan M; Waxman, David J

    2006-06-01

    Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that stimulates transcription directed by xenobiotic response elements upstream of target genes. Recently, AhR ligands were reported to induce formation of an AhR-estrogen receptor (ER) complex, which can bind to estrogen response elements (EREs) and stimulate transcription of ER target genes. Presently, we investigate the effect of the AhR ligands 3-methylcholanthrene (3MC), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 3,3',4,4',5-pentachlorobiphenyl (BZ126) on ERE-regulated luciferase reporter activity and endogenous ER target gene expression. In MCF-7 human breast cancer cells, 3MC induced transcription of ER reporter genes containing native promoter sequences of the ER-responsive genes complement 3 and pS2 and heterologous promoters regulated by isolated EREs. Dose-response studies revealed that the concentration of 3MC required to half-maximally activate transcription (EC(50)) was >100-fold higher for an ER reporter (27-57 muM) than for an AhR reporter (86-250 nM) in both MCF-7 cells and in human endometrial cancer Ishikawa cells. 3MC also stimulated expression of the endogenous ER target genes amphiregulin, cathepsin D and progesterone receptor, albeit to a much lower extent than was achieved following stimulation with 17beta-estradiol. In Ishikawa cells, 3MC, but not BZ126 or TCDD, stimulated ERalpha-dependent reporter activity but did not induce expression of endogenous ER target genes. Finally, studies carried out in the AhR-positive rat hepatoma cell line 5L and the AhR-deficient variant BP8 demonstrated that ER reporter activity could be induced by 3MC in a manner that was independent of AhR and thus distinct from the AhR-ER 'hijacking' mechanism described recently. 3MC may thus elicit estrogenic activity by multiple mechanisms.

  5. 3-methylcholanthrene induces differential recruitment of aryl hydrocarbon receptor to human promoters.

    PubMed

    Pansoy, Andrea; Ahmed, Shaimaa; Valen, Eivind; Sandelin, Albin; Matthews, Jason

    2010-09-01

    The aryl hydrocarbon receptor (AHR) is a ligand-activated protein that mediates the toxic actions of polycyclic aromatic and halogenated compounds. Identifying genes directly regulated by AHR is important in understanding the pathways regulated by this receptor. Here we used the techniques of chromatin immunoprecipitation and DNA microarrays (ChIP-chip) to detect AHR-bound genomic regions after 3-methylcholanthrene (3MC) treatment of T-47D human breast cancer cells. We identified 241 AHR-3MC-bound regions, and transcription factor-binding site analysis revealed a strong overrepresentation of the AHR-responsive element. Conventional ChIP confirmed recruitment of AHR to 26 regions with target gene responses to 3MC varying from activation to inhibition to having no effect. A comparison of identified AHR-3MC-bound regions with AHR-2,3,7,8-tetrchlorodibenzo-p-dioxin (TCDD)-bound regions from our previous study (Ahmed, S., Valen, E., Sandelin, A., and Matthews, J. (2009). Toxicol. Sci. 111, 254-266) revealed that 127 regions were common between the data sets. Time course ChIPs for six of the regions showed that 3MC induced gene-specific changes in histone H3 acetylation and methylation and induced differential oscillatory binding of AHR, with a periodicity between 1.5 and 2 h. Re-treatment of cells with 3MC failed to alter the oscillatory binding profiles of AHR or aryl hydrocarbon receptor nuclear translocator. Cells became responsive to 3MC but not TCDD after 24 h of exposure to 3MC, highlighting important differences in AHR responsiveness between the two ligands. Our results reveal a number of novel AHR-bound promoter regions and target genes that exhibit differential kinetic binding profiles and regulation by AHR.

  6. Effect of hypoxia alone or combined with inflammation and 3-methylcholanthrene on hepatic cytochrome P450 in conscious rabbits.

    PubMed

    Kurdi, J; Maurice, H; El-Kadi, A O; Ong, H; Dalkara, S; Bélanger, P M; Souich, P

    1999-09-01

    1 To investigate the effect of moderate hypoxia alone or combined with an inflammatory reaction or after 3-methylcholanthrene (3MC) pre-treatment on cytochrome P450 (P450), conscious rabbits were exposed for 24 h to a fractional concentration of inspired O2 of 10% (mean PaO2 of 34 mmHg). Hypoxia decreased theophylline metabolic clearance (ClM) from 1.73+/-0.43 to 1.48+/-0.13 ml min-1 kg-1 (P<0. 05), and reduced (P<0.05) the formation clearance of theophylline metabolites, 3-methylxanthine (3MX), 1-methyluric acid (1MU) and 1,3-dimethyluric acid (1,3DMU). Hypoxia reduced the amount of CYP1A1 and 1A2 but increased CYP3A6 proteins. 2 Turpentine-induced inflammatory reaction reduced (P<0.05) the formation clearance of 3MX, 1MU, and 1,3DMU, and diminished the amount of CYP1A1, 1A2 and 3A6 proteins. However, when combined with hypoxia, inflammation partially prevented the decrease in ClM, especially by impeding the reduction of 1,3DMU. The amount of CYP1A1 and 1A2 remained reduced but the amount of CYP3A6 protein returned to normal values. 3 Pre-treatment with 3MC augmented the ClM by 114% (P<0.05) due to the increase in the formation clearance of 3MX, 1MU and 1,3DMU. 3MC treatment increased the amount of CYP1A1 and 1A2 proteins. Pre-treatment with 3MC prevented the hypoxia-induced decrease in amount and activity of the P450. 4 It is concluded that acute moderate hypoxia and an inflammatory reaction individually reduce the amount and activity of selected apoproteins of the P450. However, the combination of hypoxia and the inflammatory reaction restores P450 activity to near normal values. On the other hand, pre-treatment with 3MC prevents the hypoxia-induced depression of the P450.

  7. Toxoplasma gondii Inhibits gamma interferon (IFN-γ)- and IFN-β-induced host cell STAT1 transcriptional activity by increasing the association of STAT1 with DNA.

    PubMed

    Rosowski, Emily E; Nguyen, Quynh P; Camejo, Ana; Spooner, Eric; Saeij, Jeroen P J

    2014-02-01

    The gamma interferon (IFN-γ) response, mediated by the STAT1 transcription factor, is crucial for host defense against the intracellular pathogen Toxoplasma gondii, but prior infection with Toxoplasma can inhibit this response. Recently, it was reported that the Toxoplasma type II NTE strain prevents the recruitment of chromatin remodeling complexes containing Brahma-related gene 1 (BRG-1) to promoters of IFN-γ-induced secondary response genes such as Ciita and major histocompatibility complex class II genes in murine macrophages, thereby inhibiting their expression. We report here that a type I strain of Toxoplasma inhibits the expression of primary IFN-γ response genes such as IRF1 through a distinct mechanism not dependent on the activity of histone deacetylases. Instead, infection with a type I, II, or III strain of Toxoplasma inhibits the dissociation of STAT1 from DNA, preventing its recycling and further rounds of STAT1-mediated transcriptional activation. This leads to increased IFN-γ-induced binding of STAT1 at the IRF1 promoter in host cells and increased global IFN-γ-induced association of STAT1 with chromatin. Toxoplasma type I infection also inhibits IFN-β-induced interferon-stimulated gene factor 3-mediated gene expression, and this inhibition is also linked to increased association of STAT1 with chromatin. The secretion of proteins into the host cell by a type I strain of Toxoplasma without complete parasite invasion is not sufficient to block STAT1-mediated expression, suggesting that the effector protein responsible for this inhibition is not derived from the rhoptries.

  8. Comparison of the peptide map and functional properties of monooxygenases induced by 3-methylcholanthrene and. beta. -naphthoflavone

    SciTech Connect

    Chasovnikova, O.B.; Mishin, V.M.; Tsyrlov, I.B.

    1987-02-20

    The similarity of the catalytic, spectral, electrophoretic, and immunochemical properties of microsomal cytochromes P-448 (molecular weight 56,000), synthesized de novo after administration of 3-methylcholanthrene and ..beta..-naphthoflavone to rats, was demonstrated. The identity of the peptide maps of the microsomal and isolated cytochrome P-448 is evidence of adequacy of the method of limited proteolysis for establishing the homogeneity and comparing the structure of the microsomal hemoproteins. The data obtained substantiate the approach for the study of the similarity and differences in the structure and enzymatic activity of various forms of monooxygenases without their preliminary isolation from the microsomal membrane.

  9. Induction of hepatic cytochrome P-450 activity in wild cotton rats (Sigmodon hispidus) by phenobarbital and 3-methylcholanthrene

    SciTech Connect

    Elangbam, C.S.; Qualls, C.W.,Jr.; Bauduy, M. )

    1989-05-01

    Wild cotton rats (Sigmodon hispidus) are ubiquitous throughout the Southeast quadrant of the United States, easy to capture, have a generation interval of less than one year and a limited range of movement (less than one hectare). This species may prove to be an excellent model for monitoring environmental contamination. Traditionally, cytochrome P-450 inducing agents are grouped into two classes. One, represented by phenobarbital, induces P-450b and P-450e; the other, represented by 3-methylcholanthrene, induces P-450c and P-450d isoenzymes. The types and amounts of cytochrome P-450 vary among species, organs, health status, sex, and stress of the animal. If the levels of cytochrome P-450 of wild cotton rats are to be used in monitoring environmental pollution, it is necessary to characterize the inducibility and concentration of cytochrome P-450 in this species. This study was designed to determine the concentration and inducibility of cytochrome P-450 in the livers of cotton rats after intraperitoneal (ip) administration of phenobarbital and 3-methylcholanthrene.

  10. Expression of cytochromes P-450 in rat hepatoma cells. Analysis by monoclonal antibodies specific for cytochromes P-450 from rat liver induced by 3-methylcholanthrene or phenobarbital.

    PubMed

    Wiebel, F J; Park, S S; Kiefer, F; Gelboin, H V

    1984-12-17

    We have studied the expression of aldrin eposidase (AE), 7-ethoxycoumarin-O-deethylase (ECDE), and aryl hydrocarbon (benzo[a]pyrene) hydroxylase (AHH) in nine differentiated or dedifferentiated cell lines derived from H4IIEC3 rat hepatoma cells. The nature of the cytochromes P-450 mediating AE, ECDE and AHH activities was analysed using monoclonal antibodies (MAb) made to the major 3-methylcholanthrene-induced cytochrome P-450 (MAb-MC) or phenobarbital-induced cytochrome P-450 (MAb-PB) from rat liver. The cells were treated with 5 microM dexamethasone for 30 h to increase the levels of the monoxygenase activities. (a) The six differentiated cell lines examined (Faza967, Fao, HF1-4, 2sFou, C2Rev7, and H4IIEC3/G-) contained MAb-PB-sensitive AE comprising 30-75% of the total AE activity. In most of these cell lines MAb-PB also markedly inhibited ECDE; however, the antibody had a considerably weaker effect on AHH. (b) MAb-PB-sensitive AHH, ECDE and AE activities were also observed in untreated and phenobarbital-treated cells. (c) MAb-MC inhibited AHH and ECDE in the two dedifferentiated lines HF1 and H5 by 50-80%. The antibody also inhibited AHH activities in the poorly differentiated line H4IIEC3/T and in the majority of the differentiated lines by 40-65%. MAb-MC-sensitive AHH was found in Fao cells after treatment with benz[a]anthracene but induced AHH in H4IIEC3/T, H4IIEC3/G-, and 2sFou cells 20-30-fold and in Faza967 and Fao cells 3-5-fold. Benz[a]anthracene remained without effect on AHH activity in C2Rev7 cells. The results show that the hepatoma cells examined express to various degrees phenobarbital-inducible cytochrome P-450 and/or 3-methylcholanthrene-inducible cytochrome P-450. These cell lines are versatile tools for studying the regulation of monooxygenase activities and analysing their role in the activation and inactivation of xenobiotics such as carcinogens, drugs and pesticides.

  11. Modulation of 3-methylcholanthrene toxicity in cultured neoplastic keratinocytes by glucocorticoids and retinoids is not accounted for by macromolecular adduct formation.

    PubMed Central

    Rubin, A L; Rice, R H

    1989-01-01

    3-Methylcholanthrene (3-MC) greatly inhibits the growth of two lines of human squamous carcinoma cells, SCC-9 and SCC-12B2. Exposure of the cells to 2,3,7,8-tetrachlorodibenzo-p-dioxin alone was much less effective and, in the presence of 3-MC, did not alter the sensitivity (EC50 = 0.3 microM) or extent of growth inhibition by the latter. The degree of 3-MC-mediated inhibition, however, was markedly alleviated by inclusion of retinoic acid (EC50 greater than or equal to 0.7 microM) and hydrocortisone (EC50 = 40 nM) or dexamethasone (EC50 = 3 nM) in the culture medium. These physiological effectors, which are known to have opposing actions on keratinocyte character in SCC cells, did not significantly alter either aryl hydrocarbon hydroxylase activity or macromolecular adduct formation. Further analysis of the cellular responses indicated that hydrocortisone and, in some experiments, retinoids increased the growth rate in 3-MC-exposed cultures, while 3-MC increased the saturation density in retinoic acid-exposed cultures, an example of interference with a physiological response of the cells. These results indicate that alteration of the differentiated state, regardless of the direction of the change, can alter the sensitivity of the cells to toxic stimuli. Further investigation of the bases of such toxic responses and their modulation by the microenvironment may enhance our understanding of the target cell specificity of polycyclic aromatic hydrocarbons. Images PMID:2468166

  12. Aryl-hydrocarbon receptor-dependent alteration of FAK/RhoA in the inhibition of HUVEC motility by 3-methylcholanthrene.

    PubMed

    Chang, Chih-Cheng; Tsai, Shih-Ying; Lin, Heng; Li, Hsiao-Fen; Lee, Yi-Hsuan; Chou, Ying; Jen, Chih-Yu; Juan, Shu-Hui

    2009-10-01

    We previously demonstrated the antiproliferative and antiangiogenic effects of 3-methylcholanthrene (3MC), an aryl-hydrocarbon receptor (AhR) agonist, in human umbilical vascular endothelial cells (HUVECs). Herein, we unraveled its molecular mechanisms in inhibiting HUVEC motility. 3MC down-regulated FAK, but up-regulated RhoA, which was rescued by AhR knockdown. It led us to identify novel AhR binding sites in the FAK/RhoA promoters. Additionally, 3MC increased RhoA activity via suppression of a negative feedback pathway of FAK/p190RhoGAP. With an increase in membrane-bound RhoA, subsequent stress fiber and focal adhesion complex formation was observed in 3MC-treated cells, and this was reversed by a RhoA inhibitor and AhR antagonists. Notably, these compounds significantly reversed 3MC-mediated anti-migration in a transwell assay. The in vitro findings were further confirmed using an animal model of Matrigel formation in Balb/c mice. Collectively, AhR's genomic regulation of FAK/RhoA, together with RhoA activation, is ascribable to the anti-migration effect of 3MC in HUVECs.

  13. Induction of 26S proteasome subunit PSMB5 by the bifunctional inducer 3-methylcholanthrene through the Nrf2-ARE, but not the AhR/Arnt-XRE, pathway

    SciTech Connect

    Kwak, Mi-Kyoung . E-mail: mkwak@yumail.ac.kr; Kensler, Thomas W.

    2006-07-14

    The 26S proteasome is responsible for degradation of abnormal intracellular proteins, including oxidatively damaged proteins and may play a role as a component of a cellular antioxidative system. However, little is known about regulation of proteasome expression. In the present study, regulation of proteasome expression by the bifunctional enzyme inducer and a specific signaling pathway for this regulation were investigated in murine neuroblastoma cells. Expression of catalytic core subunits including PSMB5 and peptidase activities of the proteasome were elevated following incubation with 3-methylcholanthrene (3-MC). Studies using reporter genes containing the murine Psmb5 promoter showed that transcriptional activity of this gene was enhanced by 3-MC. Overexpression of AhR/Arnt did not affect activation of the Pmsb5 promoter by 3-MC and deletion of the xenobiotic response elements (XREs) from this promoter exerted modest effects on inducibility in response to 3-MC. However, mutation of the proximal AREs of the Psmb5 promoter largely abrogated its inducibility by 3-MC. In addition, this promoter showed a blunted response toward 3-MC in the absence of nrf2; 3-MC incubation increased nuclear levels of Nrf2 only in wild-type cells. Collectively, these results indicate that expression of proteasome subunit PSMB5 is modulated by bifunctional enzyme inducers in a manner independent of the AhR/Arnt-XRE pathway but dependent upon the Nrf2-ARE pathway.

  14. IFNincreases efficiency of DNA vaccine in protecting ducks against infection

    PubMed Central

    Long, Jian-Er; Huang, Li-Na; Qin, Zhi-Qiang; Wang, Wen-Yi; Qu, Di

    2005-01-01

    AIM: To detect the effects of DNA vaccines in combination with duck IFN-γ gene on the protection of ducks against duck hepatitis B virus (DHBV) infection. METHODS: DuIFN-γ cDNA was cloned and expressed in COS-7 cells, and the antiviral activity of DuIFN-γ was detected and neutralized by specific antibodies. Ducks were vaccinated with DHBpreS/S DNA alone or co-immunized with plasmid expressing DuIFN-γ. DuIFN-γ mRNA in peripheral blood mononuclear cells (PBMCs) from immunized ducks was detected by semi-quantitative competitive RT-PCR. Anti-DHBpreS was titrated by enzyme-linked immunosorbent assay (ELISA). DHBV DNA in sera and liver was detected by Southern blot hybridization, after ducks were challenged with high doses of DHBV. RESULTS: DuIFN-γ expressed by COS-7 was able to protect duck fibroblasts against vesicular stomatitis virus (VSV) infection in a dose-dependent fashion, and anti-DuIFN-γ antibodies neutralized the antiviral effects. DuIFN-γ in the supernatant also inhibited the release of DHBV DNA from LMH-D2 cells. When ducks were co-immunized with DNA vaccine expressing DHBpreS/S and DuIFN-γ gene as an adjuvant, the level of DuIFN-γ mRNA in PBMCs was higher than that in ducks vaccinated with DHBpreS/S DNA alone. However, the titer of anti-DHBpreS elicited by DHBpreS/S DNA alone was higher than that co-immunized with DuIFN-γ gene and DHBpreS/S DNA. After being challenged with DHBV at high doses, the load of DHBV in sera dropped faster, and the amount of total DNA and cccDNA in the liver decreased more significantly in the group of ducks co-immunized with DuIFN-γ gene and DHBpreS/S DNA than in other groups. CONCLUSION: DHBV preS/S DNA vaccine can protect ducks against DHBV infection, DuIFN-γ gene as an immune adjuvant enhances its efficacy. PMID:16124047

  15. In vivo and in vitro biotransformation of theobromine by phenobarbital- and 3-methylcholanthrene-inducible cytochrome P-450 monooxygenases in rat liver. Role of thiol compounds.

    PubMed

    Shively, C A; Vesell, E S

    1987-01-01

    A new in vitro method was developed and applied to establish the role of the hepatic cytochrome P-450 monooxygenases in theobromine biotransformation by control and phenobarbital (PB)- and 3-methylcholanthrene (3MC)-induced Sprague-Dawley rats. In vivo theobromine metabolite formation and pharmacokinetic parameters were also determined to serve as a comparison for in vitro studies. In vivo, the major urinary metabolite was 6-amino-5-[N-methylformylamino]-1-methyluracil (3,7DAU) with lesser amounts of 3,7-dimethyluric acid (3,7DMU), 3-methylxanthine, 7-methylxanthine, 7-methyluric acid, and traces of dimethylallantoin (DMA). Following induction with 3MC, but not PB, selective increases occurred in the urinary excretion of 3,7DAU, indicating that a 3MC-inducible cytochrome P-450 isozyme plays a significant role in this metabolic pathway. Both PB and 3MC induction increased slightly urinary elimination of DMA, a minor metabolite. Pharmacokinetic studies after a single oral dose of 5 mg/kg theobromine revealed a marked effect of 3MC treatment on theobromine elimination, as evidenced by a 59% decrease in theobromine t1/2, a 75% decrease in AUC, and a 284% increase in clearance. By contrast, PB had no effect. Fecal 14C elimination accounted for approximately 5% of the administered theobromine dose, and biliary excretion studies revealed the presence of 3,7DMU, DMA, 3,7DAU, and unchanged theobromine. Studies in vitro indicated that 3,7DMU was the major theobromine metabolite produced by liver microsomes. Conversion rates in PB- and 3MC-induced rats were 2- and 11-fold higher, respectively, than in controls.(ABSTRACT TRUNCATED AT 250 WORDS)

  16. Effect of 3-methylcholanthrene induction on the distribution and DNA adduction of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) in F344 rats.

    PubMed

    Snyderwine, E G; Nouso, K; Schut, H A

    1993-06-01

    3-Methylcholanthrene (3MC) is a potent inducer of the cytochrome P450IA family of enzymes that catalyses the metabolic activation of the food mutagen/carcinogen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ). We have examined the effect of pretreatment with 3MC on the distribution and DNA adduct formation of IQ in male Fischer F344 rats. 3 hr after a single dose of [14C]IQ (10 mg/kg body weight, by gavage), the level of radioactivity in extrahepatic tissues was 30-70% less in 3MC-pretreated rats than in vehicle control rats. Although the level of radioactivity in the liver did not change after 3MC pretreatment, IQ-DNA adduct levels, measured by the 32P-postlabelling method, were 60% lower in the livers of 3MC-pretreated rats than those of control rats, and 83-97% lower in extrahepatic tissues such as the kidneys, colon, small intestine, bladder, heart and lung. IQ-DNA adducts in the testes and brain were found in control rats but were not detected in 3MC-pretreated rats. The rate of removal of IQ-DNA adducts from the livers of control and 3MC-pretreated animals was the same from 3 to 48 hr. At 48 hr, the adduct level in 3MC-pretreated rats remained lower than that seen in the control rats. The data suggest that 3MC induction of the P450IA family of cytochromes in vivo results in an increased rate of IQ detoxification.

  17. 3-Methylcholanthrene, which binds to the arylhydrocarbon receptor, inhibits proliferation and differentiation of osteoblasts in vitro and ossification in vivo.

    PubMed

    Naruse, Masae; Ishihara, Yoko; Miyagawa-Tomita, Sachiko; Koyama, Atsushi; Hagiwara, Hiromi

    2002-09-01

    3-Methylcholanthrene (3MC) is a ligand for arylhydrocarbon receptor (AhR), which binds dioxin. We examined the effects of 3MC on the proliferation and differentiation of osteoblasts using cultures of rat calvarial osteoblast-like cells (ROB cells) and mouse calvarial clonal preosteoblastic cells (MC3T3-E1 cells). Analysis by RT-PCR revealed that the mRNAs for AhR and AhR nuclear translocators were expressed in both ROB and MC3T3-E1 cells. Cell proliferation and the synthesis of DNA by ROB cells and MC3T3-E1 cells were markedly inhibited on exposure of cells to 3MC. Furthermore, 3MC reduced the activity of alkaline phosphatase and the rate of deposition of calcium by cells. The level of expression of mRNA for osteocalcin, which is a marker of osteoblastic differentiation, was also depressed by 3MC. Moreover, when 3MC (1 mg/kg body weight) was administered sc to pregnant mice at 10.5, 12.5, and 14.5 d post coitus, fetuses examined subsequently at 15.5 or 17.5 d post coitus revealed evidence of inhibition of appropriate calcification of bones. The treated metacarpals showed no subperiosteal bone matrix histologically. Our findings indicate that 3MC might have critical effects on the formation of bone both in vivo and in vitro.

  18. Effects of combined butylated hydroxyanisole and 3-methylcholanthrene treatments on the expression of p-nitro-phenol UDP-glucuronyltransferase

    SciTech Connect

    Ananaba, G.A.; Stewart, J.

    1987-01-01

    UDP-glucuronyltransferase (UDP-GT) is a very important microsomal xenobiotic detoxicating enzyme. This enzyme has been shown to be induced by a variety of xenobiotics including the carcinogen, 3-methylcholanthrene (3-MC) and the phenolic antioxidant (BHA) which is a widely used food additive. Recently, our laboratory demonstrated that simultaneous administration of 3-MC and BHA to rats results to a synergistic induction of the biotransformation enzymes including UDP-GT. We have determined the elution profile of this enzyme and its multiple forms on ion-exchange and affinity chromatography columns. To further understand the expression of this particular enzyme, we have isolated poly (A/sup +/) RNA from BHA + 3-MC induced rat livers, and in vitro translated them to demonstrate the presence of a protein similar to the authentic UDP-GT in activity and molecular weight, as well as generated cDNA library from them. We hope to immunoprecipitate specific p-nitrophenol UDP-GT from the in vitro translated poly (A/sup +/) RNA and isolate specific mRNA by polysome immunoadsorption in order to generate cDNA to further characterize this unique and important enzyme.

  19. No effect of cysteine on the pharmacokinetics of intravenous azosemide in rats with protein-calorie malnutrition by pretreatment with 3-methylcholanthrene.

    PubMed

    Kim, Y G; Cho, M K; Kwon, J W; Kim, S H; Kim, S G; Lee, M G

    2001-01-01

    The effects of cysteine on the pharmacokinetics of azosemide were investigated after intravenous administration of drug, 10 mg/kg, to male Sprague-Dawley rats pretreated with 3-methylcholanthrene fed on 23% protein diet (control rats) and 5% protein diet without (rats with protein-calorie malnutrition, PCM) or with (rats with PCMC) oral cysteine (250 mg/kg, twice daily starting from the fourth week) for 4 weeks. After intravenous administration to rats with PCM, the metabolites of azosemide excreted in urine and recovered from gastrointestinal tract decreased significantly than those in control rats, however, the plasma concentrations, total area under plasma concentration-time curve from time zero to time infinity (AUC) and time-averaged total body clearance (CL) were not significantly different between two groups of rats. It was reported that after intravenous administration of azosemide, 10 mg/kg, to rats with PCMC without pretreatment 3-methylcholanthrene, some pharmacokinetic parameters restored fully or more than the level of control rats; the time-averaged nonrenal clearance and apparent volume of distribution at steady state were comparable to those in control rats, but the terminal half-life and mean residence time were significantly shorter, AUC was significantly smaller, and time-averaged renal clearance and CL were significantly faster than those in control rats. However, the above mentioned effects of cysteine on the pharmacokinetic parameters of azosemide in rats with PCM were not observed with pretreatment with 3-methylcholanthrene.

  20. Despite Increased Type 1 IFN, Autoimmune Nonobese Diabetic Mice Display Impaired Dendritic Cell Response to CpG and Decreased Nuclear Localization of IFN-Activated STAT1

    PubMed Central

    Rahman, M. Jubayer; Rahir, Gwendoline; Dong, Matthew B.; Zhao, Yongge; Rodrigues, Kameron B.; Hotta-Iwamura, Chie; Chen, Ye; Guerrero, Alan; Tarbell, Kristin V.

    2016-01-01

    Innate immune signals help break self-tolerance to initiate autoimmune diseases such as type 1 diabetes, but innate contributions to subsequent regulation of disease progression are less clear. Most studies have measured in vitro innate responses of GM-CSF dendritic cells (DCs) that are functionally distinct from conventional DCs (cDCs) and do not reflect in vivo DC subsets. To determine whether autoimmune NOD mice have alterations in type 1 IFN innate responsiveness, we compared cDCs from prediabetic NOD and control C57BL/6 (B6) mice stimulated in vivo with the TLR9 ligand CpG, a strong type 1 IFN inducer. In response to CpG, NOD mice produce more type 1 IFN and express higher levels of CD40, and NOD monocyte DCs make more TNF. However, the overall CpG-induced transcriptional response is muted in NOD cDCs. Of relevance the costimulatory proteins CD80/CD86, signals needed for regulatory T cell homeostasis, are upregulated less on NOD cDCs. Interestingly, NOD Rag1−/− mice also display a defect in CpG-induced CD86 upregulation compared with B6 Rag1−/−, indicating this particular innate alteration precedes adaptive autoimmunity. The impaired response in NOD DCs is likely downstream of the IFN-α/β receptor because DCs from NOD and B6 mice show similar CpG-induced CD86 levels when anti–IFN-α/β receptor Ab is added. IFN-α–induced nuclear localization of activated STAT1 is markedly reduced in NOD CD11c+ cells, consistent with lower type 1 IFN responsiveness. In conclusion, NOD DCs display altered innate responses characterized by enhanced type 1 IFN and activation of monocyte-derived DCs but diminished cDC type 1 IFN response. PMID:26826238

  1. Induction time course of cytochromes P450 by phenobarbital and 3-methylcholanthrene pretreatment in liver microsomes of Alligator mississippiensis.

    PubMed

    Ertl, R P; Stegeman, J J; Winston, G W

    1998-05-01

    Alligator mississippiensis has at least two classes of inducible hepatic microsomal cytochromes P450 (CYP): (1) those induced by 3-methylcholanthrene (3MC), and (2) those induced by phenobarbital (PB). The rates of induction by these xenobiotic compounds are significantly slower than those reported for mammals. Carbon monoxide binding, western blots, and enzymatic activity measurements indicated that at least 48-72 hr are required to reach full induction. A methoxy-, ethoxy-, pentoxy, and benzyloxyphenoxazone (resorufin) O-dealkylation (MROD, EROD, PROD, and BROD) profile was indicative of substrate selectivity typical of 3MC- and PB-induced P450s. MROD and BROD showed the greatest ability to discriminate between alligator hepatic microsomes induced by 3MC and PB, respectively. This is in contrast to mammals, in which EROD is a biomarker of polycyclic aromatic hydrocarbon exposure because of its ability to discriminate the induction of CYP 1A. In a similar manner, PROD is a highly preferred activity of CYP 2B in mammals; thus, it is used to indicate CYP 2B induction. The induction of P450 by PB is a general phenomenon in mammals and birds. To the best of our knowledge, this is the first report demonstrating PB induction of P450 activities typical of the mammalian CYP 2 family isoforms in alligator or any reptilian liver. The importance of this finding to the evolution of CYP 2 family regulation by PB is heightened by the fact that induction by this xenobiotic is not common to fish and other lower vertebrates (Ertl RP and Winston GW, Comp Biochem Physiol, in press). Although indicating the presence of CYP 1A- and CYP 2B-like isoforms in alligator, it remains to be established how closely related these alligator P450s are to mammalian isoforms.

  2. Inhibition of osteoclast formation by 3-methylcholanthrene, a ligand for arylhydrocarbon receptor: suppression of osteoclast differentiation factor in osteogenic cells.

    PubMed

    Naruse, M; Otsuka, E; Naruse, M; Ishihara, Y; Miyagawa-Tomita, S; Hagiwara, H

    2004-01-01

    We investigated the effects of 3-methylcholanthrene (3MC), a ligand for arylhydrocarbon receptor (AhR), on osteoclastogenesis. Osteoclast-like cells, in cocultures with mouse spleen cells and clonal osteogenic stromal ST2 cells, are formed from spleen cells by a combination of the receptor activator of nuclear factor-kappaB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) produced by ST2 cells in response to 1alpha,25(OH)(2) Vitamin D(3). 3MC dose-dependently inhibited the formation of mono- and multinuclear osteoclast-like cells. However, 3MC did not inhibit the formation of osteoclast-like cells from mouse spleen cells which was supported by the exogenous soluble RANKL and M-CSF. 3MC did not affect the formation of an actin ring and pits on slices of dentine by osteoclast-like cells, both of which are typical indices of osteoclast activity. These results suggest that 3MC affects osteoclast-supporting cells such as ST2 cells but not osteoclast precursor cells and mature osteoclastic cells. When we measured the expression levels of RANKL mRNA in ST2 cells, 3MC dose-dependently decreased the level of this mRNA. However, 3MC did not affect levels of mRNAs for osteoprotegerin (OPG), M-CSF, and the receptor of 1alpha,25(OH)(2) Vitamin D(3) in ST2 cells. Furthermore, soluble RANKL was able to counteract the inhibitory effect of 3MC on the formation of osteoclast-like cells. Our findings indicate that 3MC inhibits osteoclastogenesis via the inhibition of RANKL expression in osteoblastic cells.

  3. Effects of 3-methylcholanthrene and aspirin co-administration on ALDH3A1 in HepG2 cells.

    PubMed

    Sotiropoulou, M; Pappas, P; Marselos, M

    2001-01-30

    The effects of two different protocols of 3-methylcholanthrene (3MC) and aspirin co-administration were studied in a well-established human hepatoma cell line (HepG2). During this work, we have performed toxicity tests for cell viability/cell proliferation as well as studies on the expression of ALDH3A1 after exposure of HepG2 cells to 3MC or/and aspirin. For the evaluation of toxic concentrations of 3MC and aspirin, the WST-1 test was used. WST-1 is a reliable cytotoxicity test which is based on the cleavage of the tetrazolium salt WST-1 to formazan by mitochondrial enzymes of living cells. A broad range of drug concentrations for either 3MC (0.25-50.0 microM) or aspirin (0.05-10.0 mM) were used for cell exposure, in several periods of time. The expression of ALDH3A1 in HepG2 cells showed typical time- and dose-response curves of induction after application of 3MC (1-5 days, 1.5-5.0 microM, respectively). When cells were firstly exposed to 3MC (2.5 and 5.0 microM) and then to aspirin (0.25 mM), the induced ALDH3A1 activity was further enhanced in a statistically significant way (P<0.05). On the contrary, when aspirin application was preceded 3MC exposuring a statistically significant decrease in ALDH3A1 inducibility was observed, as compared with the application of 3MC alone.

  4. ENU-induced mutation in USP18 causes hyperactivation of IFN-αβ signalling and suppresses STAT4-induced IFN-γ production resulting in increased susceptibility to Salmonella Typhimurium

    PubMed Central

    Richer, Etienne; Prendergast, Caitlin; Zhang, Dong-Er; Qureshi, Salman T; Vidal, Silvia M; Malo, Danielle

    2014-01-01

    In order to deepen our knowledge of the natural host response to pathogens our team undertook an in vivo screen of mutagenized 129S1 mice with Salmonella Typhimurium. One mutation affecting Salmonella susceptibility was mapped to a region of 1.3Mb on chromosome 6 that contains 15 protein-coding genes. A missense mutation was identified in the Usp18 (Ubiquitin specific peptidase 18) gene. This mutation results in an increased inflammatory response (IL-6, type 1 IFN) to Salmonella and LPS challenge while paradoxically reducing IFN-γ production during bacterial infection. Increased STAT1 phophorylation correlated with impaired STAT4 phosphorylation resulting in overwhelming IL-6 secretion but reduced IFN-γ production during infection. The reduced IFN-γ levels along with the increased inflammation rationalize the Salmonella Typhimurium susceptibility in terms of increased bacterial load in target organs and cytokine-induced septic shock and death. PMID:20693420

  5. Comparative induction of xenobiotic metabolism in rodent kidney, testis and liver by commercial mixtures of polybrominated biphenyls and polychlorinated biphenyls, phenobarbital and 3-methylcholanthrene: absolute and temporal effects.

    PubMed

    Kluwe, W M; Hook, J B

    1981-01-01

    Male Fischer 344 rats were killed at various times after single or multiple treatments with polybrominated biphenyls (PBB), polychlorinated biphenyls (PCB), sodium phenobarbital (NaPB) or 3-methylcholanthrene (3MC). p-Chloro-N-methyl-aniline N-demethylase (PCNMA) and aryl hydrocarbon hydroxylase (AHH) activities were determined in the 14 000 g supernatant fraction (postmitochondrial supernate, PMS) of renal, testicular and hepatic homogenates. Cytochrome P-450 (p-450) concentrations were determined in the 100 000 g pellet fractions of the same homogenates and the effects of enzyme induction on the sensitivities of AHH in PMS to inhibition by alpha-napthoflavone (ANF) and metyrapone (MET) in vitro were determined. Single treatments with PBB or PCB induced hepatic P-450 only, while multiple treatments with PBB, PCB or 3MC induced both renal and hepatic P-450; NaPB induced only hepatic P-450, while testicular P-450 concentration was unaffected by the inducers. Treatments with PBB, PCB or 3MC shifted the Soret maxima of renal and hepatic dithionite-reduced P-450 difference spectra to shorter wavelengths. Multiple treatments with PBB, PCB or 3MC increased renal and hepatic AHH activities, but NaPB induced hepatic AHH only. Renal AHH activity was increased more rapidly than hepatic AHH after a single treatment with PBB, PCB or 3MC and returned more rapidly to control. The renal AHH induced by PBB and PCB, like that induced by 3MC, was more sensitive to inhibition by ANF in vitro than was renal AHH from naive rats. Hepatic AHH induced by PBB and PCB, unlike that induced by NaPB or 3MC, exhibited no net alterations in sensitivities to the inhibitory effects of ANF or MET. Testicular AHH activity was not induced by PBB, PCB , NaPB or 3MC. Multiple treatments with PBB, PCB or NaPB increased hepatic, but not renal or testicular PCNMA activities. The organ-specificity and time-dependency of the effects of PBB, PCB, NaPB and 3MC on P-450 concentrations and AHH activities

  6. Molecular mechanisms of p21 and p27 induction by 3-methylcholanthrene, an aryl-hydrocarbon receptor agonist, involved in antiproliferation of human umbilical vascular endothelial cells.

    PubMed

    Pang, Pai-Huei; Lin, Ying-Hsi; Lee, Yi-Hsuan; Hou, Hsing-Han; Hsu, Sung-Po; Juan, Shu-Hui

    2008-04-01

    We previously reported that 3-methylcholanthrene (3MC), an aryl-hydrocarbon receptor (AhR) agonist, inhibits the proliferation of human umbilical vascular endothelial cells (HUVECs; Juan et al., 2006, Eur J Pharmacol 530: 1-8). Herein, pretreatment of HUVECs with p21 or p27 small interfering (si)RNA reduced 3MC-induced elimination of [(3)H]thymidine incorporation, demonstrating their essential roles in the antiproliferation of HUVECs. The molecular mechanisms of p21 and p27 involved in the antiproliferative effects of 3MC were elucidated in this study. 3MC time- and concentration-dependently increased p21 and p27 levels, and decreased the protein level of CDK2 with no apparent alteration of p53. Interestingly, 3MC-mediated p21 and p27 inductions were eliminated by resveratrol, an AhR antagonist, suggesting their AhR dependency, further confirmed by AhR siRNA. Among the relevant pathways, p38MAPK activation sustained the levels of p21 and p27 induced by 3MC, which was eliminated by AhR antagonists and N-acetylcysteine (NAC), an antioxidant. 3MC concentration-dependently enhanced not only the consensus dioxin-responsive element (DRE)-driven luciferase activity, but also the binding activity of the AhR to the putative DRE derived from the p21 and p27 promoters. A deletion of the DRE (-285/-270) in p21 (-2,300/+8) only partially alleviated the 3MC-induced luciferase activity unless NAC was added, suggesting that there may be a DRE-independent mechanism associated with oxidative stress. However, a deletion of the DRE (-660/-645) in p27 (-1,358/-100) almost completely abrogated the activation. Our study demonstrated that both the functional DRE and the phosphorylation of p38MAPK are essential for the induction of p21 and p27, resulting in the antiproliferative action of 3MC in HUVECs.

  7. Daily intake of probiotics with high IFN-γ/IL-10 ratio increases the cytotoxicity of human natural killer cells: a personalized probiotic approach.

    PubMed

    Ho, Yu-Hsuan; Lu, Yu-Chiu; Chang, Hung-Cheng; Lee, Shin-Yi; Tsai, Min-Fen; Huang, Yu-Ting; Hsu, Ting-Yuan

    2014-01-01

    A personalized probiotic microfluidic chip system has been established and used to screen the probiotics which had the highest value of IFN-γ/IL-10 or IL-10/IFN-γ among six probiotics, including L. paracasei BRAP01, L. acidophilus AD300, B. longum BA100, E. faecium BR0085, L. rhamnosus AD500, and L. reuteri BR101. One hundred volunteers were included and their PBMCs were collected and stimulated by the six probiotics. People who belonged to the IFN-γ group took the probiotics that exerted the highest ratio of IFN-γ/IL-10 and vice versa in IL-10 group. A significant increase in NK cytotoxicity of 69 volunteers in the IFN-γ group was observed compared to the IL-10 group (n = 21) and control group (n = 10). The result also showed that L. paracasei BRAP01 and L. acidophilus AD300 were the two dominant inducers in IFN-γ group which yielded higher value of IFN-γ/IL-10 than the other 4 probiotics, while L. reuteri BR101 was the most effective agent on the ratio of IL-10/IFN-γ in the IL-10 group. Our finding highlighted the concept of personalized probiotics and also provided a good foundation to investigate the probiotics with NK activity.

  8. Glucuronidation and sulfonation, in vitro, of the major endocrine-active metabolites of methoxychlor in the channel catfish, Ictalurus punctatus, and induction following treatment with 3-methylcholanthrene

    PubMed Central

    James, Margaret O.; Stuchal, Leah D.; Nyagode, Beatrice A.

    2008-01-01

    The organochlorine pesticide, methoxychlor (MXC), is metabolized in animals to phenolic mono- and bis-demethylated metabolites (OH-MXC and HPTE respectively) that interact with estrogen receptors and may be endocrine disruptors. The phase II detoxication of these compounds will influence the duration of action of the estrogenic metabolites, but has not been investigated extensively. In this study, the glucuronidation and sulfonation of OH-MXC and HPTE were investigated in subcellular fractions of liver and intestine from untreated, MXC-treated and 3-methylcholanthrene (3-MC)-treated channel catfish, Ictalurus punctatus. MXC-treated fish were given i.p. injections of 2 mg MXC/kg daily for 6 days and sacrificed 24 hr after the last dose. The 3-MC treatment was a single 10 mg/kg i.p. dose 5 days prior to sacrifice. In hepatic microsomes from control fish, the Vmax value (mean ± S.D., n=4) for glucuronidation of OH-MXC was 270 ± 50 pmol/min/mg protein, higher than found for HPTE (110 ± 20 pmol/min/mg protein). For each substrate, the Vmax values observed in intestinal microsomes were approximately twice those found in the liver. The Km values for OH-MXC and HPTE glucuronidation in control liver were not significantly different and were 0.32 ± 0.04 mM for OH-MXC and 0.26 ± 0.06 mM for HPTE. The Km for the co-substrate, UDPGA, was higher in liver (0.28 ± 0.09 mM) than intestine (0.04 ± 0.02 mM). Treatment with 3-MC but not MXC increased the Vmax for glucuronidation in liver and intestine. Glucuronidation was a more efficient pathway than sulfonation for both substrates, in both tissues. The Vmax values for sulfonation of OH-MXC and HPTE respectively in liver cytosol were 7 ± 3 and 17 ± 4 pmol/min/mg protein and in intestinal cytosol were 13 ± 3 and 30 ± 5 pmol/min/mg protein. Treatment with 3-MC but not MXC increased rates of sulfonation of OH-MXC and HPTE and the model substrate, 3-hydroxy-benzo(a)pyrene in both intestine and liver. Comparison of the kinetics

  9. Regulatory xenobiotic responsive elements in the distal 5'-flanking region of the mouse Cyp1a2 gene required for transcriptional activation by 3-methylcholanthrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin.

    PubMed

    Kawasaki, Yuki; Sakuma, Tsutomu; Goto, Yuma; Nemoto, Nobuo

    2010-10-01

    We examined the xenobiotic responsive element (XRE) responsible for induction of the mouse Cyp1a2 gene by 3-methylcholanthrene (3MC) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) using a reporter gene assay in mouse hepatocytes in primary culture. Although, the 5'-flanking region up to -9.5 kilobase pairs did not show a significant increase in transcriptional activity after treatment with 3MC or TCDD, a further distal 5'-flanking region from -13,958 to -12,520 containing 12 putative XREs (5'-GCGTG-3') demonstrated distinctive transcriptional activity after treatment with 3MC or TCDD. When a mutation was introduced into XRE14 at -12,972, the activation was decreased, and concurrent mutations in XRE14, XRE13, and XRE15 completely abolished it. However, mutations in XRE13, XRE15, XRE16, or XRE17 did not affect the inducible transcriptional activation of the mouse Cyp1a2 gene. These results suggest that XRE14 is important and that XRE13 at -12,897 and/or XRE15 at -13,061 are cooperative to the inducible transcriptional activation of the mouse Cyp1a2 gene by ligands of the aryl hydrocarbon receptor.

  10. 3-Methylcholanthrene, an AhR Agonist, Caused Cell-Cycle Arrest by Histone Deacetylation through a RhoA-Dependent Recruitment of HDAC1 and pRb2 to E2F1 Complex

    PubMed Central

    Yang, Nian-Jie; Lee, Yi-Hsuan; Juan, Shu-Hui

    2014-01-01

    We previously showed that treating vascular endothelial cells with 3-methylcholanthrene (3MC) caused cell-cycle arrest in the Go/G1 phase; this resulted from the induction of p21 and p27 and a decreased level and activity of the cyclin-dependent kinase, Cdk2. We further investigated the molecular mechanisms that modulate cell-cycle regulatory proteins through the aryl-hydrocarbon receptor (AhR)/Ras homolog gene family, member A (RhoA) dependent epigenetic modification of histone. AhR/RhoA activation mediated by 3MC was essential for the upregulation of retinoblastoma 2 (pRb2) and histone deacetylase 1 (HDAC1), whereas their nuclear translocation was primarily modulated by RhoA activation. The combination of increased phosphatase and tensin homolog (PTEN) activity and decreased phosphatidylinositide 3-kinase (PI3K) activation by 3MC led to the inactivation of the Ras-cRaf pathway, which contributed to pRb2 hypophosphorylation. Increased HDAC1/pRb2 recruitment to the E2F1 complex decreased E2F1-transactivational activity and H3/H4 deacetylation, resulting in the downregulation of cell-cycle regulatory proteins (Cdk2/4 and Cyclin D3/E). Co-immunoprecipitation and electrophoretic mobility shift assay (EMSA) results showed that simvastatin prevented the 3MC-increased binding activities of E2F1 proteins in their promoter regions. Additionally, RhoA inhibitors (statins) reversed the effect of 3MC in inhibiting DNA synthesis by decreasing the nuclear translocation of pRb2/HDAC1, leading to a recovery of the levels of cell-cycle regulatory proteins. In summary, 3MC decreased cell proliferation by the epigenetic modification of histone through an AhR/RhoA-dependent mechanism that can be rescued by statins. PMID:24658119

  11. 3-Methylcholanthrene, an AhR agonist, caused cell-cycle arrest by histone deacetylation through a RhoA-dependent recruitment of HDAC1 and pRb2 to E2F1 complex.

    PubMed

    Chang, Chih-Cheng; Sue, Yuh-Mou; Yang, Nian-Jie; Lee, Yi-Hsuan; Juan, Shu-Hui

    2014-01-01

    We previously showed that treating vascular endothelial cells with 3-methylcholanthrene (3MC) caused cell-cycle arrest in the Go/G1 phase; this resulted from the induction of p21 and p27 and a decreased level and activity of the cyclin-dependent kinase, Cdk2. We further investigated the molecular mechanisms that modulate cell-cycle regulatory proteins through the aryl-hydrocarbon receptor (AhR)/Ras homolog gene family, member A (RhoA) dependent epigenetic modification of histone. AhR/RhoA activation mediated by 3MC was essential for the upregulation of retinoblastoma 2 (pRb2) and histone deacetylase 1 (HDAC1), whereas their nuclear translocation was primarily modulated by RhoA activation. The combination of increased phosphatase and tensin homolog (PTEN) activity and decreased phosphatidylinositide 3-kinase (PI3K) activation by 3MC led to the inactivation of the Ras-cRaf pathway, which contributed to pRb2 hypophosphorylation. Increased HDAC1/pRb2 recruitment to the E2F1 complex decreased E2F1-transactivational activity and H3/H4 deacetylation, resulting in the downregulation of cell-cycle regulatory proteins (Cdk2/4 and Cyclin D3/E). Co-immunoprecipitation and electrophoretic mobility shift assay (EMSA) results showed that simvastatin prevented the 3MC-increased binding activities of E2F1 proteins in their promoter regions. Additionally, RhoA inhibitors (statins) reversed the effect of 3MC in inhibiting DNA synthesis by decreasing the nuclear translocation of pRb2/HDAC1, leading to a recovery of the levels of cell-cycle regulatory proteins. In summary, 3MC decreased cell proliferation by the epigenetic modification of histone through an AhR/RhoA-dependent mechanism that can be rescued by statins.

  12. Purification and characterization of a previously unreported form of cytochrome P-448 from the liver of 3-methylcholanthrene-pretreated rats.

    PubMed Central

    Seidel, S L; Shires, T K

    1986-01-01

    At least four hepatic isoenzymes of cytochrome P-450 were purified and characterized from rats treated with 3-methylcholanthrene. A monoclonal antibody developed against one of the forms (designated cytochrome P-450 MC-B) and polyclonal antibodies against others were used to demonstrate that form MC-B is immunologically distinct from other methylcholanthrene-inducible forms. Limited N-terminal amino acid sequencing showed that cytochrome P-450 MC-B has a primary structure that differs from the N-terminal sequences of other established rat isoenzymes. Cytochrome P-450 MC-B has a minimum Mr of 53,000, a CO-reduced spectral maximum at 448 nm, a Soret maximum of 417 nm in the absolute oxidized spectrum and a pattern of substrate preferences that differs from those of the other methylcholanthrene-induced forms. The other forms (MC-A, MC-C and MC-D) share characteristics with isoenzymes previously reported by other investigators. Images Fig. 1. Fig. 3. Fig. 4. Fig. 5. PMID:3753450

  13. Differential metabolism of acetanilide versus ethoxycoumarin and benzo[a]pyrene by two 3-methylcholanthrene-inducible forms of rat liver cytochrome P-450.

    PubMed

    Sundheimer, D W; Caveness, M B; Goldstein, J A

    1983-10-15

    The present study compares the catalytic activities of two 3-methylcholanthrene (3-MC) inducible forms of cytochrome P-450. These isozymes (P-448HCB and P-448MC) were isolated from liver microsomes of rats treated with 3,4,5,3',4',5'-hexachlorobiphenyl (HCB) and 3-MC, respectively. Catalytic activities of the isozymes were compared in a reconstituted system and by antibody inhibition studies in microsomes. In a reconstituted system, P-448HCB had very little catalytic activity toward benzo[a]pyrene or ethoxycoumarin (substrates metabolized preferentially by P-448MC). In contrast, both isozymes had high turnover numbers for aniline and acetanilide. However, catalytic activities of the purified isozymes were affected dramatically by Emulgen 911, a nonionic detergent. Since nonionic detergents used in the purification of P-450 isozymes cannot be completely removed after purification, residual amounts of detergent probably affect turnover numbers in a reconstituted system. Therefore, specific antibodies to cytochromes P-448MC and P-448HCB were used to examine the contribution of these isozymes to microsomal metabolism. Antibody inhibition studies confirmed that the majority of benzo[a]pyrene and ethoxycoumarin metabolism in 3-MC-induced microsomes was catalyzed by cytochrome P-448MC. In contrast, P-448HCB accounted for the majority of the acetanilide hydroxylase activity in 3-MC- and HCB-induced microsomes. Neither isozyme contributed appreciably to metabolism of these substrates in control microsomes.

  14. Flavin-containing monooxygenase-3: induction by 3-methylcholanthrene and complex regulation by xenobiotic chemicals in hepatoma cells and mouse liver.

    PubMed

    Celius, Trine; Pansoy, Andrea; Matthews, Jason; Okey, Allan B; Henderson, Marilyn C; Krueger, Sharon K; Williams, David E

    2010-08-15

    Flavin-containing monooxygenases often are thought not to be inducible but we recently demonstrated aryl hydrocarbon receptor (AHR)-dependent induction of FMO mRNAs in mouse liver by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (Celius et al., Drug Metab Dispos 36:2499, 2008). We now evaluated FMO induction by other AHR ligands and xenobiotic chemicals in vivo and in mouse Hepa1c1c7 hepatoma cells (Hepa-1). In mouse liver, 3-methylcholanthrene (3MC) induced FMO3 mRNA 8-fold. In Hepa-1 cells, 3MC and benzo[a]pyrene (BaP) induced FMO3 mRNA >30-fold. Induction by 3MC and BaP was AHR dependent but, surprisingly, the potent AHR agonist, TCDD, did not induce FMO3 mRNA in Hepa-1 cells nor did chromatin immunoprecipitation assays detect recruitment of AHR or ARNT to Fmo3 regulatory elements after exposure to 3MC in liver or in Hepa-1 cells. However, in Hepa-1, 3MC and BaP (but not TCDD) caused recruitment of p53 protein to a p53 response element in the 5'-flanking region of the Fmo3 gene. We tested the possibility that FMO3 induction in Hepa-1 cells might be mediated by Nrf2/anti-oxidant response pathways, but agents known to activate Nrf2 or to induce oxidative stress did not affect FMO3 mRNA levels. The protein synthesis inhibitor, cycloheximide (which causes "superinduction" of CYP1A1 mRNA in TCDD-treated cells), by itself caused dramatic upregulation (>300-fold) of FMO3 mRNA in Hepa-1 suggesting that cycloheximide prevents synthesis of a labile protein that suppresses FMO3 expression. Although FMO3 mRNA is highly induced by 3MC or TCDD in mouse liver and in Hepa-1 cells, FMO protein levels and FMO catalytic function showed only modest elevation.

  15. Flavin-containing monooxygenase-3: Induction by 3-methylcholanthrene and complex regulation by xenobiotic chemicals in hepatoma cells and mouse liver

    SciTech Connect

    Celius, Trine; Pansoy, Andrea; Matthews, Jason; Okey, Allan B.; Henderson, Marilyn C.; Krueger, Sharon K.; Williams, David E.

    2010-08-15

    Flavin-containing monooxygenases often are thought not to be inducible but we recently demonstrated aryl hydrocarbon receptor (AHR)-dependent induction of FMO mRNAs in mouse liver by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (Celius et al., Drug Metab Dispos 36:2499, 2008). We now evaluated FMO induction by other AHR ligands and xenobiotic chemicals in vivo and in mouse Hepa1c1c7 hepatoma cells (Hepa-1). In mouse liver, 3-methylcholanthrene (3MC) induced FMO3 mRNA 8-fold. In Hepa-1 cells, 3MC and benzo[a]pyrene (BaP) induced FMO3 mRNA > 30-fold. Induction by 3MC and BaP was AHR dependent but, surprisingly, the potent AHR agonist, TCDD, did not induce FMO3 mRNA in Hepa-1 cells nor did chromatin immunoprecipitation assays detect recruitment of AHR or ARNT to Fmo3 regulatory elements after exposure to 3MC in liver or in Hepa-1 cells. However, in Hepa-1, 3MC and BaP (but not TCDD) caused recruitment of p53 protein to a p53 response element in the 5'-flanking region of the Fmo3 gene. We tested the possibility that FMO3 induction in Hepa-1 cells might be mediated by Nrf2/anti-oxidant response pathways, but agents known to activate Nrf2 or to induce oxidative stress did not affect FMO3 mRNA levels. The protein synthesis inhibitor, cycloheximide (which causes 'superinduction' of CYP1A1 mRNA in TCDD-treated cells), by itself caused dramatic upregulation (> 300-fold) of FMO3 mRNA in Hepa-1 suggesting that cycloheximide prevents synthesis of a labile protein that suppresses FMO3 expression. Although FMO3 mRNA is highly induced by 3MC or TCDD in mouse liver and in Hepa-1 cells, FMO protein levels and FMO catalytic function showed only modest elevation.

  16. Increased antiviral activity of microscale-purified HuIFN alpha 8 (human interferon alpha 8) over HuIFN alpha 2b in Hep-2 cells challenged with Mengo virus.

    PubMed

    García, Julio César Sánchez; Ariza, Alejandro Miranda; Lasa, Alexis Musacchio; González, Luis Javier; Perez, Vladimir Besada

    2007-11-01

    Human proteins are not routinely expressed at high levels in Escherichia coli for, among other reasons, different codon usage. Several purification procedures have been applied to recover recombinant proteins for further biological characterization. However, the vast majority involve costly chromatography procedures. In the present study, both (Hu)IFN(alpha 2b) (human interferon alpha 2b) and (Hu)IFN(alpha 8) were expressed efficiently in E. coli BL21-codonplus-RIL. Subsequently, both recombinant proteins were purified to homogeneity by passive elution from reverse-stained SDS/PAGE gels, a cost-effective purification procedure. After purification, both recovered proteins were biologically active. The (Hu)IFN(alpha 8) subtype induced 1.46-fold more antiviral activity than (Hu)IFN(alpha 2b) using Hep-2 human laryngeal carcinoma cell challenged with Mengo virus.

  17. Hyperoxia activates NF-kappaB and increases TNF-alpha and IFN-gamma gene expression in mouse pulmonary lymphocytes.

    PubMed

    Shea, L M; Beehler, C; Schwartz, M; Shenkar, R; Tuder, R; Abraham, E

    1996-11-01

    Hyperoxia-associated production of reactive oxygen species leads to neutrophil infiltration into the lungs and increased pulmonary proinflammatory cytokine expression. However, the initial events induced by hyperoxia, and leading to acute inflammatory lung injury, remain incompletely characterized. To explore this issue, we examined nuclear transcriptional regulatory factor (NF-kappaB and NF-IL-6) activation and cytokine expression in the lungs following 12 to 48 h of hyperoxia exposure. No increases in cytokine (IL-1beta, IL-6, IL-10, TGF-beta, TNF-alpha, IFN-gamma) expression nor in NF-kappaB activation were found after 12 h of hyperoxia. Following 24 h of hyperoxia, NF-kappaB activation and increased levels of TNF-alpha mRNA were present in pulmonary lymphocytes. By 48 h of hyperoxia, amounts of IFN-gamma and TNF-alpha protein as well as mRNA were increased in the lungs, and NF-kappaB continued to show activation, even though no histologic abnormalities were present. These results show that hyperoxia activates NF-kappaB in the lungs before any increase in proinflammatory cytokine protein occurs, and suggest that NF-kappaB activation may represent an initial event in the proinflammatory sequence induced by hyperoxia.

  18. Diminished CD4+/CD25+ T cell and increased IFN-gamma levels occur in dogs vaccinated with Leishmune in an endemic area for visceral leishmaniasis.

    PubMed

    de Lima, Valéria Marçal Felix; Ikeda, Fabiana Augusta; Rossi, Cláudio N; Feitosa, Mary Marcondes; Vasconcelos, Rosemeride Oliveira; Nunes, Caris Maroni; Goto, Hiro

    2010-06-15

    The Leishmune vaccine has been used in endemic areas to prevent canine visceral leishmaniasis in Brazil, but cytokine production induced by vaccination has rarely been investigated in dogs. This study aimed to evaluate the immune response of dogs vaccinated with Leishmune FML vaccine (Fort Dodge) against total antigen of Leishmania (Leishmania) chagasi (TAg) and FML. Twenty healthy dogs from Araçatuba, São Paulo, Brazil, an endemic leishmaniasis area, received three consecutive subcutaneous injection of Leishmune vaccine at 21-day intervals. PBMC were isolated before and 10 days after completing vaccination and lymphoproliferative response and antibody production against FML or total promastigote antigen were tested. Cytokines IFN-gamma, IL-4 and TNF-alpha were measured in culture supernatant and CD4+/CD25+ and CD8+/CD25+ T cell presence was determined. Analysis of the data indicated that the vaccine conferred humoral responses (100%) against both antigens and cellular immunity to FML (85%) and total antigen (80%), the supernatant of cultured cells stimulated with TAg and FML showed an increase in IFN-gamma (P<0.05), and the vaccine reduced CD4+/CD25+ T cell presence compared to that observed before vaccination. These responses may constitute part of the immune mechanism induced by Leishmune.

  19. Induction of Cyp1a1 and Cyp1b1 and formation of DNA adducts in C57BL/6, Balb/c, and F1 mice following in utero exposure to 3-methylcholanthrene

    SciTech Connect

    Xu Mian; Nelson, Garret B.; Moore, Joseph E.; McCoy, Thomas P.; Dai, Jian; Manderville, Richard A.; Ross, Jeffrey A.; Miller, Mark Steven . E-mail: msmiller@wfubmc.edu

    2005-11-15

    Fetal mice are more sensitive to chemical carcinogens than are adults. Previous studies from our laboratory demonstrated differences in the mutational spectrum induced in the Ki-ras gene from lung tumors isolated from [D2 x B6D2F1]F2 mice and Balb/c mice treated in utero with 3-methylcholanthrene (MC). We thus determined if differences in metabolism, adduct formation, or adduct repair influence strain-specific responses to transplacental MC exposure in C57BL/6 (B6), Balb/c (BC), and reciprocal F1 crosses between these two strains of mice. The induction of Cyp1a1 and Cyp1b1 in fetal lung and liver tissue was determined by quantitative fluorescent real-time PCR. MC treatment caused maximal induction of Cyp1a1 and Cyp1b1 RNA 2-8 h after injection in both organs. RNA levels for both genes then declined in both fetal organs, but a small biphasic, secondary increase in Cyp1a1 was observed specifically in the fetal lung 24-48 h after MC exposure in all four strains. Cyp1a1 induction by MC at 4 h was 2-5 times greater in fetal liver (7000- to 16,000-fold) than fetal lung (2000- to 6000-fold). Cyp1b1 induction in both fetal lung and liver was similar and much lower than that observed for Cyp1a1, with induction ratios of 8- to 18-fold in fetal lung and 10- to 20-fold in fetal liver. The overall kinetics and patterns of induction were thus very similar across the four strains of mice. The only significant strain-specific effect appeared to be the relatively poor induction of Cyp1b1 in the parental strain of B6 mice, especially in fetal lung tissue. We also measured the levels of MC adducts and their disappearance from lung tissue by the P{sup 32} post-labeling assay on gestation days 18 and 19 and postnatal days 1, 4, 11, and 18. Few differences were seen between the different strains of mice; the parental strain of B6 mice had nominally higher levels of DNA adducts 2 (gestation day 19) and 4 (postnatal day 1) days after injection, although this was not statistically

  20. Increased TNF-α/IFN-γ/IL-2 and Decreased TNF-α/IFN-γ Production by Central Memory T Cells Are Associated with Protective Responses against Bovine Tuberculosis Following BCG Vaccination

    PubMed Central

    Maggioli, Mayara F.; Palmer, Mitchell V.; Thacker, Tyler C.; Vordermeier, Hans Martin; McGill, Jodi L.; Whelan, Adam O.; Larsen, Michelle H.; Jacobs, William R.; Waters, W. Ray

    2016-01-01

    Central memory T cell (Tcm) and polyfunctional CD4 T cell responses contribute to vaccine-elicited protection with both human and bovine tuberculosis (TB); however, their combined role in protective immunity to TB is unclear. To address this question, we evaluated polyfunctional cytokine responses by CD4 T cell effector/memory populations from bacille Calmette–Guerin (BCG) vaccinated and non-vaccinated calves by flow cytometry prior to and after aerosol challenge with virulent Mycobacterium bovis. Polyfunctional cytokine expression patterns in the response by Tcm, effector memory, and effector T cell subsets were similar between BCG-vaccinated and M. bovis-infected calves, only differing in magnitude (i.e., infected > vaccinated). BCG vaccination, however, did alter the kinetics of the ensuing response to virulent M. bovis infection. Early after challenge (3 weeks post-infection), non-vaccinates had greater antigen-specific interferon-γ (IFN-γ)/tumor necrosis factor-α (TNF-α) and lesser IFN-γ/TNF-α/IL-2 responses by Tcm cells than did vaccinated animals. Importantly, these differences were also associated with mycobacterial burden upon necropsy. Polyfunctional responses to ESAT-6:CFP10 (antigens not synthesized by BCG strains) were detected in memory subsets, as well as in effector cells, as early as 3 weeks after challenge. These findings suggest that cell fate divergence may occur early after antigen priming in the response to bovine TB and that memory and effector T cells may expand concurrently during the initial phase of the immune response. In summary, robust IFN-γ/TNF-α response by Tcm cells is associated with greater mycobacterial burden, while IFN-γ/TNF-α/IL-2 response by Tcm cells are indicative of a protective response to bovine TB. PMID:27799930

  1. Absence of MyD88 results in enhanced TLR3-dependent phosphorylation of IRF3 and increased IFN-β and RANTES production.

    PubMed

    Siednienko, Jakub; Gajanayake, Thusitha; Fitzgerald, Katherine A; Moynagh, Paul; Miggin, Sinéad M

    2011-02-15

    Toll-like receptors are a group of pattern-recognition receptors that play a crucial role in "danger" recognition and induction of the innate immune response against bacterial and viral infections. TLR3 has emerged as a key sensor of viral dsRNA, resulting in the induction of the anti-viral molecule, IFN-β. Thus, a clearer understanding of the biological processes that modulate TLR3 signaling is essential. Previous studies have shown that the TLR adaptor, Mal/TIRAP, an activator of TLR4, inhibits TLR3-mediated IFN-β induction through a mechanism involving IRF7. In this study, we sought to investigate whether the TLR adaptor, MyD88, an activator of all TLRs except TLR3, has the ability to modulate TLR3 signaling. Although MyD88 does not significantly affect TLR3 ligand-induced TNF-α induction, MyD88 negatively regulates TLR3-, but not TLR4-, mediated IFN-β and RANTES production; this process is mechanistically distinct from that employed by Mal/TIRAP. We show that MyD88 inhibits IKKε-, but not TBK1-, induced activation of IRF3. In doing so, MyD88 curtails TLR3 ligand-induced IFN-β induction. The present study shows that while MyD88 activates all TLRs except TLR3, MyD88 also functions as a negative regulator of TLR3. Thus, MyD88 is essential in restricting TLR3 signaling, thereby protecting the host from unwanted immunopathologies associated with the excessive production of IFN-β. Our study offers a new role for MyD88 in restricting TLR3 signaling through a hitherto unknown mechanism whereby MyD88 specifically impairs IKKε-mediated induction of IRF3 and concomitant IFN-β and RANTES production.

  2. Protozoan Parasites and Type I IFNs.

    PubMed

    Silva-Barrios, Sasha; Stäger, Simona

    2017-01-01

    For many years, the role of interferon (IFN)-I has been characterized primarily in the context of viral infections. However, regulatory functions mediated by IFN-I have also been described against bacterial infections and in tumor immunology. Only recently, the interest in understanding the immune functions mediated by IFN-I has dramatically increased in the field of protozoan infections. In this review, we discuss the discrete role of IFN-I in the immune response against major protozoan infections: Plasmodium, Leishmania, Trypanosoma, and Toxoplasma.

  3. Protozoan Parasites and Type I IFNs

    PubMed Central

    Silva-Barrios, Sasha; Stäger, Simona

    2017-01-01

    For many years, the role of interferon (IFN)-I has been characterized primarily in the context of viral infections. However, regulatory functions mediated by IFN-I have also been described against bacterial infections and in tumor immunology. Only recently, the interest in understanding the immune functions mediated by IFN-I has dramatically increased in the field of protozoan infections. In this review, we discuss the discrete role of IFN-I in the immune response against major protozoan infections: Plasmodium, Leishmania, Trypanosoma, and Toxoplasma. PMID:28154565

  4. 60 Tear IFN-G is Increased After Sublingual Immunotherapy in Allergic Conjunctivitis Patients and Correlates With Clinical Improvement

    PubMed Central

    Aguilar-Velazquez, Gustavo; Santacruz, Concepcion; Hernandez, Pedro M.; Chavez, Raul; Ayala-Balboa, Julio; Estrada-Parra, Sergio; Perez-Tapia, Mayra; Jiménez-Martínez, Maria C.

    2012-01-01

    Background Despite success of sublingual immunotherapy (SLIT) in the treatment of allergy diseases, more research is needed related to ocular allergy. Thus, the aim of this work was to analyze the ocular microenvironment provided by tear cytokines in allergic conjunctivitis (AC) patients treated with SLIT and to correlate tear and serum cytokines with ocular findings. Methods 19 AC-patients were included in this study. AC diagnosis was based on a clinical history and full ophthalmologic examination according to the diagnosis standards of the American Academy of Ophthalmology. Routine immunological studies were performed to corroborate allergic status Negative coproparasitoscopic results were documented. This study was approved by Scientific and Ethics Committees if Institute of Ophthalmology “Conde de la Valenciana,” Mexico City an all subjects gave their informed consent to obtain samples. Tear and serum samples were collected to determine cytokines IL2, IL-4, IL-5, IFN-g, TNF-a, IL-10 by cytometric bead arrays (CBA), following manufacturer's instructions. Results After 6 months of treatment with SLIT we observed significant higher IFN-g concentration, without significant changes in IL2, IL4, IL5, TNFa or IL10. We observed significant clinical improvement since 3 months of treatment and it was maintained until the end of 6 months. Clinical improvement correlated with IFN-g concentration. Conclusions Clinical outcome in AC-patients treated with SLIT could be tear IFN-g dependent.

  5. Toxoplasma gondii-skeletal muscle cells interaction increases lipid droplet biogenesis and positively modulates the production of IL-12, IFN-g and PGE2

    PubMed Central

    2014-01-01

    Background The interest in the mechanisms involved in Toxoplasma gondii lipid acquisition has steadily increased during the past few decades, but it remains not completely understood. Here, we investigated the biogenesis and the fate of lipid droplets (LD) of skeletal muscle cells (SkMC) during their interaction with T. gondii by confocal and electron microscopy. We also evaluated whether infected SkMC modulates the production of prostaglandin E2 (PGE2), cytokines interleukin-12 (IL-12) and interferon-gamma (INF-g), and also the cyclooxygenase-2 (COX-2) gene induction. Methods Primary culture of skeletal muscle cells were infected with tachyzoites of T. gondii and analysed by confocal microscopy for observation of LD. Ultrastructural cytochemistry was also used for lipid and sarcoplasmatic reticulum (SR) detection. Dosage of cytokines (IL-12 and INF-g) by ELISA technique and enzyme-linked immunoassay (EIA) for PGE2 measurement were employed. The COX-2 gene expression analysis was performed by real time reverse transcriptase polymerase chain reaction (qRT-PCR). Results We demonstrated that T. gondii infection of SkMC leads to increase in LD number and area in a time course dependent manner. Moreover, the ultrastructural analysis demonstrated that SR and LD are in direct contact with parasitophorous vacuole membrane (PVM), within the vacuolar matrix, around it and interacting directly with the membrane of parasite, indicating that LD are recruited and deliver their content inside the parasitophorous vacuole (PV) in T. gondii-infected SkMC. We also observed a positive modulation of the production of IL-12 and IFN-g, increase of COX-2 mRNA levels in the first hour of T. gondii-SkMC interaction and an increase of prostaglandin E2 (PGE2) synthesis from 6 h up to 48 h of infection. Conclusions Taken together, the close association between SR and LD with PV could represent a source of lipids as well as other nutrients for the parasite survival, and together with the

  6. Severe South American Ocular Toxoplasmosis Is Associated with Decreased Ifn-γ/Il-17a and Increased Il-6/Il-13 Intraocular Levels

    PubMed Central

    de-la-Torre, Alejandra; Sauer, Arnaud; Pfaff, Alexander W.; Bourcier, Tristan; Brunet, Julie; Speeg-Schatz, Claude; Ballonzoli, Laurent; Villard, Odile; Ajzenberg, Daniel; Sundar, Natarajan; Grigg, Michael E.

    2013-01-01

    In a cross sectional study, 19 French and 23 Colombian cases of confirmed active ocular toxoplasmosis (OT) were evaluated. The objective was to compare clinical, parasitological and immunological responses and relate them to the infecting strains. A complete ocular examination was performed in each patient. The infecting strain was characterized by genotyping when intraocular Toxoplasma DNA was detectable, as well as by peptide-specific serotyping for each patient. To characterize the immune response, we assessed Toxoplasma protein recognition patterns by intraocular antibodies and the intraocular profile of cytokines, chemokines and growth factors. Significant differences were found for size of active lesions, unilateral macular involvement, unilateral visual impairment, vitreous inflammation, synechiae, and vasculitis, with higher values observed throughout for Colombian patients. Multilocus PCR-DNA sequence genotyping was only successful in three Colombian patients revealing one type I and two atypical strains. The Colombian OT patients possessed heterogeneous atypical serotypes whereas the French were uniformly reactive to type II strain peptides. The protein patterns recognized by intraocular antibodies and the cytokine patterns were strikingly different between the two populations. Intraocular IFN-γ and IL-17 expression was lower, while higher levels of IL-13 and IL-6 were detected in aqueous humor of Colombian patients. Our results are consistent with the hypothesis that South American strains may cause more severe OT due to an inhibition of the protective effect of IFN-γ. PMID:24278490

  7. Modifications in rat testicular morphology and increases in IFN-gamma serum levels by the oral administration of subtoxic doses of mercuric chloride.

    PubMed

    Penna, Salvador; Pocino, Marisol; Marval, Maria Josefina; Lloreta, José; Gallardo, Luis; Vila, Joan

    2009-01-01

    Mercury induces structural and functional damage in several organs, however the effects of subtoxic doses of the metal on the male reproductive system are not well defined. In order to analyze testicular and epididymal morphological alterations and changes in IL-4 or IFN-gamma serum levels, adult male Sprague-Dawley rats received 0.01, 0.05 or 0.1 microg/ml of mercuric chloride (HgCl(2)) in deionized water for 1 to 7 months by oral route. Controls received deionized water alone. Twenty rats, separated in four groups of five animals each, were used per time of exposure. Progressive degenerative lesions consisting of lack of germ cell cohesion and desquamation, arrest at spermatocyte stage and hypospermatogenesis were observed in seminiferous epithelium by light and electron microscopy. Leydig cells showed cytoplasmic vacuolation and nuclear signs of cell death. Loss of peritubular cell aggregation was evidenced in the epididymis. Mercury accumulation was detected in both organs by mass spectroscopy. Rats showed enhanced IFN-gamma serum levels as compared to controls but only reached significance after 7 months of mercury administration. Subtoxic doses of inorganic mercury could lead to reproductive and immunological alterations. The results demonstrate that sublethal concentrations of mercuric chloride are enough to induce morphological and ultrastructural modifications in male reproductive organs. These contribute to functional alterations of spermatogenesis with arrest at spermatocyte stage, hypospermatogenesis and possibly impaired steroidogenesis which together could affect male fertility.

  8. Protective effect of Ocimum sanctum on 3-methylcholanthrene, 7,12-dimethylbenz(a)anthracene and aflatoxin B1 induced skin tumorigenesis in mice

    SciTech Connect

    Rastogi, Shipra; Shukla, Yogeshwer; Paul, Bhola N.; Chowdhuri, D. Kar; Khanna, Subhash K.; Das, Mukul

    2007-11-01

    A study on the protective effect of alcoholic extract of the leaves of Ocimum sanctum on 3-mthylcholanthrene (MCA), 7,12-dimethylbenzanthracene (DMBA) and aflatoxin B{sub 1} (AFB{sub 1}) induced skin tumorigenesis in a mouse model has been investigated. The study involved pretreatment of mice with the leaf extract prior to either MCA application or tetradecanoyl phorbol acetate (TPA) treatment in a two-stage tumor protocol viz a viz, DMBA/TPA and AFB1/TPA. The results of the present study indicate that the pretreatment with alcoholic extract of the leaves of O. sanctum decreased the number of tumors in MCA, DMBA/TPA and AFB1/TPA treated mice. The skin tumor induced animals pretreated with alcoholic extract led to a decrease in the expression of cutaneous {gamma}-glutamyl transpeptidase (GGT) and glutathione-S-transferase-P (GST-P) protein. The histopathological examination of skin tumors treated with leaf extract showed increased infiltration of polymorphonuclear, mononuclear and lymphocytic cells, decreased ornithine decarboxylase activity with concomitant enhancement of interleukin-1{beta} (IL-1{beta}) and tumor necrosis factor-{alpha} (TNF-{alpha}) in the serum, implying the in vivo antiproliferative and immunomodulatory activity of leaf extract. The decrease in cutaneous phase I enzymes and elevation of phase II enzymes in response to topical application of leaf extract prior to MCA, AFB1, DMBA/TPA and AFB1/TPA treatment indicate the possibility of impairment in reactive metabolite(s) formation and thereby reducing skin carcinogenicity. Furthermore, pretreatment of leaf extract in the carcinogen induced animals resulted in elevation of glutathione levels and decrease in lipid peroxidation along with heat shock protein expression, indicating a scavenging or antioxidant potential of the extract during chemical carcinogenesis. Thus it can be concluded that leaf extract of O. sanctum provides protection against chemical carcinogenesis in one or more of the

  9. Extracellular UDP and P2Y6 function as a danger signal to protect mice from vesicular stomatitis virus infection through an increase in IFN-β production.

    PubMed

    Li, Ruimei; Tan, Binghe; Yan, Yan; Ma, Xiaobin; Zhang, Na; Zhang, Zhi; Liu, Mingyao; Qian, Min; Du, Bing

    2014-11-01

    Extracellular nucleotides that constitute a "danger signal" play an important role in the regulation of immune responses. However, the function and mechanism of extracellular UDP and P2Y6 in antiviral immunity remain unknown. In this study, we demonstrated the in vitro and in vivo protection of UDP/P2Y6 signaling in vesicular stomatitis virus (VSV) infection. First, we demonstrated that VSV-infected cells secrete UDP from the cytoplasm as a danger signal to arouse surrounding cells. Meanwhile, expression of the UDP-specific receptor P2Y6 also was enhanced by VSV. Consequently, UDP protects RAW 264.7 cells, murine embryonic fibroblasts, bone marrow-derived macrophages, and L929 cells from VSV and GFP lentivirus infection. This protection can be blocked by the P2Y6 selective antagonist MRS2578 or IFN-α/β receptor-blocking Ab. VSV-induced cell death and virus replication were both enhanced significantly by knocking down and knocking out P2Y6 in different cells. Mechanistically, UDP facilitates IFN-β secretion through the p38/JNK- and ATF-2/c-Jun-signaling pathways, which are crucial in promoting antiviral immunity. Interestingly, UDP was released through a caspase-cleaved pannexin-1 channel in VSV-induced apoptotic cells and protected cells from infection through P2Y6 receptor in an autocrine or paracrine manner. Furthermore, UDP also protected mice from VSV infection through P2Y6 receptors in an acute neurotropic infection mouse model. Taken together, these results demonstrate the important role of extracellular UDP and P2Y6 as a danger signal in antiviral immune responses and suggest a potential therapeutic role for UDP/P2Y6 in preventing and controlling viral diseases.

  10. Strength and Aerobic Physical Exercises Are Able to Increase Survival of Toxoplasma gondii-Infected C57BL/6 Mice by Interfering in the IFN-γ Expression.

    PubMed

    Bortolini, Miguel J S; Silva, Murilo V; Alonso, Fábio M; Medeiros, Luciana A; Carvalho, Fernando R; Costa, Lourenço F; Silva, Neide M; Penha-Silva, Nilson; Mineo, Tiago W P; Mineo, José R

    2016-01-01

    Physical exercise has been implicated in several immunophysiological improvements, particularly during the aging process, when an immunocompromised status could be established. Toxoplasma gondii is a protozoan parasite that causes a widespread opportunistic infection, which may present severe consequences, mainly to the fetus and immunocompromised patients. It is estimated that one-third of the human population worldwide has been infected by this parasite, being the reactivation during immunesenescence an unexplored public health issue. The major purpose of the present study was to observe the immunophysiological differences between exercised vs. sedentary C57BL/6 male mice that have been experimentally infected by T. gondii. In the first set of experiments, the animals were infected after exercising and three groups were set up: experimental groups-infected sedentary (IS, n = 6); infected exercised (IEx, n = 6) and control group-non-infected sedentary (NIS, n = 6). When stimulated in vitro by T. gondii-soluble tachyzoite antigen, it was found that splenocytes from exercised group produced higher levels of IFN-γ, as well as of IFN-γ/IL-10 ratios in comparison with splenocytes from sedentary animals (P < 0.001). However, it was not found significant differences concerning quantification of T. gondii genomic DNA by qRT-PCR and immunohistochemistry analysis in brain cysts from both group of animals (P > 0.05). In order to further investigate the consequences of these data for the host, a second set of experiments was performed, when the animals were infected before exercising and four groups of animals were established for comparison purpose, as follows: experimental groups-infected sedentary (IS, n = 7); infected exercised (IEx, n = 6) and control groups-non-infected sedentary (NIS, n = 6) and non-infected exercised (NIEx, n = 6). It was found significant differences in the survival rates of the exercised group the animals, as they survived longer than sedentary

  11. Strength and Aerobic Physical Exercises Are Able to Increase Survival of Toxoplasma gondii-Infected C57BL/6 Mice by Interfering in the IFN-γ Expression

    PubMed Central

    Bortolini, Miguel J. S.; Silva, Murilo V.; Alonso, Fábio M.; Medeiros, Luciana A.; Carvalho, Fernando R.; Costa, Lourenço F.; Silva, Neide M.; Penha-Silva, Nilson; Mineo, Tiago W. P.; Mineo, José R.

    2016-01-01

    Physical exercise has been implicated in several immunophysiological improvements, particularly during the aging process, when an immunocompromised status could be established. Toxoplasma gondii is a protozoan parasite that causes a widespread opportunistic infection, which may present severe consequences, mainly to the fetus and immunocompromised patients. It is estimated that one-third of the human population worldwide has been infected by this parasite, being the reactivation during immunesenescence an unexplored public health issue. The major purpose of the present study was to observe the immunophysiological differences between exercised vs. sedentary C57BL/6 male mice that have been experimentally infected by T. gondii. In the first set of experiments, the animals were infected after exercising and three groups were set up: experimental groups—infected sedentary (IS, n = 6); infected exercised (IEx, n = 6) and control group—non-infected sedentary (NIS, n = 6). When stimulated in vitro by T. gondii-soluble tachyzoite antigen, it was found that splenocytes from exercised group produced higher levels of IFN-γ, as well as of IFN-γ/IL-10 ratios in comparison with splenocytes from sedentary animals (P < 0.001). However, it was not found significant differences concerning quantification of T. gondii genomic DNA by qRT-PCR and immunohistochemistry analysis in brain cysts from both group of animals (P > 0.05). In order to further investigate the consequences of these data for the host, a second set of experiments was performed, when the animals were infected before exercising and four groups of animals were established for comparison purpose, as follows: experimental groups—infected sedentary (IS, n = 7); infected exercised (IEx, n = 6) and control groups—non-infected sedentary (NIS, n = 6) and non-infected exercised (NIEx, n = 6). It was found significant differences in the survival rates of the exercised group the animals, as they survived longer than

  12. IFN-α, IFN-β, and IFN-γ Have Different Effect on the Production of Proinflammatory Factors Deposited in Weibel-Palade Bodies of Endothelial Cells Infected with Herpes Simplex Virus Type 1.

    PubMed

    Shcheglovitova, O N; Boldyreva, N V; Sklyankina, N N; Babayants, A A; Frolova, I S

    2016-06-01

    We demonstrated similarities and differences in the effects of IFN-α and IFN-β compared to IFN-γ on the production of factors deposited in the Weibel-Palade bodies in cultures of endothelial cells (intact and infected with herpes simplex virus 1). IFN-α and IFN-β reduced the content of von Willebrand factor, endothelin-1, and soluble P-selectin and increased IL-8 concentration in the culture medium of human umbilical vein endothelial cells. IFN-γ reduced the content of all studied factors in the endothelial cell culture medium. Possible mechanisms of these effects are discussed.

  13. Absence of IFNincreases brain pathology in experimental autoimmune encephalomyelitis-susceptible DRB1*0301.DQ8 HLA transgenic mice through secretion of proinflammatory cytokine IL-17 and induction of pathogenic monocytes/microglia into the central nervous system.

    PubMed

    Mangalam, Ashutosh K; Luo, Ningling; Luckey, David; Papke, Louisa; Hubbard, Alyssa; Wussow, Arika; Smart, Michele; Giri, Shailendra; Rodriguez, Moses; David, Chella

    2014-11-15

    Multiple sclerosis is an inflammatory, demyelinating disease of the CNS of presumed autoimmune origin. Of all the genetic factors linked with multiple sclerosis, MHC class II molecules have the strongest association. Generation of HLA class II transgenic (Tg) mice has helped to elucidate the role of HLA class II genes in chronic inflammatory and demyelinating diseases. We have shown that the human HLA-DRB1*0301 gene predisposes to proteolipid protein (PLP)-induced experimental autoimmune encephalomyelitis (EAE), whereas HLA-DQβ1*0601 (DQ6) was resistant. We also showed that the DQ6 molecule protects from EAE in DRB1*0301.DQ6 double-Tg mice by producing anti-inflammatory IFN-γ. HLA-DQβ1*0302 (DQ8) Tg mice were also resistant to PLP(91-110)-induced EAE, but production of proinflammatory IL-17 exacerbated disease in DRB1*0301.DQ8 mice. To further confirm the role of IFN-γ in protection, we generated DRB1*0301.DQ8 mice lacking IFN-γ (DRB1*0301.DQ8.IFN-γ(-/-)). Immunization with PLP(91-110) peptide caused atypical EAE in DRB1*0301.DQ8.IFN-γ(-/-) mice characterized by ataxia, spasticity, and dystonia, hallmarks of brain-specific disease. Severe brain-specific inflammation and demyelination in DRB1*0301.DQ8.IFN-γ(-/-) mice with minimal spinal cord pathology further confirmed brain-specific pathology. Atypical EAE in DRB1*0301.DQ8.IFN-γ(-/-) mice was associated with increased encephalitogenicity of CD4 T cells and their ability to produce greater levels of IL-17 and GM-CSF compared with DRB1*0301.DQ8 mice. Further, areas with demyelination showed increased presence of CD68(+) inflammatory cells, suggesting an important role for monocytes/microglia in causing brain pathology. Thus, our study supports a protective role for IFN-γ in the demyelination of brain through downregulation of IL-17/GM-CSF and induction of neuroprotective factors in the brain by monocytes/microglial cells.

  14. Genomic effects of IFN-beta in multiple sclerosis patients.

    PubMed

    Weinstock-Guttman, Bianca; Badgett, Darlene; Patrick, Kara; Hartrich, Laura; Santos, Roseane; Hall, Dennis; Baier, Monika; Feichter, Joan; Ramanathan, Murali

    2003-09-01

    The purpose of this report was to characterize the dynamics of the gene expression cascades induced by an IFN-beta-1a treatment regimen in multiple sclerosis patients and to examine the molecular mechanisms potentially capable of causing heterogeneity in response to therapy. In this open-label pharmacodynamic study design, peripheral blood was obtained from eight relapsing-remitting multiple sclerosis patients just before and at 1, 2, 4, 8, 24, 48, 120, and 168 h after i.m. injection of 30 micro g of IFN-beta-1a. The total RNA was isolated from monocyte-depleted PBL and analyzed using cDNA microarrays containing probes for >4000 known genes. IFN-beta-1a treatment resulted in selective, time-dependent effects on multiple genes. The mRNAs for genes implicated in the anti-viral response, e.g., double-stranded RNA-dependent protein kinase, myxovirus resistance proteins 1 and 2, and guanylate binding proteins 1 and 2 were rapidly induced within 1-4 h of IFN-beta treatment. The mRNAs for several genes involved in IFN-beta signaling, such as IFN-alpha/beta receptor-2 and Stat1, were also increased. The mRNAs for lymphocyte activation markers, such as IFN-induced transmembrane protein 1 (9-27), IFN-induced transmembrane protein 2 (1-8D), beta(2)-microglobulin, and CD69, were also increased in a time-dependent manner. The findings demonstrate that IFN-beta treatment induces specific and time-dependent changes in multiple mRNAs in lymphocytes of multiple sclerosis patients that could provide a framework for rapid monitoring of the response to therapy.

  15. Increasing the ex vivo antigen-specific IFN-γ production in subpopulations of T cells and NKp46+ cells by anti-CD28, anti-CD49d and recombinant IL-12 costimulation in cattle vaccinated with recombinant proteins from Mycobacterium avium subspecies paratuberculosis.

    PubMed

    Thakur, Aneesh; Riber, Ulla; Davis, William C; Jungersen, Gregers

    2013-10-01

    T cells, which encounter specific antigen (Ag), require additional signals to mount a functional immune response. Here, we demonstrate activation of signal 2, by anti-CD28 mAb (aCD28) and other costimulatory molecules (aCD49d, aCD5), and signal 3, by recombinant IL-12, enhance Ag-specific IFN-γ secretion by CD4, CD8, γδ T cells and NK cells. Age matched male jersey calves, experimentally infected with Mycobacterium avium subsp. paratuberculosis (MAP), were vaccinated with a cocktail of recombinant MAP proteins or left unvaccinated. Vaccine induced ex vivo recall responses were measured through Ag-specific IFN-γ production by ELISA and flow cytometry. There was a significant increase in production of IFN-γ by T cell subsets or NKp46+ cells cultured in the presence of Ag and aCD28/aCD49d. The increase was accompanied by an increase in the integrated median fluorescence intensity (iMFI) of activated T cells. Addition of rIL-12 induced a significant additive effect leading to a maximum increase in responder frequency of Ag-specific T cell subsets or NKp46+ cells with a heavy bias toward IFN-γ production by CD4 T cells. We provide the first description of using aCD28/aCD49d costimulation to potentiate an Ag-specific increase in the production of IFN-γ in bovine immunology. The study also shows the degree of signaling in T cells is regulated by the costimulatory environment.

  16. Type I IFN Inhibits Alternative Macrophage Activation during Mycobacterium tuberculosis Infection and Leads to Enhanced Protection in the Absence of IFN-γ Signaling

    PubMed Central

    Sousa, Jeremy; McNab, Finlay W.; Torrado, Egídio; Cardoso, Filipa; Machado, Henrique; Castro, Flávia; Cardoso, Vânia; Gaifem, Joana; Wu, Xuemei; Appelberg, Rui; Castro, António Gil; O’Garra, Anne; Saraiva, Margarida

    2016-01-01

    Tuberculosis causes ∼1.5 million deaths every year, thus remaining a leading cause of death from infectious diseases in the world. A growing body of evidence demonstrates that type I IFN plays a detrimental role in tuberculosis pathogenesis, likely by interfering with IFN-γ–dependent immunity. In this article, we reveal a novel mechanism by which type I IFN may confer protection against Mycobacterium tuberculosis infection in the absence of IFN-γ signaling. We show that production of type I IFN by M. tuberculosis–infected macrophages induced NO synthase 2 and inhibited arginase 1 gene expression. In vivo, absence of both type I and type II IFN receptors led to strikingly increased levels of arginase 1 gene expression and protein activity in infected lungs, characteristic of alternatively activated macrophages. This correlated with increased lung bacterial burden and pathology and decreased survival compared with mice deficient in either receptor. Increased expression of other genes associated with alternatively activated macrophages, as well as increased expression of Th2-associated cytokines and decreased TNF expression, were also observed. Thus, in the absence of IFN-γ signaling, type I IFN suppressed the switching of macrophages from a more protective classically activated phenotype to a more permissive alternatively activated phenotype. Together, our data support a model in which suppression of alternative macrophage activation by type I IFN during M. tuberculosis infection, in the absence of IFN-γ signaling, contributes to host protection. PMID:27849167

  17. TBX21-1993T/C (rs4794067) polymorphism is associated with increased risk of chronic periodontitis and increased T-bet expression in periodontal lesions, but does not significantly impact the IFN-g transcriptional level or the pattern of periodontophatic bacterial infection

    PubMed Central

    Cavalla, Franco; Biguetti, Claudia Cristina; Colavite, Priscila Maria; Silveira, Elcia Varise; Martins, Walter; Letra, Ariadne; Trombone, Ana Paula Favaro; Silva, Renato Menezes; Garlet, Gustavo Pompermaier

    2015-01-01

    Th1-polarized host response, mediated by IFN-γ, has been associated with increased severity of periodontal disease as well as control of periodontal infection. The functional polymorphism TBX21-1993T/C (rs4794067) increases the transcriptional activity of the TBX21 gene (essential for Th1 polarization) resulting in a predisposition to a Th-1 biased immune response. Thus, we conducted a case-control study, including a population of healthy controls (H, n = 218), chronic periodontitis (CP, n = 197), and chronic gingivitis patients (CG, n = 193), to investigate if genetic variations in TBX21 could impact the development of Th1 responses, and consequently influence the pattern of bacterial infection and periodontitis outcome. We observed that the polymorphic allele T was significantly enriched in the CP patients compared to CG subjects, while the H controls demonstrated and intermediate genotype. Also, investigating the putative functionality TBX21-1993T/C in the modulation of local response, we observed that the transcripts levels of T-bet, but not of IFN-γ, were upregulated in homozygote and heterozygote polymorphic subjects. In addition, TBX21-1993T/C did not influence the pattern of bacterial infection or the clinical parameters of disease severity, being the presence/absence of red complex bacteria the main factor associated with the disease status and the subrogate variable probing depth (PD) in the logistic regression analysis. PMID:25832120

  18. IFN-γ ameliorates autoimmune encephalomyelitis by limiting myelin lipid peroxidation

    PubMed Central

    Sosa, Rebecca A.; Murphey, Cathi; Robinson, Rachel R.; Forsthuber, Thomas G.

    2015-01-01

    Evidence has suggested both a pathogenic and a protective role for the proinflammatory cytokine IFN-γ in experimental autoimmune encephalomyelitis (EAE). However, the mechanisms underlying the protective role of IFN-γ in EAE have not been fully resolved, particularly in the context of CNS antigen-presenting cells (APCs). In this study we examined the role of IFN-γ in myelin antigen uptake by CNS APCs during EAE. We found that myelin antigen colocalization with APCs was decreased substantially and that EAE was significantly more severe and showed a chronic-progressive course in IFN-γ knockout (IFN-γ−/−) or IFN-γ receptor knockout (IFN-γR−/−) mice as compared with WT animals. IFN-γ was a critical regulator of phagocytic/activating receptors on CNS APCs. Importantly, “free” myelin debris and lipid peroxidation activity at CNS lesions was increased in mice lacking IFN-γ signaling. Treatment with N-acetyl-l-cysteine, a potent antioxidant, abolished lipid peroxidation activity and ameliorated EAE in IFN-γ–signaling-deficient mice. Taken together the data suggest a protective role for IFN-γ in EAE by regulating the removal of myelin debris by CNS APCs and thereby limiting the substrate available for the generation of neurotoxic lipid peroxidation products. PMID:26305941

  19. Negatively charged phospholipids suppress IFN-{gamma} production in T cells

    SciTech Connect

    Yotsumoto, Satoshi; Kakiuchi, Terutaka; Aramaki, Yukihiko . E-mail: aramaki@ps.toyaku.ac.jp

    2005-12-30

    The effect of phospholipids on IFN-{gamma} production in mouse T cells was investigated. Phosphatidylserine (PS), which has a negatively charged head group, completely inhibited IFN-{gamma} production in splenic naive T cells and antigen-dependent IFN-{gamma} production in Th1 clone 42-6A cells, whereas other phospholipids, which have neutrally charged head group, had no effect. The structural requirements for IFN-{gamma} inhibitory effects by PS were investigated, and dimyristoyl-PS (C14: 0) and dipalmitoyl-PS (C16: 0) had no effect on IFN-{gamma} production, and interestingly, distearoyl-PS (18: 0) increased IFN-{gamma} production. Dioleoyl-PS (C18: 1), dilinoleoyl-PS (C18: 2), and oleoyl-lyso-PS (C18: 1) completely inhibited IFN-{gamma} production. To clarify this mechanism, we focused on the stability of IFN-{gamma} mRNA, and the treatment of splenic naive T cells with PS brought about 40% reductions in IFN-{gamma} mRNA expression in the presence of actinomycin D. Collectively, IFN-{gamma} inhibitory effects by PS are highly dependent on the molecular structure of PS and involve the decreasing of the stability of IFN-{gamma} mRNA.

  20. Human IFN-gamma up-regulates IL-2 receptors in mitogen-activated T lymphocytes.

    PubMed Central

    Rodriguez, M A; De Sanctis, J B; Blasini, A M; Leon-Ponte, M; Abadi, I

    1990-01-01

    This study examined the role of human recombinant interferon-gamma (rIFN-gamma) in the expression of interleukin-2 receptors (IL-2R) by human T lymphocytes. rIFN-gamma enhanced total numbers of IL-2R in mitogen-activated but not resting T cells. Scatchard plot analysis indicated that rIFN-gamma increased both high- and low-affinity receptors, with a predominant effect on the latter. Phytohaemagglutinin (PHA)-activated T cells treated with IFN-gamma showed higher IL-2 binding and greater IL-2 internalization and degradation than cells treated with PHA alone. There was a corresponding increase of mitogen-driven proliferative responses, indicating an increase of functional receptors in IFN-treated cultures. IFN-gamma may influence T-cell activation and proliferation by enhancing expression of IL-2R and promoting IL-2 uptake by mitogen-activated lymphocytes. PMID:2110548

  1. [Anti-HIV effects of IFN-tau in human macrophages: role of cellular antiviral factors and interleukin-6].

    PubMed

    Maneglier, B; Rogez-Kreuz, C; Dereuddre-Bosquet, N; Martal, J; Devillier, P; Dormont, D; Clayette, P

    2008-01-01

    Tau interferon (IFN-tau) was shown to inhibit human immunodeficiency virus (HIV) replication in vitro more strongly than human IFN-alpha, particularly in human macrophages. IFN-tau efficiently inhibited the early steps of HIV biological cycle, decreasing intracellular HIV RNA and inhibiting the initiation of the reverse transcription of viral RNA into proviral DNA. In this study, the in vitro immunomodulatory effects of IFN-tau were explored in human macrophages. We found that IFN-tau increased the synthesis of the cellular antiviral factors, such as 2',5'-oligoadenylate synthetase/RNase L and MxA protein. These results suggested that IFN-tau induces the same antiviral pathways in macrophages as other type I IFNs. We found that IFN-tau increased the production of interleukins (IL)-10 and IL-6, but not of IL-1ss or TNF-alpha, in not infected and in in vitro HIV-1/Ba-L-infected macrophages. We also found that the neutralization of IL-6 biological activity in the cell culture supernatants of IFN-tau-treated macrophages led to a decrease in the antiretroviral effects of IFN-tau towards HIV RNA. In conclusion, anti-HIV effects of IFN-tau are mediated by several modes of action, mediated either directly by IFN-tau or via other cytokines, such as IL-6, also known to be induced by IFN-alpha.

  2. IFN-gamma AU-rich element removal promotes chronic IFN-gamma expression and autoimmunity in mice

    PubMed Central

    Hodge, Deborah L.; Berthet, Cyril; Coppola, Vincenzo; Kastenmüller, Wolfgang; Buschman, Matthew D.; Schaughency, Paul M.; Shirota, Hidekazu; Scarzello, Anthony J.; Subleski, Jeff J.; Anver, Miriam R.; Ortaldo, John R.; Lin, Fanching; Reynolds, Della A.; Sanford, Michael E.; Kaldis, Philipp; Tessarollo, Lino; Klinman, Dennis M.; Young, Howard A.

    2014-01-01

    We generated a mouse model with a 162 nt AU-rich element (ARE) region deletion in the 3′ untranslated region (3′UTR) of the interferon-gamma (IFN-γ) gene that results in chronic circulating serum IFN-γ levels. Mice homozygous for the ARE deletion (ARE-Del) −/− present both serologic and cellular abnormalities typical of patients with systemic lupus erythematosus (SLE). ARE-Del−/− mice display increased numbers of pDCs in bone marrow and spleen. Addition of IFN-γ to Flt3-ligand (Flt3L) treated in vitro bone marrow cultures results in a 2-fold increase in pDCs with concurrent increases in IRF8 expression. Marginal zone B (MZB) cells and marginal zone macrophages (MZMs) are absent in ARE-Del−/− mice. ARE-Del+/− mice retain both MZB cells and MZMs and develop no or mild autoimmunity. However, low dose clodronate treatment in ARE-Del+/− mice specifically eliminates MZMs and promotes anti-DNA antibody development and glomerulonephritis. Our findings demonstrate the consequences of a chronic IFN-γ milieu on B220+ cell types and in particular the impact of MZB cell loss on MZM function in autoimmunity. Furthermore, similarities between disease states in ARE-Del−/− mice and SLE patients suggest that IFN-γ may not only be a product of SLE but may be critical for disease onset and progression. PMID:24583068

  3. IFN-gamma AU-rich element removal promotes chronic IFN-gamma expression and autoimmunity in mice.

    PubMed

    Hodge, Deborah L; Berthet, Cyril; Coppola, Vincenzo; Kastenmüller, Wolfgang; Buschman, Matthew D; Schaughency, Paul M; Shirota, Hidekazu; Scarzello, Anthony J; Subleski, Jeff J; Anver, Miriam R; Ortaldo, John R; Lin, Fanching; Reynolds, Della A; Sanford, Michael E; Kaldis, Philipp; Tessarollo, Lino; Klinman, Dennis M; Young, Howard A

    2014-09-01

    We generated a mouse model with a 162 nt AU-rich element (ARE) region deletion in the 3' untranslated region (3'UTR) of the interferon-gamma (IFN-γ) gene that results in chronic circulating serum IFN-γ levels. Mice homozygous for the ARE deletion (ARE-Del) (-/-) present both serologic and cellular abnormalities typical of patients with systemic lupus erythematosus (SLE). ARE-Del(-/-) mice display increased numbers of pDCs in bone marrow and spleen. Addition of IFN-γ to Flt3-ligand (Flt3L) treated in vitro bone marrow cultures results in a 2-fold increase in pDCs with concurrent increases in IRF8 expression. Marginal zone B (MZB) cells and marginal zone macrophages (MZMs) are absent in ARE-Del(-/-) mice. ARE-Del(+/-) mice retain both MZB cells and MZMs and develop no or mild autoimmunity. However, low dose clodronate treatment in ARE-Del(+/-) mice specifically eliminates MZMs and promotes anti-DNA antibody development and glomerulonephritis. Our findings demonstrate the consequences of a chronic IFN-γ milieu on B220(+) cell types and in particular the impact of MZB cell loss on MZM function in autoimmunity. Furthermore, similarities between disease states in ARE-Del(-/-) mice and SLE patients suggest that IFN-γ may not only be a product of SLE but may be critical for disease onset and progression.

  4. HSV-2 immediate-early protein US1 inhibits IFN-β production by suppressing association of IRF-3 with IFN-β promoter.

    PubMed

    Zhang, Mudan; Liu, Yalan; Wang, Ping; Guan, Xinmeng; He, Siyi; Luo, Sukun; Li, Chang; Hu, Kai; Jin, Wei; Du, Tao; Yan, Yan; Zhang, Zhenfeng; Zheng, Zhenhua; Wang, Hanzhong; Hu, Qinxue

    2015-04-01

    HSV-2 is the major cause of genital herpes, and its infection increases the risk of HIV-1 acquisition and transmission. After initial infection, HSV-2 can establish latency within the nervous system and thus maintains lifelong infection in humans. It has been suggested that HSV-2 can inhibit type I IFN signaling, but the underlying mechanism has yet to be determined. In this study, we demonstrate that productive HSV-2 infection suppresses Sendai virus (SeV) or polyinosinic-polycytidylic acid-induced IFN-β production. We further reveal that US1, an immediate-early protein of HSV-2, contributes to such suppression, showing that US1 inhibits IFN-β promoter activity and IFN-β production at both mRNA and protein levels, whereas US1 knockout significantly impairs such capability in the context of HSV-2 infection. US1 directly interacts with DNA binding domain of IRF-3, and such interaction suppresses the association of nuclear IRF-3 with the IRF-3 responsive domain of IFN-β promoter, resulting in the suppression of IFN-β promoter activation. Additional studies demonstrate that the 217-414 aa domain of US1 is critical for the suppression of IFN-β production. Our results indicate that HSV-2 US1 downmodulates IFN-β production by suppressing the association of IRF-3 with the IRF-3 responsive domain of IFN-β promoter. Our findings highlight the significance of HSV-2 US1 in inhibiting IFN-β production and provide insights into the molecular mechanism by which HSV-2 evades the host innate immunity, representing an unconventional strategy exploited by a dsDNA virus to interrupt type I IFN signaling pathway.

  5. Limiting Cholesterol Biosynthetic Flux Spontaneously Engages Type I IFN Signaling.

    PubMed

    York, Autumn G; Williams, Kevin J; Argus, Joseph P; Zhou, Quan D; Brar, Gurpreet; Vergnes, Laurent; Gray, Elizabeth E; Zhen, Anjie; Wu, Nicholas C; Yamada, Douglas H; Cunningham, Cameron R; Tarling, Elizabeth J; Wilks, Moses Q; Casero, David; Gray, David H; Yu, Amy K; Wang, Eric S; Brooks, David G; Sun, Ren; Kitchen, Scott G; Wu, Ting-Ting; Reue, Karen; Stetson, Daniel B; Bensinger, Steven J

    2015-12-17

    Cellular lipid requirements are achieved through a combination of biosynthesis and import programs. Using isotope tracer analysis, we show that type I interferon (IFN) signaling shifts the balance of these programs by decreasing synthesis and increasing import of cholesterol and long chain fatty acids. Genetically enforcing this metabolic shift in macrophages is sufficient to render mice resistant to viral challenge, demonstrating the importance of reprogramming the balance of these two metabolic pathways in vivo. Unexpectedly, mechanistic studies reveal that limiting flux through the cholesterol biosynthetic pathway spontaneously engages a type I IFN response in a STING-dependent manner. The upregulation of type I IFNs was traced to a decrease in the pool size of synthesized cholesterol and could be inhibited by replenishing cells with free cholesterol. Taken together, these studies delineate a metabolic-inflammatory circuit that links perturbations in cholesterol biosynthesis with activation of innate immunity.

  6. Limiting cholesterol biosynthetic flux spontaneously engages type I IFN signaling

    PubMed Central

    York, Autumn G.; Williams, Kevin J.; Argus, Joseph P.; Zhou, Quan D.; Brar, Gurpreet; Vergnes, Laurent; Gray, Elizabeth E.; Zhen, Anjie; Wu, Nicholas C.; Yamada, Douglas H.; Cunningham, Cameron R.; Tarling, Elizabeth J.; Wilks, Moses Q.; Casero, David; Gray, David H.; Yu, Amy K.; Wang, Eric S.; Brooks, David G.; Sun, Ren; Kitchen, Scott G.; Wu, Ting-Ting; Reue, Karen; Stetson, Daniel B.; Bensinger, Steven J.

    2015-01-01

    Summary Cellular lipid requirements are achieved through a combination of biosynthesis and import programs. Using isotope tracer analysis, we show that type I interferon (IFN) signaling shifts the balance of these programs by decreasing synthesis and increasing import of cholesterol and long chain fatty acids. Genetically enforcing this metabolic shift in macrophages is sufficient to render mice resistant to viral challenge, demonstrating the importance of reprogramming the balance of these two metabolic pathways in vivo. Unexpectedly, mechanistic studies reveal that limiting flux through the cholesterol biosynthetic pathway spontaneously engages a type I IFN response in a STING-dependent manner. The upregulation of type I IFNs was traced to a decrease in the pool size of synthesized cholesterol, and could be inhibited by replenishing cells with free cholesterol. Taken together, these studies delineate a metabolic-inflammatory circuit that links perturbations in cholesterol biosynthesis with activation of innate immunity. PMID:26686653

  7. Posttranscriptional regulation of IL-23 expression by IFN-γ through tristetraprolin

    PubMed Central

    Qian, Xuesong; Ning, Huan; Zhang, Jidong; Hoft, Daniel F.; Stumpo, Deborah J.; Blackshear, Perry J.; Liu, Jianguo

    2014-01-01

    Interleukin-23 (IL-23) plays an essential role in maintenance of IL-17-producing T helper (Th17) cells that are involved in the pathogenesis of several autoimmune diseases. Regulation of Th17 cells is tightly controlled by multiple factors such as IL-27 and IFN-γ. However, the detailed mechanisms responsible for IFN-γ-mediated Th17 cell inhibition are still largely unknown. In this study, we demonstrate that IFN-γ differentially regulates IL-12 and IL-23 production in both dendritic cells and macrophages. IFN-γ suppresses IL-23 expression by selectively targeting p19 mRNA stability through its 3′untranslated region (3′UTR). Furthermore, IFN-γ enhances LPS-induced tristetraprolin (TTP) mRNA expression and protein production. Overexpression of TTP suppresses IL-23 p19 mRNA expression and p19 3′UTR-dependent luciferase activity. In addition, deletion of TTP completely abolishes IFN-γ-mediated p19 mRNA degradation. We further demonstrate that IFN-γ suppresses LPS-induced p38 phosphorylation and blockade of p38 MAPK signaling pathway with SB203580 inhibits IFN-γ and LPS induced p19 mRNA expression whereas overexpression of p38 increases p19 mRNA expression via reducing TTP binding to the p19 3′UTR. Finally, inhibition of p38 phosphorylation by IFN-γ leads to TTP dephosphorylation that could result in stronger binding of the TTP to the adenosine/uridine-rich elements in the p19 3′UTR and p19 mRNA degradation. In summary, our results reveal a direct link among TTP, IFN-γ and IL-23, indicating that IFN-γ-mediated Th17 cell suppression might act through TTP by increasing p19 mRNA degradation and therefore IL-23 inhibition. PMID:21515794

  8. Stimulator of IFN gene is critical for induction of IFN-beta during Chlamydia muridarum infection.

    PubMed

    Prantner, Daniel; Darville, Toni; Nagarajan, Uma M

    2010-03-01

    Type I IFN signaling has recently been shown to be detrimental to the host during infection with Chlamydia muridarum in both mouse lung and female genital tract. However, the pattern recognition receptor and the signaling pathways involved in chlamydial-induced IFN-beta are unclear. Previous studies have demonstrated no role for TLR4 and a partial role for MyD88 in chlamydial-induced IFN-beta. In this study, we demonstrate that mouse macrophages lacking TLR3, TRIF, TLR7, or TLR9 individually or both TLR4 and MyD88, still induce IFN-beta equivalent to wild type controls, leading to the hypothesis that TLR-independent cytosolic pathogen receptor pathways are crucial for this response. Silencing nucleotide-binding oligomerization domain 1 in HeLa cells partially decreased chlamydial-induced IFN-beta. Independently, small interfering RNA-mediated knockdown of the stimulator of IFN gene (STING) protein in HeLa cells and mouse oviduct epithelial cells significantly decreased IFN-beta mRNA expression, suggesting a critical role for STING in chlamydial-induced IFN-beta induction. Conversely, silencing of mitochondria-associated antiviral signaling proteins and the Rig-I-like receptors, RIG-I, and melanoma differentiation associated protein 5, had no effect. In addition, induction of IFN-beta depended on the downstream transcription IFN regulatory factor 3, and on activation of NF-kappaB and MAPK p38. Finally, STING, an endoplasmic reticulum-resident protein, was found to localize in close proximity to the chlamydial inclusion membrane during infection. These results indicate that C. muridarum induces IFN-beta via stimulation of nucleotide-binding oligomerization domain 1 pathway, and TLR- and Rig-I-like receptor-independent pathways that require STING, culminating in activation of IFN regulatory factor 3, NF-kappaB, and p38 MAPK.

  9. Silencing IFN-γ binding/signaling in astrocytes vs. microglia leads to opposite effects on CNS autoimmunity

    PubMed Central

    Ding, Xiaoli; Yan, Yaping; Li, Xing; Li, Ke; Ciric, Bogoljub; Yang, Jingxian; Zhang, Yuan; Wu, Shuai; Xu, Hui; Chen, Wanjun; Lovett-Racke, Amy E.; Zhang, Guang-Xian; Rostami, Abdolmohamad

    2015-01-01

    IFN-γ, the hallmark cytokine of Th1 cells, plays an important role in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). Thus far, the role of IFN-γ in EAE has been largely studied through its effects on immune cells, while much less is known about its effects on central nervous system (CNS) cells, especially in vivo. In this study we dissected the in vivo effects and mechanisms of IFN-γ binding/signaling in astrocytes and microglia and found that IFN-γ signaling in these cell types has opposite effects in EAE pathogenesis. Silencing IFN-γ binding/signaling in astrocytes ameliorated EAE, while in microglia, and likely in some infiltrating macrophages, it increased disease severity. Silencing IFN-γ signaling in astrocytes resulted in diminished expression of chemokines and fewer inflammatory cells infiltrating into the CNS, while blocking IFN-γ binding/signaling in microglia, probably infiltrating macrophages as well, increased disease severity through augmented activation and proliferation of microglia. Further, blocking IFN-γ binding/signaling in astrocytes ameliorated both Th1- and Th17-mediated adoptive EAE, indicating an important role for IFN-γ signaling in astrocytes in autoimmune CNS inflammation. Thus, our study defines novel mechanisms of action of IFN-γ in EAE pathogenesis, and also highlights an opportunity for development of MS therapies directed at CNS cells. PMID:25795755

  10. In vivo kinetics and characterization of IFN-gamma-producing cells during a thymus-independent immune response.

    PubMed

    van den Eertwegh, A J; Fasbender, M J; Schellekens, M M; van Oudenaren, A; Boersma, W J; Claassen, E

    1991-07-15

    Using immunohistochemical techniques, we studied IFN-gamma-producing cells (IFN-gamma-PC) in vivo during immune responses to thymus-independent type-2 (TI-2) Ag. Detection of IFN-gamma-PC in cryostat sections of spleen-tissue was performed with an enzyme labeled mAb directed against IFN-gamma. After TNP-Ficoll immunization, IFN-gamma-PC and TNP-specific antibody-forming cells (TNP-AFC) displayed similar kinetics reaching a maximum number at day 5 to 7. The IFN-gamma-PC were localized in the same compartment as TNP-AFC and a part of them in juxtaposition to TNP-AFC. Immunization with other TI-2 Ag resulted also in a significant increase of the number of IFN-gamma-PC. In a parallel experiment we found both in vivo and in an ELISA-spot assay a significant increase of the number of IFN-gamma-PC and IFN-gamma-spot-forming-cells, respectively, in spleens of mice 6 to 7 days after TNP-Ficoll immunization. Double staining of spleen sections for IFN-gamma and surface Ag revealed that 5 to 7 days after TNP-Ficoll immunization, +/- 40% of the IFN-gamma-PC expressed the MT4 Ag (CD4), +/- 50% the Lyt-2+ Ag (CD8) and +/- 10% the asialo-GM1 Ag (NK cell). This study represents the first description of the in vivo activity and characterization of IFN-gamma-PC during a TI-2 immune response. Moreover, the presented data confirm suggestions from in vitro investigations that IFN-gamma and T cells may play a direct role in the in vivo regulation of a primary immune response against a TI-2 Ag.

  11. IFN-λ cancer immunotherapy: new kid on the block.

    PubMed

    Lasfar, Ahmed; Gogas, Helen; Zloza, Andrew; Kaufman, Howard L; Kirkwood, John M

    2016-07-01

    Interferon-lambda (IFN-λ) is a new IFN type, related to IFN-α, that is commonly used in the clinic. However, significant side effects accompanying IFN-α treatment limit enthusiasm for IFN-α. In this review, we discuss the current landscape of IFN-α use in oncology and describe the biologic characteristics of IFN-λ. IFN-λ offers unique advantages, including a more tumor cell selective targeting, lower off-target binding and an ability to generate both innate and adaptive immune responses. IFN-λ has also demonstrated therapeutic benefit in murine cancer models. IFN-λ may be used in clinic as a single agent or in combination with other immunotherapy agents, such as immune checkpoint inhibitors. Further clinical trials will be needed to fully elucidate the potential of this novel agent in oncology.

  12. Curcumin modulation of IFN-β and IL-12 signalling and cytokine induction in human T cells

    PubMed Central

    Fahey, Angela J; Adrian Robins, R; Constantinescu, Cris S

    2007-01-01

    Abstract Curcumin is a polyphenol derived from the dietary spice turmeric. It possesses diverse anti-inflammatory and anti-cancer properties. Curcumin has been shown to exhibit an inhibitory effect on the production of inflammatory cytokines by human monocytes and has inhibited the animal model of multiple sclerosis (MS), experimental autoimmune encephalomyelitis (EAE) in association with a decrease in interleukin 12 (IL-12) production and signal transducer and activator of transcription 4 (STAT4) activation. The type I interferon (IFN) IFN-has the ability to suppress IL-12. Both IL-12 and IFN-α/β signal through the activation by phosphorylation of STAT4. Our aim was to investigate the effects of curcumin on the ability of T cells to respond to IL-12 or IFN-α/β. We report that curcumin decreases IL-12-induced STAT4 phosphorylation, IFN-γ production, and IL-12 Rβ1 and β2 expression. IFN-β-induced STAT4 phosphorylation, IL-10 production and IFN receptor (IFNAR) subunits 1 and 2 expression were enhanced by curcumin. Curcumin increased IFN-α-induced IL-10 and IFNAR1 expression. Prior exposure to curcumin decreased IFN-α-induced IFNAR2 expression and did not modify the level of IFN-α-induced pSTAT4 generation. Thus, the effect of curcumin on STAT4 activation in T cells is dependent upon the stimulus to which the T cells have been exposed. PMID:17979888

  13. HELZ2 Is an IFN Effector Mediating Suppression of Dengue Virus

    PubMed Central

    Fusco, Dahlene N.; Pratt, Henry; Kandilas, Stephen; Cheon, Scarlett Se Yun; Lin, Wenyu; Cronkite, D. Alex; Basavappa, Megha; Jeffrey, Kate L.; Anselmo, Anthony; Sadreyev, Ruslan; Yapp, Clarence; Shi, Xu; O'Sullivan, John F.; Gerszten, Robert E.; Tomaru, Takuya; Yoshino, Satoshi; Satoh, Tetsurou; Chung, Raymond T.

    2017-01-01

    Flaviviral infections including dengue virus are an increasing clinical problem worldwide. Dengue infection triggers host production of the type 1 IFN, IFN alpha, one of the strongest and broadest acting antivirals known. However, dengue virus subverts host IFN signaling at early steps of IFN signal transduction. This subversion allows unbridled viral replication which subsequently triggers ongoing production of IFN which, again, is subverted. Identification of downstream IFN antiviral effectors will provide targets which could be activated to restore broad acting antiviral activity, stopping the signal to produce endogenous IFN at toxic levels. To this end, we performed a targeted functional genomic screen for IFN antiviral effector genes (IEGs), identifying 56 IEGs required for antiviral effects of IFN against fully infectious dengue virus. Dengue IEGs were enriched for genes encoding nuclear receptor interacting proteins, including HELZ2, MAP2K4, SLC27A2, HSP90AA1, and HSP90AB1. We focused on HELZ2 (Helicase With Zinc Finger 2), an IFN stimulated gene and IEG which encodes a promiscuous nuclear factor coactivator that exists in two isoforms. The two unique HELZ2 isoforms are both IFN responsive, contain ISRE elements, and gene products increase in the nucleus upon IFN stimulation. Chromatin immunoprecipitation-sequencing revealed that the HELZ2 complex interacts with triglyceride-regulator LMF1. Mass spectrometry revealed that HELZ2 knockdown cells are depleted of triglyceride subsets. We thus sought to determine whether HELZ2 interacts with a nuclear receptor known to regulate immune response and lipid metabolism, AHR, and identified HELZ2:AHR interactions via co-immunoprecipitation, found that AHR is a dengue IEG, and that an AHR ligand, FICZ, exhibits anti-dengue activity. Primary bone marrow derived macrophages from HELZ2 knockout mice, compared to wild type controls, exhibit enhanced dengue infectivity. Overall, these findings reveal that IFN antiviral

  14. HELZ2 Is an IFN Effector Mediating Suppression of Dengue Virus.

    PubMed

    Fusco, Dahlene N; Pratt, Henry; Kandilas, Stephen; Cheon, Scarlett Se Yun; Lin, Wenyu; Cronkite, D Alex; Basavappa, Megha; Jeffrey, Kate L; Anselmo, Anthony; Sadreyev, Ruslan; Yapp, Clarence; Shi, Xu; O'Sullivan, John F; Gerszten, Robert E; Tomaru, Takuya; Yoshino, Satoshi; Satoh, Tetsurou; Chung, Raymond T

    2017-01-01

    Flaviviral infections including dengue virus are an increasing clinical problem worldwide. Dengue infection triggers host production of the type 1 IFN, IFN alpha, one of the strongest and broadest acting antivirals known. However, dengue virus subverts host IFN signaling at early steps of IFN signal transduction. This subversion allows unbridled viral replication which subsequently triggers ongoing production of IFN which, again, is subverted. Identification of downstream IFN antiviral effectors will provide targets which could be activated to restore broad acting antiviral activity, stopping the signal to produce endogenous IFN at toxic levels. To this end, we performed a targeted functional genomic screen for IFN antiviral effector genes (IEGs), identifying 56 IEGs required for antiviral effects of IFN against fully infectious dengue virus. Dengue IEGs were enriched for genes encoding nuclear receptor interacting proteins, including HELZ2, MAP2K4, SLC27A2, HSP90AA1, and HSP90AB1. We focused on HELZ2 (Helicase With Zinc Finger 2), an IFN stimulated gene and IEG which encodes a promiscuous nuclear factor coactivator that exists in two isoforms. The two unique HELZ2 isoforms are both IFN responsive, contain ISRE elements, and gene products increase in the nucleus upon IFN stimulation. Chromatin immunoprecipitation-sequencing revealed that the HELZ2 complex interacts with triglyceride-regulator LMF1. Mass spectrometry revealed that HELZ2 knockdown cells are depleted of triglyceride subsets. We thus sought to determine whether HELZ2 interacts with a nuclear receptor known to regulate immune response and lipid metabolism, AHR, and identified HELZ2:AHR interactions via co-immunoprecipitation, found that AHR is a dengue IEG, and that an AHR ligand, FICZ, exhibits anti-dengue activity. Primary bone marrow derived macrophages from HELZ2 knockout mice, compared to wild type controls, exhibit enhanced dengue infectivity. Overall, these findings reveal that IFN antiviral

  15. The inhibitory effect of IFN-γ on protease HTRA1 expression in rheumatoid arthritis.

    PubMed

    Hou, Yuzhu; Lin, Haijiang; Zhu, Linnan; Liu, Zhaoting; Hu, Fanlei; Shi, Jianfeng; Yang, Tao; Shi, Xiaoyun; Guo, Huifang; Tan, Xiaotian; Zhang, Lianfeng; Wang, Qiang; Li, Zhanguo; Zhao, Yong

    2014-07-01

    The high temperature requirement A1 (HTRA1) is a potent protease involved in many diseases, including rheumatoid arthritis (RA). However, the regulatory mechanisms that control HTRA1 expression need to be determined. In this study, we demonstrated that IFN-γ significantly inhibited the basal and LPS-induced HTRA1 expression in fibroblasts and macrophages, which are two major cells for HTRA1 production in RA. Importantly, the inhibitory effect of IFN-γ on HTRA1 expression was evidenced in collagen-induced arthritis (CIA) mouse models and in human RA synovial cells. In parallel with the enhanced CIA incidence and pathological changes in IFN-γ-deficient mice, HTRA1 expression in the joint tissues was also increased as determined by real-time PCR and Western blots. IFN-γ deficiency increased the incidence of CIA and the pathological severity in mice. Neutralization of HTRA1 by Ab significantly reversed the enhanced CIA frequency and severity in IFN-γ-deficient mice. Mechanistically, IFN-γ negatively controls HTRA1 expression through activation of p38 MAPK/STAT1 pathway. Dual luciferase reporter assay and chromatin immunoprecipitation analysis showed that STAT1 could directly bind to HTRA1 promoter after IFN-γ stimulation. This study offers new insights into the molecular regulation of HTRA1 expression and its role in RA pathogenesis, which may have significant impact on clinical therapy for RA and possibly other HTRA1-related diseases, including osteoarthritis, age-related macular degeneration, and cancer.

  16. The biological effects of five feline IFN-alpha subtypes.

    PubMed

    Baldwin, Susan L; Powell, Tim D; Sellins, Karen S; Radecki, Steven V; Cohen, J John; Milhausen, Michael J

    2004-06-01

    IFN-alpha has been shown to induce both antiviral and antiproliferative activities in animals. This report describes the biological activity of five recently identified feline IFN-alpha subtypes expressed in the Chinese hamster ovary (CHO) cell line (rfeIFN-alpha1[CHO], rfeIFN-alpha2[CHO], rfeIFN-alpha3[CHO], rfeIFN-alpha5[CHO] and rfeIFN-alpha6[CHO]) and the feIFN-alpha6 subtype expressed in and purified from Pichia pastoris (rfeIFN-alpha6[P. pastoris]). The rfeIFN-alpha[CHO] subtypes were tested for antiviral activity against either Vesicular stomatitis virus (VSV) or feline calicivirus (FCV) infected feline embryonic fibroblast cell line (AH927) or Crandell feline kidney cell line (CRFK). Antiviral activity was induced against both VSV and FCV infected AH927 cells and VSV infected CRFK cells by all five of the rfeIFN-alpha[CHO] subtypes and rfeIFN-alpha6[P. pastoris]. In addition, the IFN-alpha inducible Mx gene (associated with antiviral activity) was upregulated in vivo 24 h following treatment with rfeIFN-alpha6[P. pastoris], compared to baseline levels seen prior to treatment. All of the rfeIFN-alpha[CHO] subtypes and rfeIFN-alpha6[P. pastoris] exhibited antiproliferative activity in the FeT-J cell line (an IL-2 independent feline T-cell line). Both necrosis and apoptosis were observed in rfeIFN-alpha6[P. pastoris]-treated FeT-J cells. The rfeIFN-alpha3[CHO] subtype consistently exhibited lower antiviral and antiproliferative activity compared to that observed with the other four rfeIFN-alpha[CHO] subtypes. In summary, this paper demonstrates that five previously described feIFN-alpha subtypes induce both antiviral and antiproliferative activities in vitro and are capable of upregulating the feMx gene in vivo.

  17. IFN-λ determines the intestinal epithelial antiviral host defense

    PubMed Central

    Pott, Johanna; Mahlakõiv, Tanel; Mordstein, Markus; Duerr, Claudia U.; Michiels, Thomas; Stockinger, Silvia; Staeheli, Peter; Hornef, Mathias W.

    2011-01-01

    Type I and type III IFNs bind to different cell-surface receptors but induce identical signal transduction pathways, leading to the expression of antiviral host effector molecules. Despite the fact that type III IFN (IFN-λ) has been shown to predominantly act on mucosal organs, in vivo infection studies have failed to attribute a specific, nonredundant function. Instead, a predominant role of type I IFN was observed, which was explained by the ubiquitous expression of the type I IFN receptor. Here we comparatively analyzed the role of functional IFN-λ and type I IFN receptor signaling in the innate immune response to intestinal rotavirus infection in vivo, and determined viral replication and antiviral gene expression on the cellular level. We observed that both suckling and adult mice lacking functional receptors for IFN-λ were impaired in the control of oral rotavirus infection, whereas animals lacking functional receptors for type I IFN were similar to wild-type mice. Using Mx1 protein accumulation as marker for IFN responsiveness of individual cells, we demonstrate that intestinal epithelial cells, which are the prime target cells of rotavirus, strongly responded to IFN-λ but only marginally to type I IFN in vivo. Systemic treatment of suckling mice with IFN-λ repressed rotavirus replication in the gut, whereas treatment with type I IFN was not effective. These results are unique in identifying a critical role of IFN-λ in the epithelial antiviral host defense. PMID:21518880

  18. Feedback regulation of IFN-α/β signaling by Axl receptor tyrosine kinase modulates HBV immunity.

    PubMed

    Huang, Miao-Tzu; Liu, Wei-Liang; Lu, Chun-Wei; Huang, Jian-Jhih; Chuang, Hsiao-Li; Huang, Yen-Te; Horng, Jau-Haw; Liu, Peng; Han, Dai-Shu; Chiang, Bor-Luen; Shih, Chiaho; Chen, Pei-Jer; Chen, Ding-Shinn

    2015-06-01

    Hepatitis B virus (HBV) is known to cause age-dependent infection outcomes wherein most infections during young age result in chronicity. The mechanism underlying the differential outcome remains elusive. By using hydrodynamic injection of the replication-competent pAAV-HBV, we established a mouse model in which HBV persistence was generated in 4-5 w/o C57BL/6 young mice, but not in adult mice over 10 w/o. HBV-tolerant young mice expressed higher interferon (IFN)-α/β levels in hepatocytes and intrahepatic plasmacytoid DCs (pDCs) than adult mice after pAAV-HBV injection. Excessive IFN-α/β expression in young mice was associated with induction of the Axl regulatory pathway and expansion of intrahepatic Treg cells. In line with these findings, augmented IFN-β expression increased Axl expression in the liver and HBV persistence in adult mice, whereas IFN-α/β signaling blockage decreased Axl expression and HBV persistence in young mice. Accordingly, Axl overexpression decreased HBV clearance of adult mice whereas Axl silencing enhanced HBV clearance of young mice. In vitro, IFN-β priming of pDCs and Axl-overexpressing macrophages enhanced Treg-cell differentiation. These findings suggest that age-dependent HBV chronicity is attributed to IFN-β-Axl immune regulation, which is selectively induced in young mice by excessive IFN-α/β production at early stage of HBV infection.

  19. TNF-alpha/IFN-gamma-induced iNOS expression increased by prostaglandin E2 in rat primary astrocytes via EP2-evoked cAMP/PKA and intracellular calcium signaling.

    PubMed

    Hsiao, Han-Yun; Mak, Oi-Tong; Yang, Chung-Shi; Liu, Yu-Peng; Fang, Kuan-Ming; Tzeng, Shun-Fen

    2007-01-15

    Astrocytes, the most abundant glia in the central nervous system (CNS), produce a large amount of prostaglandin E(2) (PGE(2)) in response to proinflammatory mediators after CNS injury. However, it is unclear whether PGE(2) has a regulatory role in astrocytic activity under the inflamed condition. In the present work, we showed that PGE(2) increased inducible nitric oxide synthase (iNOS) production by tumor necrosis factor-alpha and interferon-gamma (T/I) in astrocytes. Pharmacological and RNA interference approaches further indicated the involvement of the receptor EP2 in PGE(2)-induced iNOS upregulation in T/I-treated astrocytes. Quantitative real-time polymerase chain reaction and gel mobility shift assays also demonstrated that PGE(2) increased iNOS transcription through EP2-induced cAMP/protein kinase A (PKA)-dependent pathway. Consistently, the effect of EP2 was significantly attenuated by the PKA inhibitor KT-5720 and partially suppressed by the inhibitor (SB203580) of p38 mitogen-activated protein kinase (p38MAPK), which serves as one of the downstream components of the PKA-dependent pathway. Interestingly, EP2-mediated PKA signaling appeared to increase intracellular Ca(2+) release through inositol triphosphate (IP3) receptor activation, which might in turn stimulate protein kinase C (PKC) activation to promote iNOS production in T/I-primed astrocytes. By analyzing the expression of astrocytic glial fibrillary acidic protein (GFAP), we found that PGE(2) alone only triggered the EP2-induced cAMP/PKA/p38MAPK signaling pathway in astrocytes. Collectively, PGE(2) may enhance T/I-induced astrocytic activation by augmenting iNOS/NO production through EP2-mediated cross-talk between cAMP/PKA and IP3/Ca(2+) signaling pathways.

  20. IFN-γ stimulates autophagy-mediated clearance of Burkholderia cenocepacia in human cystic fibrosis macrophages.

    PubMed

    Assani, Kaivon; Tazi, Mia F; Amer, Amal O; Kopp, Benjamin T

    2014-01-01

    Burkholderia cenocepacia is a virulent pathogen that causes significant morbidity and mortality in patients with cystic fibrosis (CF), survives intracellularly in macrophages, and uniquely causes systemic infections in CF. Autophagy is a physiologic process that involves engulfing non-functional organelles and proteins and delivering them for lysosomal degradation, but also plays a role in eliminating intracellular pathogens, including B. cenocepacia. Autophagy is defective in CF but can be stimulated in murine CF models leading to increased clearance of B. cenocepacia, but little is known about autophagy stimulation in human CF macrophages. IFN-γ activates macrophages and increases antigen presentation while also inducing autophagy in macrophages. We therefore, hypothesized that treatment with IFN-γ would increase autophagy and macrophage activation in patients with CF. Peripheral blood monocyte derived macrophages (MDMs) were obtained from CF and non-CF donors and subsequently infected with B. cenocepacia. Basal serum levels of IFN-γ were similar between CF and non-CF patients, however after B. cenocepacia infection there is deficient IFN-γ production in CF MDMs. IFN-γ treated CF MDMs demonstrate increased co-localization with the autophagy molecule p62, increased autophagosome formation, and increased trafficking to lysosomes compared to untreated CF MDMs. Electron microscopy confirmed IFN-γ promotes double membrane vacuole formation around bacteria in CF MDMs, while only single membrane vacuoles form in untreated CF cells. Bacterial burden is significantly reduced in autophagy stimulated CF MDMs, comparable to non-CF levels. IL-1β production is decreased in CF MDMs after IFN-γ treatment. Together, these results demonstrate that IFN-γ promotes autophagy-mediated clearance of B. cenocepacia in human CF macrophages.

  1. IFN-γ-mediated hematopoietic cell destruction in murine models of immune-mediated bone marrow failure

    PubMed Central

    Feng, Xingmin; Desierto, Marie J.; Keyvanfar, Keyvan; Young, Neal S.

    2015-01-01

    Interferon gamma (IFN-γ) has been reported to have both negative and positive activity on hematopoietic cells, adding complexity to the interpretation of its pleiotropic functions. We examined the effects of IFN-γ on murine hematopoietic stem cells (HSCs) and progenitors in vitro and in vivo by using mouse models. IFN-γ treatment expanded bone marrow (BM) c-Kit+Sca1+Lin– (KSL) cell number but reduced BM KLCD150+ and KLCD150+CD48– cells. IFN-γ-expanded KSL cells engrafted poorly when tested by competitive repopulation in vivo. KSL, KLCD150+, and KLCD150+CD48– cells from IFN-γ-treated animals all showed significant upregulation in Fas expression. When cocultured with activated T cells in vitro, KSL and KLCD150+ cells from IFN-γ-treated donors showed increased apoptosis relative to those from untreated animals, and infusion of activated CD8 T cells into IFN-γ-injected animals in vivo led to partial elimination of KSL cells. Exposure of BM cells or KSL cells to IFNincreased expression of Fas, caspases, and related proapoptotic genes and decreased expression of Ets-1 and other hematopoietic genes. In mouse models of BM failure, mice genetically deficient in IFN-γ receptor expression showed attenuation of immune-mediated marrow destruction, whereas effector lymphocytes from IFN-γ-deficient donors were much less potent in initiating BM damage. We conclude that the activity of IFN-γ on murine hematopoiesis is context dependent. IFN-γ-augmented apoptotic gene expression facilitates destruction of HSCs and progenitors in the presence of activated cytotoxic T cells, as occurs in human BM failure. PMID:26491068

  2. IFN-γ-mediated hematopoietic cell destruction in murine models of immune-mediated bone marrow failure.

    PubMed

    Chen, Jichun; Feng, Xingmin; Desierto, Marie J; Keyvanfar, Keyvan; Young, Neal S

    2015-12-10

    Interferon gamma (IFN-γ) has been reported to have both negative and positive activity on hematopoietic cells, adding complexity to the interpretation of its pleiotropic functions. We examined the effects of IFN-γ on murine hematopoietic stem cells (HSCs) and progenitors in vitro and in vivo by using mouse models. IFN-γ treatment expanded bone marrow (BM) c-Kit(+)Sca1(+)Lin(-) (KSL) cell number but reduced BM KLCD150(+) and KLCD150(+)CD48(-) cells. IFN-γ-expanded KSL cells engrafted poorly when tested by competitive repopulation in vivo. KSL, KLCD150(+), and KLCD150(+)CD48(-) cells from IFN-γ-treated animals all showed significant upregulation in Fas expression. When cocultured with activated T cells in vitro, KSL and KLCD150(+) cells from IFN-γ-treated donors showed increased apoptosis relative to those from untreated animals, and infusion of activated CD8 T cells into IFN-γ-injected animals in vivo led to partial elimination of KSL cells. Exposure of BM cells or KSL cells to IFNincreased expression of Fas, caspases, and related proapoptotic genes and decreased expression of Ets-1 and other hematopoietic genes. In mouse models of BM failure, mice genetically deficient in IFN-γ receptor expression showed attenuation of immune-mediated marrow destruction, whereas effector lymphocytes from IFN-γ-deficient donors were much less potent in initiating BM damage. We conclude that the activity of IFN-γ on murine hematopoiesis is context dependent. IFN-γ-augmented apoptotic gene expression facilitates destruction of HSCs and progenitors in the presence of activated cytotoxic T cells, as occurs in human BM failure.

  3. Elevated expression of IFN-gamma in the HIV-1 infected brain.

    PubMed

    Shapshak, Paul; Duncan, Robert; Minagar, Alireza; Rodriguez de la Vega, Pura; Stewart, Renée V; Goodkin, Karl

    2004-05-01

    We determined the extent of expression of three cytokines (IFN-gamma, IL-4, and TNF-alpha ) in brain tissue infected with human immunodeficiency virus-1 (HIV-1). The selections were IFN-gamma as a Th1 cytokine, IL- 4 as a Th2 cytokine, and TNF-alpha as a pro-inflammatory cytokine (and because of its prior implication in brain tissue damage due to HIV-1 infection). Based on current models for pathogenesis of HIV-1-associated dementia (HAD), in the periphery, Th1 cytokines are considered to be salutary, whereas Th2 cytokines are regarded as deleterious. However, we hypothesized that in the CNS these roles are reversed. Post-mortem temporal lobe tissue specimens from 16 HIV-1-seropositive patients and 11 HIV-1-seronegative controls were stained for IFN-gamma, IL-4, and TNF-alpha utilizing immunohistochemistry and alkaline phosphatase. HIV-1 infection causes alterations of brain cytokine expression that include increased IFN-gamma expression for HIV-1-seropositive vs. HIV-1-seronegative individuals. There was increased expression of IFN-gamma for HIV-1-seropositive individuals with or without HAD, with or without the broader category of neuropsychiatric impairment (NPI), and with or without opportunistic infections (OIs) compared to HIV-1-seronegatives. A significant inverse correlation between IFN-gamma vs. IL-4 in HIV-1-seropositives with HAD and in seronegative individuals was observed. There was an inverse correlation in seropositives between IFN-gamma vs. TNF-alpha, a positive trend with HAD, significant without HAD, significant with NPI and significant without OIs. Between IL-4 vs. TNF-alpha there was a correlation (trend) in seropositives, a trend with NPI, significant without NPI, and a trend without OI. Due to HIV-1 infection of the brain and neurological disease there is a prominent increased expression of IFN-gamma, an inverse expression of IFN-gamma vs. TNF-alpha, and TNF-alpha vs. IL-4. The inverse correlation between increased IFN-gamma and decreased IL-4

  4. T helper type 1 and 17 cells Determine Efficacy of IFN-β in Multiple Sclerosis and Experimental Encephalomyelitis

    PubMed Central

    Axtell, Robert C.; de Jong, Brigit A.; Boniface, Katia; van der Voort, Laura F.; Bhat, Roopa; De Sarno, Patrizia; Naves, Rodrigo; Han, May; Zhong, Franklin; Castellanos, Jim G.; Mair, Robert; Christakos, Athena; Kolkowitz, Ilan; Katz, Liat; Killestein, Joep; Polman, Chris H.; de Waal Malefyt, René; Steinman, Lawrence; Raman, Chander

    2010-01-01

    Interferon-β is the major treatment for multiple sclerosis (MS). However, this treatment is not always effective. Here we see congruence in outcome between responses to IFN-β in experimental autoimmune encephalomyelitis (EAE) and relapsing-remitting MS (RRMS). IFN-β is effective in reducing EAE induced by TH1 cells, but exacerbated disease induced by TH17. Effective treatment in TH1 EAE correlated with increased IL-10 in the spleen. In TH17 disease, the amount of IL-10 was unaltered by treatment, though unexpectedly IFN-β still reduced IL-17 without benefit. Both inhibition of IL-17 and induction of IL-10 depended on IFN-γ. In the absence of IFN-γ signaling, IFN-β therapy was ineffective in EAE. In RRMS, IFN-β non-responders had higher IL-17F in serum compared to responders. Non-responders had worse disease with more steroid usage and more relapses than responders. Hence, IFN-β is pro-inflammatory in TH17 induced EAE. Moreover, high IL-17F in the serum of RRMS patients is associated with non-responsiveness to therapy with IFN-β. PMID:20348925

  5. IFN-ε protects primary macrophages against HIV infection

    PubMed Central

    Tasker, Carley; Subbian, Selvakumar; Gao, Pan; Couret, Jennifer; Levine, Carly; Ghanny, Saleena; Soteropoulos, Patricia; Zhao, Xilin; Landau, Nathaniel; Lu, Wuyuan

    2016-01-01

    IFN-ε is a unique type I IFN that is not induced by pattern recognition response elements. IFN-ε is constitutively expressed in mucosal tissues, including the female genital mucosa. Although the direct antiviral activity of IFN-ε was thought to be weak compared with IFN-α, IFN-ε controls Chlamydia muridarum and herpes simplex virus 2 in mice, possibly through modulation of immune response. We show here that IFN-ε induces an antiviral state in human macrophages that blocks HIV-1 replication. IFN-ε had little or no protective effect in activated CD4+ T cells or transformed cell lines unless activated CD4+ T cells were infected with replication-competent HIV-1 at a low MOI. The block to HIV infection of macrophages was maximal after 24 hours of treatment and was reversible. IFN-ε acted on early stages of the HIV life cycle, including viral entry, reverse transcription, and nuclear import. The protection did not appear to operate through known type I IFN-induced HIV host restriction factors, such as APOBEC3A and SAMHD1. IFN-ε–stimulated immune mediators and pathways had the signature of type I IFNs but were distinct from IFN-α in macrophages. IFN-ε induced significant phagocytosis and ROS, which contributed to the block to HIV replication. These findings indicate that IFN-ε induces an antiviral state in macrophages that is mediated by different factors than those induced by IFN-α. Understanding the mechanism of IFN-ε–mediated HIV inhibition through immune modulation has implications for prevention. PMID:27942584

  6. Induction of ceruloplasmin synthesis by IFN-gamma in human monocytic cells

    NASA Technical Reports Server (NTRS)

    Mazumder, B.; Mukhopadhyay, C. K.; Prok, A.; Cathcart, M. K.; Fox, P. L.

    1997-01-01

    Ceruloplasmin is a 132-kDa glycoprotein abundant in human plasma. It has multiple in vitro activities, including copper transport, lipid pro- and antioxidant activity, and oxidation of ferrous ion and aromatic amines; however, its physiologic role is uncertain. Although ceruloplasmin is synthesized primarily by the liver in adult humans, production by cells of monocytic origin has been reported. We here show that IFN-gamma is a potent inducer of ceruloplasmin synthesis by monocytic cells. Activation of human monoblastic leukemia U937 cells with IFN-gamma increased the production of ceruloplasmin by at least 20-fold. The identity of the protein was confirmed by plasmin fingerprinting. IFN-gamma also increased ceruloplasmin mRNA. Induction followed a 2- to 4-h lag and was partially blocked by cycloheximide, indicating a requirement for newly synthesized factors. Ceruloplasmin induction in monocytic cells was agonist specific, as IL-1, IL-4, IL-6, IFN-alpha, IFN-beta, TNF-alpha, and LPS were completely ineffective. The induction was also cell type specific, as IFN-gamma did not induce ceruloplasmin synthesis in endothelial or smooth muscle cells. In contrast, IFN-gamma was stimulatory in other monocytic cells, including THP-1 cells and human peripheral blood monocytes, and also in HepG2 cells. Ceruloplasmin secreted by IFN-gamma-stimulated U937 cells had ferroxidase activity and was, in fact, the only secreted protein with this activity. Monocytic cell-derived ceruloplasmin may contribute to defense responses via its ferroxidase activity, which may drive iron homeostasis in a direction unfavorable to invasive organisms.

  7. Induction of interferon-gamma (IFN-gamma) and T helper 1 (Th1) immune response by bitter gourd extract.

    PubMed

    Ike, Kazunori; Uchida, Yuko; Nakamura, Tomohiko; Imai, Soichi

    2005-05-01

    Mice were inoculated intraperitoneally wih 34 different types of vegetable juices, and interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) were measured as markers for the induction of Th1 and Th2 cells, respectively. Serum IFN-gamma level was markedly increased in mice inoculated with bitter gourd (Momordica charantia) juice, but IL-4 levels were not increased with any of the 34 vegetable juices. Testing of the various components of bitter gourd, including peel, pulp, and seed, showed that the pulp induced the highest levels of IFN-gamma. Trial immunogen including the heat extract of the pulp induced specific IgG(2a) antibody of the mice serum inoculated with this immunogen. These results demonstrate that bitter gourd pulp induced IFN-gamma production and show its promise as a means of effective immunostimulatory therapy specific for Th1 cells and IFN-gamma production.

  8. IFN-γ and its receptors in a reptile reveal the evolutionary conservation of type II IFNs in vertebrates.

    PubMed

    Chen, Shan Nan; Huang, Bei; Zhang, Xiao Wen; Li, Ye; Zhao, Li Juan; Li, Nan; Gao, Qian; Nie, P

    2013-12-01

    In this study, interferon gamma (IFN-γ) and interferon gamma receptor (IFN-γR) genes have been identified in non-avian reptile, the North American green anole lizard (Anolis carolinensis). Like their counterparts from other jawed vertebrates, lizard IFN-γ, IFN-γR1 and IFN-γR2 show conserved features in genomic organizations, gene loci and protein sequences. The IFN-γ gene has the full cDNA sequence of 936 bp, with 522 bp open reading frame (ORF) encoding 174 amino acids, and has the genomic organization of four exons and three introns as observed in IFN-γ genes of other classes of vertebrates. The receptors, IFN-γR1 and IFN-γR2 have the ORF of 1278 and 984 bp, coding for 425 and 327 aa, respectively, with the genome organization of seven exons and six introns. In the gene loci of IFN-γ, DYRK2, IL22, IL26 and MDM1 are found with conserved synteny in vertebrates, and similar genes adjacent to IFN-γR1 and IFN-γR2 were also found. These receptors also contain conserved motifs, such as the membrane-proximal region and the C-terminal five residue motif in IFN-γR1, and intracellular conservative sequence in IFN-γR2, which have been confirmed to mediate down-stream JAK-STAT signaling pathway in mammals. IFN-γ and its receptors, IFN-γR1 and IFN-γR2 were constitutively expressed in organs/tissues examined in the lizard, and up-regulated expression of IFN-γ was observed in organs/tissues examined following the poly(I:C) stimulation, suggesting its antiviral role in lizards. The conserved features of IFN-γ and its receptors, IFN-γR1 and IFN-γR2, in gene organization and gene locus as well as in functional domain or motif may imply that the function of type II IFN system is evolutionarily conserved in the green anole lizard, as observed in other classes of vertebrates.

  9. Regression of infancy hemangiomas with recombinant IFN-alpha 2b.

    PubMed

    Garmendía, G; Miranda, N; Borroso, S; Longchong, M; Martínez, E; Ferrero, J; Porrero, P; López-Saura, P

    2001-01-01

    Interferon-alpha (IFN-alpha) has antitumor and antiangiogenic effects. The purpose of this work was to evaluate its efficacy and safety in the treatment of infancy hemangioma and to monitor the appearance of anti-IFN antibodies in these patients. Thirty-nine children (29 girls) aged 1.5-158 months, with 19 younger than 1 year and 9 older than 5, were treated with 3 x 10(6) IU/m(2) IFN-alpha 2b, subcutaneously (s.c.) daily. Inclusion criteria were life-threatening or life-limiting hemangioma and parents' informed consent. Regression was considered if tumor size diminished by 50% or more. Of the 38 patients who completed 6 months of treatment, 27 (71.1%) had regression and 11 (28.9%) had stable disease. No patient experienced progression. Regression was more frequent (100%) among patients between 1 and 5 years old, but it was particularly important (68%) among those under 1 year old, when spontaneous regression is rare. The main side effects were the IFN-related flulike syndrome (79%), increase in serum alanine aminotransferase (ALT) (28%), anorexia (19%), and mild inflammation at the injection site (19%). There was no effect on psychomotor or physical development. On the contrary, 1 patient with neurologic symptoms improved remarkably, including seizure disappearance. Eight patients developed anti-IFN-alpha 2 neutralizing antibodies, and 7 of them responded to IFN treatment. IFN-alpha 2b is a safe and efficacious treatment of infancy hemangioma. Further work should look for other treatment schedules and ways of administration and carefully monitor anti-IFN neutralizing antibodies, which does not seem to interfere with response.

  10. ZNF395 Is an Activator of a Subset of IFN-Stimulated Genes

    PubMed Central

    Schroeder, Linda; Herwartz, Christine; Jordanovski, Darko

    2017-01-01

    Activation of the interferon (IFN) pathway in response to infection with pathogens results in the induction of IFN-stimulated genes (ISGs) including proinflammatory cytokines, which mount the proper antiviral immune response. However, aberrant expression of these genes is pathogenic to the host. In addition to IFN-induced transcription factors non-IFN-regulated factors contribute to the transcriptional control of ISGs. Here, we show by genome wide expression analysis, siRNA-mediated suppression and Doxycycline-induced overexpression that the cellular transcription factor ZNF395 activates a subset of ISGs including the chemokines CXCL10 and CXCL11 in keratinocytes. We found that ZNF395 acts independently of IFN but enhances the IFN-induced expression of CXCL10 and CXCL11. Luciferase reporter assays revealed a requirement of intact NFκB-binding sites for ZNF395 to stimulate the CXCL10 promoter. The transcriptional activation of CXCL10 and CXCL11 by ZNF395 was abolished after inhibition of IKK by BMS-345541, which increased the stability of ZNF395. ZNF395 encodes at least two motifs that mediate the enhanced degradation of ZNF395 in response to IKK activation. Thus, IKK is required for ZNF395-mediated activation of transcription and enhances its turn-over to keep the activity of ZNF395 low. Our results support a previously unrecognized role of ZNF395 in the innate immune response and inflammation. PMID:28316371

  11. Polymorphism of IFN-γ (+874 T/A) in Syrian patients with chronic hepatitis B

    PubMed Central

    Al Kadi, Mohamad; Monem, Fawza

    2017-01-01

    Aim: This study aimed to investigate the association of IFN- γ +874 (T/A) polymorphism with susceptibility to chronic HBV infection in the Syrian population. Background: Accumulating evidence indicate that the inadequate immune responses are responsible for HBV persistency. Therefore, polymorphisms in genes encoding the cytokines, which are responsible for regulation of the immune response, can affect the course and outcome of the infection. The IFN-γ +874 T/A polymorphism affects the expression of IFN-γ, which has been shown to be crucial to HBV clearance. Methods: In this case-control study, 140 samples were collected (70 healthy individuals, 70 chronic HBV patients), and genomic DNA was isolated. Sequencing and ARMS-PCR were performed to genotype the IFN-γ +874 T/A polymorphism. Results: Results of this study showed an association between IFN- γ +874 T/A polymorphism and the susceptibility to chronic HBV infection (P < 0.05). In addition, results showed that the AA genotype increased the risk of chronicity (OR = 3.05, 95% CI = 1.35 – 6.89), whereas the AT and TT genotypes reduced the risk of chronicity (OR = 0.33, 95% CI = 0.150 – 0.753). Conclusion: Results of this study conclude that the IFN- γ +874 T/A polymorphism may be associated with the chronic HBV infection, according to the genetic model AA vs. AT&TT. PMID:28331562

  12. A peptide mimetic of human interferon (IFN)-beta.

    PubMed Central

    Sato, Atsushi; Sone, Saburo

    2003-01-01

    Type I interferons (IFNs) are cytokines that are used clinically as antiviral and antitumour agents. The interaction of IFNs with their heterodimeric type I IFN receptor comprised of IFNAR1 and IFNAR2 is a first step to inducing biological actions. Here, we describe the successful mimicry of IFN-beta by a peptide isolated by phage-display screening using a neutralizing anti-IFN-beta monoclonal antibody. The 15-mer peptide, designated SYR6, was shown to compete with IFN-beta for binding to type I IFN receptor in a concentration-dependent manner, and was shown to elicit antiviral activity on cultured cells. This antiviral activity was not eliminated in the presence of neutralizing monoclonal antibodies to IFN-alpha, -beta and -gamma, and a low concentration of soluble type I IFN receptor, suggesting that it was not due to IFN contamination or the induction of endogenous IFNs by SYR6. This peptide might be a potent agonist to provide a mechanism of activating heterodimeric cytokine receptors. PMID:12542398

  13. Cloning and characterization of a novel feline IFN-omega.

    PubMed

    Yang, Li-Min; Xue, Qing-Hua; Sun, Lei; Zhu, Yi-Ping; Liu, Wen-Jun

    2007-02-01

    The interferons (IFNs) are a large family of multifunctional secreted protein involved in antiviral defense, cell growth regulation, and immune activation. The human IFNs are used worldwide as antiviral drugs. Here, we present cDNAs encoding 13 novel feline IFN-omega (FeIFN-omega) subtypes that share 95%-99% amino acid sequence identity. FeIFN-omega2 and FeIFN-omega4 have seven additional amino acids at position 109 that are not present in other subtypes. Sequence identity of the present FeIFN proteins encoded by the 13 subtypes is approximately 57% compared with human IFN-omega (HuIFN-omega). All 13 FeIFN-omega subtypes were expressed in Escherichia coli using a periplasmic expression system. The antiviral activity of each product was evaluated in vitro. In addition, subtype FeIFN-omega2 was cytoplasm expressed in E. coli and secretion expressed in Pichia pastoris. The purified mature recombinant protein demonstrated significant antiviral activity on both homologous and heterologous animal cells in vitro.

  14. B cell IFN-γ receptor signaling promotes autoimmune germinal centers via cell-intrinsic induction of BCL-6

    PubMed Central

    Jackson, Shaun W.; Jacobs, Holly M.; Arkatkar, Tanvi; Dam, Elizabeth M.; Scharping, Nicole E.; Kolhatkar, Nikita S.; Hou, Baidong; Buckner, Jane H.

    2016-01-01

    Dysregulated germinal center (GC) responses are implicated in the pathogenesis of human autoimmune diseases, including systemic lupus erythematosus (SLE). Although both type 1 and type 2 interferons (IFNs) are involved in lupus pathogenesis, their respective impacts on the establishment of autoimmune GCs has not been addressed. In this study, using a chimeric model of B cell-driven autoimmunity, we demonstrate that B cell type 1 IFN receptor signals accelerate, but are not required for, lupus development. In contrast, B cells functioning as antigen-presenting cells initiate CD4+ T cell activation and IFN-γ production, and strikingly, B cell–intrinsic deletion of the IFN-γ receptor (IFN-γR) abrogates autoimmune GCs, class-switched autoantibodies (auto-Abs), and systemic autoimmunity. Mechanistically, although IFN-γR signals increase B cell T-bet expression, B cell–intrinsic deletion of T-bet exerts an isolated impact on class-switch recombination to pathogenic auto-Ab subclasses without impacting GC development. Rather, in both mouse and human B cells, IFN-γ synergized with B cell receptor, toll-like receptor, and/or CD40 activation signals to promote cell-intrinsic expression of the GC master transcription factor, B cell lymphoma 6 protein. Our combined findings identify a novel B cell–intrinsic mechanism whereby IFN signals promote lupus pathogenesis, implicating this pathway as a potential therapeutic target in SLE. PMID:27069113

  15. Prevention of SHIV transmission by topical IFN-β treatment

    PubMed Central

    Veazey, Ronald S.; Pilch Cooper, Heather A.; Hope, Thomas J.; Alter, Galit; Carias, Ann M.; Sips, Magdalena; Wang, Xiaolei; Rodriguez, Benigno; Sieg, Scott F.; Reich, Adrian; Wilkinson, Peter; Cameron, Mark J.; Lederman, Michael M.

    2015-01-01

    Understanding vaginal and rectal HIV transmission and protective cellular and molecular mechanisms is critical for designing new prevention strategies, including those required for an effective vaccine. The determinants of protection against HIV infection are, however, poorly understood. Increasing evidence suggest that innate immune defenses may help protect mucosal surfaces from HIV transmission in highly exposed, uninfected subjects 1. More recent studies suggest that systemically administered type 1 interferon protects against simian immunodeficiency virus infection of macaques 2. Here we hypothesized that topically applied type 1 interferons might stimulate vaginal innate responses that could protect against HIV transmission. We therefore applied a recombinant human type 1 interferon (IFN-β) to the vagina of rhesus macaques and vaginally challenged them with pathogenic simian/human immunodeficiency virus (SHIV). Vaginal administration of IFN-β resulted in marked local changes in immune cell phenotype, increasing immune activation and HIV coreceptor expression, yet provided significant protection from SHIV acquisition as interferon response genes (IRGs) were also upregulated. These data suggest that protection from vaginal HIV acquisition may be achieved by activating innate mucosal defenses. PMID:26838048

  16. Human alloreactive CTL interactions with gliomas and with those having upregulated HLA expression from exogenous IFN-gamma or IFN-gamma gene modification.

    PubMed

    Read, Susana B; Kulprathipanja, Nisha V; Gomez, German G; Paul, David B; Winston, Ken R; Robbins, Joan M; Kruse, Carol A

    2003-07-01

    By flow cytometry, a panel of 18 primary glioma cell explants exhibited high expression of class I HLA-A, B, C, but class II HLA-DR expression was absent. Freshly isolated normal brain cells displayed little or no HLA antigens. Alloreactive cytotoxic T lymphocytes (aCTL), sensitized to the HLA of the patient, were generated in a one-way mixed lymphocyte response (MLR). The specificity of aCTL was confirmed to be to target cells (patient glioma cells or lymphoblasts) expressing the relevant HLA antigens. However, nontumor patient-specific aCTL did not lyse normal brain cells. Titration of antibodies to HLA class I into cytotoxicity assays blocked lysis of gliomas by aCTL, confirming aCTL T cell receptor (TCR) interactions with the class I antigen on gliomas. Furthermore, aCTL interactions with glioma cells caused their apoptosis. Coincubations of aCTL with gliomas resulted in upregulated cytokine secretion. Importantly, dexamethasone, an immunosuppressive steroid used for brain edema, did not affect aCTL lytic function against tumor, indicating that steroid-dependent patients may benefit from the immunotherapy. We also explored the use of interferon-gamma (IFN-gamma) to increase aCTL tumor recognition. Coincubation of gliomas with exogenous IFN-gamma (500 U/ml, 48 h) caused a 3-fold upregulation of HLA class I and a slight induction of class II antigen expression. Gene-modified glioma cells producing IFN-gamma similarly displayed upregulated HLA expression. Glioma cells incubated with exogenous IFN-gamma or IFN-gamma-transduced glioma cells were more susceptible to lysis by aCTL than their parental counterparts, thus supporting the concept of combining IFN-gamma cytokine gene therapy with adoptive aCTL immunotherapy for brain tumor treatment.

  17. Differential IFN-gamma stimulation of HLA-A gene expression through CRM-1-dependent nuclear RNA export.

    PubMed

    Browne, Sarah K; Roesser, James R; Zhu, Sheng Zu; Ginder, Gordon D

    2006-12-15

    IFNs regulate most MHC class I genes by stimulating transcription initiation. As shown previously, IFN-gamma controls HLA-A expression primarily at the posttranscriptional level. We have defined two 8-base sequences in a 39-nucleotide region in the 3'-transcribed region of the HLA-A gene that are required for the posttranscriptional response to IFN-gamma. Stimulation of HLA-A expression by IFN-gamma requires nuclear export of HLA-A mRNA by chromosome maintenance region 1 (CRM-1). Treatment of cells with leptomycin B, a specific inhibitor of CRM-1, completely inhibited IFN-gamma induction of HLA-A. Expression of a truncated, dominant-negative form of the nucleoporin NUP214/CAN, DeltaCAN, that specifically interacts with CRM-1, also prevented IFN-gamma stimulation of HLA-A, providing confirmation of the role of CRM-1. Increased expression of HLA-A induced by IFN-gamma also requires protein methylation, as shown by the fact that treatment of SK-N-MC cells or HeLa cells with the PRMT1 inhibitor 5'-methyl-5'-thioadenosine abolished the cellular response to IFN-gamma. In contrast with HLA-A, IFN-gamma-induced expression of the HLA class Ib gene, HLA-E, was not affected by either 5'-methyl-5'-thioadenosine or leptomycin B. These results provide proof of principle that it is possible to differentially modulate the IFN-gamma-induced expression of the HLA-E and HLA-A genes, whose products often mediate opposing effects on cellular immunity to tumor cells, pathogens, and autoantigens.

  18. Ablation of Type-1 IFN Signaling in Hematopoietic Cells Confers Protection Following Traumatic Brain Injury.

    PubMed

    Karve, Ila P; Zhang, Moses; Habgood, Mark; Frugier, Tony; Brody, Kate M; Sashindranath, Maithili; Ek, C Joakim; Chappaz, Stephane; Kile, Ben T; Wright, David; Wang, Hong; Johnston, Leigh; Daglas, Maria; Ates, Robert C; Medcalf, Robert L; Taylor, Juliet M; Crack, Peter J

    2016-01-01

    Type-1 interferons (IFNs) are pleiotropic cytokines that signal through the type-1 IFN receptor (IFNAR1). Recent literature has implicated the type-1 IFNs in disorders of the CNS. In this study, we have investigated the role of type-1 IFNs in neuroinflammation following traumatic brain injury (TBI). Using a controlled cortical impact model, TBI was induced in 8- to 10-week-old male C57BL/6J WT and IFNAR1(-/-) mice and brains were excised to study infarct volume, inflammatory mediator release via quantitative PCR analysis and immune cell profile via immunohistochemistry. IFNAR1(-/-) mice displayed smaller infarcts compared with WT mice after TBI. IFNAR1(-/-) mice exhibited an altered anti-inflammatory environment compared with WT mice, with significantly reduced levels of the proinflammatory mediators TNFα, IL-1β and IL-6, an up-regulation of the anti-inflammatory mediator IL-10 and an increased activation of resident and peripheral immune cells after TBI. WT mice injected intravenously with an anti-IFNAR1 blocking monoclonal antibody (MAR1) 1 h before, 30 min after or 30 min and 2 d after TBI displayed significantly improved histological and behavioral outcome. Bone marrow chimeras demonstrated that the hematopoietic cells are a peripheral source of type-1 IFNs that drives neuroinflammation and a worsened TBI outcome. Type-1 IFN mRNA levels were confirmed to be significantly altered in human postmortem TBI brains. Together, these data demonstrate that type-1 IFN signaling is a critical pathway in the progression of neuroinflammation and presents a viable therapeutic target for the treatment of TBI.

  19. Ablation of Type-1 IFN Signaling in Hematopoietic Cells Confers Protection Following Traumatic Brain Injury123

    PubMed Central

    Karve, Ila P.; Zhang, Moses; Habgood, Mark; Frugier, Tony; Brody, Kate M.; Sashindranath, Maithili; Ek, C. Joakim; Kile, Ben T.; Wright, David; Wang, Hong; Johnston, Leigh; Daglas, Maria; Ates, Robert C.; Medcalf, Robert L.; Taylor, Juliet M.

    2016-01-01

    Abstract Type-1 interferons (IFNs) are pleiotropic cytokines that signal through the type-1 IFN receptor (IFNAR1). Recent literature has implicated the type-1 IFNs in disorders of the CNS. In this study, we have investigated the role of type-1 IFNs in neuroinflammation following traumatic brain injury (TBI). Using a controlled cortical impact model, TBI was induced in 8- to 10-week-old male C57BL/6J WT and IFNAR1−/− mice and brains were excised to study infarct volume, inflammatory mediator release via quantitative PCR analysis and immune cell profile via immunohistochemistry. IFNAR1−/− mice displayed smaller infarcts compared with WT mice after TBI. IFNAR1−/− mice exhibited an altered anti-inflammatory environment compared with WT mice, with significantly reduced levels of the proinflammatory mediators TNFα, IL-1β and IL-6, an up-regulation of the anti-inflammatory mediator IL-10 and an increased activation of resident and peripheral immune cells after TBI. WT mice injected intravenously with an anti-IFNAR1 blocking monoclonal antibody (MAR1) 1 h before, 30 min after or 30 min and 2 d after TBI displayed significantly improved histological and behavioral outcome. Bone marrow chimeras demonstrated that the hematopoietic cells are a peripheral source of type-1 IFNs that drives neuroinflammation and a worsened TBI outcome. Type-1 IFN mRNA levels were confirmed to be significantly altered in human postmortem TBI brains. Together, these data demonstrate that type-1 IFN signaling is a critical pathway in the progression of neuroinflammation and presents a viable therapeutic target for the treatment of TBI. PMID:27022620

  20. Contraction of the type I IFN locus and unusual constitutive expression of IFN-α in bats

    PubMed Central

    Zhou, Peng; Tachedjian, Mary; Wynne, James W.; Boyd, Victoria; Smith, Ina; Cowled, Christopher; Ng, Justin H. J.; Mok, Lawrence; Michalski, Wojtek P.; Mendenhall, Ian H.; Tachedjian, Gilda; Baker, Michelle L.

    2016-01-01

    Bats harbor many emerging and reemerging viruses, several of which are highly pathogenic in other mammals but cause no clinical signs of disease in bats. To determine the role of interferons (IFNs) in the ability of bats to coexist with viruses, we sequenced the type I IFN locus of the Australian black flying fox, Pteropus alecto, providing what is, to our knowledge, the first gene map of the IFN region of any bat species. Our results reveal a highly contracted type I IFN family consisting of only 10 IFNs, including three functional IFN-α loci. Furthermore, the three IFN-α genes are constitutively expressed in unstimulated bat tissues and cells and their expression is unaffected by viral infection. Constitutively expressed IFN-α results in the induction of a subset of IFN-stimulated genes associated with antiviral activity and resistance to DNA damage, providing evidence for a unique IFN system that may be linked to the ability of bats to coexist with viruses. PMID:26903655

  1. Reduction of cell viability induced by IFN-alpha generates impaired data on antiviral assay using Hep-2C cells.

    PubMed

    de Oliveira, Edson R A; Lima, Bruna M M P; de Moura, Wlamir C; Nogueira, Ana Cristina M de A

    2013-12-31

    Type I interferons (IFNs) exert an array of important biological functions on the innate immune response and has become a useful tool in the treatment of various diseases. An increasing demand in the usage of recombinant IFNs, mainly due to the treatment of chronic hepatitis C infection, augmented the need of quality control for this biopharmaceutical. A traditional bioassay for IFN potency assessment is the cytopathic effect reduction antiviral assay where a given cell line is preserved by IFN from a lytic virus activity using the cell viability as a frequent measure of end point. However, type I IFNs induce other biological effects such as cell-cycle arrest and apoptosis that can influence directly on viability of many cell lines. Here, we standardized a cytopathic effect reduction antiviral assay using Hep-2C cell/mengovirus combination and studied a possible impact of cell viability variations caused by IFN-alpha 2b on responses generated on the antiviral assay. Using the four-parameter logistic model, we observed less correlation and less linearity on antiviral assay when responses from IFN-alpha 2b 1000 IU/ml were considered in the analysis. Cell viability tests with MTT revealed a clear cell growth inhibition of Hep-2C cells under stimulation with IFN-alpha 2b. Flow cytometric cell-cycle analysis and apoptosis assessment showed an increase of S+G2 phase and higher levels of apoptotic cells after treatment with IFN-alpha 2b 1000 IU/ml under our standardized antiviral assay procedure. Considering our studied dose range, we also observed strong STAT1 activation on Hep-2C cells after stimulation with the higher doses of IFN-alpha 2b. Our findings showed that the reduction of cell viability driven by IFN-alpha can cause a negative impact on antiviral assays. We assume that the cell death induction and the cell growth inhibition effect of IFNs should also be considered while employing antiviral assay protocols in a quality control routine and emphasizes the

  2. IFN-inducible GTPases in Host Defense

    PubMed Central

    Kim, Bae-Hoon; Shenoy, Avinash R.; Kumar, Pradeep; Bradfield, Clinton J.; MacMicking, John D.

    2012-01-01

    From plants to humans, the ability to control infection at the level of an individual cell – a process termed cell-autonomous immunity – equates firmly with survival of the species. Recent work has begun to unravel this programmed cell-intrinsic response and the central roles played by IFN-inducible GTPases in defending the mammalian cell’s interior against a diverse group of invading pathogens. These immune GTPases regulate vesicular traffic and protein complex assembly to stimulate oxidative, autophagic, membranolytic and inflammasome-related antimicrobial activities within the cytosol as well as on pathogen-containing vacuoles. Moreover, human genome-wide association studies (GWAS) and disease-related transcriptional profiling have linked mutations in the Immunity-Related GTPase M (IRGM) locus and altered expression of Guanylate Binding Proteins (GBPs) with tuberculosis susceptibility and Crohn’s colitis. PMID:23084913

  3. Preparation and characterization of latex films photo-immobilized with IFN-α.

    PubMed

    Wu, Lifang; Hu, Kaikai; Zhang, Li; Chen, Wuya; Chen, Xiaohui; You, Rong; Yin, Liang; Guan, Yan-Qing

    2016-09-01

    We developed a biomaterial by photo-immobilizing interferon-α (IFN-α) on the surface of latex condom films for the prevention and treatment of cervicitis, cervical cancers and diseases caused by cervical virus. The IFN-α modification by photoactive N-(4-azidobenzoyloxy) succinimide was characterized on a nano-scale by spectroscopy analysis and micro morphology. The anti-bacterial, anti-cancer, and anti-viral effects of the modified bioactive latex films were evaluated by antibacterial susceptibility testing, Gram staining, flow cytometry, immunofluorescence, and Western blotting. Our results showed that the photo-immobilized IFN-α latex films effectively inhibited the growth of both Neisseria gonorrhoeae and human cervical cancer HeLa cells. Moreover, the expression of anti-viral proteins, including P56, MxA, and 2', 5'-OAS, in the human cervical epithelial cell line NC104 was significantly increased by photo-immobilized IFN-α latex films. Taken together, these results suggest that photo-immobilized IFN-α latex films may have therapeutic effects against cervicitis, cervical cancers, and cervical virus.

  4. Development of monoclonal antibodies (MAbs) to feline interferon (fIFN)-γ as tools to evaluate cellular immune responses to feline infectious peritonitis virus (FIPV).

    PubMed

    Satoh, Ryoichi; Kaku, Ayumi; Satomura, Megumi; Kohori, Michiyo; Noura, Kanako; Furukawa, Tomoko; Kotake, Masako; Takano, Tomomi; Hohdatsu, Tsutomu

    2011-06-01

    Feline infectious peritonitis virus (FIPV) can cause a lethal disease in cats, feline infectious peritonitis (FIP). The antibody-dependent enhancement (ADE) of FIPV infection has been recognised in experimentally infected cats, and cellular immunity is considered to play an important role in preventing the onset of FIP. To evaluate the importance of cellular immunity for FIPV infection, monoclonal antibodies (MAbs) against feline interferon (fIFN)-γ were first created to establish fIFN-γ detection systems using the MAbs. Six anti-fIFN-γ MAbs were created. Then, the difference in epitope which those MAbs recognise was demonstrated by competitive enzyme-linked immunosorbent assay (ELISA) and IFN-γ neutralisation tests. Detection systems for fIFN-γ (sandwich ELISA, ELISpot assay, and two-colour flow cytometry) were established using anti-fIFN-γ MAbs that recognise different epitopes. In all tests, fIFN-γ production from peripheral blood mononuclear cells (PBMCs) obtained from cats experimentally infected with an FIPV isolate that did not develop the disease was significantly increased by heat-inactivated FIPV stimulation in comparison with medium alone. Especially, CD8(+)fIFN-γ(+) cells, but not CD4(+)fIFN-γ(+) cells, were increased. In contrast, fIFN-γ production from PBMCs isolated from cats that had developed FIP and specific pathogen-free (SPF) cats was not increased by heat-inactivated FIPV stimulation. These results suggest that cellular immunity plays an important role in preventing the development of FIP. Measurement of fIFN-γ production with the anti-fIFN-γ MAbs created in this study appeared to be useful in evaluating cellular immunity in cats.

  5. Identification of differentially expressed genes that potentially confer pest resistance in transgenic ChIFN-γ tobacco.

    PubMed

    Wu, Yong-Jun; Wu, Yu-Jun; Luo, Xi; Shen, Xi-Long; Zhao, De-Gang

    2014-06-15

    Chicken interferon-γ (ChIFN-γ) is both an inhibitor of viral replication and a regulator of numerous immunological functions. However, since little is known about the mechanisms underlying the insect-resistance of transgenic ChIFN-γ, a transgenic ChIFN-γ tobacco line was employed in the present study to explore this mechanism. A cDNA microarray (with 43,760 unigenes) was used to analyze the gene expression profiles of transgenic and wild-type (WT) tobacco leaves at two different growth stages. Compared with the WT, 1529 and 405 expressed sequence tags were significantly up- or downregulated on days 119 and 147, respectively. The differentially expressed genes (DEGs) are involved in metabolic regulation, cell division and differentiation, material synthesis and transport, signal transduction, and protein synthesis and degradation. Candidate genes that may increase cell density, thicken cell walls, promote secondary metabolite synthesis, and mediate plant hormone-induced resistance responses were used to identify the ChIFN-γ-mediated insect-resistance mechanisms. The insect-resistance of transgenic ChIFN-γ tobacco possibly involves unknown signaling pathways, which may directly or indirectly affect DEG expression-mediating genes. The degree of pest resistance increased as the plants grew. Three genes likely to be related to jasmonic acid- or salicylic acid-dependent plant defense responses, including CAF 1, Cop 8/CSN, and HD, are implicated in the insect-resistance of the transgenic plants. The mechanism of transgenic ChIFN-γ tobacco resistance also involves RPS20 and other genes that induce microRNA-based gene regulation. The ChIFN-γ-mediated DGEs contribute to insect-resistance in transgenic ChIFN-γ tobacco, which provides new insight into the role of ChIFN-γ.

  6. G-rich DNA-induced stress response blocks type-I-IFN but not CXCL10 secretion in monocytes

    PubMed Central

    Herzner, Anna-Maria; Wolter, Steven; Zillinger, Thomas; Schmitz, Saskia; Barchet, Winfried; Hartmann, Gunther; Bartok, Eva; Schlee, Martin

    2016-01-01

    Excessive inflammation can cause damage to host cells and tissues. Thus, the secretion of inflammatory cytokines is tightly regulated at transcriptional, post-transcriptional and post-translational levels and influenced by cellular stress responses, such as endoplasmic reticulum (ER) stress or apoptosis. Here, we describe a novel type of post-transcriptional regulation of the type-I-IFN response that was induced in monocytes by cytosolic transfection of a short immunomodulatory DNA (imDNA), a G-tetrad forming CpG-free derivative of the TLR9 agonist ODN2216. When co-transfected with cytosolic nucleic acid stimuli (DNA or 3P-dsRNA), imDNA induced caspase-3 activation, translational shutdown and upregulation of stress-induced genes. This stress response inhibited the type-I-IFN induction at the translational level. By contrast, the induction of most type-I-IFN-associated chemokines, including Chemokine (C-X-C Motif) Ligand (CXCL)10 was not affected, suggesting a differential translational regulation of chemokines and type-I-IFN. Pan-caspase inhibitors could restore IFN-β secretion, yet, strikingly, caspase inhibition did not restore global translation but instead induced a compensatory increase in the transcription of IFN-β but not CXCL10. Altogether, our data provide evidence for a differential regulation of cytokine release at both transcriptional and post-transcriptional levels which suppresses type-I-IFN induction yet allows for CXCL10 secretion during imDNA-induced cellular stress. PMID:27941826

  7. Role of Baicalin in Anti-Influenza Virus A as a Potent Inducer of IFN-Gamma

    PubMed Central

    Chu, Ming; Xu, Lan; Zhang, Ming-bo; Chu, Zheng-yun; Wang, Yue-dan

    2015-01-01

    Baicalin (BA) is a flavonoid compound purified from Scutellaria baicalensis Georgi and has been shown to possess a potent inhibitory activity against viruses. However, the role of BA in anti-influenza virus has not been extensively studied, and the immunological mechanism of BA in antiviral activity remains unknown. Here, we observed that BA could protect mice from infection by influenza virus A/PR/8/34 (H1N1), associated with increasing IFN-γ production, but presented no effects in IFN-γ or IFN-γ receptor deficient mice. Further study indicated that BA could inhibit A/PR/8/34 replication through IFN-γ in human PBMC. Moreover, BA can directly induce IFN-γ production in human CD4+ and CD8+ T cells and NK cells, and activate JAK/STAT-1 signaling pathway. Collectively, BA exhibited anti-influenza virus A (H1N1) activity in vitro and in vivo as a potent inducer of IFN-γ in major IFN-γ producing cells. PMID:26783516

  8. Polo-like Kinase 1 (PLK1) Regulates Interferon (IFN) Induction by MAVS*

    PubMed Central

    Vitour, Damien; Dabo, Stéphanie; Ahmadi Pour, Malek; Vilasco, Myriam; Vidalain, Pierre-Olivier; Jacob, Yves; Mezel-Lemoine, Mariana; Paz, Suzanne; Arguello, Meztli; Lin, Rongtuan; Tangy, Frédéric; Hiscott, John; Meurs, Eliane F.

    2009-01-01

    The mitochondria-bound adapter MAVS participates in IFN induction by recruitment of downstream partners such as members of the TRAF family, leading to activation of NF-κB, and the IRF3 pathways. A yeast two-hybrid search for MAVS-interacting proteins yielded the Polo-box domain (PBD) of the mitotic Polo-like kinase PLK1. We showed that PBD associates with two different domains of MAVS in both dependent and independent phosphorylation events. The phosphodependent association requires the phosphopeptide binding ability of PBD. It takes place downstream of the proline-rich domain of MAVS, within an STP motif, characteristic of the binding of PLK1 to its targets, where the central Thr234 residue is phosphorylated. Its phosphoindependent association takes place at the C terminus of MAVS. PLK1 strongly inhibits the ability of MAVS to activate the IRF3 and NF-κB pathways and to induce IFN. Reciprocally, depletion of PLK1 can increase IFN induction in response to RIG-I/SeV or RIG-I/poly(I)-poly(C) treatments. This inhibition is dependent on the phosphoindependent association of PBD at the C terminus of MAVS where it disrupts the association of MAVS with its downstream partner TRAF3. IFN induction was strongly inhibited in cells arrested in G2/M by nocodazole, which provokes increased expression of endogenous PLK1. Interestingly, depletion of PLK1 from these nocodazole-treated cells could restore, at least partially, IFN induction. Altogether, these data demonstrate a new function for PLK1 as a regulator of IFN induction and provide the basis for the development of inhibitors preventing the PLK1/MAVS association to sustain innate immunity. PMID:19546225

  9. Sub-optimal dose of Sodium Antimony Gluconate (SAG)-diperoxovanadate combination clears organ parasites from BALB/c mice infected with antimony resistant Leishmania donovani by expanding antileishmanial T-cell repertoire and increasing IFN-gamma to IL-10 ratio.

    PubMed

    Haldar, Arun Kumar; Banerjee, Subha; Naskar, Kshudiram; Kalita, Diganta; Islam, Nashreen S; Roy, Syamal

    2009-06-01

    We demonstrate that the combination of sub-optimal doses of Sodium Antimony Gluconate (SAG) and the diperoxovanadate compound K[VO(O2)2(H2O)], also designated as PV6, is highly effective in combating experimental infection of BALB/c mice with antimony resistant (Sb(R)) Leishmania donovani (LD) as evident from the significant reduction in organ parasite burden where SAG is essentially ineffective. Interestingly, such treatment also allowed clonal expansion of antileishmanial T-cells coupled with robust surge of IFN-c and concomitant decrease in IL-10 production. The splenocytes from the treated animals generated significantly higher amounts of IFN-c inducible parasiticidal effector molecules like superoxide and nitric oxide as compared to the infected group. Our study indicates that the combination of sub-optimal doses of SAG and PV6 may be beneficial for the treatment of SAG resistant visceral leishmaniasis patients.

  10. Intracranial AAV-IFN-β gene therapy eliminates invasive xenograft glioblastoma and improves survival in orthotopic syngeneic murine model.

    PubMed

    GuhaSarkar, Dwijit; Neiswender, James; Su, Qin; Gao, Guangping; Sena-Esteves, Miguel

    2017-02-01

    The highly invasive property of glioblastoma (GBM) cells and genetic heterogeneity are largely responsible for tumor recurrence after the current standard-of-care treatment and thus a direct cause of death. Previously, we have shown that intracranial interferon-beta (IFN-β) gene therapy by locally administered adeno-associated viral vectors (AAV) successfully treats noninvasive orthotopic glioblastoma models. Here, we extend these findings by testing this approach in invasive human GBM xenograft and syngeneic mouse models. First, we show that a single intracranial injection of AAV encoding human IFN-β eliminates invasive human GBM8 tumors and promotes long-term survival. Next, we screened five AAV-IFN-β vectors with different promoters to drive safe expression of mouse IFN-β in the brain in the context of syngeneic GL261 tumors. Two AAV-IFN-β vectors were excluded due to safety concerns, but therapeutic studies with the other three vectors showed extensive tumor cell death, activation of microglia surrounding the tumors, and a 56% increase in median survival of the animals treated with AAV/P2-Int-mIFN-β vector. We also assessed the therapeutic effect of combining AAV-IFN-β therapy with temozolomide (TMZ). As TMZ affects DNA replication, an event that is crucial for second-strand DNA synthesis of single-stranded AAV vectors before active transcription, we tested two TMZ treatment regimens. Treatment with TMZ prior to AAV-IFN-β abrogated any benefit from the latter, while the reverse order of treatment doubled the median survival compared to controls. These studies demonstrate the therapeutic potential of intracranial AAV-IFN-β therapy in a highly migratory GBM model as well as in a syngeneic mouse model and that combination with TMZ is likely to enhance its antitumor potency.

  11. IL-12 Enhances the Antitumor Actions of Trastuzumab via NK Cell IFN-γ Production

    PubMed Central

    Jaime-Ramirez, Alena Cristina; Mundy-Bosse, Bethany L.; Kondadasula, SriVidya; Jones, Natalie B.; Roda, Julie M.; Mani, Aruna; Parihar, Robin; Karpa, Volodymyr; Papenfuss, Tracey L.; LaPerle, Krista M.; Biller, Elizabeth; Lehman, Amy; Chaudhury, Abhik Ray; Jarjoura, David; Burry, Richard W.; Carson, William E.

    2013-01-01

    The antitumor effects of therapeutic mAbs may depend on immune effector cells that express FcRs for IgG. IL-12 is a cytokine that stimulates IFN-γ production from NK cells and T cells. We hypothesized that coadministration of IL-12 with a murine anti-HER2/neu mAb (4D5) would enhance the FcR-dependent immune mechanisms that contribute to its antitumor activity. Thrice-weekly therapy with IL-12 (1 μg) and 4D5 (1 mg/kg) significantly suppressed the growth of a murine colon adenocarcinoma that was engineered to express human HER2 (CT-26HER2/neu) in BALB/c mice compared with the result of therapy with IL-12, 4D5, or PBS alone. Combination therapy was associated with increased circulating levels of IFN-γ, monokine induced by IFN-γ, and RANTES. Experiments with IFN-γ–deficient mice demonstrated that this cytokine was necessary for the observed antitumor effects of therapy with IL-12 plus 4D5. Immune cell depletion experiments showed that NK cells (but not CD4+ or CD8+ T cells) mediated the antitumor effects of this treatment combination. Therapy of HER2/neu-positive tumors with trastuzumab plus IL-12 induced tumor necrosis but did not affect tumor proliferation, apoptosis, vascularity, or lymphocyte infiltration. In vitro experiments with CT-26HER2/neu tumor cells revealed that IFN-γ induced an intracellular signal but did not inhibit cellular proliferation or induce apoptosis. Taken together, these data suggest that tumor regression in response to trastuzumab plus IL-12 is mediated through NK cell IFN-γ production and provide a rationale for the coadministration of NK cell-activating cytokines with therapeutic mAbs. PMID:21321106

  12. Neutralizing Antibodies against IFN-[Beta] in Multiple Sclerosis: Antagonization of IFN-[Beta] Mediated Suppression of MMPs

    ERIC Educational Resources Information Center

    Gilli, Francesca; Bertolotto, Antonio; Sala, Arianna; Hoffmann, Francine; Capobianco, Marco; Malucchi, Simona; Glass, Tracy; Kappos, Ludwig; Lindberg, Raija L. P.; Leppert, David

    2004-01-01

    Neutralizing antibodies (NAb) against interferon-[Beta] (IFN-Beta) develop in about a third of treated multiple sclerosis patients and are believed to reduce therapeutic efficacy of IFN-[Beta] on clinical and MRI measures. The expression of the interferon acute-response protein, myxovirus resistance protein A (MxA) is a sensitive measure of the…

  13. Enrichment of IFN-γ producing cells in different murine adipose tissue depots upon infection with an apicomplexan parasite

    PubMed Central

    Teixeira, Luzia; Marques, Raquel M.; Ferreirinha, Pedro; Bezerra, Filipa; Melo, Joana; Moreira, João; Pinto, Ana; Correia, Alexandra; Ferreira, Paula G.; Vilanova, Manuel

    2016-01-01

    Here we report that lean mice infected with the intracellular parasite Neospora caninum show a fast but sustained increase in the frequency of IFN-γ-producing cells noticeable in distinct adipose tissue depots. Moreover, IFN-γ-mediated immune memory could be evoked in vitro in parasite antigen-stimulated adipose tissue stromal vascular fraction cells collected from mice infected one year before. Innate or innate-like cells such as NK, NK T and TCRγδ+ cells, but also CD4+ and CD8+ TCRβ+ lymphocytes contributed to the IFN-γ production observed since day one of infection. This early cytokine production was largely abrogated in IL-12/IL23 p40-deficient mice. Moreover, production of IFN-γ by stromal vascular fraction cells isolated from these mice was markedly lower than that of wild-type counterparts upon stimulation with parasite antigen. In wild-type mice the increased IFN-γ production was concomitant with up-regulated expression of genes encoding interferon-inducible GTPases and nitric oxide synthase, which are important effector molecules in controlling intracellular parasite growth. This increased gene expression was markedly impaired in the p40-deficient mice. Overall, these results show that NK cells but also diverse T cell populations mediate a prompt and widespread production of IFN-γ in the adipose tissue of N. caninum infected mice. PMID:27001522

  14. Induction of IFN-α Subtypes and Their Antiviral Activity in Mumps Virus Infection

    PubMed Central

    Šantak, Maja; Košutić-Gulija, Tanja; Jergović, Mladen; Jug, Renata; Forčić, Dubravko

    2014-01-01

    Abstract Human type I interferons (IFNs) comprise one IFN-β, -ω, -κ, and -ɛ and 12 different IFN-α subtypes, which play an important role in early host antiviral response. Despite their high structural homology and signaling through the same receptor, IFN-α subtypes exhibit different antiviral, antiproliferative, and immunomodulatory activities. Differences in the production of IFN-α subtypes therefore determine the quality of an antiviral response. In this study, we investigated the pattern of IFN-α subtypes induced in infection with different mumps virus (MuV) strains and examined the MuV sensitivity to the action of IFN-α subtypes. We found that all IFN-α subtypes are being expressed in response to MuV infection with a highly similar IFN-α subtype pattern between the virus strains. We assessed an antiviral activity of several IFN-α subtypes: IFN-α1, IFN-α2, IFN-α4, IFN-α6, IFN-α8, IFN-α14, IFN-α17, and IFN-α21. Although they were all effective in suppressing MuV replication, the intensity and pattern of their action varied between MuV strains. Our results indicate that the overall IFN antiviral activity as well as the activity of specific IFN-α subtypes against MuV depend on a virus strain. PMID:25361048

  15. Early IFN type I response: Learning from microbial evasion strategies.

    PubMed

    Coccia, Eliana M; Battistini, Angela

    2015-03-01

    Type I interferon (IFN) comprises a class of cytokines first discovered more than 50 years ago and initially characterized for their ability to interfere with viral replication and restrict locally viral propagation. As such, their induction downstream of germ-line encoded pattern recognition receptors (PRRs) upon recognition of pathogen-associated molecular patterns (PAMPs) is a hallmark of the host antiviral response. The acknowledgment that several PAMPs, not just of viral origin, may induce IFN, pinpoints at these molecules as a first line of host defense against a number of invading pathogens. Acting in both autocrine and paracrine manner, IFN interferes with viral replication by inducing hundreds of different IFN-stimulated genes with both direct anti-pathogenic as well as immunomodulatory activities, therefore functioning as a bridge between innate and adaptive immunity. On the other hand an inverse interference to escape the IFN system is largely exploited by pathogens through a number of tactics and tricks aimed at evading, inhibiting or manipulating the IFN pathway, that result in progression of infection or establishment of chronic disease. In this review we discuss the interplay between the IFN system and some selected clinically important and challenging viruses and bacteria, highlighting the wide array of pathogen-triggered molecular mechanisms involved in evasion strategies.

  16. Role of IFN-γ and LPS on neuron/glial co-cultures infected by Neospora caninum

    PubMed Central

    De Jesus, Erica Etelvina Viana; Santos, Alex Barbosa Dos; Ribeiro, Catia Suse Oliveira; Pinheiro, Alexandre Moraes; Freire, Songeli Menezes; El-Bachá, Ramon Santos; Costa, Silvia Lima; de Fatima Dias Costa, Maria

    2014-01-01

    Neospora caninum causes cattle abortion and neurological symptoms in dogs. Although infection is usually asymptomatic, classical neurological symptoms of neosporosis may be associated with encephalitis. This parasite can grow in brain endothelial cells without markedly damages, but it can modulate the cellular environment to promote its survival in the brain. In previous studies, we described that IFN-γ decreased the parasite proliferation and down regulated nitric oxide (NO) production in astrocyte/microglia cultures. However, it remains unclear how glial cells respond to N. caninum in the presence of neurons. Therefore, we evaluated the effect of 300 IU/mL IFN-γ or 1.0 mg/mL of LPS on infected rat neuron/glial co-cultures. After 72 h of infection, LPS did not affect the mitochondrial dehydrogenase activity. However, IFN-γ decreased this parameter by 15.5 and 12.0% in uninfected and infected cells, respectively. The number of tachyzoites decreased 54.1 and 44.3% in cells stimulated with IFN-γ and LPS, respectively. Infection or LPS treatment did not change NO production. On the other hand, IFN-γ induced increased nitrite release in 55.7%, but the infection reverted this induction. IL-10 levels increased only in infected cultures (treated or not), meanwhile PGE2 release was improved in IFN-γ/infected or LPS/infected cells. Although IFN-γ significantly reduced the neurite length in uninfected cultures (42.64%; p < 0.001), this inflammatory cytokine reverted the impairment of neurite outgrowth induced by the infection (81.39%). The results suggest a neuroprotective potential response of glia to N. caninum infection under IFN-γ stimulus. This observation contributes to understand the immune mediated mechanisms of neosporosis in central nervous system (CNS). PMID:25386119

  17. Type I IFN induces protein ISGylation to enhance cytokine expression and augments colonic inflammation

    PubMed Central

    Fan, Jun-Bao; Miyauchi-Ishida, Sayuri; Arimoto, Kei-ichiro; Liu, Dan; Yan, Ming; Liu, Chang-Wei; Győrffy, Balázs; Zhang, Dong-Er

    2015-01-01

    Type I IFNs have broad activity in tissue inflammation and malignant progression that depends on the expression of IFN-stimulated genes (ISGs). ISG15, one such ISG, can form covalent conjugates to many cellular proteins, a process termed “protein ISGylation.” Although type I IFNs are involved in multiple inflammatory disorders, the role of protein ISGylation during inflammation has not been evaluated. Here we report that protein ISGylation exacerbates intestinal inflammation and colitis-associated colon cancer in mice. Mechanistically, we demonstrate that protein ISGylation negatively regulates the ubiquitin–proteasome system, leading to increased production of IFN-induced reactive oxygen species (ROS). The increased cellular ROS then enhances LPS-induced activation of p38 MAP kinase and the expression of inflammation-related cytokines in macrophages. Thus our studies reveal a regulatory role for protein ISGylation in colonic inflammation and its related malignant progression, indicating that targeting ubiquitin-activating enzyme E1 homolog has therapeutic potential in treating inflammatory diseases. PMID:26515094

  18. IFN-γ-Dependent Recruitment of CD4+ T Cells and Macrophages Contributes to Pathogenesis During Leishmania amazonensis Infection

    PubMed Central

    Carneiro, Matheus Batista Heitor; Lopes, Mateus Eustáquio de Moura; Vaz, Leonardo Gomes; Sousa, Louisa Maria Andrade; dos Santos, Liliane Martins; de Souza, Carolina Carvalho; Campos, Ana Carolina de Angelis; Gomes, Dawidson Assis; Gonçalves, Ricardo; Tafuri, Wagner Luiz

    2015-01-01

    Interferon gamma (IFN-γ) is a key factor in the protection of hosts against intracellular parasites. This cytokine induces parasite killing through nitric oxide and reactive oxygen species production by phagocytes. Surprisingly, during Leishmania amazonensis infection, IFN-γ plays controversial roles. During in vitro infections, IFN-γ induces the proliferation of the amastigote forms of L. amazonensis. However, this cytokine is not essential at the beginning of an in vivo infection. It is not clear why IFN-γ does not mediate protection during the early stages of infection. Thus, the aim of our study was to investigate the role of IFN-γ during L. amazonensis infection. We infected IFN-γ−/− mice in the footpad and followed the development of leishmaniasis in these mice compared with that in WT mice. CD4+ T lymphocytes and macrophages migrated earlier to the site of infection in the WT mice, and the earlier migration of these 2 cell types was associated with lesion development and parasite growth, respectively. These differences in the infiltrate populations were explained by the increased expression of chemokines in the lesions of the WT mice. Thus, we propose that IFN-γ plays a dual role during L. amazonensis infection; it is an important inducer of effector mechanisms, particularly through inducible nitric oxide synthase expression, and conversely, it is a mediator of inflammation and pathogenesis through the induction of the expression of chemokines. Our data provided evidence for a pathogenic effect of IFN-γ production during leishmaniasis that was previously unknown. PMID:26401717

  19. Stimulation of the primary anti-HIV antibody response by IFN-{alpha} in patients with acute HIV-1 infection

    PubMed Central

    Adalid-Peralta, Laura; Godot, Véronique; Colin, Céline; Krzysiek, Roman; Tran, Thi; Poignard, Pascal; Venet, Alain; Hosmalin, Anne; Lebon, Pierre; Rouzioux, Christine; Chêne, Geneviève; Emilie, Dominique

    2008-01-01

    Type I IFNs are needed for the production of antiviral antibodies in mice; whether they also stimulate primary antibody responses in vivo during human viral infections is unknown. This was assessed in patients acutely infected with HIV-1 and treated with IFN-α2b. Patients with acute HIV-1 infection were randomized to receive anti-retroviral therapy alone (Group A, n=60) or combined for 14 weeks with pegylated-IFN-α2b (Group B, n=30). Emergence of anti-HIV antibodies was monitored during 32 weeks by Western blot (WB) analyses of serum samples. IFN-α2b treatment stimulated the production of anti-HIV antibodies. On Week 32, 19 weeks after the last IFN-α2b administration, there were 8.5 (6.5–10.0) HIV WB bands (median, interquartile range) in Group B and 7.0 (5.0–10.0) bands in Group A (P=0.054), and band intensities were stronger in Group B (P<0.05 for p18, p24, p34, p40, and p55 HIV antigens). IFN-α2b treatment also increased circulating concentrations of the B cell-activating factor of the TNF family (P<0.001) and ex vivo production of IL-12 (P<0.05), reflecting its effect on innate immune cells. Withdrawal of antiretroviral treatment on Week 36 resulted in a lower rebound of HIV replication in Group B than in Group A (P<0.05). Therefore, type I IFNs stimulate the emerging anti-HIV immune response in patients with acute HIV-1 infection, resulting in an improved control of HIV replication. Type I IFNs are thus critical in the development of efficient antiviral immune responses in humans, including the production of antiviral antibodies. PMID:18182457

  20. Stimulation of the primary anti-HIV antibody response by IFN-alpha in patients with acute HIV-1 infection.

    PubMed

    Adalid-Peralta, Laura; Godot, Véronique; Colin, Céline; Krzysiek, Roman; Tran, Thi; Poignard, Pascal; Venet, Alain; Hosmalin, Anne; Lebon, Pierre; Rouzioux, Christine; Chene, Genevieve; Emilie, Dominique

    2008-04-01

    Type I IFNs are needed for the production of antiviral antibodies in mice; whether they also stimulate primary antibody responses in vivo during human viral infections is unknown. This was assessed in patients acutely infected with HIV-1 and treated with IFN-alpha2b. Patients with acute HIV-1 infection were randomized to receive antiretroviral therapy alone (Group A, n=60) or combined for 14 weeks with pegylated-IFN-alpha2b (Group B, n=30). Emergence of anti-HIV antibodies was monitored during 32 weeks by Western blot (WB) analyses of serum samples. IFN-alpha2b treatment stimulated the production of anti-HIV antibodies. On Week 32, 19 weeks after the last IFN-alpha2b administration, there were 8.5 (6.5-10.0) HIV WB bands (median, interquartile range) in Group B and 7.0 (5.0-10.0) bands in Group A (P=0.054), and band intensities were stronger in Group B (P<0.05 for p18, p24, p34, p40, and p55 HIV antigens). IFN-alpha2b treatment also increased circulating concentrations of the B cell-activating factor of the TNF family (P<0.001) and ex vivo production of IL-12 (P<0.05), reflecting its effect on innate immune cells. Withdrawal of antiretroviral treatment on Week 36 resulted in a lower rebound of HIV replication in Group B than in Group A (P<0.05). Therefore, type I IFNs stimulate the emerging anti-HIV immune response in patients with acute HIV-1 infection, resulting in an improved control of HIV replication. Type I IFNs are thus critical in the development of efficient antiviral immune responses in humans, including the production of antiviral antibodies.

  1. Involvement of Differential Relationship between HCV Replication and Hepatic PRR Signaling Gene Expression in Responsiveness to IFN-Based Therapy.

    PubMed

    Yuki, Nobukazu; Matsumoto, Shinji; Kato, Michio; Yamaguchi, Toshikazu

    2013-01-01

    Aim. To gain an insight into the effect of HCV replication-associated interference with the IFN system on hepatic mRNA expression involved in IFN production. Methods. Relative mRNA expression of TLR3/RIG-I signaling genes involved in IFN- β production was correlated with positive- and negative-strand HCV RNAs in pretreatment liver tissues responsive and nonresponsive to peginterferon and ribavirin for chronic hepatitis C genotype 1. Treatment response was analyzed for per protocol population at weeks 12 (n = 45) and 24 (n = 40) and at 24 weeks aftertreatment (n = 38). Results. HCV replication had no relation to the expression of TLR3, RIG-I, TRIF, IPS-1, IRF3, and IFN- β mRNAs in responders. In striking contrast, positive- and/or negative-strand HCV showed positive correlations with TLR3, RIG-I, TRIF, IPS-1, and IRF3 mRNAs in week-12 nonresponders; with RIG-I, TRIF, IPS-1, and IRF3 mRNAs in week-24 nonresponders; and with TLR3, RIG-I, and IRF3 mRNAs in posttreatment nonresponders. Thus mRNA expression of TLR3/RIG-I signaling genes was increased in relation to viral replication in nonresponders. Conclusions. The findings in IFN nonresponders may imply a host feedback response to severe impairment of the IFN system associated with HCV replication.

  2. Antigen expression determines adenoviral vaccine potency independent of IFN and STING signaling

    PubMed Central

    Quinn, Kylie M.; Zak, Daniel E.; Costa, Andreia; Yamamoto, Ayako; Kastenmuller, Kathrin; Hill, Brenna J.; Lynn, Geoffrey M.; Darrah, Patricia A.; Lindsay, Ross W.B.; Wang, Lingshu; Cheng, Cheng; Nicosia, Alfredo; Folgori, Antonella; Colloca, Stefano; Cortese, Riccardo; Gostick, Emma; Price, David A.; Gall, Jason G.D.; Roederer, Mario; Aderem, Alan; Seder, Robert A.

    2015-01-01

    Recombinant adenoviral vectors (rAds) are lead vaccine candidates for protection against a variety of pathogens, including Ebola, HIV, tuberculosis, and malaria, due to their ability to potently induce T cell immunity in humans. However, the ability to induce protective cellular immunity varies among rAds. Here, we assessed the mechanisms that control the potency of CD8 T cell responses in murine models following vaccination with human-, chimpanzee-, and simian-derived rAds encoding SIV-Gag antigen (Ag). After rAd vaccination, we quantified Ag expression and performed expression profiling of innate immune response genes in the draining lymph node. Human-derived rAd5 and chimpanzee-derived chAd3 were the most potent rAds and induced high and persistent Ag expression with low innate gene activation, while less potent rAds induced less Ag expression and robustly induced innate immunity genes that were primarily associated with IFN signaling. Abrogation of type I IFN or stimulator of IFN genes (STING) signaling increased Ag expression and accelerated CD8 T cell response kinetics but did not alter memory responses or protection. These findings reveal that the magnitude of rAd-induced memory CD8 T cell immune responses correlates with Ag expression but is independent of IFN and STING and provide criteria for optimizing protective CD8 T cell immunity with rAd vaccines. PMID:25642773

  3. Type I IFN promotes NK cell expansion during viral infection by protecting NK cells against fratricide.

    PubMed

    Madera, Sharline; Rapp, Moritz; Firth, Matthew A; Beilke, Joshua N; Lanier, Lewis L; Sun, Joseph C

    2016-02-08

    Type I interferon (IFN) is crucial in host antiviral defense. Previous studies have described the pleiotropic role of type I IFNs on innate and adaptive immune cells during viral infection. Here, we demonstrate that natural killer (NK) cells from mice lacking the type I IFN-α receptor (Ifnar(-/-)) or STAT1 (which signals downstream of IFNAR) are defective in expansion and memory cell formation after mouse cytomegalovirus (MCMV) infection. Despite comparable proliferation, Ifnar(-/-) NK cells showed diminished protection against MCMV infection and exhibited more apoptosis compared with wild-type NK cells. Furthermore, we show that Ifnar(-/-) NK cells express increased levels of NK group 2 member D (NKG2D) ligands during viral infection and are susceptible to NK cell-mediated fratricide in a perforin- and NKG2D-dependent manner. Adoptive transfer of Ifnar(-/-) NK cells into NK cell-deficient mice reverses the defect in survival and expansion. Our study reveals a novel type I IFN-dependent mechanism by which NK cells evade mechanisms of cell death after viral infection.

  4. CRAM-A indicates IFN-γ-associated inflammatory response in breast cancer.

    PubMed

    Sarmadi, Parisa; Tunali, Gurcan; Esendagli-Yilmaz, Guldal; Yilmaz, Kerim Bora; Esendagli, Gunes

    2015-12-01

    Atypical chemokine receptors (ACKRs) function as endpoint regulators of chemokine gradients. These non-signaling receptors that are transiently expressed under inflammatory conditions have critical roles in the control or maintenance of immune responses. Alternatively, here, CCRL2 (ACKR5) expression was determined to be constitutive in breast cancer cells. Increased amount of CCRL2 was also found in breast tumor tissues with high immune infiltration. Its expression was upregulated in the presence of pro-inflammatory cytokines, IL-1β, TNF-α, IL-6, and especially IFN-γ⋅ Moreover, an alternative transcript of CCRL2 gene, CRAM-A, was specifically expressed in a transient fashion under the influence of IFN-γ. CRAM-A expression was also positively correlated with the presence of IFN-γ mRNA in patient samples. CCRL2-associated chemotactic molecules, chemerin, CCL19 and CCL5, were also detected in cancer tissues and CCL5 mRNA level was correlated with that of CRAM-A and IFN-γ. Hence, in breast cancer, CRAM-A becomes specifically upregulated under inflammatory stimuli and may serve as a potential marker of immune response.

  5. Programmed death-1 receptor suppresses γ-IFN producing NKT cells in human tuberculosis.

    PubMed

    Singh, Amar; Dey, Aparajit Ballav; Mohan, Anant; Mitra, Dipendra Kumar

    2014-05-01

    IFN-γ biased Th1 effector immune response is crucial for containment of Mycobacterium tuberculosis infection. Various T cell subsets with regulatory function dictate the generation of Th1 like cells. NKT cells are a specialized T cell subset known to be activated early in immune response and control T cell response via release of immunoregulatory cytokines like IFN-γ, IL-4 and IL-10. M. tuberculosis, with abundance of its cell wall lipids may potently activate NKT cells resulting in cytokine production and PD-1 expression. In this study, among 49 treatment naive active pulmonary tuberculosis patients, we found a higher percentage of PD1(+) NKT cells correlating with sputum bacillary load. Furthermore, blocking PD-1 increased the number of IFN-γ producing NKT cells by inhibiting their apoptosis. Moreover, peripheral frequency of NKT cells declined with therapy suggesting their role in host T cell response. In this study, we concluded that PD-1 preferentially induces apoptosis of IFN-γ producing NKT cells while sparing NKT cells that produce IL-4. Such a polarized NKT cell function may impose a Th2 bias on the ensuing effector T cell response leading to inefficient clearance of M. tuberculosis. Inhibiting PD-1 may therefore alter the T cell response in favor of the host by rescuing type 1 NKT cells from apoptosis and boosting Th1 effector T cell functions against M. tuberculosis.

  6. Cloning and expression of pigeon IFN-γ gene.

    PubMed

    Fringuelli, Elena; Urbanelli, Lorena; Tharuni, Omar; Proietti, Patrizia Casagrande; Bietta, Annalisa; Davidson, Irit; Franciosini, Maria Pia

    2010-12-01

    This is the first paper describing the cloning of pigeon IFN-γ gene (PiIFN-γ) and the analysis of the in vitro expressed recombinant protein. The PiIFN-γ gene was identified by RT-PCR as a 498bp, fragment coding for a precursor protein of 165 amino acids instead of 164 amino acids, as observed in the other avian species. The recombinant protein was expressed in vitro by an eukaryotic system and the biological properties of the cytokine were tested using a chicken macrophage cell line. The high degree of amino acid and nucleotide identity, shared with the ChIFN-γ, and the fact that the pigeon protein was functional on chicken cells, indicates a cross-reactivity between pigeon and chicken IFN-γ. The detection of the PiIFN-γ could represent an useful instrument in understanding the role played by this cytokine in immune response related to vaccinations and infectious diseases in the pigeon.

  7. Antigen-Specific IFN-γ Responses Correlate with the Activity of M. tuberculosis Infection but Are Not Associated with the Severity of Tuberculosis Disease

    PubMed Central

    Nikitina, Irina Yu.; Panteleev, Alexander V.; Karpina, Natalya L.; Bagdasarian, Tatef R.; Burmistrova, Irina A.; Andreevskaya, Sofia N.; Chernousova, Larisa N.; Vasilyeva, Irina A.

    2016-01-01

    IFN-γ is a key cytokine in antituberculosis (TB) defense. However, how the levels of its secretion affect M. tuberculosis (Mtb) infection is not clear. We have analyzed associations between IFN-γ responses measured in QuantiFERON®-TB Gold In-tube (QFT) assay, TB disease severity, and Mtb infection activity. TB severity was evaluated based on the results of radiological, microbiological, and clinical examinations. Antigen-driven IFN-γ secretion did not correlate with TB severity. Mitogen-induced IFN-γ secretion correlated inversely with the form of pulmonary pathology and the area of affected pulmonary tissue; the levels of spontaneous IFN-γ secretion correlated with patients' age (r = 0.395, p = 0.001). Mtb infection activity was evaluated based on radiological data of lung tissue infiltration, destruction, dissemination or calcification, and condensation. The rate of positive QFT results and the levels of antigen-driven IFN-γ secretion increased in a row: patients with residual TB lesions < patients with low TB activity < patients with high TB activity. Thus, antigen-driven IFN-γ secretion and QFT results did not associate with TB severity but associated with the infection activity. The results suggest that quantitative parameters of IFN-γ secretion play a minor role in determining the course of TB disease but mirror the activity of the infectious process. PMID:28042583

  8. Mechanism of IFN-γ-induced Endocytosis of Tight Junction Proteins: Myosin II-dependent Vacuolarization of the Apical Plasma Membrane

    PubMed Central

    Utech, Markus; Ivanov, Andrei I.; Samarin, Stanislav N.; Bruewer, Matthias; Turner, Jerrold R.; Mrsny, Randall J.; Parkos, Charles A.; Nusrat, Asma

    2005-01-01

    Disruption of epithelial barrier by proinflammatory cytokines such as IFN-γ represents a major pathophysiological consequence of intestinal inflammation. We have previously shown that IFNincreases paracellular permeability in model T84 epithelial cells by inducing endocytosis of tight junction (TJ) proteins occludin, JAM-A, and claudin-1. The present study was designed to dissect mechanisms of IFN-γ-induced endocytosis of epithelial TJ proteins. IFN-γ treatment of T84 cells resulted in internalization of TJ proteins into large actin-coated vacuoles that originated from the apical plasma membrane and resembled the vacuolar apical compartment (VAC) previously observed in epithelial cells that lose cell polarity. The IFN-γ dependent formation of VACs required ATPase activity of a myosin II motor but was not dependent on rapid turnover of F-actin. In addition, activated myosin II was observed to colocalize with VACs after IFN-γ exposure. Pharmacological analyses revealed that formation of VACs and endocytosis of TJ proteins was mediated by Rho-associated kinase (ROCK) but not myosin light chain kinase (MLCK). Furthermore, IFN-γ treatment resulted in activation of Rho GTPase and induced expressional up-regulation of ROCK. These results, for the first time, suggest that IFN-γ induces endocytosis of epithelial TJ proteins via RhoA/ROCK-mediated, myosin II-dependent formation of VACs. PMID:16055505

  9. IFN-τ Displays Anti-Inflammatory Effects on Staphylococcus aureus Endometritis via Inhibiting the Activation of the NF-κB and MAPK Pathways in Mice

    PubMed Central

    Zhang, Zhenbiao; Guo, Yingfang; Liu, Yuzhu; Li, Chengye

    2017-01-01

    The aim of the present study was to determine the anti-inflammatory effect of IFN-τ on endometritis using a mouse model of S. aureus-induced endometritis and to elucidate the mechanism of action underlying these effects. In the present study, the effect of IFN-τ on S. aureus growth was monitored by turbidimeter at 600 nm. IFN-τ did not affect S. aureus growth. The histopathological changes indicated that IFN-τ had a protective effect on uterus tissues with S. aureus infection. The ELISA and qPCR results showed the production of the proinflammatory cytokines TNF-α, IL-1β, and IL-6 was decreased with IFN-τ treatment. In contrast, the level of the anti-inflammatory cytokine IL-10 was increased. We further studied the signaling pathway associated with these observations, and the qPCR results showed that the expression of TLR2 was repressed by IFN-τ. Furthermore, the western blotting results showed the phosphorylation of IκB, NF-κB p65, and MAPKs (p38, JNK, and ERK) was inhibited by IFN-τ treatment. The results suggested that IFN-τ may be a potential drug for the treatment of uterine infection due to S. aureus or other infectious inflammatory diseases. PMID:28331850

  10. Antigen-Specific IFN-γ Responses Correlate with the Activity of M. tuberculosis Infection but Are Not Associated with the Severity of Tuberculosis Disease.

    PubMed

    Nikitina, Irina Yu; Panteleev, Alexander V; Sosunova, Ekaterina V; Karpina, Natalya L; Bagdasarian, Tatef R; Burmistrova, Irina A; Andreevskaya, Sofia N; Chernousova, Larisa N; Vasilyeva, Irina A; Lyadova, Irina V

    2016-01-01

    IFN-γ is a key cytokine in antituberculosis (TB) defense. However, how the levels of its secretion affect M. tuberculosis (Mtb) infection is not clear. We have analyzed associations between IFN-γ responses measured in QuantiFERON®-TB Gold In-tube (QFT) assay, TB disease severity, and Mtb infection activity. TB severity was evaluated based on the results of radiological, microbiological, and clinical examinations. Antigen-driven IFN-γ secretion did not correlate with TB severity. Mitogen-induced IFN-γ secretion correlated inversely with the form of pulmonary pathology and the area of affected pulmonary tissue; the levels of spontaneous IFN-γ secretion correlated with patients' age (r = 0.395, p = 0.001). Mtb infection activity was evaluated based on radiological data of lung tissue infiltration, destruction, dissemination or calcification, and condensation. The rate of positive QFT results and the levels of antigen-driven IFN-γ secretion increased in a row: patients with residual TB lesions < patients with low TB activity < patients with high TB activity. Thus, antigen-driven IFN-γ secretion and QFT results did not associate with TB severity but associated with the infection activity. The results suggest that quantitative parameters of IFN-γ secretion play a minor role in determining the course of TB disease but mirror the activity of the infectious process.

  11. Evidence for a regulatory loop between IFN-γ and IL-33 in skin inflammation.

    PubMed

    Seltmann, Jenny; Werfel, Thomas; Wittmann, Miriam

    2013-02-01

    Interleukin-33 has recently gained much attention due to its role in allergic responses. It has been shown to amplify Th2 responses and to act as a damage-associated molecular pattern. IL-33 acts on a broad range of cells and has been proposed to link innate and adaptive features of allergic responses. It was the aim of this study to investigate this property of IL-33 in the inflammatory response characterising atopic dermatitis (AD). We have analysed the response of skin-resident cells derived from patients with AD and healthy donors with regard to the expression of IL-33 and its receptor ST2. The functional impact of IL-33 on CD4+ T cells was investigated. Keratinocytes and dermal fibroblasts clearly differ in their regulation of IL-33. In fibroblasts, the concerted action of TNF-α and IL-1β was the strongest inducer, whereas IFN-γ is clearly the key molecule that upregulates IL-33 in keratinocytes with a more pronounced response of cells derived from patients with AD. Keratinocytes from patients with AD showed a markedly higher constitutive expression level of surface ST2. CD4+ T cells respond to IL-33. Unexpectedly, IL-33 failed to induce a significant secretion of IL-5 or IL-13. By contrast, high amounts of IFN-γ were detectable if IL-33 was added to the T-cell receptor-stimulated cells or in combination with IL-12. These results suggest that IL-33 and IFN-γ are closely interlinked in epidermal AD inflammation. IFN-γ induces IL-33 in keratinocytes and IL-33 acts on activated T cells to further increase the release of IFN-γ, therefore contributing to drive skin inflammation towards chronic responses.

  12. IFN-γ differentially modulates memory-related processes under basal and chronic stressor conditions

    PubMed Central

    Litteljohn, Darcy; Nelson, Eric; Hayley, Shawn

    2014-01-01

    Cytokines are inflammatory messengers that orchestrate the brain’s response to immunological challenges, as well as possibly even toxic and psychological insults. We previously reported that genetic ablation of the pro-inflammatory cytokine, interferon-gamma (IFN-γ), attenuated some of the corticosteroid, cytokine, and limbic dopaminergic variations induced by 6 weeks of exposure to an unpredictable psychologically relevant stressor. Presently, we sought to determine whether a lack of IFN-γ would likewise modify the impact of chronic stress on hippocampus-dependent memory function and related neurotransmitter and neurotrophin signaling systems. As predicted, chronic stress impaired spatial recognition memory (Y-maze task) in the wild-type animals. In contrast, though the IFN-γ knockouts (KOs) showed memory disturbances in the basal state, under conditions of chronic stress these mice actually exhibited facilitated memory performance. Paralleling these findings, while overall the KOs displayed altered noradrenergic and/or serotonergic activity in the hippocampus and locus coeruleus, norepinephrine utilization in both of these memory-related brain regions was selectively increased among the chronically stressed KOs. However, contrary to our expectations, neither IFN-γ deletion nor chronic stressor exposure significantly affected nucleus accumbens dopaminergic neurotransmission or hippocampal brain-derived neurotrophic factor protein expression. These findings add to a growing body of evidence implicating cytokines in the often differential regulation of neurobehavioral processes in health and disease. Whereas in the basal state IFN-γ appears to be involved in sustaining memory function and the activity of related brain monoamine systems, in the face of ongoing psychologically relevant stress the cytokine may, in fact, act to restrict potentially adaptive central noradrenergic and spatial memory responses. PMID:25477784

  13. IDO1 and TGF-β Mediate Protective Effects of IFN-α in Antigen-Induced Arthritis

    PubMed Central

    Pallotta, Maria Teresa; Narendra, Sudeep Chenna; Carlsson, Björn; Iacono, Alberta; Namale, Joanitah; Boon, Louis; Grohmann, Ursula; Magnusson, Mattias

    2016-01-01

    IFN-α prevents Ag-induced arthritis (AIA), and in this study we investigated the role of IDO1 and TGF-β signaling for this anti-inflammatory property of IFN-α. Arthritis was induced by methylated BSA (mBSA) in mBSA-sensitized wild-type (WT), Ido1−/−, or Ifnar−/− mice, treated or not with IFN-α or the IDO1 product kynurenine (Kyn). Enzymatic IDO1 activity, TGF-β, and plasmacytoid dendritic cells (pDC) were neutralized by 1-methyltryptophan and Abs against TGF-β and pDC, respectively. IDO1 expression was determined by RT-PCR, Western blot, and FACS, and enzymatic activity by HPLC. Proliferation was measured by 3H-thymidine incorporation and TGF-β by RT-PCR and ELISA. WT but not Ido1−/− mice were protected from AIA by IFN-α, and Kyn, the main IDO1 product, also prevented AIA, both in WT and Ifnar−/− mice. Protective treatment with IFNincreased the expression of IDO1 in pDC during AIA, and Ab-mediated depletion of pDC, either during mBSA sensitization or after triggering of arthritis, completely abrogated the protective effect of IFN-α. IFN-α treatment also increased the enzymatic IDO1 activity (Kyn/tryptophan ratio), which in turn activated production of TGF-β. Neutralization of enzymatic IDO1 activity or TGF-β signaling blocked the protective effect of IFN-α against AIA, but only during sensitization and not after triggering of arthritis. Likewise, inhibition of the IDO1 enzymatic activity in the sensitization phase, but not after triggering of arthritis, subdued the IFN-α–induced inhibition of mBSA-induced proliferation. In conclusion, presence of IFN-α at Ag sensitization activates an IDO1/TGF-β–dependent anti-inflammatory program that upon antigenic rechallenge prevents inflammation via pDC. PMID:27647832

  14. Systemic Activation of the IFN System by Exposure of Natural Epithelia to Physiologic (Low Dose) Levels of IFN

    DTIC Science & Technology

    1990-06-20

    concentrations of IFN interacted with the peritoneal epiti.elium and transferred amplified antiviral resistance to the mouse. This raised the...infecting organisms. The potential efficacy of using low concentrations of IFN for therapeutic treatment of feline leukemia virus and human...Command Air Force Ofc of Scient. Res. National Naval Medical Center Bolling Air Force Base Bethesda, MD 20814 Washington, DC 20332 Director, Infectious

  15. Type I IFN Signaling Is Dispensable during Secondary Viral Infection

    PubMed Central

    Hosking, Martin P.; Flynn, Claudia T.; Whitton, J. Lindsay

    2016-01-01

    Innate immune responses in general, and type I interferons (T1IFNs) in particular, play an important and often essential role during primary viral infections, by directly combatting the virus and by maximizing the primary adaptive immune response. Several studies have suggested that T1IFNs also contribute very substantially to the secondary (recall) response; they are thought (i) to be required to drive the early attrition of memory T cells, (ii) to support the subsequent expansion of surviving virus-specific memory cells, and (iii) to assist in the suppression and clearance of the infectious agent. However, many of these observations were predicated upon models in which T1IFN signaling was interrupted prior to a primary immune response, raising the possibility that the resulting memory cells might be intrinsically abnormal. We have directly addressed this by using an inducible-Cre model system in which the host remains genetically-intact during the primary response to infection, and in which T1IFN signaling can be effectively ablated prior to secondary viral challenge. We report that, in stark contrast to primary infection, T1IFN signaling is not required during the recall response. IFNαβR-deficient memory CD8+ and CD4+ memory T cells undergo attrition and expansion with kinetics that are indistinguishable from those of receptor-sufficient cells. Moreover, even in the absence of functional T1IFN signaling, the host’s immune capacity to rapidly suppress, and then to eradicate, a secondary infection remains intact. Thus, this study shows that T1IFN signaling is dispensable during the recall response to a virus infection. Moreover, two broader implications may be drawn. First, a T cell’s requirement for a cytokine is highly dependent on the cell’s maturation / differentiation status. Consequently, second, these data underscore the importance of evaluating a gene’s impact by modulating its expression or function in a temporally-controllable manner. PMID

  16. Wild type measles virus attenuation independent of type I IFN

    PubMed Central

    Druelle, Johan; Sellin, Caroline I; Waku-Kouomou, Diane; Horvat, Branka; Wild, Fabian T

    2008-01-01

    Background Measles virus attenuation has been historically performed by adaptation to cell culture. The current dogma is that attenuated virus strains induce more type I IFN and are more resistant to IFN-induced protection than wild type (wt). Results The adaptation of a measles virus isolate (G954-PBL) by 13 passages in Vero cells induced a strong attenuation of this strain in vivo. The adapted virus (G954-V13) differs from its parental strain by only 5 amino acids (4 in P/V/C and 1 in the M gene). While a vaccine strain, Edmonston Zagreb, could replicate equally well in various primate cells, both G954 strains exhibited restriction to the specific cell type used initially for their propagation. Surprisingly, we observed that both G954 strains induced type I IFN, the wt strain inducing even more than the attenuated ones, particularly in human plasmacytoid Dendritic Cells. Type I IFN-induced protection from the infection of both G954 strains depended on the cell type analyzed, being less efficient in the cells used to grow the viral strain. Conclusion Thus, mutations in M and P/V/C proteins can critically affect MV pathogenicity, cellular tropism and lead to virus attenuation without interfering with the α/β IFN system. PMID:18241351

  17. Mechanism of IFN-γ in regulating OPN/Th17 pathway during vascular collagen remodeling of hypertension induced by ANG II.

    PubMed

    Jiang, Lei; He, Pengcheng; Liu, Yong; Chen, Jiyan; Wei, Xuebiao; Tan, Ning

    2015-01-01

    More and more researches show that hypertensive vascular remodeling is closely related to the imbalance of immune system in recent years. IFN-γ is natural protein with the function of immune regulation and has resistance effect on vascular remodeling. However, the mechanism of IFN-γ is to be defined. This paper is to explore the mechanism of IFN-γ in regulating OPN/Th17 pathway. In this research, animal models of vascular collagen remodeling were established by inducing hypertensive mice with ANG II. There was no statistical significance when the systolic blood pressures and the percentages of wall thickness/lumen diameter in both groups of WT + AngII + IFN-γ and WT + PBS were compared (P=0.219>0.05, P=0.118>0.05). The concentration of serum precollagen-type I and III and their ratio in WT + AngII + IFN-γ group were decreased after the IFN-γ being given (P<0.01). Expression of OPN within tissue in WT + Ang II group was relatively high, but lowered after treated by IFN-γ. Th17 cell ratio was decreased in WT + AngII + IFN-γ group (P<0.01). Expressions of RORα and RORγt mRNA within Th17 cell were decreased (P<0.01). The content of IL-23 in WT + AngII + IFN-γ group was increased, while IL-10 and TGF-β decreased. It has proved that IFN-γ can regulate the hypertensive vascular collagen remodeling induced by ANG II, lower the systolic pressure and reduce the pathological damage of vascular collagen remodeling and the collagen synthesis. The mechanism may that the differentiation of Th17 is inhibited by suppressing the OPN expression and regulating the secretion of inflammatory cytokines.

  18. An essential role for IFN-β in the induction of IFN-stimulated gene expression by LPS in macrophages.

    PubMed

    Sheikh, Faruk; Dickensheets, Harold; Gamero, Ana M; Vogel, Stefanie N; Donnelly, Raymond P

    2014-10-01

    TLR agonists such as LPS and poly(I:C) induce expression of type I IFNs, such as IFN-α and -β, by macrophages. To examine the role of IFN-β in the induction of ISGs by LPS, we compared the ability of LPS to induce ISGF3 activity and ISG expression in bone marrow-derived macrophages from WT and Ifnb1(-/-) mice. We found that LPS treatment activated ISGF3 and induced expression of ISGs such as Oas1, Mx1, Ddx58 (RIG-I), and Ifih1 (MDA5) in WT macrophages, but not in macrophages derived from Ifnb1(-/-) mice or Ifnar1(-/-) mice. The inability of LPS to induce activation of ISGF3 and ISG expression in Ifnb1(-/-) macrophages correlated with the failure of LPS to induce activation of STAT1 and -2 in these cells. Consistent with these findings, LPS treatment also failed to induce ISG expression in bone marrow-derived macrophages from Stat2 KO mice. Although activation of ISGF3 and induction of ISG expression by LPS was abrogated in Ifnb1(-/-) and Ifnar1(-/-) macrophages, activation of NF-κB and induction of NF-κB-responsive genes, such as Tnf (TNF-α) and Il1b (IL-1β), were not affected by deletion of either the IFN-β or IFN-αR1 genes. These findings demonstrate that induction of ISGF3 activity and ISG expression by LPS is critically dependent on intermediate production of IFN-β and autocrine signaling through type I IFN receptors.

  19. Complement component C5a permits the coexistence of pathogenic Th17 cells and type I IFN in lupus.

    PubMed

    Pawaria, Sudesh; Ramani, Kritika; Maers, Kelly; Liu, Youhua; Kane, Lawrence P; Levesque, Marc C; Biswas, Partha S

    2014-10-01

    Systemic lupus erythematosus (SLE) is a type I IFN (IFN-I)-driven autoimmune disorder with exaggerated B and Th cell responses. Th17 cells, a recently identified Th cell subset, have been strongly implicated in the pathogenesis of SLE. Because IFN-I suppresses the generation and expansion of Th17 cells in an IL-27-dependent manner, it is unclear how pathogenic Th17 cells are generated in SLE in the presence of an environment characterized by high IFN-I levels. In this study, we showed that activation of c5aR on murine macrophages blocked IFN-I-mediated IL-27 production, thus permitting the development of Th17 cells. C5aR activation on IFN-I-responsive macrophages inhibits IRF-1-mediated transactivation of IL-27 gene expression via the PI3K/Akt pathway. Consistently, C5aR-deficient mice exhibited increased IL-27 expression and fewer Th17 cells and consequently developed reduced lupus nephritis in comparison with wild-type mice. In support of these findings in mice, we found that C5a inhibited IFN-I-induced IL-27 production from macrophages of lupus subjects. Moreover, the level of serum C5a correlated with Th17 frequency in peripheral blood. Collectively, these data indicate an essential role for C5a in the generation of pathogenic Th17 responses in SLE. Thus, therapeutic strategies to block C5aR activation may be beneficial for controlling pathogenic Th17-mediated inflammation in SLE.

  20. Taenia crassiceps infection and its excreted/secreted products inhibit STAT1 activation in response to IFN-γ.

    PubMed

    Becerra-Díaz, Mireya; Terrazas, Luis I

    2014-08-01

    It is well understood that helminth infections modulate the immune responses of their hosts but the mechanisms involved in this modulation are not fully known. Macrophages and dendritic cells appear to be consistently affected during this type of infection and are common target cells for helminth-derived molecules. In this report, we show that macrophages obtained from chronically Taenia crassiceps-infected mice displayed an impaired response to recombinant murine IFN-γ, but not to recombinant murine IL-4, as measured based on the phosphorylation of STAT1 and STAT6, respectively. These macrophages expressed high levels of SOCS3. However, the inhibition of phosphatase activity by orthovanadate restored the IFN-γ response of these macrophages by increasing STAT1 phosphorylation without affecting SOCS3 expression. Therefore, we aimed to identify the phosphatases associated with IFN-γ signaling inhibition and found that macrophages from T. crassiceps-infected mice displayed enhanced SHP-1 expression. Interestingly, the exposure of naïve macrophages to T. crassiceps excreted/secreted products similarly interfered with IFN-γ-induced STAT1 phosphorylation. Moreover, macrophages exposed to T. crassiceps excreted/secreted products expressed high levels of SOCS3 as well as SHP-1. Strikingly, human peripheral blood mononuclear cells that were exposed to T. crassiceps excreted/secreted products in vitro also displayed impaired STAT1 phosphorylation in response to IFN-γ; again, phosphatase inhibition abrogated the T. crassiceps excreted/secreted product-altered IFN-γ signaling. These data demonstrate a new mechanism by which helminth infection and the products derived during this infection target intracellular pathways to block the response to inflammatory cytokines such as IFN-γ in both murine and human cells.

  1. Myeloid cells, BAFF, and IFN-γ establish an inflammatory loop that exacerbates autoimmunity in Lyn-deficient mice

    PubMed Central

    Scapini, Patrizia; Hu, Yongmei; Chu, Ching-Liang; Migone, Thi-Sau; DeFranco, Anthony L.; Cassatella, Marco A.

    2010-01-01

    Autoimmunity is traditionally attributed to altered lymphoid cell selection and/or tolerance, whereas the contribution of innate immune cells is less well understood. Autoimmunity is also associated with increased levels of B cell–activating factor of the TNF family (BAFF; also known as B lymphocyte stimulator), a cytokine that promotes survival of self-reactive B cell clones. We describe an important role for myeloid cells in autoimmune disease progression. Using Lyn-deficient mice, we show that overproduction of BAFF by hyperactive myeloid cells contributes to inflammation and autoimmunity in part by acting directly on T cells to induce the release of IFN-γ. Genetic deletion of IFN-γ or reduction of BAFF activity, achieved by either reducing myeloid cell hyperproduction or by treating with an anti-BAFF monoclonal antibody, reduced disease development in lyn−/− mice. The increased production of IFN-γ in lyn−/− mice feeds back on the myeloid cells to further stimulate BAFF release. Expression of BAFF receptor on T cells was required for their full activation and IFN-γ release. Overall, our data suggest that the reciprocal production of BAFF and IFN-γ establishes an inflammatory loop between myeloid cells and T cells that exacerbates autoimmunity in this model. Our findings uncover an important pathological role of BAFF in autoimmune disorders. PMID:20624892

  2. MicroRNA-302d targets IRF9 to regulate the IFN-induced gene expression in SLE.

    PubMed

    Smith, Siobhán; Fernando, Thilini; Wu, Pei Wen; Seo, Jane; Ní Gabhann, Joan; Piskareva, Olga; McCarthy, Eoghan; Howard, Donough; O'Connell, Paul; Conway, Richard; Gallagher, Phil; Molloy, Eamonn; Stallings, Raymond L; Kearns, Grainne; Forbess, Lindsy; Ishimori, Mariko; Venuturupalli, Swamy; Wallace, Daniel; Weisman, Michael; Jefferies, Caroline A

    2017-05-01

    Systemic lupus erythematosus (SLE) is a complex disease targeting multiple organs as a result of overactivation of the type I interferon (IFN) system, a feature currently being targeted by multiple biologic therapies against IFN-α. We have identified an estrogen-regulated microRNA, miR-302d, whose expression is decreased in SLE patient monocytes and identify its target as interferon regulatory factor (IRF)-9, a critical component of the transcriptional complex that regulates expression of interferon-stimulated genes (ISGs). In keeping with the reduced expression of miR-302d in SLE patient monocytes, IRF9 levels were increased, as was expression of a number of ISGs including MX1 and OAS1. In vivo evaluation revealed that miR-302d protects against pristane-induced inflammation in mice by targeting IRF9 and hence ISG expression. Importantly, patients with enhanced disease activity have markedly reduced expression of miR-302d and enhanced IRF9 and ISG expression, with miR-302d negatively correlating with IFN score. Together these findings identify miR-302d as a key regulator of type I IFN driven gene expression via its ability to target IRF9 and regulate ISG expression, underscoring the importance of non-coding RNA in regulating the IFN pathway in SLE.

  3. Role of IFN-gamma in regulating T2 immunity and the development of alternatively activated macrophages during allergic bronchopulmonary mycosis.

    PubMed

    Arora, Shikha; Hernandez, Yadira; Erb-Downward, John R; McDonald, Roderick A; Toews, Galen B; Huffnagle, Gary B

    2005-05-15

    Pulmonary Cryptococcus neoformans infection of C57BL/6 mice is an established model of a chronic pulmonary fungal infection accompanied by an "allergic" response (T2) to the infection, i.e., a model of an allergic bronchopulmonary mycosis. Our objective was to determine whether IFN-gamma plays a role in regulating the pulmonary T2 immune response in C. neoformans-infected C57BL/6 mice. Long-term pulmonary fungistasis was lost in IFN-gamma knockout (KO) mice, resulting in an increased pulmonary burden of fungi at wk 3. IFN-gamma was required for the early influx of leukocytes into the lungs but was not required later in the infection. By wk 3, eosinophil and macrophage numbers were elevated in the absence of IFN-gamma. The inducible NO synthase to arginase ratio was lower in the lungs of IFN-gamma KO mice and the macrophages had increased numbers of intracellular cryptococci and YM1 crystals, indicative of alternatively activated macrophages in these mice. There was evidence of pulmonary fibrosis in both wild-type and IFN-gamma KO mice by 5 wk postinfection. IFN-gamma production was not required for the development of T2 cytokine (IL-4, IL-5, IL-13) producing cells in the lungs and lung-associated lymph nodes or induction of an IgE response. At a number of time points, T2 cytokine production was enhanced in IFN-gamma KO mice. Thus, in the absence of IFN-gamma, C57BL/6 mice develop an augmented allergic response to C. neoformans, including enhanced generation of alternatively activated macrophages, which is accompanied by a switch from a chronic to a progressive pulmonary cryptococcal infection.

  4. ORF7-encoded accessory protein 7a of feline infectious peritonitis virus as a counteragent against IFN-α-induced antiviral response.

    PubMed

    Dedeurwaerder, Annelike; Olyslaegers, Dominique A J; Desmarets, Lowiese M B; Roukaerts, Inge D M; Theuns, Sebastiaan; Nauwynck, Hans J

    2014-02-01

    The type I IFN-mediated immune response is the first line of antiviral defence. Coronaviruses, like many other viruses, have evolved mechanisms to evade this innate response, ensuring their survival. Several coronavirus accessory genes play a central role in these pathways, but for feline coronaviruses this has never to our knowledge been studied. As it has been demonstrated previously that ORF7 is essential for efficient replication in vitro and virulence in vivo of feline infectious peritonitis virus (FIPV), the role of this ORF in the evasion of the IFN-α antiviral response was investigated. Deletion of ORF7 from FIPV strain 79-1146 (FIPV-Δ7) rendered the virus more susceptible to IFN-α treatment. Given that ORF7 encodes two proteins, 7a and 7b, it was further explored which of these proteins is active in this mechanism. Providing 7a protein in trans rescued the mutant FIPV-Δ7 from IFN sensitivity, which was not achieved by addition of 7b protein. Nevertheless, addition of protein 7a to FIPV-Δ3Δ7, a FIPV mutant deleted in both ORF3 and ORF7, could no longer increase the replication capacity of this mutant in the presence of IFN. These results indicate that FIPV 7a protein is a type I IFN antagonist and protects the virus from the antiviral state induced by IFN, but it needs the presence of ORF3-encoded proteins to exert its antagonistic function.

  5. IFN-γ is essential for the inhibition of B16BL6 melanoma lung metastasis in chronic alcohol drinking mice.

    PubMed

    Zhang, Hui; Zhu, Zhaohui; McKinley, Jenifer M; Meadows, Gary G

    2011-03-01

    We previously found that chronic alcohol consumption (20% w/v in drinking water) that models the level consumed by human alcoholics, when administered to female C57BL/6 mice inhibits B16BL6 melanoma metastasis to the lung; however, the mechanism is not known. Chronic alcohol consumption increases IFN-γ producing NK, NKT, CD4(+), and CD8(+) T cells. To examine the impact of IFN-γ on metastasis, we inoculated B16BL6 melanoma cells i.v. into control and chronic alcohol drinking IFN-γ knockout (KO) mice. Knockout of the ifn-γ gene abrogated the anti-metastatic effects associated with alcohol consumption. We examined metastasis in common gamma-chain (γC) KO mice, which are deficient in NK, NKT and CD8(+) T cells, and in Vα14Jα281(-/-) KO mice, which are deficient in invariant NKT (iNKT) cells, in order to assess the importance of specific IFN-γ producing cell types to this effect. We found that the antimetastatic effect of alcohol was still present in γC KO mice and also in γC KO mice depleted of Gr-1(+) cells. Knockout of iNKT cells reduced the degree but not the antimetastatic effect associated with alcohol. These results indicate that the antimetastatic effect induced by chronic alcohol consumption is IFN-γ dependent and that multiple IFN-γ producing cell types contribute to this effect.

  6. The Natural Product Phyllanthusmin C Enhances IFN-γ Production by Human Natural Killer Cells through Upregulation of TLR-Mediated NF-κB Signaling

    PubMed Central

    Deng, Youcai; Chu, Jianhong; Ren, Yulin; Fan, Zhijin; Ji, Xiaotian; Mundy, Bethany; Yuan, Shunzong; Hughes, Tiffany; Zhang, Jianying; Cheema, Baljash; Camardo, Andrew T.; Xia, Yong; Wu, Lai-Chu; Wang, Li-Shu; He, Xiaoming; Kinghorn, A. Douglas; Li, Xiaohui; Caligiuri, Michael A; Yu, Jianhua

    2014-01-01

    Natural products are a major source for cancer drug development. NK cells are a critical component of innate immunity with the capacity to destroy cancer cells, cancer initiating cells, and clear viral infections. However, few reports describe a natural product that selectively stimulates NK cell IFN-γ production and unravel a mechanism of action. In this study, through screening, we found that a natural product, phyllanthusmin C (PL-C), alone enhanced IFN-γ production by human NK cells. PL-C also synergized with IL-12, even at the low cytokine concentration of 0.1 mg/ml, and stimulated IFN-γ production in both human CD56bright and CD56dim NK cell subsets. Mechanistically, TLR1 and/or TLR6 mediated PL-C’s activation of the NF-κB p65 subunit that in turn bound to the proximal promoter of IFNG and subsequently resulted in increased IFN-γ production in NK cells. However, IL-12/IL-15 receptors and their related STAT signaling pathways were not significantly modulated by PL-C. PL-C induced little or no T cell IFN-γ production or NK cell cytotoxicity. Collectively, we identify a natural product with the capacity to selectively activate human NK cell IFN-γ. Given the role of IFN-γ in immune surveillance, additional studies to understand the role of this natural product in prevention of cancer or infection in select populations are warranted. PMID:25122922

  7. A Novel Model for IFN-γ–Mediated Autoinflammatory Syndromes

    PubMed Central

    Reinhardt, R. Lee; Liang, Hong-Erh; Bao, Katherine; Price, April E.; Mohrs, Markus; Kelly, Ben L.

    2015-01-01

    Autoinflammatory disease and hyperinflammatory syndromes represent a growing number of diseases associated with inappropriately controlled inflammation in multiple organs. Systemic inflammation commonly results from dysregulated activation of innate immune cells, and therapeutic targeting of the IL-1β pathway has been used to ameliorate some of these diseases. Some hyperinflammatory syndromes, however, such as hemophagocytic lymphohistiocytosis and the newly classified proteasome disability syndromes, are refractory to such treatments, suggesting that other factors or environmental stressors may be contributing. In comparing two cytokine reporter mouse strains, we identify IFN-γ as a mediator of systemic autoinflammatory disease. Chronically elevated levels of IFN-γ resulted in progressive multiorgan inflammation and two copies of the mutant allele resulted in increased mortality accompanied by myeloproliferative disease. Disease was alleviated by genetic deletion of T-bet. These studies raise the possibility that therapeutics targeting the IFN-γ pathway might be effective in hyperinflammatory conditions refractory to IL-1β–targeted therapies. PMID:25637019

  8. Host immune responses after hypoxic reactivation of IFN-γ induced persistent Chlamydia trachomatis infection

    PubMed Central

    Jerchel, Stefan; Kaufhold, Inga; Schuchardt, Larissa; Shima, Kensuke; Rupp, Jan

    2014-01-01

    Genital tract infections with Chlamydia trachomatis (C. trachomatis) are the most frequent sexually transmitted disease worldwide. Severe clinical sequelae such as pelvic inflammatory disease (PID), tubal occlusion, and tubal infertility are linked to inflammatory processes of chronically infected tissues. The oxygen concentrations in the female urogenital tract are physiologically low and further diminished (0.5–5% O2, hypoxia) during an ongoing inflammation. However, little is known about the effect of a low oxygen environment on genital C. trachomatis infections. In this study, we investigated the host immune responses during reactivation of IFN-γ induced persistent C. trachomatis infection under hypoxia. For this purpose, the activation of the MAP-kinases p44/42 and p38 as well as the induction of the pro-inflammatory cytokines IL-1β, IL-6, IL-8, and MCP-1 were analyzed. Upon hypoxic reactivation of IFN-γ induced persistent C. trachomatis infection, the phosphorylation of the p44/42 but not of the p38 MAP-kinase was significantly diminished compared to IFN-γ induced chlamydial persistence under normoxic condition. In addition, significantly reduced IL-6 and IL-8 mRNA expression levels were observed for reactivated Chlamydiae under hypoxia compared to a persistent chlamydial infection under normoxia. Our findings indicate that hypoxia not only reactivates IFN-γ induced persistent C. trachomatis infections resulting in increased bacterial growth and progeny but also dampens inflammatory host immune signaling responses that are normally observed in a normoxic environment. PMID:24783060

  9. The exopolysaccharide alginate protects Pseudomonas aeruginosa biofilm bacteria from IFN-gamma-mediated macrophage killing.

    PubMed

    Leid, Jeff G; Willson, Carey J; Shirtliff, Mark E; Hassett, Daniel J; Parsek, Matthew R; Jeffers, Alyssa K

    2005-12-01

    The ability of Pseudomonas aeruginosa to form biofilms and cause chronic infections in the lungs of cystic fibrosis patients is well documented. Numerous studies have revealed that P. aeruginosa biofilms are highly refractory to antibiotics. However, dramatically fewer studies have addressed P. aeruginosa biofilm resistance to the host's immune system. In planktonic, unattached (nonbiofilm) P. aeruginosa, the exopolysaccharide alginate provides protection against a variety of host factors yet the role of alginate in protection of biofilm bacteria is unclear. To address this issue, we tested wild-type strains PAO1, PA14, the mucoid cystic fibrosis isolate, FRD1 (mucA22+), and the respective isogenic mutants which lacked the ability to produce alginate, for their susceptibility to human leukocytes in the presence and absence of IFN-gamma. Human leukocytes, in the presence of recombinant human IFN-gamma, killed biofilm bacteria lacking alginate after a 4-h challenge at 37 degrees C. Bacterial killing was dependent on the presence of IFN-gamma. Killing of the alginate-negative biofilm bacteria was mediated through mononuclear cell phagocytosis since treatment with cytochalasin B, which prevents actin polymerization, inhibited leukocyte-specific bacterial killing. By direct microscopic observation, phagocytosis of alginate-negative biofilm bacteria was significantly increased in the presence of IFN-gamma vs all other treatments. Addition of exogenous, purified alginate to the alginate-negative biofilms restored resistance to human leukocyte killing. Our results suggest that although alginate may not play a significant role in bacterial attachment, biofilm development, and formation, it may play an important role in protecting mucoid P. aeruginosa biofilm bacteria from the human immune system.

  10. Failure to activate the IFN-β promoter by a paramyxovirus lacking an interferon antagonist.

    PubMed

    Killip, M J; Young, D F; Ross, C S; Chen, S; Goodbourn, S; Randall, R E

    2011-06-20

    It is generally thought that pathogen-associated molecular patterns (PAMPs) responsible for triggering interferon (IFN) induction are produced during virus replication and, to limit the activation of the IFN response by these PAMPs, viruses encode antagonists of IFN induction. Here we have studied the induction of IFN by parainfluenza virus type 5 (PIV5) at the single-cell level, using a cell line expressing GFP under the control of the IFN-β promoter. We demonstrate that a recombinant PIV5 (termed PIV5-VΔC) that lacks a functional V protein (the viral IFN antagonist) does not activate the IFN-β promoter in the majority of infected cells. We conclude that viral PAMPs capable of activating the IFN induction cascade are not produced or exposed during the normal replication cycle of PIV5, and suggest instead that defective viruses are primarily responsible for inducing IFN during PIV5 infection in this system.

  11. Recombinant hIFN-α2b-BCG inhibits tumor growth in a mouse model of bladder cancer.

    PubMed

    Sun, Erlin; Fan, Xiaodong; Wang, Lining; Lei, Mingde; Zhou, Xiaodong; Liu, Chunyu; Lu, Bingxin; Nian, Xuewu; Sun, Yan; Han, Ruifa

    2015-07-01

    Bacillus Calmette-Guérin (BCG) reduces the recurrence and progression of non-muscle invasive bladder cancer. The present study aimed to investigate the impact of a recombinant hIFN-α2b-secreting BCG (rBCG) on the mouse bladder MB49 cell line and an orthotopic mouse model of bladder cancer. MB49 cells were cultivated in the presence or absence of rBCG, BCG or BCG+hIFN-α2b. Cellular morphology and viability were assessed by microscopy and CCK-8 assay, respectively. Apoptosis was assessed by acridine orange, Hoechst 33258 staining and flow cytometry. MHC-I expression was assessed by flow cytometry. MB49 cells were transplanted into the bladders of C57BL/6 mice administered BCG, rBCG or BCG+hIFN-α2b. Local tissue Fas expression and T cell subsets were assessed by immunohistochemistry. Peripheral blood TNF-α and IL-12 levels were measured by ELISA, and circulating T lymphocyte subsets by flow cytometry. BCG, rBCG and BCG+hIFN-α2b increased the distortion and death of MB49 cells, yet rBCG reduced the proliferation and enhanced apoptosis most substantially. Apoptosis was increased after a 24-h co-culture with rBCG or BCG+hIFN-α2b. Mice administered rBCG survived longer than mice administered BCG (p<0.001), yet this result was not significantly different from mice administered BCG+hIFN-α2b. The average bladder weight was reduced by administration of rBCG (p<0.001). Fas expression and peripheral blood mTNF-α and mIL-12, cell counts of polymorphonuclear leukocytes, monocytes, T lymphocytes and CD4+/CD8+ ratios were significantly increased by all BCG treatments (p≤0.05), yet monocyte and T lymphocyte counts were higher in mice administered rBCG than in mice treated with BCG or BCG+hIFN-α2b (p=0.000). These results indicate that in an orthotopic murine bladder cancer model rBCG possesses superior antitumor activity to BCG+hIFN-α2b.

  12. LAP-like process as an immune mechanism downstream of IFN-γ in control of the human malaria Plasmodium vivax liver stage

    PubMed Central

    Boonhok, Rachasak; Rachaphaew, Nattawan; Duangmanee, Apisak; Chobson, Pornpimol; Pattaradilokrat, Sittiporn; Utaisincharoen, Pongsak; Sattabongkot, Jetsumon

    2016-01-01

    IFN-γ is a major regulator of immune functions and has been shown to induce liver-stage Plasmodium elimination both in vitro and in vivo. The molecular mechanism responsible for the restriction of liver-stage Plasmodium downstream of IFN-γ remains uncertain, however. Autophagy, a newly described immune defense mechanism, was recently identified as a downstream pathway activated in response to IFN-γ in the control of intracellular infections. We thus hypothesized that the killing of liver-stage malarial parasites by IFN-γ involves autophagy induction. Our results show that whereas IFN-γ treatment of human hepatocytes activates autophagy, the IFN-γ–mediated restriction of liver-stage Plasmodium vivax depends only on the downstream autophagy-related proteins Beclin 1, PI3K, and ATG5, but not on the upstream autophagy-initiating protein ULK1. In addition, IFN-γ enhanced the recruitment of LC3 onto the parasitophorous vacuole membrane (PVM) and increased the colocalization of lysosomal vesicles with P. vivax compartments. Taken together, these data indicate that IFN-γ mediates the control of liver-stage P. vivax by inducing a noncanonical autophagy pathway resembling that of LC3-associated phagocytosis, in which direct decoration of the PVM with LC3 promotes the fusion of P. vivax compartments with lysosomes and subsequent killing of the pathogen. Understanding the hepatocyte response to IFN-γ during Plasmodium infection and the roles of autophagy-related proteins may provide an urgently needed alternative strategy for the elimination of this human malaria. PMID:27185909

  13. Abrasive Endoprosthetic Wear Particles Inhibit IFN-γ Secretion in Human Monocytes Via Upregulating TNF-α-Induced miR-29b.

    PubMed

    Bu, Yan-Min; Zheng, De-Zhi; Wang, Lei; Liu, Jun

    2017-02-01

    The adverse biological responses to prostheses wear particles commonly led to the failure of total hip arthroplasty. Among the released cytokines, interferon-γ (IFN-γ) has been found to be a critical functional factor during osteoclast differentiation. However, the molecular mechanism underlying the regulation of IFN-γ in wear particles-induced cells still needs to be determined. Four kinds of abrasive endoprosthetic wear particle were used to treat THP-1 cells, including polymethylmethacrylate (PMMA), zirconiumoxide (ZrO2), commercially pure titanium (cpTi), and titanium alloy (Ti-6Al-7Nb), with a concentration of 0.01, 0.05, 0.1, or 0.2 mg/ml for 48 h. The expression of IFN-γ and miR-29b was detected by real-time RT-PCR or ELISA. Luciferase reporter assay was performed to determine the regulation of miR-29b on IFN-γ. The effect of miR-29b inhibitor on the expression of wear particle-induced IFN-γ was detected. The expression of miR-29b was examined in THP-1 cells treated with tumor necrosis factor-alpha (TNF-α). The expression of IFN-γ was downregulated and the level of miR-29b was increased in THP-1 cells pretreated with wear particles. IFN-γ was a target of miR-29b. Wear particles inhibited the expression of IFN-γ through miR-29b. The expression of miR-29b was significantly reduced in THP-1 cells treated with TNF-α neutralizing antibody and particles comparing to that in the cells treated with particles alone. Wear particles inhibit the IFN-γ secretion in human monocytes, which was associated with the upregulating TNF-α-induced miR-29b.

  14. LAP-like process as an immune mechanism downstream of IFN-γ in control of the human malaria Plasmodium vivax liver stage.

    PubMed

    Boonhok, Rachasak; Rachaphaew, Nattawan; Duangmanee, Apisak; Chobson, Pornpimol; Pattaradilokrat, Sittiporn; Utaisincharoen, Pongsak; Sattabongkot, Jetsumon; Ponpuak, Marisa

    2016-06-21

    IFN-γ is a major regulator of immune functions and has been shown to induce liver-stage Plasmodium elimination both in vitro and in vivo. The molecular mechanism responsible for the restriction of liver-stage Plasmodium downstream of IFN-γ remains uncertain, however. Autophagy, a newly described immune defense mechanism, was recently identified as a downstream pathway activated in response to IFN-γ in the control of intracellular infections. We thus hypothesized that the killing of liver-stage malarial parasites by IFN-γ involves autophagy induction. Our results show that whereas IFN-γ treatment of human hepatocytes activates autophagy, the IFN-γ-mediated restriction of liver-stage Plasmodium vivax depends only on the downstream autophagy-related proteins Beclin 1, PI3K, and ATG5, but not on the upstream autophagy-initiating protein ULK1. In addition, IFN-γ enhanced the recruitment of LC3 onto the parasitophorous vacuole membrane (PVM) and increased the colocalization of lysosomal vesicles with P. vivax compartments. Taken together, these data indicate that IFN-γ mediates the control of liver-stage P. vivax by inducing a noncanonical autophagy pathway resembling that of LC3-associated phagocytosis, in which direct decoration of the PVM with LC3 promotes the fusion of P. vivax compartments with lysosomes and subsequent killing of the pathogen. Understanding the hepatocyte response to IFN-γ during Plasmodium infection and the roles of autophagy-related proteins may provide an urgently needed alternative strategy for the elimination of this human malaria.

  15. FACTORS INFLUENCING AGE AND STRAIN-RELATED SUSCEPTIBILITY TO 3-METHYLCHOLANTHRENE CARCINOGENICITY

    EPA Science Inventory

    Fetal mice are more sensitive to chemical carcinogens than are adults. Further, some strains of mice are more susceptible to chemical carcinogens than others. We have been conducting studies to understand the interactions between age and genetic background underlying these suscep...

  16. Gene therapy with plasmids encoding IFN-β or IFN-α14 confers long-term resistance to HIV-1 in humanized mice

    PubMed Central

    Abraham, Sojan; Choi, Jang-Gi; Ortega, Nora M.; Zhang, Junli; Shankar, Premlata; Swamy, N. Manjunath

    2016-01-01

    Because endogenous interferon type I (IFN-I) produced by HIV-1 infection might complicate the analysis of therapeutically administered IFN-I, we tested different humanized mouse models for induction of IFN-I during HIV-1 infection. While HIV-1 induced high levels of IFN-α in BLT mice, IFN-I was undetectable following infection in the Hu-PBL mouse model, in which only T cells expand. We therefore tested the effect of treatment with Pegylated IFN-2 (pegasys), in Hu-PBL mice. Pegasys prevented CD4 T cell depletion and reduced the viral load for 10 days, but the effect waned thereafter. We next expressed IFN-I subsets (IFN-α2, −α6, −α8, −α14, and −β) in Hu-PBL mice by hydrodynamic injection of plasmids encoding them and 2 days later infected the mice with HIV-1. CD4 T cell depletion was prevented in all subtypes of IFN-I-expressing mice by day 10. However, at day 40 post-infection, protection was seen in IFN-β- and IFN-α14-expressing mice, but not the others. The viral load followed an inverse pattern and was highest in control mice and lowest in IFN-β- and IFN-α14-expressing mice until day 40 after infection. These results show that gene therapy with plasmids encoding IFN-β and −α14, but not the commonly used −α2, confers long-term suppression of HIV-1 replication. PMID:27729616

  17. A Novel Glycolipid Antigen for NKT Cells That Preferentially Induces IFN-γ Production.

    PubMed

    Birkholz, Alysia M; Girardi, Enrico; Wingender, Gerhard; Khurana, Archana; Wang, Jing; Zhao, Meng; Zahner, Sonja; Illarionov, Petr A; Wen, Xiangshu; Li, Michelle; Yuan, Weiming; Porcelli, Steven A; Besra, Gurdyal S; Zajonc, Dirk M; Kronenberg, Mitchell

    2015-08-01

    In this article, we characterize a novel Ag for invariant NKT (iNKT) cells capable of producing an especially robust Th1 response. This glycosphingolipid, DB06-1, is similar in chemical structure to the well-studied α-galactosylceramide (αGalCer), with the only change being a single atom: the substitution of a carbonyl oxygen with a sulfur atom. Although DB06-1 is not a more effective Ag in vitro, the small chemical change has a marked impact on the ability of this lipid Ag to stimulate iNKT cells in vivo, with increased IFN-γ production at 24 h compared with αGalCer, increased IL-12, and increased activation of NK cells to produce IFN-γ. These changes are correlated with an enhanced ability of DB06-1 to load in the CD1d molecules expressed by dendritic cells in vivo. Moreover, structural studies suggest a tighter fit into the CD1d binding groove by DB06-1 compared with αGalCer. Surprisingly, when iNKT cells previously exposed to DB06-1 are restimulated weeks later, they have greatly increased IL-10 production. Therefore, our data are consistent with a model whereby augmented and or prolonged presentation of a glycolipid Ag leads to increased activation of NK cells and a Th1-skewed immune response, which may result, in part, from enhanced loading into CD1d. Furthermore, our data suggest that strong antigenic stimulation in vivo may lead to the expansion of IL-10-producing iNKT cells, which could counteract the benefits of increased early IFN-γ production.

  18. A novel glycolipid antigen for NKT cells that preferentially induces IFN-γ production

    PubMed Central

    Birkholz, Alysia M.; Girardi, Enrico; Wingender, Gerhard; Khurana, Archana; Wang, Jing; Zhao, Meng; Zahner, Sonja; Illarionov, Petr A.; Wen, Xiangshu; Li, Michelle; Yuan, Weiming; Porcelli, Steven A.; Besra, Gurdyal S.; Zajonc, Dirk M.; Kronenberg, Mitchell

    2015-01-01

    Here we characterize a novel Ag for invariant natural killer T-cells (iNKT cells) capable of producing an especially robust Th1 response. This glycosphingolipid (GSL), DB06-1, is similar in chemical structure to the well-studied α-galactosylceramide (αGalCer), the only change being in a single atom, the substitution of a carbonyl oxygen with a sulfur atom. Although DB06-1 is not a more effective Ag in vitro, the small chemical change has a marked impact on the ability of this lipid Ag to stimulate iNKT cells in vivo, with increased IFN-γ production at 24 h compared to αGalCer, increased IL-12, and increased activation of NK cells to produce IFN-γ. These changes are correlated with an enhanced ability of DB06-1 to load in the CD1d molecules expressed by DCs in vivo. Moreover, structural studies suggest a tighter fit into the CD1d binding groove by DB061 compared to αGalCer. Surprisingly, when iNKT cells previously exposed to DB06-1 are restimulated weeks later, they have greatly increased IL-10 production. Our data are therefore consistent with a model whereby augmented and or prolonged presentation of a glycolipid Ag leads to increased activation of NK cells and a Th1-skewed immune response, which may result in part from enhanced loading into CD1d. Furthermore, our data suggest that strong antigenic stimulation in vivo may lead to the expansion of IL-10 producing iNKT cells, which could counteract the benefits of increased, early IFN-γ production. PMID:26078271

  19. Association of the IFN-γ (+874A/T) Genetic Polymorphism with Paranoid Schizophrenia in Tunisian Population.

    PubMed

    Jemli, Achraf; Eshili, Awatef; Trifa, Fatma; Mechri, Anouar; Zaafrane, Ferid; Gaha, Lotfi; Juckel, George; Tensaout, Besma Bel Hadj Jrad

    2017-02-01

    Since growing evidence suggests a significant role of chronic low-grade inflammation in the physiopathology of schizophrenia, we have hypothesized that functional genetic variant of the IFN gamma (IFN-γ; +874A/T; rs2430561) gene may be involved in the predisposition to schizophrenia. This research is based on a case-control study which aims to identify whether polymorphism of the IFN-γ gene is a risk factor for the development of schizophrenia. The RFLP-PCR genotyping of the IFN-γ gene was conducted on a Tunisian population composed of 218 patients and 162 controls. The IFN-γ (+874A/T) polymorphism analysis showed higher frequencies of minor homozygous genotype (TT) and allele (T) in all patients compared with controls (11.5 vs. 4.9%; p = 0.03, OR = 2.64 and 30.7 vs. 24.1%, p = 0.04, OR = 1.4, respectively). This correlation was confirmed for male but not for female patients. Also, the T allele was significantly more common among patients with paranoid schizophrenia when compared with controls (25.8 vs. 4.9%, p = 0.0001; OR = 6.7). Using the binary regression analysis to eliminate confounding factors as age and sex, only this last association remained significant (p = 0.03; OR = 1.76, CI = 1.05-2.93). In conclusion, our results showed a significant association between +874A/T polymorphism of IFN-γ and paranoid schizophrenia, suggesting that this single nucleotide polymorphism (SNP) or another at proximity could predispose to paranoid schizophrenia. Since the minor allele of this polymorphism was correlated with an increased expression of their product, our study validates the hypothesis of excessive pro-inflammatory cytokine in the physiopathology of paranoid schizophrenia.

  20. Histone deacetylase inhibitor MS-275 augments expression of a subset of IFN-γ-regulated genes in Toxoplasma gondii-infected macrophages but does not improve parasite control.

    PubMed

    Sumpf, Kristina; Nast, Roswitha; Downie, Bryan; Salinas, Gabriela; Lüder, Carsten G K

    2017-02-09

    Toxoplasma gondii is a ubiquitous apicomplexan parasite of mammals and birds and an important pathogen of humans. IFN-γ is the major mediator of host resistance against T. gondii but intriguingly, parasite-infected host cells including macrophages are severely impaired to respond to IFN-γ due to defective transcriptional activation of target genes. Here, we tested the possibility that the impaired responsiveness of T. gondii-infected macrophages to IFN-γ can be restored by inhibiting histone deacetylases (HDACs) using the class I-specific inhibitor MS-275. Treatment of RAW264.7 cells with MS-275 indeed increased MHC class II surface expression in infected and non-infected cells and largely abolished the inhibition of IFN-γ-regulated MHC class II expression exerted by T. gondii. Genome-wide transcriptome profiling revealed that MS-275 increased mean mRNA levels of IFN-γ-regulated genes particularly in non-infected macrophages. Transcript levels of 33% of IFN-γ secondary response genes but only those of a few primary response genes were also increased by MS-275 in T. gondii-infected cells. Importantly, the unresponsiveness of parasite-infected cells to IFN-γ was however not abolished by MS-275. Furthermore, MS-275 also up-regulated several anti-inflammatory cytokines or signaling molecules in T. gondii-infected macrophages. It additionally regulated expression of more than 2500 genes in non-infected macrophages expression of which was surprisingly counteracted by prior infection with T. gondii. FACS analysis and immunofluorescence microscopy revealed that MS-275 did not considerably diminish the number of parasite-positive cells or the intracellular replication in macrophages stimulated or not with IFN-γ. Thus, a supportive therapy using MS-275 appears inappropriate for treatment of toxoplasmosis.

  1. Effects of IRF1 and IFN-β interaction on the M1 polarization of macrophages and its antitumor function.

    PubMed

    Xie, Changli; Liu, Cuiying; Wu, Bitao; Lin, Yan; Ma, Tingting; Xiong, Haiyu; Wang, Qin; Li, Ziwei; Ma, Chenyu; Tu, Zhiguang

    2016-07-01

    Macrophages that differentiate from precursor monocytes can be polarized into a classically activated (M1) or alternatively activated (M2) status depending on different stimuli. Generally, interferon (IFN)-γ and lipopolysaccharide (LPS) are considered the classical stimuli with which to establish M1 polarization. IFN regulatory factor (IRF)1 and IFN-β are two crucial molecules involved in IFN-γ- and LPS-initialed signaling. However, the association between IRF1 and IFN-β in the context of the M1 polarization of macrophages is not yet fully understood. In this study, we demonstrate that U937-derived macrophages, in response to IFN-γ and LPS stimulation, readily acquire an M1 status, indicated by the increased expression of interleukin (IL)-12, IL-6, IL-23, tumor necrosis factor (TNF)-α and the M1-specific cell surface antigen, CD86, and the decreased expression of the M2-specific mannose receptor, CD206. However, the knockdown of IRF1 in U937-derived macrophages led to an impaired M1 status, as indicated by the decreased expression of the above-mentioned M1 markers, and the increased expression of the M2 markers, CD206 and IL-10. A similar phenomenon was observed in the M1 macrophages in which IFN-β was inhibited. Furthermore, we demonstrated that IRF1 and IFN-β may interact with each other in the IFN-γ- and LPS-initiated signaling pathway, and contribute to the IRF5 regulation of M1 macrophages. In addition, the conditioned medium collected from the M1 macrophages in which IRF1 or IFN-β were inhibited, exerted pro-tumor effects on the HepG2 and SMMC-7721 cells, as indicated by an increase in proliferation, the inhibition of apoptosis and an enhanced invasion capability. The findings of our study suggest that the interactions of IRF1, IFN-β and IRF5 are involved in the M1 polarization of macrophages and have antitumor functions. These data may provide a novel antitumor strategy for targeted cancer therapy.

  2. Phenotypic and ultrastructural characteristics of bronchoalveolar lavage cells of lentivirus-infected lambs treated with recombinant ovine IFN-tau.

    PubMed

    Singh, B; Ott, T L; Bazer, F W; de la Concha-Bermejillo, A

    2001-09-01

    Ovine lentivirus (OvLV) belongs to the family Retroviridae and closely resembles the human immunodeficiency virus (HIV). Pulmonary lesions in OvLV-infected sheep consist of lymphoid interstitial pneumonia (LIP) and lymphocytic alveolitis. Similar pulmonary lesions occur in up to 40% of HIV-infected children and in some adults with AIDS. Interferon-tau (IFN-tau), a type I IFN, is produced by trophectoderm of ruminant conceptuses and is the pregnancy recognition signal in these species. To evaluate changes in phenotypes of bronchoalveolar lavage (BAL) cells of OvLV-infected lambs treated with recombinant ovine IFN-tau (rOvIFN-tau), 24 lambs were randomly allocated to one of four groups (n = 6 per group): 1, no virus + placebo (NVP); 2, no virus + rOvIFN-tau (NVI); 3, virus + placebo (VP); 4, virus + rOvIFN-tau (VI). The BAL cells from 3 lambs in each group were labeled with monoclonal antibodies (mAb) to cell surface markers at 16 weeks of treatment, and cells from the remaining 3 lambs in each group were labeled with mAb at 34 weeks of treatment. After labeling, BAL cells were analyzed by flow cytometry. The morphology of BAL cells from all experimental lambs was examined by transmission electron microscopy (TEM). At week 16, no differences in the relative proportions of BAL cell phenotypes were detected among the experimental groups. At week 34, VI lambs had higher proportions of CD8(+), gammadelta(+), MHC class II(+), and L-selectin (LS(+)) BAL cells compared with VP lambs. Higher proportions of CD14(+) and CD44(+) cells were found in VP lambs compared with NVP lambs at 34 weeks. OvLV-like particles were detected only in bronchoalveolar macrophages of VP lambs. In this study, rOvIFN-tau increased the proportions of primary antiviral gammadelta(+) and CD8(+) immune cells in OvLV-infected lambs. This may represent a cellular mechanism to explain the antiviral and therapeutic efficacy of this cytokine, in addition to its direct antiviral effect. However, because the

  3. Biological monitoring of IFN-β therapy in Multiple Sclerosis.

    PubMed

    Bertolotto, A; Granieri, L; Marnetto, F; Valentino, P; Sala, A; Capobianco, M; Malucchi, S; Di Sapio, A; Malentacchi, M; Matta, M; Caldano, M

    2015-04-01

    Multiple Sclerosis (MS) is a heterogeneous disease and a variable percentage of patients are non-responders to common treatment. Early diagnosis of non-responders allows change to a more useful therapy for the patient and better allocates a large amount of financial resources. Quantification of Neutralizing antibodies (Nabs) and of biological activity of IFN-β are recognized approaches to identify immuno-pharmacological non-responders. A consistent number of studies have demonstrated that quantification of Myxovirus-induced protein A (MxA) is a valid biomarker to detect immune-pharmacological non responders after one year of treatment. Persistent high titre of Nabs and absence of biological activity predict abolition of IFN-β effects in disease activity measured through MRI, number of relapses and disability. Guidelines and flow-charts including both Nabs and MxA quantification are presented.

  4. Neuronal IFN signaling is dispensable for the establishment of HSV-1 latency.

    PubMed

    Rosato, Pamela C; Katzenell, Sarah; Pesola, Jean M; North, Brian; Coen, Donald M; Leib, David A

    2016-10-01

    IFN responses control acute HSV infection, but their role in regulating HSV latency is poorly understood. To address this we used mice lacking IFN signaling specifically in neural tissues. These mice supported a higher acute viral load in nervous tissue and delayed establishment of latency. While latent HSV-1 genome copies were equivalent, ganglia from neuronal IFN signaling-deficient mice unexpectedly supported reduced reactivation. IFNβ promoted survival of primary sensory neurons after infection with HSV-1, indicating a role for IFN signaling in sustaining neurons. We observed higher levels of latency associated transcripts (LATs) per HSV genome in mice lacking neuronal IFN signaling, consistent with a role for IFN in regulating LAT expression. These data show that neuronal IFN signaling modulates the expression of LAT and may conserve the pool of neurons available to harbor latent HSV-1 genome. The data also show that neuronal IFN signaling is dispensable for the establishment of latency.

  5. Immunomodulation of bivalent Newcastle disease DNA vaccine induced immune response by co-delivery of chicken IFN-γ and IL-4 genes.

    PubMed

    Sawant, P M; Verma, P C; Subudhi, P K; Chaturvedi, U; Singh, M; Kumar, Rajeev; Tiwari, A K

    2011-11-15

    The basic objective of this study was to enumerate whether co-administration of interferon-γ (IFN-γ) and/or interleukin-4 (IL-4) gene along with a bivalent Newcastle disease (ND) DNA vaccine construct could modulate the immune response to the DNA vaccine in chickens. pVIVO2 vector carrying Haemaglutinin-Neuraminidase (HN) and Fusion (F) genes of Newcastle disease virus (NDV) at its two cloning sites was used as a DNA vaccine. The same vector was used to clone the chicken IFN-γ and IL-4 genes at the multiple cloning site-1 separately. In vitro expression of IFN-γ and IL-4 gene constructs was assessed by reverse transcriptase-polymerase chain reaction (RT-PCR) and that of HN and F genes by indirect fluorescent antibody technique (IFAT) in addition to RT-PCR. The chickens were immunized thrice intramuscularly at 21, 36 and 46 days of age with the bivalent DNA vaccine alone, or in combination with IFN-γ/IL-4 or both cytokine gene constructs. The bivalent DNA vaccine led to increase in both NDV specific antibodies as assessed by enzyme linked immunosorbent assay (ELISA) and haemagglutination inhibition test (HI) and cell mediated immune (CMI) response as assessed by lymphocyte transformation test (LTT) employing MTT assay. Co-administration of the DNA vaccine with IL-4 gene resulted in highest IgY levels while IFN-γ produced highest CMI response. The DNA vaccine alone could afford only 10% protection against challenge infection by velogenic NDV. This protection was increased to 40% when IL-4 gene construct was co-administered with the DNA vaccine. Co-injection of IFN-γ as well as the combination of IFN-γ and IL-4 gene constructs with the DNA vaccine yielded 20% protection. Our study suggests that IL-4 may prove to be more appropriate as a genetic adjuvant than IFN-γ for ND DNA vaccine.

  6. Noncanonical IFN Signaling: Mechanistic Linkage of Genetic and Epigenetic Events

    PubMed Central

    Ahmed, Chulbul M.

    2016-01-01

    The canonical model of cytokine signaling via the JAK/STAT pathway dominates our view of signal transduction but provides no insight into the significance of the simultaneous presence of activated JAKs and STATs in the nucleus of cells treated with cytokines. Such a mechanistic shortcoming challenges the usefulness of the model in its present form. Focusing on the interferon (IFN) cytokines, we have developed a noncanonical model of IFN signaling that naturally connects activated JAKs and STATs at or near response elements of genes that are activated by the IFNs. Specifically, cells treated with IFNγ showed association of activated STAT1α and JAK2 at the GAS element of genes activated by IFNγ. For IFNα treated cells, the association involved activated STAT1α and TYK2 JAK kinase at the ISRE promoter. The power of the noncanonical model is that it provides mechanistic insight into specific gene activation at the level of the associated epigenetics, akin to that of steroid/steroid receptor signaling. PMID:28077919

  7. [Neutralizing Monoclonal and Chimeric Antibodies to Human IFN-γ].

    PubMed

    Larina, M V; Aliev, T K; Solopova, O N; Pozdnyakova, L P; Korobova, S V; Yakimov, S A; Sveshnikov, P G; Dolgikh, D A; Kirpichnikov, M P

    2015-01-01

    Autoiminune disorders are chronic diseases characterized by abnormal immune response directed against self-antigens that leads to tissue damage and violation of its normal functioning. Such diseases often result in disability or even death of patients. Nowadays a number of monoclonal antibodies to pro-inflammatory cytokines and their receptors are successfully used for the targeted treatment of autoimmune diseases. One of the perspective targets in autoimmune disease therapy is interferon gamma, a key cytokine in Th1 cells differentiation, activation of macrophages, and inflammation. In the present work, 5 monoclonal antibodies to human IFN-γ were obtained. For the development of potential therapeutic agent, we have performed neutralizing activity and affinity analysis of the antibodies. Based on the data obtained, the monoclonal antibody F1 was selected. This antibody has a dissociation constant 1.7 x 10(-9) M and IC90 = 8.9 ± 2.0 nM measured upon antibody inhibition of the IFN-γ-induced HLA-DR expression on the surface of U937 cells. We have constructed a bicistronic vector for the production of recombinant chimeric Fab fragment F1 chim in E. coli cells. The recombinant chimeric Fab fragment Fl chim neutralizes IFN-γ activity in vitro and has a dissociation constant 1.8 x 10(-9) M.

  8. The HPV-16 E7 oncogene sensitizes malignant cells to IFN-alpha-induced apoptosis

    SciTech Connect

    Wang, Yisong

    2005-10-01

    Interferons (IFNs) exert antitumor effects in several human malignancies, but their mechanism of action is unclear. There is a great variability in sensitivity to IFN treatment depending on both tumor type and the individual patient. The reason for this variable sensitivity is not known. The fact that several IFN-induced anticellular effects are exerted through modulation of proto-oncogenes and tumor suppressor genes may indicate that the malignant genotype may be decisive in the cell's sensitivity to IFN. To determine if a deregulated oncogene could alter the cellular response to IFN, a mouse lymphoma cell line (J3D) was stably transfected with the viral human papillomavirus-16 (HPV-16) E7 oncogene. The E7-transfected cells and their respective mock-transfected sister clones were treated with IFN-{alpha} and examined for possible IFN-induced anticellular effects. We found that the E7-transfected clones were greatly sensitized to IFN-{alpha}-induced apoptosis compared with their mock-transfected counterparts. Induction of apoptosis in the transfected cells correlated with the ability of IFN to activate parts of the proapoptotic machinery specifically in these cells, including activation of caspases and the proapoptotic protein Bak. In summary, our data suggest that transfection of malignant cells with the E7 oncogene can sensitize them to IFN-{alpha}-induced apoptosis. This demonstrates that an oncogenic event may alter the cellular sensitivity to IFN and might also have implications for treatment of HPV related diseases with IFN.

  9. Endogenous IL-22 Plays a Dual Role in Arthritis: Regulation of Established Arthritis via IFN-γ Responses

    PubMed Central

    Justa, Shivali; Zhou, Xiaoqun; Sarkar, Sujata

    2014-01-01

    Objective IL-22 is elevated in patients with inflammatory arthritis and correlates with disease activity. IL-22 deficient mice have reduced incidence of arthritis. Recombinant IL-22 restrains progression of arthritis via increase in IL-10 responses when administered prior to onset of arthritis. These findings imply a possible dual role of IL-22 in inflammatory arthritis depending on the phase of arthritis. Experiments outlined here were designed to elucidate the contribution of endogenous IL-22 before and after the onset of arthritis. Methods Collagen induced arthritis (CIA) was induced in DBA1 or IFN-γ deficient mice following immunization with collagen and complete Freund's adjuvant. Anti-IL-22 antibody or isotype control were administered prior to or after onset of arthritis and disease progression assessed by clinical scoring and histopathology. IL-22, IL-17 and IFN-γ responses were measured by ELISA and flowcytometry. Anti-collagen antibody responses were analyzed by ELISA. Expression of IL-22R1 in CD4+ cells was elucidated by flowcytometry and real time PCR. Results Collagen specific IL-22 responses were expanded during arthritis and IL-22 producing cells were discrete from IL-17 or IFN-γ producing cells. Neutralization of IL-22 after onset of arthritis resulted in significant increase in Th1 responses and significantly reduced severity of arthritis. CD4+ cells from arthritic mice showed increased surface expression of IL-22R1. In vitro, CD4+T cells cultured with antigen presenting cells in the presence or absence of IL-22 suppressed or induced IFN-γ, respectively. The protective effect of anti-IL-22 was reversed in IFN-γ deficient mice. Moreover, administration of anti-IL-22 prior to onset of arthritis augmented arthritis severity. Conclusion We show for the first time that IL-22 plays a dual role: protective prior to the onset of arthritis and pathogenic after onset of arthritis. The pathogenic effect of IL-22 is dependent on suppression of IFN

  10. Low concentration of PDGF-AB shows synergism with IFN-α in induction of IFN-β and -γ in MRC5 fibroblasts.

    PubMed

    Šantak, G; Šantak, M; Forčić, D

    2013-11-01

    Platelet-derived growth factor (PDGF) is a potent mediator of fibroblast proliferation and chemotaxis. Also it has been reported as a strong suppressor of interferon (IFN) expression. IFN-α has opposite effect on fibroblast function and IFN induction. Here is our early report on the effect of low concentration of PDGF-AB alone or in combination with IFN-α on IFN mRNA production in MRC5 fibroblasts. MRC5 cells incubated with IFN-α or PDGF-AB, alone or in combination, produced significant induction of IFN-α, -β and -γ mRNA in comparison with untreated cells. The induction was dose-dependent, with higher effect in cells treated with lower concentrations of PDGF-AB. Also, low concentration of PDGF-AB showed synergism with IFN-α in IFN-β and -γ induction. Results presented here open new possibilities in multi-cytokine therapy and expand previous results on PDGF activity.

  11. The natural product phyllanthusmin C enhances IFN-γ production by human NK cells through upregulation of TLR-mediated NF-κB signaling.

    PubMed

    Deng, Youcai; Chu, Jianhong; Ren, Yulin; Fan, Zhijin; Ji, Xiaotian; Mundy-Bosse, Bethany; Yuan, Shunzong; Hughes, Tiffany; Zhang, Jianying; Cheema, Baljash; Camardo, Andrew T; Xia, Yong; Wu, Lai-Chu; Wang, Li-Shu; He, Xiaoming; Kinghorn, A Douglas; Li, Xiaohui; Caligiuri, Michael A; Yu, Jianhua

    2014-09-15

    Natural products are a major source for cancer drug development. NK cells are a critical component of innate immunity with the capacity to destroy cancer cells, cancer-initiating cells, and clear viral infections. However, few reports describe a natural product that stimulates NK cell IFN-γ production and unravel a mechanism of action. In this study, through screening, we found that a natural product, phyllanthusmin C (PL-C), alone enhanced IFN-γ production by human NK cells. PL-C also synergized with IL-12, even at the low cytokine concentration of 0.1 ng/ml, and stimulated IFN-γ production in both human CD56(bright) and CD56(dim) NK cell subsets. Mechanistically, TLR1 and/or TLR6 mediated PL-C's activation of the NF-κB p65 subunit that in turn bound to the proximal promoter of IFNG and subsequently resulted in increased IFN-γ production in NK cells. However, IL-12 and IL-15Rs and their related STAT signaling pathways were not responsible for the enhanced IFN-γ secretion by PL-C. PL-C induced little or no T cell IFN-γ production or NK cell cytotoxicity. Collectively, we identify a natural product with the capacity to selectively enhance human NK cell IFN-γ production. Given the role of IFN-γ in immune surveillance, additional studies to understand the role of this natural product in prevention of cancer or infection in select populations are warranted.

  12. In vivo IFN-γ secretion by NK cells in response to Salmonella typhimurium requires NLRC4 inflammasomes.

    PubMed

    Kupz, Andreas; Curtiss, Roy; Bedoui, Sammy; Strugnell, Richard A

    2014-01-01

    Natural killer (NK) cells are a critical part of the innate immune defense against viral infections and for the control of tumors. Much less is known about how NK cells contribute to anti-bacterial immunity. NK cell-produced interferon gamma (IFN-γ) contributes to the control of early exponential replication of bacterial pathogens, however the regulation of these events remains poorly resolved. Using a mouse model of invasive Salmonellosis, here we report that the activation of the intracellular danger sensor NLRC4 by Salmonella-derived flagellin within CD11c+ cells regulates early IFN-γ secretion by NK cells through the provision of interleukin 18 (IL-18), independently of Toll-like receptor (TLR)-signaling. Although IL18-signalling deficient NK cells improved host protection during S. Typhimurium infection, this increased resistance was inferior to that provided by wild-type NK cells. These findings suggest that although NLRC4 inflammasome-driven secretion of IL18 serves as a potent activator of NK cell mediated IFN-γ secretion, IL18-independent NK cell-mediated mechanisms of IFN-γ secretion contribute to in vivo control of Salmonella replication.

  13. Type I IFN Induction via Poly-ICLC Protects Mice against Cryptococcosis.

    PubMed

    Sionov, Edward; Mayer-Barber, Katrin D; Chang, Yun C; Kauffman, Keith D; Eckhaus, Michael A; Salazar, Andres M; Barber, Daniel L; Kwon-Chung, Kyung J

    2015-08-01

    Cryptococcus neoformans is the most common cause of fungal meningoencephalitis in AIDS patients. Depletion of CD4 cells, such as occurs during advanced AIDS, is known to be a critical risk factor for developing cryptococcosis. However, the role of HIV-induced innate inflammation in susceptibility to cryptococcosis has not been evaluated. Thus, we sought to determine the role of Type I IFN induction in host defense against cryptococci by treatment of C. neoformans (H99) infected mice with poly-ICLC (pICLC), a dsRNA virus mimic. Unexpectedly, pICLC treatment greatly extended survival of infected mice and reduced fungal burdens in the brain. Protection from cryptococcosis by pICLC-induced Type I IFN was mediated by MDA5 rather than TLR3. PICLC treatment induced a large, rapid and sustained influx of neutrophils and Ly6Chigh monocytes into the lung while suppressing the development of eosinophilia. The pICLC-mediated protection against H99 was CD4 T cell dependent and analysis of CD4 T cell polyfunctionality showed a reduction in IL-5 producing CD4 T cells, marginal increases in Th1 cells and dramatic increases in RORγt+ Th17 cells in pICLC treated mice. Moreover, the protective effect of pICLC against H99 was diminished in IFNγ KO mice and by IL-17A neutralization with blocking mAbs. Furthermore, pICLC treatment also significantly extended survival of C. gattii infected mice with reduced fungal loads in the lungs. These data demonstrate that induction of type I IFN dramatically improves host resistance against the etiologic agents of cryptococcosis by beneficial alterations in both innate and adaptive immune responses.

  14. Transcriptional Dysregulation of Upstream Signaling of IFN Pathway in Chronic HCV Type 4 Induced Liver Fibrosis

    PubMed Central

    Ibrahim, Marwa K.; Salum, Ghada Maher; Bader El Din, Noha G.; Dawood, Reham M.; Barakat, Ahmed; Khairy, Ahmed; El Awady, Mostafa K.

    2016-01-01

    IFN orchestrates the expression of various genes to halt hepatitis C virus (HCV) replication with the possibility of either reduced or increased liver fibrosis; due to controlled viral replication or overproduction of inflammatory mediators, repectively. In this study, we examined the transcriptional profiling of type I IFN related genes in HCV-chronically infected patients with varying degrees of liver fibrosis. PCR array was used to examine the expression of 84 type I IFN related genes in peripheral blood mononuclear cells (PBMCs) RNA from 12 treatment-naïve chronic HCV patients (5 F0-F1 and 7 F2-F4) and 5 healthy subjects. We further validated our results by quantitative real time PCR (qRT-PCR) in 103 treatment-naïve chronic HCV patients (43 F0-F1 and 60 F2-F4) and 15 controls. PCR array data revealed dysregulation in TLR7 pathway. The expression of TLR7 was decreased by 4 folds and MyD88 was increased by 3 folds in PBMCs of F2-F4 patients when compared to the healthy volunteers (p = 0.03 and 0.002, respectively). In addition, IRF7 and TLR7 showed dramatic downregulation (6 and 8 folds, respectively) in F2-F4 patients when compared to F0-F1 ones. qRT-PCR confirmed the altered expression patterns of TLR7 and MyD88 in F2-F4 patients when compared to either controls or F0-F1 patients. However, by qRT-PCR, IRF7 and NF-κB1 (TLR7 pathway transcription factors) exhibited similar mRNA abundance among F2-F4 and F0-F1 patients. These results suggest that TLR7 and MyD88 are possible candidates as biomarkers for the progression of HCV-induced liver fibrosis and/ or targets for therapeutic intervention. PMID:27135246

  15. Transcriptional Dysregulation of Upstream Signaling of IFN Pathway in Chronic HCV Type 4 Induced Liver Fibrosis.

    PubMed

    Ibrahim, Marwa K; Salum, Ghada Maher; Bader El Din, Noha G; Dawood, Reham M; Barakat, Ahmed; Khairy, Ahmed; El Awady, Mostafa K

    2016-01-01

    IFN orchestrates the expression of various genes to halt hepatitis C virus (HCV) replication with the possibility of either reduced or increased liver fibrosis; due to controlled viral replication or overproduction of inflammatory mediators, repectively. In this study, we examined the transcriptional profiling of type I IFN related genes in HCV-chronically infected patients with varying degrees of liver fibrosis. PCR array was used to examine the expression of 84 type I IFN related genes in peripheral blood mononuclear cells (PBMCs) RNA from 12 treatment-naïve chronic HCV patients (5 F0-F1 and 7 F2-F4) and 5 healthy subjects. We further validated our results by quantitative real time PCR (qRT-PCR) in 103 treatment-naïve chronic HCV patients (43 F0-F1 and 60 F2-F4) and 15 controls. PCR array data revealed dysregulation in TLR7 pathway. The expression of TLR7 was decreased by 4 folds and MyD88 was increased by 3 folds in PBMCs of F2-F4 patients when compared to the healthy volunteers (p = 0.03 and 0.002, respectively). In addition, IRF7 and TLR7 showed dramatic downregulation (6 and 8 folds, respectively) in F2-F4 patients when compared to F0-F1 ones. qRT-PCR confirmed the altered expression patterns of TLR7 and MyD88 in F2-F4 patients when compared to either controls or F0-F1 patients. However, by qRT-PCR, IRF7 and NF-κB1 (TLR7 pathway transcription factors) exhibited similar mRNA abundance among F2-F4 and F0-F1 patients. These results suggest that TLR7 and MyD88 are possible candidates as biomarkers for the progression of HCV-induced liver fibrosis and/ or targets for therapeutic intervention.

  16. Gamma interferon (IFN-γ) receptor restricts systemic dengue virus replication and prevents paralysis in IFN-α/β receptor-deficient mice.

    PubMed

    Prestwood, Tyler R; Morar, Malika M; Zellweger, Raphaël M; Miller, Robyn; May, Monica M; Yauch, Lauren E; Lada, Steven M; Shresta, Sujan

    2012-12-01

    We previously reported that mice lacking alpha/beta and gamma interferon receptors (IFN-α/βR and -γR) uniformly exhibit paralysis following infection with the dengue virus (DENV) clinical isolate PL046, while only a subset of mice lacking the IFN-γR alone and virtually no mice lacking the IFN-α/βR alone develop paralysis. Here, using a mouse-passaged variant of PL046, strain S221, we show that in the absence of the IFN-α/βR, signaling through the IFN-γR confers approximately 140-fold greater resistance against systemic vascular leakage-associated dengue disease and virtually complete protection from dengue-induced paralysis. Viral replication in the spleen was assessed by immunohistochemistry and flow cytometry, which revealed a reduction in the number of infected cells due to IFN-γR signaling by 2 days after infection, coincident with elevated levels of IFN-γ in the spleen and serum. By 4 days after infection, IFN-γR signaling was found to restrict DENV replication systemically. Clearance of DENV, on the other hand, occurred in the absence of IFN-γR, except in the central nervous system (CNS) (brain and spinal cord), where clearance relied on IFN-γ from CD8(+) T cells. These results demonstrate the roles of IFN-γR signaling in protection from initial systemic and subsequent CNS disease following DENV infection and demonstrate the importance of CD8(+) T cells in preventing DENV-induced CNS disease.

  17. Type I IFN signaling in CD8– DCs impairs Th1-dependent malaria immunity

    PubMed Central

    Haque, Ashraful; Best, Shannon E.; Montes de Oca, Marcela; James, Kylie R.; Ammerdorffer, Anne; Edwards, Chelsea L.; de Labastida Rivera, Fabian; Amante, Fiona H.; Bunn, Patrick T.; Sheel, Meru; Sebina, Ismail; Koyama, Motoko; Varelias, Antiopi; Hertzog, Paul J.; Kalinke, Ulrich; Gun, Sin Yee; Rénia, Laurent; Ruedl, Christiane; MacDonald, Kelli P.A.; Hill, Geoffrey R.; Engwerda, Christian R.

    2014-01-01

    Many pathogens, including viruses, bacteria, and protozoan parasites, suppress cellular immune responses through activation of type I IFN signaling. Recent evidence suggests that immune suppression and susceptibility to the malaria parasite, Plasmodium, is mediated by type I IFN; however, it is unclear how type I IFN suppresses immunity to blood-stage Plasmodium parasites. During experimental severe malaria, CD4+ Th cell responses are suppressed, and conventional DC (cDC) function is curtailed through unknown mechanisms. Here, we tested the hypothesis that type I IFN signaling directly impairs cDC function during Plasmodium infection in mice. Using cDC-specific IFNAR1-deficient mice, and mixed BM chimeras, we found that type I IFN signaling directly affects cDC function, limiting the ability of cDCs to prime IFN-γ–producing Th1 cells. Although type I IFN signaling modulated all subsets of splenic cDCs, CD8– cDCs were especially susceptible, exhibiting reduced phagocytic and Th1-promoting properties in response to type I IFNs. Additionally, rapid and systemic IFN-α production in response to Plasmodium infection required type I IFN signaling in cDCs themselves, revealing their contribution to a feed-forward cytokine-signaling loop. Together, these data suggest abrogation of type I IFN signaling in CD8– splenic cDCs as an approach for enhancing Th1 responses against Plasmodium and other type I IFN–inducing pathogens. PMID:24789914

  18. Cellular sources and targets of IFN-γ-mediated protection against viral demyelination and neurological deficits

    PubMed Central

    Murray, Paul D.; McGavern, Dorian B.; Pease, Larry R.; Rodriguez, Moses

    2017-01-01

    IFN-γ is an anti-viral and immunomodulatory cytokine critical for resistance to multiple pathogens. Using mice with targeted disruption of the gene for IFN-γ, we previously demonstrated that this cytokine is critical for resistance to viral persistence and demyelination in the Theiler’s virus model of multiple sclerosis. During viral infections, IFN-γ is produced by natural killer (NK) cells, CD4+ and CD8+ T cells; however, the proportions of lymphocyte subsets responding to virus infection influences the contributions to IFN-γ-mediated protection. To determine the lymphocyte subsets that produce IFN-γ to maintain resistance, we used adoptive transfer strategies to generate mice with lymphocyte-specific deficiencies in IFN-γ-production. We demonstrate that IFN-γ production by both CD4+ and CD8+ T cell subsets is critical for resistance to Theiler’s murine encephalomyelitis virus (TMEV)-induced demyelination and neurological disease, and that CD4+ T cells make a greater contribution to IFN-γ-mediated protection. To determine the cellular targets of IFN-γ-mediated responses, we used adoptive transfer studies and bone marrow chimerism to generate mice in which either hematopoietic or somatic cells lacked the ability to express IFN-γ receptor. We demonstrate that IFN-γ receptor must be present on central nervous system glia, but not bone marrow-derived lymphocytes, in order to maintain resistance to TMEV-induced demyelination. PMID:11857334

  19. NKG2D+ IFN-γ+ CD8+ T Cells Are Responsible for Palladium Allergy

    PubMed Central

    Kawano, Mitsuko; Nakayama, Masafumi; Aoshima, Yusuke; Nakamura, Kyohei; Ono, Mizuho; Nishiya, Tadashi; Nakamura, Syou; Takeda, Yuri; Dobashi, Akira; Takahashi, Akiko; Endo, Misato; Ito, Akiyo; Ueda, Kyosuke; Sato, Naoki; Higuchi, Shigehito; Kondo, Takeru; Hashimoto, Suguru; Watanabe, Masamichi; Watanabe, Makoto; Takahashi, Tetsu; Sasaki, Keiichi; Nakamura, Masanori; Sasazuki, Takehiko; Narushima, Takayuki; Suzuki, Ryuji; Ogasawara, Kouetsu

    2014-01-01

    Nickel, cobalt, and chromium are well known to be causal agents of allergic contact dermatitis. Palladium (Pd) can also cause allergic disease and exposure results from wide use of this metal in dental restorations and jewelry. Metal allergy is categorized as a delayed-type hypersensitivity, and metal-responsive T cell clones have been isolated from allergic patients. However, compared to nickel, little is known about the pathology of allergic disease mediated by Pd, and pathogenic T cells are poorly understood. To identify the pathogenic T cells that are responsible for onset of Pd allergy, we enriched metal-responsive lymphocytes by sequential adoptive transfer of involved lymph node cells. Here we show that sequential adoptive transfer gradually increased the incidence and the intensity of Pd allergy, and CD8+ T cells are responsible for the disease as CD8+ T cell-depleted mice and β2-microglobulin-deficient mice did not develop Pd allergy. In addition, we found that draining lymph node cells skewed toward CD8+ T cells in response to Pd challenge in 8th adoptive transferred recipient mice. The CD8+ T cells expressed NKG2D, a costimulatory molecule involved in the production of IFN-γ. NKG2D ligand was also induced in Pd-injected tissues. Furthermore, both NKG2D ligand-transgenic mice, where NKG2D is downmodulated, and IFN-γ-deficient mice showed impaired Pd allergy. Taken together, these results indicate that IFN-γ-producing NKG2D+ CD8+ T cells are responsible for Pd allergy and suggest that NKG2D is a potential therapeutic target for treatment of metal allergy. PMID:24533050

  20. PAR2 activation alters colonic paracellular permeability in mice via IFN-γ-dependent and -independent pathways

    PubMed Central

    Cenac, Nicolas; Chin, Alex C; Garcia-Villar, Rafael; Salvador-Cartier, Christel; Ferrier, Laurent; Vergnolle, Nathalie; Buret, André G; Fioramonti, Jean; Bueno, Lionel

    2004-01-01

    Activation of colonic proteinase-activated receptor-2 (PAR2) caused inflammation and increased mucosal permeability in mouse colon. The present study was aimed at characterizing the possible links between these two phenomena. We evaluated the effects of intracolonic infusion of PAR2-activating peptide, SLIGRL, on colonic paracellular permeability and inflammation at two different doses, 5 and 100 μg per mouse, in an attempt to discriminate between both PAR2-mediated effects. We further investigated the possible involvement of interferon γ (IFN-γ) and calmodulin-dependent activation of myosin light chain kinase (MLCK), and alterations of zonula occludens-1 (ZO-1) localization in PAR2-induced responses. Thus, at the lower dose, SLIGRL increased colonic permeability without causing inflammation. Western blotting showed phosphorylation of mucosal myosin light chain (MLC) expression after both doses of SLIGRL. Moreover, while the MLCK inhibitor, ML-7, abolished the permeability effects of the low dose of SLIGRL, it only partially inhibited that of the high dose. In IFN-γ-deficient mice (B6 ifng−/−), the increases in permeability were similar for both doses of SLIGRL and prevented by ML-7. In addition, MLCK immunoprecipitation revealed an increase of calmodulin binding to MLCK in the mucosa of mice treated with either dose of SLIGRL. Finally, we have shown that direct activation of PAR2 on enterocytes is responsible for increased permeability and ZO-1 disruption. Moreover, SLIGRL at a dose that does not produce inflammation increases permeability via calmodulin activation, which binds and activates MLCK. The resulting tight junction opening does not depend upon IFN-γ secretion, while the increased permeability in response to the high dose of PAR2 agonist involves IFN-γ secretion. PMID:15194744

  1. ISG12a inhibits HCV replication and potentiates the anti-HCV activity of IFN-α through activation of the Jak/STAT signaling pathway independent of autophagy and apoptosis.

    PubMed

    Chen, Yanzhao; Jiao, Baihai; Yao, Min; Shi, Xuezhen; Zheng, Zhebin; Li, Shilin; Chen, Limin

    2017-01-02

    Interferon stimulated (sensitive) genes (ISGs) are the effector molecules downstream of type I/III interferon (IFN) signaling pathways in host innate immunity. ISG12a can be induced by IFN-α. Although ISG12a has been reported to inhibit the replication of HCV, the exact mechanism remains to be determined. In this study, we investigated the possible mechanisms of ISG12a anti- HCV property by exploring the production of type I IFN and the activation of Janus kinase/signal transducer and activator of transcription (Jak/STAT) signaling pathway, apoptosis and autophagy in Huh7.5.1 cells transiently transfected with ISG12a over-expression plasmid. Interestingly, we found that ISG12a inhibited HCV replication in both Con1b replicon and the HCV JFH1-based cell culture system and potentiated the anti-HCV activity of IFN-α. ISG12a promoted the production of IFN α/β and activated the type I IFN signaling pathway as shown by increased p-STAT1 level, higher Interferon sensitive response element (ISRE) activity and up-regulated ISG levels. However, ISG12a over-expression did not affect cell autophagy and apoptosis. Data from our current study collectively indicated that ISG12a inhibited HCV replication and potentiated the anti-HCV activity of IFN-α possibly through induced production of type I IFNs and activation of Jak/STAT signaling pathway independent of autophagy and cell apoptosis.

  2. IFN-{gamma} gene expression in pancreatic islet-infiltrating mononuclear cells correlates with autoimmune diabetes in nonobese diabetic mice

    SciTech Connect

    Rabinovitch, A.; Suarez-Pinzon, W.L.; Sorensen, O.

    1995-05-01

    Insulin-dependent diabetes mellitus in nonobese diabetic (NOD) mice results from selective destruction of pancreatic islet {beta}-cells following islet filtration by mononuclear leukocytes. Cytokines produced by islet-infiltrating mononuclear cells may be involved in {beta}-cell destruction. Therefore, we analyzed cytokine mRNA expression, by reverse-transcriptase PCR (RT-PCR) assay, in mononuclear leukocytes isolated from pancreatic islets of four groups of mice: diabetes-prone female NOD mice; female NOD mice protected from diabetes by injection of CFA at an early age; male NOD mice with a low diabetes incidence; and female BALB/c mice that do not develop diabetes. We found that mRNA levels of IL-1{beta}, IL-2, IL-4, IL-10, and IFN-{gamma} in mononuclear cells from islets of diabetes-prone female NOD mice increased progressively as these cells infiltrated the islets from age 5 wk to diabetes onset (>13 wk). However, only IFN-{gamma} mRNA levels were significantly higher in islet mononuclear cells from 12-wk-old diabetes-prone female NOD mice than from less diabetes-prone NOD mice (CFA-treated females, and males) and normal mice (BALB/c). In contrast, IL-4 mRNA levels were lower in islet mononuclear cells from diabetes-prone female NOD mice than from NOD mice with low diabetes incidence (CFA-treated females and males). Splenic cell mRNA levels of IFN-{gamma} and IL-4 were not different in the four groups of mice. These results suggest that islet {beta}-cell destruction and diabetes in female NOD mice are dependent upon intra-islet IFN-{gamma} production by mononuclear cells, and that CFA-treated female NOD mice and male NOD mice may be protected from diabetes development by down-regulation of IFN-{gamma} production in the islets. 56 refs., 4 figs., 3 tabs.

  3. Essential involvement of cross-talk between IFN-gamma and TNF-alpha in CXCL10 production in human THP-1 monocytes.

    PubMed

    Qi, Xu-Feng; Kim, Dong-Heui; Yoon, Yang-Suk; Jin, Dan; Huang, Xue-Zhu; Li, Jian-Hong; Deung, Young-Kun; Lee, Kyu-Jae

    2009-09-01

    Interferon (IFN)-gamma-induced protein 10 (IP-10/CXCL10), a CXC chemokine, has been documented in several inflammatory and autoimmune disorders including atopic dermatitis and bronchial asthma. Although CXCL10 could be induced by IFN-gamma depending on cell type, the mechanisms regulating CXCL10 production following treatment with combination of IFN-gamma and TNF-alpha have not been adequately elucidated in human monocytes. In this study, we showed that TNF-alpha had more potential than IFN-gamma to induce CXCL10 production in THP-1 monocytes. Furthermore, IFN-gamma synergistically enhanced the production of CXCL10 in parallel with the activation of NF-kappaB in TNF-alpha-stimulated THP-1 cells. Blockage of STAT1 or NF-kappaB suppressed CXCL10 production. JAKs inhibitors suppressed IFN-gamma plus TNF-alpha-induced production of CXCL10 in parallel with activation of STAT1 and NF-kappaB, while ERK inhibitor suppressed production of CXCL10 as well as activation of NF-kappaB, but not that of STAT1. IFN-gamma-induced phosphorylation of JAK1 and JAK2, whereas TNF-alpha induced phosphorylation of ERK1/2. Interestingly, IFN-gamma alone had no effect on phosphorylation and degradation of IkappaB-alpha, whereas it significantly promoted TNF-alpha-induced phosphorylation and degradation of IkappaB-alpha. These results suggest that TNF-alpha induces CXCL10 production by activating NF-kappaB through ERK and that IFN-gamma induces CXCL10 production by increasing the activation of STAT1 through JAKs pathways. Of note, TNF-alpha-induced NF-kappaB may be the primary pathway contributing to CXCL10 production in THP-1 cells. IFN-gamma potentiates TNF-alpha-induced CXCL10 production in THP-1 cells by increasing the activation of STAT1 and NF-kappaB through JAK1 and JAK2.

  4. Differential involvement of IFN-beta in Toll-like receptor-stimulated dendritic cell activation.

    PubMed

    Hoshino, Katsuaki; Kaisho, Tsuneyasu; Iwabe, Tomio; Takeuchi, Osamu; Akira, Shizuo

    2002-10-01

    Toll-like receptor (TLR) can activate dendritic cells (DC) through common signaling pathways requiring a cytoplasmic adapter, MyD88. However, the signaling is differentially regulated among TLR family members. TLR4 can activate MyD88-deficient bone marrow-derived DC (BMDC), and lead to induction of IFN-inducible genes and up-regulation of co-stimulatory molecules such as CD40, implying that the MyD88-independent signaling pathway functions downstream of TLR4. Because these effects can also be induced by type I IFN, we have analyzed whether type I IFN is involved in TLR4-induced responses. In response to lipopolysaccharide (LPS), IFN-beta gene expression was augmented in both wild-type and MyD88-deficient BMDC. Expression of all IFN-inducible genes except immune-responsive gene 1 (IRG1) was abolished and CD40 up-regulation was decreased in LPS-stimulated BMDC lacking either IFN-alpha/beta receptor (IFN-alpha/betaR) or signal transducer and activator of transcription 1 (STAT-1). Similar to the LPS response, TLR9 signaling can also induce expression of IFN-beta and IFN-inducible genes, and up-regulation of CD40. However, all these effects were MyD88 dependent. Thus, in TLR4 signaling, IFN-beta expression can be induced either by the MyD88-dependent or -independent pathway, whereas, in TLR9 signaling, it is dependent on MyD88. In CpG DNA-stimulated DC, expression of IFN-inducible genes except IRG1 was dependent on type I IFN signaling as in LPS-stimulated DC. However, in contrast to TLR4 signaling, TLR9 signaling requires type I IFN signaling for CD40 up-regulation. Taken together, this study demonstrates differential involvement of type I IFN in TLR4- and TLR9-induced effects on DC.

  5. Intron-containing type I and type III IFN coexist in amphibians: refuting the concept that a retroposition event gave rise to type I IFNs.

    PubMed

    Qi, Zhitao; Nie, Pin; Secombes, Chris J; Zou, Jun

    2010-05-01

    Type I and III IFNs are structurally related cytokines with similar antiviral functions. They have different genomic organizations and bind to distinct receptor complexes. It has been vigorously debated whether the recently identified intron containing IFN genes in fish and amphibians belong to the type I or III IFN family or diverged from a common ancestral gene, that subsequently gave rise to both types. In this report, we have identified intron containing type III IFN genes that are tandemly linked in the Xenopus tropicalis genome and hence demonstrate for the first time that intron containing type I and III genes diverged relatively early in vertebrate evolution, and at least by the appearance of early tetrapods, a transition period when vertebrates migrated from an aquatic environment to land. Our data also suggest that the intronless type I IFN genes seen in reptiles, birds, and mammals have originated from a type I IFN transcript via a retroposition event that led to the disappearance of intron-containing type I IFN genes in modern vertebrates. In vivo and in vitro studies in this paper show that the Xenopus type III IFNs and their cognate receptor are ubiquitously expressed in tissues and primary splenocytes and can be upregulated by stimulation with synthetic double-stranded RNA, suggesting they are involved in antiviral defense in amphibians.

  6. Rb selectively inhibits innate IFN-β production by enhancing deacetylation of IFN-β promoter through HDAC1 and HDAC8.

    PubMed

    Meng, Jun; Liu, Xingguang; Zhang, Peng; Li, Dong; Xu, Sheng; Zhou, Qingqing; Guo, Meng; Huai, Wanwan; Chen, Xiang; Wang, Quanxing; Li, Nan; Cao, Xuetao

    2016-09-01

    Type I IFN production is tightly controlled by host to generate efficient viral clearance without harmful immunopathology or induction of autoimmune disorders. Epigenetic regulation of type I IFN production in innate immunity and inflammatory disorders remains to be fully understood. Several tumor suppressors have been shown to regulate immune response and inflammation. However, the non-classical functions of tumor suppressors in innate immunity and inflammatory diseases need further identification. Here we report retinoblastoma protein (Rb) deficiency selectively enhanced TLR- and virus-triggered production of IFN-β which thus induced more IFN-α generation in the later phase of innate stimuli, but had no effect on the production of TNF, IL-6 and early phase IFN-α in macrophages. Rb1(fl/fl)Lyz2cre(+) Rb-deficient mice exhibited more resistant to lethal virus infection and more effective clearance of influenza virus. Rb selectively bound Ifnb1 enhancer region, but not the promoter of Ifna4, Tnf and Il6, by interacting with c-Jun, the component of IFN-β enhanceosome. Then Rb recruited HDAC1 and HDAC8 to attenuate acetylation of Histone H3/H4 in Ifnb1 promoter, resulting in suppression of Ifnb1 transcription. Therefore, Rb selectively inhibits innate IFN-β production by enhancing deacetylation of Ifnb1 promoter, exhibiting a previous unknown non-classical role in innate immunity, which also suggests a role of Rb in the regulation of type I IFN production in inflammatory or autoimmune diseases.

  7. Evaluation of gamma interferon (IFN-gamma)-induced protein 10 (IP-10) responses for detection of cattle infected with Mycobacterium bovis: comparisons to IFN-gamma responses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Gamma interferon (IFN-gamma)-induced protein 10 (IP-10) has recently shown promise as a diagnostic biomarker of Mycobacterium tuberculosis infection of humans. The aim of the current study was to compare IP-10 and IFN-gamma responses upon Mycobacterium bovis infection in cattle using archived sample...

  8. Respiratory syncytial virus infection suppresses IFN-γ production of γδ T cells

    PubMed Central

    AOYAGI, M; SHIMOJO, N; SEKINE, K; NISHIMUTA, T; KOHNO, Y

    2003-01-01

    The immunological mechanisms by which respiratory syncytial virus (RSV) contributes to the development of asthma are poorly understood. γδ T cells are important in mucosal defence, and may contribute to the establishment of primary immune responses by producing cytokines early during respiratory infections. Thus, we used flow cytometry and intracellular cytokine staining to investigate the expression of interferon (IFN)-γ and interleukin (IL)-4 by mitogen-stimulated γδ T cells from the peripheral blood of 15 hospitalized infants with RSV bronchiolitis, seven rotavirus-infected infants and eight normal controls. γδ T cells from RSV-infected infants had a lower proportion of IFN-γ-producing cells (median, 4.00%; range, 0.58–6.60%) and a slightly but significantly higher proportion of IL-4-producing cells (median, 0.40%; range, 0.13–2.76%) than rotavirus-infected infants (median, 32.10%; range, 14.43–61.21%; P < 0·01, median, 0.00%; range, 0.00–0.00%; P < 0·05) in the acute phase. By contrast, differences in cytokine production by total CD3+ T cells did not differ significantly between patient groups. Thus, reduced IFN-γ-production by γδ T cells in the peripheral blood of RSV-infected infants is accompanied by increased Th2 cytokine production during the acute phase of disease. At follow-up, eight children had recurrent episodes of wheezing. The frequencies of IFN-γ-producing γδ T cells were significantly lower in patients who developed recurrent wheezing (median, 0.65%; range, 0.02–1.75%) than in patients without recurrent wheezing (median, 6.90%; range, 5.25–10.98%; P < 0·005). Cytokine production by γδ T cells may therefore be important in the pathogenesis of acute RSV disease, and play a part in the development of recurrent childhood wheezing after bronchilolitis. PMID:12562394

  9. Induction of multiple programmed cell death pathways by IFN-beta in human non-small-cell lung cancer cell lines.

    PubMed

    Zhang, H; Koty, P P; Mayotte, J; Levitt, M L

    1999-02-25

    Tissue transglutaminase (tTG) and keratinocyte transglutaminase (kTG), as well as the cross-linked envelopes (CLE) that they form, have been associated with squamous differentiation and programmed cell death in epithelial cells. When interferon-beta (IFN-beta) was used to stimulate differentiation and programmed cell death in the human lung cancer cell lines NCI-H596 and NCI-H226, the cells underwent a decrease in cellular density. In NCI-H596 IFN-beta caused an increase in kTG activity and DNA fragmentation in the lower density cells, which were significantly slower growing than control cells. However, in the higher density cells, which were only slightly slower growing than control cells, IFN-beta caused an increase in tTG activity and CLE competence. Dual-parameter flow cytometry demonstrated that IFN-beta-induced squamous differentiation preceded programmed cell death. Treatment of NCI-H596 cells with monodansylcadaverine, a transglutaminase inhibitor, prevented the increase in CLE competence, but did not inhibit DNA fragmentation. These results suggest that IFN-beta can induce NCI-H596 cells to enter multiple cell death pathways and that these pathways are not only differentiation related, but may also be growth driven.

  10. IFN-λ: A New Inducer of Local Immunity against Cancer and Infections

    PubMed Central

    Lasfar, Ahmed; Zloza, Andrew; de la Torre, Andrew; Cohen-Solal, Karine A.

    2016-01-01

    IFN-λ is the newly established type III IFN with unique immunomodulatory functions. In contrast to the IFN-α/β family and to some extent IFN-γ, IFN-λ is apparently acting in specific areas of the body to activate resident immune cells and induces a local immunity, instrumental in preventing particular infections and also keeping transformed cells under control. Mucosal areas of lung and gastrointestinal tracts are now under scrutiny to elucidate the immune mechanisms triggered by IFN-λ and leading to viral protection. New evidence also indicates the crucial role of IFN-λ in promoting innate immunity in solid cancer models. Based on its unique biological activities among the IFN system, new immunotherapeutic approaches are now emerging for the treatment of cancer, infection, and autoimmune diseases. In the present review, we highlight the recent advances of IFN-λ immunomodulatory functions. We also discuss the perspectives of IFN-λ as a therapeutic agent. PMID:28018361

  11. Interferon (IFN) production by peripheral blood mononuclear (PBM) cells of an elderly population

    SciTech Connect

    Nelson, B.J.; Murasko, D.M.

    1986-03-05

    Previous investigations in the laboratory have reported decreased mitogen responses of PBM's from elderly individuals compared to responses of young adults to PHA and ConA. Current studies have investigated the role of IFN in this decreased T cell responsiveness of the elderly. Supernatants of PBM's from 80 elderly (mean age 85) and 50 young individuals (mean age 28) were assayed for antiviral activity, after incubation with optimum and supraoptimum concentrations of mitogen for 24-120 hrs. IFN levels were maximum for both elderly and young populations at 72 hrs coinciding with time of maximum proliferation as determined by uptake of /sup 3/H thymidine. IFN levels declined with longer incubation periods. All IFN produced was IFN-gamma as determined by sensitivity to pH 2 and by neutralizations with monoclonal antibody specific for human IFN-gamma and polyclonal antiserum specific for IFN-alpha. The elderly population's mean IFN titers for both PHA and ConA were about 39% of the mean titers of the young (p less than or equal to 0.02). Both elderly and young groups displayed significant positive correlation between the amount of IFN produced and the level of proliferation in response to the mitogens (p less than or equal to 0.036). Therefore, the above data suggests the decreased levels of IFN produced by elderly PBM's may be one of the factors responsible for the observed decreased proliferative response to mitogens.

  12. Self-priming determines high type I IFN production by plasmacytoid dendritic cells

    PubMed Central

    Kim, Sarah; Kaiser, Vera; Beier, Esther; Bechheim, Matthias; Guenthner-Biller, Margit; Ablasser, Andrea; Berger, Michael; Endres, Stefan

    2015-01-01

    Plasmacytoid dendritic cells (pDCs) are responsible for the robust and immediate production of type I IFNs during viral infection. pDCs employ TLR7 and TLR9 to detect RNA and CpG motifs present in microbial genomes. CpG-A was the first synthetic stimulus available that induced large amounts of IFN-α (type I IFN) in pDCs. CpG-B, however, only weakly activates pDCs to produce IFN-α. Here, we demonstrate that differences in the kinetics of TLR9 activation in human pDCs are essential for the understanding of the functional difference between CpG-A and CpG-B. While CpG-B quickly induces IFN-α production in pDCs, CpG-A stimulation results in delayed yet maximal IFN-α induction. Constitutive production of low levels of type I IFN in pDCs, acting in a paracrine and autocrine fashion, turned out to be the key mechanism responsible for this phenomenon. At high cell density, pDC-derived, constitutive type I IFN production primes pDCs for maximal TLR responsiveness. This accounts for the high activity of higher structured TLR agonists that trigger type I IFN production in a delayed fashion. Altogether, these data demonstrate that high type I IFN production by pDCs cannot be simply ascribed to cell-autonomous mechanisms, yet critically depends on the local immune context. PMID:24338737

  13. Impaired HCV Clearance in HIV/HCV Coinfected Subjects Treated with PegIFN and RBV Due to Interference of IFN Signaling by IFNαR2a

    PubMed Central

    Lee, Yu-Jin; Zhang, Xiaozhen; Vazquez, Estefania; Shivasabesan, Gayathri; Young, Howard A.; Murphy, Alison; Wang, Honghui; Suffredini, Anthony F.; Siebenlist, Ulrich

    2014-01-01

    Enhanced endogenous interferon (IFN) stimulated gene (ISG) signature has been associated with nonresponsiveness to hepatitis C treatment using pegylated-IFNα (pegIFNα) and ribavirin (RBV) in human immunodeficiency virus/hepatitis C virus (HIV/HCV) coinfected patients. Using a proteomic approach, we identified high levels of IFNα receptor 2a (IFNαR2a) in the serum of null responders to pegIFNα/RBV. IFNαR2a inhibited antiviral activity of all formulations of IFNα in JFH/Huh7.5 cells. Furthermore, serum from null responders, but not from those who achieved sustained virologic response, suppressed IFN-signaling and ISG expression in IFNα-stimulated PBMCs of healthy donors in an IFNαR2a specific fashion. An IFNαR2a transgenic mice model (C57BL/6) was generated that had significantly higher levels of IFNαR2a in the serum than the controls (P=0.001). Total ISG expression in the lymph nodes was significantly higher compared to wild-type mice (P value=0.0016). In addition, IFITM1 and SP110 had significantly increased expression in the liver, IFITM1 and ISG15 in the lymph node, and ISG15 and PLSCR1 in the spleen (P value<0.05). The underlying mechanism of resistance to hepatitis C treatment may involve transsignaling of the JAK/STAT pathway by the sIFNαR2a-IFNα/β complex and result in the enhanced ISG signature observed in null responders. In this regard, the transgenic mice model simulated nonresponders to IFNα therapy and provides valuable insights into the role of sIFNαR2a-IFNα interactions in vivo. PMID:24171456

  14. Impaired HCV clearance in HIV/HCV coinfected subjects treated with PegIFN and RBV due to interference of IFN signaling by IFNαR2a.

    PubMed

    Lee, Yu-Jin; Zhang, Xiaozhen; Vazquez, Estefania; Shivasabesan, Gayathri; Young, Howard A; Murphy, Alison; Wang, Honghui; Suffredini, Anthony F; Siebenlist, Ulrich; Kottilil, Shyam

    2014-01-01

    Enhanced endogenous interferon (IFN) stimulated gene (ISG) signature has been associated with nonresponsiveness to hepatitis C treatment using pegylated-IFNα (pegIFNα) and ribavirin (RBV) in human immunodeficiency virus/hepatitis C virus (HIV/HCV) coinfected patients. Using a proteomic approach, we identified high levels of IFNα receptor 2a (IFNαR2a) in the serum of null responders to pegIFNα/RBV. IFNαR2a inhibited antiviral activity of all formulations of IFNα in JFH/Huh7.5 cells. Furthermore, serum from null responders, but not from those who achieved sustained virologic response, suppressed IFN-signaling and ISG expression in IFNα-stimulated PBMCs of healthy donors in an IFNαR2a specific fashion. An IFNαR2a transgenic mice model (C57BL/6) was generated that had significantly higher levels of IFNαR2a in the serum than the controls (P=0.001). Total ISG expression in the lymph nodes was significantly higher compared to wild-type mice (P value=0.0016). In addition, IFITM1 and SP110 had significantly increased expression in the liver, IFITM1 and ISG15 in the lymph node, and ISG15 and PLSCR1 in the spleen (P value<0.05). The underlying mechanism of resistance to hepatitis C treatment may involve transsignaling of the JAK/STAT pathway by the sIFNαR2a-IFNα/β complex and result in the enhanced ISG signature observed in null responders. In this regard, the transgenic mice model simulated nonresponders to IFNα therapy and provides valuable insights into the role of sIFNαR2a-IFNα interactions in vivo.

  15. Effects of parturition and dexamethasone on DNA methylation patterns of IFN-γ and IL-4 promoters in CD4+ T-lymphocytes of Holstein dairy cows

    PubMed Central

    Paibomesai, Marlene; Hussey, Brendan; Nino-Soto, Maria; Mallard, Bonnie A.

    2013-01-01

    This study investigated epigenetic mechanisms by which DNA methylation affects the function of bovine adaptive immune system cells, particularly during the peripartum period, when shifts in type 1 and type 2 immune response (IR) biases are thought to occur. Stimulation of CD4+ T-lymphocytes isolated from 5 Holstein dairy cows before and after parturition with concanavalin A (ConA) and stimulation of CD4+ T-lymphocytes isolated from 3 Holstein dairy cows in mid-lactation with ConA alone or ConA plus dexamethasone (Dex) had significant effects on production of the cytokines interferon gamma (IFN-γ, type 1) and interleukin 4 (IL-4, type 2) that were consistent with DNA methylation profiles of the IFN-γ gene promoter region but not consistent for the IL-4 promoter region. ConA stimulation increased the production of both cytokines before and after parturition. It decreased DNA methylation in the IFN-γ promoter region but increased for IL-4 promoter region. Parturition was associated with an increase in IFN-γ production in ConA-stimulated cells that approached significance. Overall, DNA methylation in both promoter regions increased between the prepartum and postpartum periods, although this did not correlate with secreted cytokine concentrations. Dexamethasone treated cells acted in a manner consistent with the glucocorticoid’s immunosuppressive activity, which mimicked the change at the IFN-γ promoter region observed during parturition. These results support pregnancy as type 2 IR biased, with increases of IFN-γ occurring after parturition and an increase in IL-4 production before calving. It is likely that these changes may be epigenetically controlled. PMID:23814356

  16. Effects of parturition and dexamethasone on DNA methylation patterns of IFN-γ and IL-4 promoters in CD4+ T-lymphocytes of Holstein dairy cows.

    PubMed

    Paibomesai, Marlene; Hussey, Brendan; Nino-Soto, Maria; Mallard, Bonnie A

    2013-01-01

    This study investigated epigenetic mechanisms by which DNA methylation affects the function of bovine adaptive immune system cells, particularly during the peripartum period, when shifts in type 1 and type 2 immune response (IR) biases are thought to occur. Stimulation of CD4+ T-lymphocytes isolated from 5 Holstein dairy cows before and after parturition with concanavalin A (ConA) and stimulation of CD4+ T-lymphocytes isolated from 3 Holstein dairy cows in mid-lactation with ConA alone or ConA plus dexamethasone (Dex) had significant effects on production of the cytokines interferon gamma (IFN-γ, type 1) and interleukin 4 (IL-4, type 2) that were consistent with DNA methylation profiles of the IFN-γ gene promoter region but not consistent for the IL-4 promoter region. ConA stimulation increased the production of both cytokines before and after parturition. It decreased DNA methylation in the IFN-γ promoter region but increased for IL-4 promoter region. Parturition was associated with an increase in IFN-γ production in ConA-stimulated cells that approached significance. Overall, DNA methylation in both promoter regions increased between the prepartum and postpartum periods, although this did not correlate with secreted cytokine concentrations. Dexamethasone treated cells acted in a manner consistent with the glucocorticoid's immunosuppressive activity, which mimicked the change at the IFN-γ promoter region observed during parturition. These results support pregnancy as type 2 IR biased, with increases of IFN-γ occurring after parturition and an increase in IL-4 production before calving. It is likely that these changes may be epigenetically controlled.

  17. Engagement of TLR3, TLR7, and NKG2D regulate IFN-gamma secretion but not NKG2D-mediated cytotoxicity by human NK cells stimulated with suboptimal doses of IL-12.

    PubMed

    Girart, María V; Fuertes, Mercedes B; Domaica, Carolina I; Rossi, Lucas E; Zwirner, Norberto W

    2007-09-15

    NK cells express different TLRs, such as TLR3, TLR7, and TLR9, but little is known about their role in NK cell stimulation. In this study, we used specific agonists (poly(I:C), loxoribine, and synthetic oligonucleotides containing unmethylated CpG sequences to stimulate human NK cells without or with suboptimal doses of IL-12, IL-15, or IFN-alpha, and investigated the secretion of IFN-gamma, cytotoxicity, and expression of the activating receptor NKG2D. Poly(I:C) and loxoribine, in conjunction with IL-12, but not IL-15, triggered secretion of IFN-gamma. Inhibition of IFN-gamma secretion by chloroquine suggested that internalization of the TLR agonists was necessary. Also, secretion of IFN-gamma was dependent on MEK1/ERK, p38 MAPK, p70(S6) kinase, and NF-kappaB, but not on calcineurin. IFN-alpha induced a similar effect, but promoted lesser IFN-gamma secretion. However, cytotoxicity (51Cr release assays) against MHC class I-chain related A (MICA)- and MICA+ tumor targets remained unchanged, as well as the expression of the NKG2D receptor. Excitingly, IFN-gamma secretion was significantly increased when NK cells were stimulated with poly(I:C) or loxoribine and IL-12, and NKG2D engagement was induced by coculture with MICA+ tumor cells in a PI3K-dependent manner. We conclude that resting NK cells secrete high levels of IFN-gamma in response to agonists of TLR3 or TLR7 and IL-12, and this effect can be further enhanced by costimulation through NKG2D. Hence, integration of the signaling cascades that involve TLR3, TLR7, IL-12, and NKG2D emerges as a critical step to promote IFN-gamma-dependent NK cell-mediated effector functions, which could be a strategy to promote Th1-biased immune responses in pathological situations such as cancer.

  18. IFN-τ inhibits S. aureus-induced inflammation by suppressing the activation of NF-κB and MAPKs in RAW 264.7 cells and mice with pneumonia.

    PubMed

    Zhao, Gan; Wu, Haichong; Jiang, Kangfeng; Rui, Guangze; Zhu, Zhe; Qiu, Changwei; Guo, Mengyao; Deng, Ganzhen

    2016-06-01

    Staphylococcus aureus (S. aureus), a significant cause of pneumonia, leads to severe inflammation. Few effective treatments or drugs have been reported for S. aureus infection. Interferon tau (IFN-τ) is a type I interferon with low cellular toxicity even at high doses. Previous studies have reported that IFN-τ could significantly mitigate tissue inflammation; however, IFN-τ treatment in S. aureus-induced pneumonia has not been well reported. Thus, the aim of this study was to identify the anti-inflammatory mechanism of IFN-τ in S. aureus-induced pneumonia in mice. A S. aureus-induced pneumonia model and RAW 264.7 cells were used in this research. The histopathological as well as lung wet to dry ratio (W/D) and myeloperoxidase (MPO) activity results showed that IFN-τ could protect the lung from S. aureus damage. In addition, ELISA and qPCR revealed that IFN-τ treatment led to a decreased expression of pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6) in both the cells and mouse model, but IL-10 was increased. TLR2, which is involved in the response during S. aureus infection, was also down-regulated by IFN-τ treatment and directly affected NF-κB and MAPK pathway activation. Then, we examined the phosphorylation of IκBα, NF-κB p65 and MAPKs by western blotting, and the results displayed that the phosphorylation of IκBα, NF-κB p65 and MAPKs was inhibited upon IFN-τ treatment in both the cells and mouse model. These findings indicate that IFN-τ has anti-inflammatory properties in vitro and in vivo through the inhibition of NF-κB and MAPK activation, suggesting that IFN-τ may have potential as a therapeutic agent against S. aureus-induced inflammatory diseases.

  19. Cucurbita ficifolia (Cucurbitaceae) modulates inflammatory cytokines and IFN-γ in obese mice.

    PubMed

    Fortis-Barrera, Á; García-Macedo, R; Almanza-Perez, J C; Blancas-Flores, G; Zamilpa-Alvarez, A; Flores-Sáenz, J L; Cruz, M; Román-Ramos, R; Alarcón-Aguilar, F J

    2017-02-01

    This study investigated the effect of aqueous extract of Cucurbita ficifolia Bouché on systemic chronic inflammation in an obesity model induced by monosodium glutamate (MSG) via modulating the expression of adipokines (TNF-α, IL-6, resistin, and adiponectin) and immune-regulatory cytokines (IFN-γ and IL-10). Cucurbita ficifolia extract was administered daily by gavage to lean and MSG-obese mice for 30 days. At the end of treatment, cytokine mRNA expression in adipose tissue was determined by real-time polymerase chain reaction (PCR), and the protein levels of these cytokines were also quantified by enzyme-linked immunosorbent assay (ELISA). Cucurbita ficifolia extract decreased body mass and inflammation in MSG-obese mice by reducing the expression of TNF-α and IL-6; these decreases were parallel to significant reductions in protein levels. The extract also increased protein levels of IL-10 in lean mice and IFN-γ in both lean and MSG-obese mice. In conclusion, C. ficifolia extract modulates systemic chronic inflammation in MSG-obese mice and could have a beneficial effect on the adaptive immune system in obesity.

  20. Identification of Two Subgroups of Type I IFNs in Perciforme Fish Large Yellow Croaker Larimichthys crocea Provides Novel Insights into Function and Regulation of Fish Type I IFNs

    PubMed Central

    Ding, Yang; Ao, Jingqun; Huang, Xiaohong; Chen, Xinhua

    2016-01-01

    Like mammals, fish possess an interferon regulatory factor (IRF) 3/IRF7-dependent type I IFN responses, but the exact mechanism by which IRF3/IRF7 regulate the type I IFNs remains largely unknown. In this study, we identified two type I IFNs in the Perciforme fish large yellow croaker Larimichthys crocea, one of which belongs to the fish IFNd subgroup and the other is assigned to a novel subgroup of group I IFNs in fish, tentatively termed IFNh. The two IFN genes are constitutively expressed in all examined tissues, but with varied expression levels. Both IFN genes can be rapidly induced in head kidney and spleen tissues by polyinosinic–polycytidylic acid. The recombinant IFNh was shown to be more potent to trigger a rapid induction of the antiviral genes MxA and protein kinase R than the IFNd, suggesting that they may play distinct roles in regulating early antiviral immunity. Strikingly, IFNd, but not IFNh, could induce the gene expression of itself and IFNh through a positive feedback loop mediated by the IFNd-dependent activation of IRF3 and IRF7. Furthermore, our data demonstrate that the induction of IFNd can be enhanced by the dimeric formation of IRF3 and IRF7, while the IFNh expression mainly involves IRF3. Taken together, our findings demonstrate that the IFN responses are diverse in fish and are likely to be regulated by distinct mechanisms. PMID:27656183

  1. Inhibiting autophagy potentiates the anticancer activity of IFN1@/IFNα in chronic myeloid leukemia cells.

    PubMed

    Zhu, Shan; Cao, Lizhi; Yu, Yan; Yang, Liangchun; Yang, Minghua; Liu, Ke; Huang, Jun; Kang, Rui; Livesey, Kristen M; Tang, Daolin

    2013-03-01

    IFN1@ (interferon, type 1, cluster, also called IFNα) has been extensively studied as a treatment for patients with chronic myeloid leukemia (CML). The mechanism of anticancer activity of IFN1@ is complex and not well understood. Here, we demonstrate that autophagy, a mechanism of cellular homeostasis for the removal of dysfunctional organelles and proteins, regulates IFN1@-mediated cell death. IFN1@ activated the cellular autophagic machinery in immortalized or primary CML cells. Activation of JAK1-STAT1 and RELA signaling were required for IFN1@-induced expression of BECN1, a key regulator of autophagy. Moreover, pharmacological and genetic inhibition of autophagy enhanced IFN1@-induced apoptosis by activation of the CASP8-BID pathway. Taken together, these findings provide evidence for an important mechanism that links autophagy to immunotherapy in leukemia.

  2. IFN-λ Inhibits Drug-Resistant HIV Infection of Macrophages

    PubMed Central

    Wang, Xu; Wang, He; Liu, Man-Qing; Li, Jie-Liang; Zhou, Run-Hong; Zhou, Yu; Wang, Yi-Zhong; Zhou, Wang; Ho, Wen-Zhe

    2017-01-01

    Type III interferons (IFN-λs) have been demonstrated to inhibit a number of viruses, including HIV. Here, we further examined the anti-HIV effect of IFN-λs in macrophages. We found that IFN-λs synergistically enhanced anti-HIV activity of antiretrovirals [azidothymidine (AZT), efavirenz, indinavir, and enfuvirtide] in infected macrophages. Importantly, IFN-λs could suppress HIV infection of macrophages with the drug-resistant strains, including AZT-resistant virus (A012) and reverse transcriptase inhibitor-resistant virus (TC49). Mechanistically, IFN-λs were able to induce the expression of several important anti-HIV cellular factors, including myxovirus resistance 2 (Mx2), a newly identified HIV post-entry inhibitor and tetherin, a restriction factor that blocks HIV release from infected cells. These observations provide additional evidence to support the potential use of IFN-λs as therapeutics agents for the treatment of HIV infection. PMID:28321215

  3. In vitro anti-influenza A activity of interferon (IFN)-λ1 combined with IFN-β or oseltamivir carboxylate.

    PubMed

    Ilyushina, Natalia A; Donnelly, Raymond P

    2014-11-01

    Influenza viruses, which can cross species barriers and adapt to new hosts, pose a constant potential threat to human health. The influenza pandemic of 2009 highlighted the rapidity with which an influenza virus can spread worldwide. Currently available antivirals have a number of limitations against influenza, and novel antiviral strategies, including novel drugs and drug combinations, are urgently needed. Here, we evaluated the in vitro effects of interferon (IFN)-β, IFN-λ1, oseltamivir carboxylate (a neuraminidase (NA) inhibitor), and combinations of these agents against two seasonal (i.e., H1N1 and H3N2) influenza A viruses. We observed that A/California/04/09 (H1N1) and A/Panama/2007/99 (H3N2) isolates were equally sensitive to the antiviral activity of IFN-β and oseltamivir carboxylate in A549 and Calu-3 cells. In contrast, IFN-λ1 exhibited substantially lower protective potential against the H1N1 strain (64-1030-fold ↓, P<0.05), and was ineffective against H3N2 virus in both cell lines. Three dimensional analysis of drug-drug interactions revealed that IFN-λ1 interacted with IFN-β and oseltamivir carboxylate in an additive or synergistic manner, respectively, to inhibit influenza A virus replication in human airway epithelial cells. Overall, the present study demonstrated that anti-influenza agents with different mechanisms of action (e.g., a NA inhibitor combined with IFN-λ1) exerted a significantly greater (P<0.05) synergistic effect compared to co-treatment with drugs that target the same signaling pathway (i.e., IFN-β plus IFN-λ1) in vitro. Our findings provide support for the combined use of interferon plus oseltamivir as a potential means for treating influenza infections.

  4. IFN-inducible GTPases in host cell defense.

    PubMed

    Kim, Bae-Hoon; Shenoy, Avinash R; Kumar, Pradeep; Bradfield, Clinton J; MacMicking, John D

    2012-10-18

    From plants to humans, the ability to control infection at the level of an individual cell-a process termed cell-autonomous immunity-equates firmly with survival of the species. Recent work has begun to unravel this programmed cell-intrinsic response and the central roles played by IFN-inducible GTPases in defending the mammalian cell's interior against a diverse group of invading pathogens. These immune GTPases regulate vesicular traffic and protein complex assembly to stimulate oxidative, autophagic, membranolytic, and inflammasome-related antimicrobial activities within the cytosol, as well as on pathogen-containing vacuoles. Moreover, human genome-wide association studies and disease-related transcriptional profiling have linked mutations in the Immunity-Related GTPase M (IRGM) locus and altered expression of guanylate binding proteins (GBPs) with tuberculosis susceptibility and Crohn's colitis.

  5. A new electronic device for subcutaneous injection of IFN-β-1a.

    PubMed

    Exell, Simon; Verdun, Elisabetta; Driebergen, Reinoud

    2011-09-01

    Disease-modifying drugs (DMDs) can provide important benefits for patients with multiple sclerosis (MS), but nonadherence to treatment is associated with an increased risk of relapse. All first-line DMDs used in MS require regular injection, but injection-related problems are common barriers to treatment adherence. Autoinjectors that allow automatic injection at the press of a button have increased the ease and convenience of injection, compared with manual injection. A new electronic autoinjector has recently been introduced for the administration of subcutaneous IFN-β-1a. This device is the first electronic autoinjector for use with any MS therapy, and includes several innovative features that may be advantageous to patients. One of these features is an accurate electronic dosing log, which can be viewed by the patient and the healthcare provider. This article discusses this new electronic device in the context of other autoinjectors currently used to self-inject first-line DMDs in MS.

  6. HCV-specific immune responses induced by CIGB-230 in combination with IFN-α plus ribavirin

    PubMed Central

    Amador-Cañizares, Yalena; Martínez-Donato, Gillian; Álvarez-Lajonchere, Liz; Vasallo, Claudia; Dausá, Mariacarla; Aguilar-Noriega, Daylen; Valenzuela, Carmen; Raíces, Ivette; Dubuisson, Jean; Wychowski, Czeslaw; Cinza-Estévez, Zurina; Castellanos, Marlén; Núñez, Magdalys; Armas, Anny; González, Yaimé; Revé, Ismariley; Guerra, Ivis; Pérez Aguiar, Ángel; Dueñas-Carrera, Santiago

    2014-01-01

    AIM: To analyze hepatitis C virus (HCV)-specific immune responses in chronically infected patients under triple therapy with interferon-α (IFN-α) plus ribavirin and CIGB-230. METHODS: CIGB-230 was administered in different schedules with respect to IFN-α plus ribavirin therapy. Paired serum and peripheral blood mononuclear cells (PBMC) samples from baseline and end of treatment were analyzed. The HCV-specific humoral response was tested by enzyme-linked immunosorbent assay, neutralizing antibodies were evaluated by cell culture HCV neutralization assays, PBMC proliferation was assayed by carboxyfluorescein succinimidyl ester staining and IFN-γ secretion was assessed by enzyme-linked immunospot. Data on virological and histological response and their association with immune variables are also provided. RESULTS: From week 12 to week 48, all groups of patients showed a significant reduction in mean leukocyte counts. Statistically significant reductions in antibody titers were frequent, but only individuals immunized with CIGB-230 as early add-on treatment sustained the core-IgG response, and the neutralizing antibody response was enhanced only in patients receiving CIGB-230. Cell-mediated immune responses also tended to decline, but significant reductions in IFN-γ secretion and total absence of core-specific lymphoproliferation were exclusive of the control group. Only CIGB-230-immunized individuals showed de novo induced lymphoproliferative responses against the structural antigens. Importantly, it was demonstrated that the quality of the CIGB-230-induced immune response depended on the number of doses and timing of administration in relation to the antiviral therapy. Specifically, the administration of 6 doses of CIGB-230 as late add-on to therapy increased the neutralizing antibody activity and the de novo core-specific IFN-γ secretion, both of which were associated with the sustained virological response. CONCLUSION: CIGB-230, combined with IFN

  7. A transcriptional signaling pathway in the IFN system mediated by 2′-5′-oligoadenylate activation of RNase L

    PubMed Central

    Malathi, Krishnamurthy; Paranjape, Jayashree M.; Bulanova, Elena; Shim, Minsub; Guenther-Johnson, Jeanna M.; Faber, Pieter W.; Eling, Thomas E.; Williams, Bryan R. G.; Silverman, Robert H.

    2005-01-01

    Virus replication in higher vertebrates is restrained by IFNs that cause cells to transcribe genes encoding antiviral proteins, such as 2′-5′ oligoadenylate synthetases. 2′-5′ oligoadenylate synthetase is stimulated by dsRNA to produce 5′-phosphorylated, 2′-5′-linked oligoadenylates (2-5A), whose function is to activate RNase L. Although RNase L is required for a complete IFN antiviral response and mutations in the RNase L gene (RNASEL or HPC1) increase prostate cancer rates, it is unknown how 2-5A affects these biological endpoints through its receptor, RNase L. Presently, we show that 2-5A activation of RNase L produces a remarkable stimulation of transcription (≥20-fold) for genes that suppress virus replication and prostate cancer. Unexpectedly, exposure of DU145 prostate cancer cells to physiologic levels of 2-5A (0.1 μM) induced approximately twice as many RNA species as it down-regulated. Among the 2-5A-induced genes are several IFN-stimulated genes, including IFN-inducible transcript 1/P56, IFN-inducible transcript 2/P54, IL-8, and IFN-stimulated gene 15. 2-5A also potently elevated RNA for macrophage inhibitory cytokine-1/nonsteroidal antiinflammatory drug-activated gene-1, a TGF-β superfamily member implicated as an apoptotic suppressor of prostate cancer. Transcriptional signaling to the macrophage inhibitory cytokine-1/nonsteroidal antiinflammatory drug-activated gene-1 promoter by 2-5A was deficient in HeLa cells expressing a nuclease-dead mutant of RNase L and was dependent on the mitogen-activated protein kinases c-Jun N-terminal kinase and extracellular signal-regulated kinase, both of which were activated in response to 2-5A treatments. Because 2-5A and RNase L participate in defenses against viral infections and prostate cancer, our findings have implications for basic cellular mechanisms that control major pathogenic processes. PMID:16203993

  8. A transcriptional signaling pathway in the IFN system mediated by 2'-5'-oligoadenylate activation of RNase L.

    PubMed

    Malathi, Krishnamurthy; Paranjape, Jayashree M; Bulanova, Elena; Shim, Minsub; Guenther-Johnson, Jeanna M; Faber, Pieter W; Eling, Thomas E; Williams, Bryan R G; Silverman, Robert H

    2005-10-11

    Virus replication in higher vertebrates is restrained by IFNs that cause cells to transcribe genes encoding antiviral proteins, such as 2'-5' oligoadenylate synthetases. 2'-5' oligoadenylate synthetase is stimulated by dsRNA to produce 5'-phosphorylated, 2'-5'-linked oligoadenylates (2-5A), whose function is to activate RNase L. Although RNase L is required for a complete IFN antiviral response and mutations in the RNase L gene (RNASEL or HPC1) increase prostate cancer rates, it is unknown how 2-5A affects these biological endpoints through its receptor, RNase L. Presently, we show that 2-5A activation of RNase L produces a remarkable stimulation of transcription (>/=20-fold) for genes that suppress virus replication and prostate cancer. Unexpectedly, exposure of DU145 prostate cancer cells to physiologic levels of 2-5A (0.1 muM) induced approximately twice as many RNA species as it down-regulated. Among the 2-5A-induced genes are several IFN-stimulated genes, including IFN-inducible transcript 1/P56, IFN-inducible transcript 2/P54, IL-8, and IFN-stimulated gene 15. 2-5A also potently elevated RNA for macrophage inhibitory cytokine-1/nonsteroidal antiinflammatory drug-activated gene-1, a TGF-beta superfamily member implicated as an apoptotic suppressor of prostate cancer. Transcriptional signaling to the macrophage inhibitory cytokine-1/nonsteroidal antiinflammatory drug-activated gene-1 promoter by 2-5A was deficient in HeLa cells expressing a nuclease-dead mutant of RNase L and was dependent on the mitogen-activated protein kinases c-Jun N-terminal kinase and extracellular signal-regulated kinase, both of which were activated in response to 2-5A treatments. Because 2-5A and RNase L participate in defenses against viral infections and prostate cancer, our findings have implications for basic cellular mechanisms that control major pathogenic processes.

  9. Comparative Functional Analysis of 12 Mammalian IFN-λ4 Orthologs

    PubMed Central

    Paquin, Ashley; Onabajo, Olusegun O.; Tang, Wei

    2016-01-01

    IFN-λ4 is a novel type-III interferon with strong clinical significance in humans. Only a subset of individuals—up to 10% of Asians, 50% of Europeans, and 90% of Africans—carry the ΔG allele of a genetic variant rs368234815-TT/ΔG and are genetically able to produce IFN-λ4 protein. Carriers of the ΔG allele have impaired ability to clear infection with hepatitis C virus (HCV). IFN-λ4 is also predicted to exist and be functionally important in several nonhuman mammals. In this study, we present the first comparative analysis of 12 mammalian IFN-λ4 orthologs in a human hepatic cell line, HepG2, which supports signaling of the human IFN-λ4. We show that despite differences in protein sequences, functional properties of the recombinant human and nonhuman IFN-λ4 proteins are comparable—they are all expressed as predominantly cytoplasmic proteins that are biologically active for induction of interferon signaling. We show that several IFN-λ4 orthologs can be detected by Western blotting, flow cytometry, and confocal imaging using a monoclonal antibody developed for the human IFN-λ4. Studies of IFN-λ4 in animals should help improve our understanding of the biology of this novel clinically important interferon in normal and disease conditions. PMID:26308395

  10. MIP-T3 is a negative regulator of innate type I IFN response.

    PubMed

    Ng, Ming-Him James; Ho, Ting-Hin; Kok, Kin-Hang; Siu, Kam-Leung; Li, Jun; Jin, Dong-Yan

    2011-12-15

    TNFR-associated factor (TRAF) 3 is an important adaptor that transmits upstream activation signals to protein kinases that phosphorylate transcription factors to induce the production of type I IFNs, the important effectors in innate antiviral immune response. MIP-T3 interacts specifically with TRAF3, but its function in innate IFN response remains unclear. In this study, we demonstrated a negative regulatory role of MIP-T3 in type I IFN production. Overexpression of MIP-T3 inhibited RIG-I-, MDA5-, VISA-, TBK1-, and IKKε-induced transcriptional activity mediated by IFN-stimulated response elements and IFN-β promoter. MIP-T3 interacted with TRAF3 and perturbed in a dose-dependent manner the formation of functional complexes of TRAF3 with VISA, TBK1, IKKε, and IFN regulatory factor 3. Consistent with this finding, retinoic acid-inducible gene I- and TBK1-induced phosphorylation of IFN regulatory factor 3 was significantly diminished when MIP-T3 was overexpressed. Depletion of MIP-T3 facilitated Sendai virus-induced activation of IFN production and attenuated the replication of vesicular stomatitis virus. In addition, MIP-T3 was found to be dissociated from TRAF3 during the course of Sendai virus infection. Our findings suggest that MIP-T3 functions as a negative regulator of innate IFN response by preventing TRAF3 from forming protein complexes with critical downstream transducers and effectors.

  11. Interdependent IL-7 and IFN-γ signalling in T-cell controls tumour eradication by combined α-CTLA-4+α-PD-1 therapy

    PubMed Central

    Shi, Lewis Zhichang; Fu, Tihui; Guan, Baoxiang; Chen, Jianfeng; Blando, Jorge M.; Allison, James P.; Xiong, Liangwen; Subudhi, Sumit K.; Gao, Jianjun; Sharma, Padmanee

    2016-01-01

    Combination therapy with α-CTLA-4 and α-PD-1 has shown significant clinical responses in different types of cancer. However, the underlying mechanisms remain elusive. Here, combining detailed analysis of human tumour samples with preclinical tumour models, we report that concomitant blockade of CTLA-4 and PD-1 improves anti-tumour immune responses and synergistically eradicates tumour. Mechanistically, combination therapy relies on the interdependence between IL-7 and IFN-γ signalling in T cells, as lack of either pathway abrogates the immune-boosting and therapeutic effects of combination therapy. Combination treatment increases IL-7Rα expression on tumour-infiltrating T cells in an IFN-γ/IFN-γR signalling-dependent manner, which may serve as a potential biomarker for clinical trials with immune checkpoint blockade. Our data suggest that combining immune checkpoint blockade with IL-7 signalling could be an effective modality to improve immunotherapeutic efficacy. Taken together, we conclude that combination therapy potently reverses immunosuppression and eradicates tumours via an intricate interplay between IFN-γ/IFN-γR and IL-7/IL-7R pathways. PMID:27498556

  12. Inflammatory impact of IFN-γ in CD8+ T cell-mediated lung injury is mediated by both Stat1-dependent and -independent pathways

    PubMed Central

    Ramana, Chilakamarti V.; DeBerge, Matthew P.; Kumar, Aseem; Alia, Christopher S.; Durbin, Joan E.

    2015-01-01

    Influenza infection results in considerable pulmonary pathology, a significant component of which is mediated by CD8+ T cell effector functions. To isolate the specific contribution of CD8+ T cells to lung immunopathology, we utilized a nonviral murine model in which alveolar epithelial cells express an influenza antigen and injury is initiated by adoptive transfer of influenza-specific CD8+ T cells. We report that IFN-γ production by adoptively transferred influenza-specific CD8+ T cells is a significant contributor to acute lung injury following influenza antigen recognition, in isolation from its impact on viral clearance. CD8+ T cell production of IFN-γ enhanced lung epithelial cell expression of chemokines and the subsequent recruitment of inflammatory cells into the airways. Surprisingly, Stat1 deficiency in the adoptive-transfer recipients exacerbated the lung injury that was mediated by the transferred influenza-specific CD8+ T cells but was still dependent on IFN-γ production by these cells. Loss of Stat1 resulted in sustained activation of Stat3 signaling, dysregulated chemokine expression, and increased infiltration of the airways by inflammatory cells. Taken together, these data identify important roles for IFN-γ signaling and Stat1-independent IFN-γ signaling in regulating CD8+ T cell-mediated acute lung injury. This is the first study to demonstrate an anti-inflammatory effect of Stat1 on CD8+ T cell-mediated lung immunopathology without the complication of differences in viral load. PMID:25617378

  13. IFN-γ-producing NKT cells exacerbate sepsis by enhancing C5a generation via IL-10-mediated inhibition of CD55 expression on neutrophils.

    PubMed

    Kim, Ji Hyung; Oh, Sae Jin; Ahn, Sehee; Chung, Doo Hyun

    2014-07-01

    A role for NKT cells has been implicated in sepsis, but the mechanism by which NKT cells contribute to sepsis remains unclear. Here, we examined WT and NKT-cell-deficient mice of C57BL/6 background during cecal ligation and puncture-induced sepsis. The levels of C5a, IFN-γ, and IL-10 were higher in the serum and peritoneal fluid of WT mice than in those of CD1d(-/-) mice, while the mortality rate was lower in CD1d(-/-) mice than in WT mice. C5a blockade decreased mortality of WT mice during sepsis, whereas it did not alter that of CD1d(-/-) mice. As assessed by intracellular staining, NKT cells expressed IFN-γ, while neutrophils expressed IL-10. Upon coculture, IL-10-deficient NKT cells enhanced IL-10 production by WT, but not IFN-γR-deficient, neutrophils. Meanwhile, CD1d(-/-) mice exhibited high CD55 expression on neutrophils during sepsis, whereas those cells from WT mice expressed minimal levels of CD55. Recombinant IL-10 administration into CD1d(-/-) mice reduced CD55 expression on neutrophils. Furthermore, adoptive transfer of sorted WT, but not IFN-γ-deficient, NKT cells into CD1d(-/-) mice suppressed CD55 expression on neutrophils, but increased IL-10 and C5a levels. Taken together, IFN-γ-producing NKT cells enhance C5a generation via IL-10-mediated inhibition of CD55 expression on neutrophils, thereby exacerbating sepsis.

  14. Characterization of increased drug metabolism activity in dimethyl sulfoxide (DMSO)-treated Huh7 hepatoma cells

    PubMed Central

    Choi, S.; Sainz, B.; Corcoran, P.; Uprichard, S.; Jeong, H.

    2010-01-01

    1. The objective of this study was to characterize Huh7 cells' baseline capacity to metabolize drugs and to investigate whether the drug metabolism was enhanced upon treatment with dimethyl sulfoxide (DMSO). 2. The messenger RNA (mRNA) levels of major Phase I and Phase II enzymes were determined by quantitative real-time-polymerase chain reaction (RT-PCR), and activities of major drug-metabolizing enzymes were examined using probe drugs by analysing relevant metabolite production rates. 3. The expression levels of drug-metabolizing enzymes in control Huh7 cells were generally very low, but DMSO treatment dramatically increased the mRNA levels of most drug-metabolizing enzymes as well as other liver-specific proteins. Importantly, functionality assays confirmed concomitant increases in drug-metabolizing enzyme activity. Additionally, treatment of the Huh7 cells with 3-methylcholanthrene induced cytochrome P450 (CYP) 1A1 expression. 4. The results indicate that DMSO treatment of Huh7 cells profoundly enhances their differentiation state, thus improving the usefulness of this common cell line as an in vitro hepatocyte model. PMID:19280519

  15. Characterization of increased drug metabolism activity in dimethyl sulfoxide (DMSO)-treated Huh7 hepatoma cells.

    PubMed

    Choi, S; Sainz, B; Corcoran, P; Uprichard, S; Jeong, H

    2009-03-01

    The objective of this study was to characterize Huh7 cells' baseline capacity to metabolize drugs and to investigate whether the drug metabolism was enhanced upon treatment with dimethyl sulfoxide (DMSO). The messenger RNA (mRNA) levels of major Phase I and Phase II enzymes were determined by quantitative real-time-polymerase chain reaction (RT-PCR), and activities of major drug-metabolizing enzymes were examined using probe drugs by analysing relevant metabolite production rates. The expression levels of drug-metabolizing enzymes in control Huh7 cells were generally very low, but DMSO treatment dramatically increased the mRNA levels of most drug-metabolizing enzymes as well as other liver-specific proteins. Importantly, functionality assays confirmed concomitant increases in drug-metabolizing enzyme activity. Additionally, treatment of the Huh7 cells with 3-methylcholanthrene induced cytochrome P450 (CYP) 1A1 expression. The results indicate that DMSO treatment of Huh7 cells profoundly enhances their differentiation state, thus improving the usefulness of this common cell line as an in vitro hepatocyte model.

  16. Expression of the chemokine MIG is a sensitive and predictive marker for antigen-specific, genetically restricted IFN-gamma production and IFN-gamma-secreting cells.

    PubMed

    Brice, G T; Graber, N L; Hoffman, S L; Doolan, D L

    2001-11-01

    The evaluation of antigen-specific immune responses is critical for understanding the mechanisms of immune protection and for establishing the efficacy of candidate vaccines. Here, we describe a novel assay for IFN-gamma activity which is based on the flow cytometric detection of the chemokine, monokine induced by gamma interferon (MIG) as a sensitive and predictive measure of IFN-gamma-mediated effector function, and a surrogate marker for IFN-gamma-producing cells. Upregulation of MIG expression was demonstrated following in vitro activation of peripheral blood mononuclear cells (PBMCs) with defined CD8+ T-cell epitopes derived from influenza virus, cytomegalovirus (CMV), or Epstein-Barr virus (EBV) and was antigen-specific, genetically restricted and dependent on both CD8+ T cells and IFN-gamma. Furthermore, antigen-specific MIG expression was also demonstrated with Plasmodium falciparum circumsporozoite protein (CSP) peptides, using PBMCs from volunteers immunized with irradiated P. falciparum sporozoites. In multiple parallel experiments, the MIG assay was compared to conventional IFN-gamma ELISPOT, IFN-gamma ELISA, MIG ELISA and intracellular cytokine staining assays. The level of MIG expression was shown to be directly associated with the number of IFN-gamma spot-forming cells (SFCs) detected by ELISPOT (r2=0.94). Moreover, in all instances where cultures were considered positive by ELISPOT, a higher stimulation index was noted with the MIG assay as compared with the ELISPOT assay (on average at least threefold higher) and, in some cases, responses as detected by the MIG assay were significant, but the corresponding response as measured by ELISPOT was not significant. Finally, the flow-based MIG assay offers a number of practical and technical advantages over the ELISPOT assay. Our data validate this novel method for the detection of low as well as high levels of antigen-specific and genetically restricted IFN-gamma activity.

  17. Palmitoylation of Interferon-α (IFN-α) Receptor Subunit IFNAR1 Is Required for the Activation of Stat1 and Stat2 by IFN-α*

    PubMed Central

    Claudinon, Julie; Gonnord, Pauline; Beslard, Emilie; Marchetti, Marta; Mitchell, Keith; Boularan, Cédric; Johannes, Ludger; Eid, Pierre; Lamaze, Christophe

    2009-01-01

    Type I interferons (IFNs) bind IFNAR receptors and activate Jak kinases and Stat transcription factors to stimulate the transcription of genes downstream from IFN-stimulated response elements. In this study, we analyze the role of protein palmitoylation, a reversible post-translational lipid modification, in the functional properties of IFNAR. We report that pharmacological inhibition of protein palmitoylation results in severe defects of IFN receptor endocytosis and signaling. We generated mutants of the IFNAR1 subunit of the type I IFN receptor, in which each or both of the two cysteines present in the cytoplasmic domain are replaced by alanines. We show that cysteine 463 of IFNAR1, the more proximal of the two cytoplasmic cysteines, is palmitoylated. A thorough microscopic and biochemical analysis of the palmitoylation-deficient IFNAR1 mutant revealed that IFNAR1 palmitoylation is not required for receptor endocytosis, intracellular distribution, or stability at the cell surface. However, the lack of IFNAR1 palmitoylation affects selectively the activation of Stat2, which results in a lack of efficient Stat1 activation and nuclear translocation and IFN-α-activated gene transcription. Thus, receptor palmitoylation is a previously undescribed mechanism of regulating signaling activity by type I IFNs in the Jak/Stat pathway. PMID:19561067

  18. Predictive Value of Serum IFN-γ inducible Protein-10 and IFN-γ/IL-4 Ratio for Liver Fibrosis Progression in CHB Patients

    PubMed Central

    Wang, Yadong; Yu, Weiyan; Shen, Chuan; Wang, Wei; Zhang, Li; Liu, Fang; Sun, Hui; Zhao, Yajuan; Che, Honghao; Zhao, Caiyan

    2017-01-01

    Noninvasive serum markers for assessment of liver fibrosis in chronic hepatitis B (CHB) patients have not been well-studied. The present study was to evaluate the predictive value of serum interferon gamma-inducible protein-10 (IP-10/CXCL10) and the interferon (IFN)-γ/interleukin (IL)-4 ratio for liver fibrosis progression in CHB patients. A total of 180 CHB patients were categorized into four groups: no fibrosis, mild fibrosis, moderate fibrosis, and severe fibrosis. Serum and intrahepatic levels of IP-10, IFN-γ, and IL-4 were examined, from which the IFN-γ/IL-4 ratio was calculated. We found that the serum IP-10 levels were positively correlated with the severity of liver fibrosis, whereas the IFN-γ/IL-4 ratio was negatively associated with the progression of hepatic fibrosis. Multivariate logistic regression analysis revealed that the serum IP-10 was an independent predictor for significant fibrosis. For predicting significant fibrosis, the IP-10 cut-off value of 300 ng/mL had a sensitivity of 92.7% and a specificity of 68.6%. When the IP-10 level was combined with the IFN-γ/IL-4 ratio, the specificity and positive predictive value were 93.8% and 94.6%, respectively; thus, the discriminatory ability was much improved. In conclusion, the serum IP-10 level and the IFN-γ/IL-4 ratio have great potential to predict significant fibrosis among CHB patients. PMID:28067328

  19. p21 mediates macrophage reprogramming through regulation of p50-p50 NF-κB and IFN

    PubMed Central

    Hernández-Jiménez, Enrique; Shokri, Rahman; Carmona-Rodríguez, Lorena; Mañes, Santos; Álvarez-Mon, Melchor; López-Collazo, Eduardo; Martínez-A, Carlos

    2016-01-01

    M1 and M2 macrophage phenotypes, which mediate proinflammatory and antiinflammatory functions, respectively, represent the extremes of immunoregulatory plasticity in the macrophage population. This plasticity can also result in intermediate macrophage states that support a balance between these opposing functions. In sepsis, M1 macrophages can compensate for hyperinflammation by acquiring an M2-like immunosuppressed status that increases the risk of secondary infection and death. The M1 to M2 macrophage reprogramming that develops during LPS tolerance resembles the pathological antiinflammatory response to sepsis. Here, we determined that p21 regulates macrophage reprogramming by shifting the balance between active p65-p50 and inhibitory p50-p50 NF-κB pathways. p21 deficiency reduced the DNA-binding affinity of the p50-p50 homodimer in LPS-primed and -rechallenged macrophages, impairing their ability to attenuate IFN-β production and acquire an M2-like hyporesponsive status. High p21 levels in sepsis patients correlated with low IFN-β expression, and p21 knockdown in human monocytes corroborated its role in IFN-β regulation. The data demonstrate that p21 adjusts the equilibrium between p65-p50 and p50-p50 NF-κB pathways to mediate macrophage plasticity in LPS tolerance. Identifying p21-related pathways involved in monocyte reprogramming may lead to potential targets for sepsis treatment. PMID:27427981

  20. Inducible Interleukin 32 (IL-32) Exerts Extensive Antiviral Function via Selective Stimulation of Interferon λ1 (IFN-λ1)*

    PubMed Central

    Li, Yongkui; Xie, Jiajia; Xu, Xiupeng; Liu, Li; Wan, Yushun; Liu, Yingle; Zhu, Chengliang; Zhu, Ying

    2013-01-01

    Interleukin (IL)-32 has been recognized as a proinflammatory cytokine that participates in responses to viral infection. However, little is known about how IL-32 is induced in response to viral infection and the mechanisms of IL-32-mediated antiviral activities. We discovered that IL-32 is elevated by hepatitis B virus (HBV) infection both in vitro and in vivo and that HBV induced IL-32 expression at the level of both transcription and post-transcription. Furthermore, microRNA-29b was found to be a key factor in HBV-regulated IL-32 expression by directly targeting the mRNA 3′-untranslated region of IL-32. Antiviral analysis showed that IL-32 was not sufficient to alter HBV replication in HepG2.2.15 cells. To mimic the viremic phase of viral infection, freshly isolated peripheral blood mononuclear cells were treated with IL-32γ, the secretory isoform, and the supernatants were used for antiviral assays. Surprisingly, these supernatants exhibited extensive antiviral activity against multiplex viruses besides HBV. Thus, we speculated that the IL-32γ-treated peripheral blood mononuclear cells produced and secreted an unknown antiviral factor. Using antibody neutralization assays, we identified the factor as interferon (IFN)-λ1 and not IFN-α. Further studies indicated that IL-32γ effectively inhibited HBV replication in a hydrodynamic injection mouse model. Clinical data showed that elevated levels of IFN-λ1 both in serum and liver tissue of HBV patients were positively correlated to the increased levels of IL-32. Our results demonstrate that elevated IL-32 levels during viral infection mediate antiviral effects by stimulating the expression of IFN-λ1. PMID:23729669

  1. Mullerian Inhibiting Substances (MIS) Augments IFN-gamma Mediated Inhibition of Breast Cancer Cell Growth

    DTIC Science & Technology

    2006-06-01

    transgenic mouse model carries the SV40 large T antigen targeted to the epithelium of the mammary and prostate glands andprogression of disease in these...mIFN-g 3) 100 ng mIFN-g each day for 5 days a week for 5 weeks. Two weeks after the start of treament animals started having palpable tumors which

  2. Definition of IFN-γ-related pathways critical for chemically-induced systemic autoimmunity.

    PubMed

    Pollard, K Michael; Hultman, Per; Toomey, Christopher B; Cauvi, David M; Hoffman, Hal M; Hamel, John C; Kono, Dwight H

    2012-12-01

    IFN-γ is essential for idiopathic and murine mercury-induced systemic autoimmunity (mHgIA), and heterozygous IFN-γ(+/-) mice also exhibit reduced disease. This suggests that blocking specific IFN-γ-related pathways that may only partially inhibit IFN-γ production or function will also suppress autoimmunity. To test this hypothesis, mice deficient in genes regulating IFN-γ expression (Casp1, Nlrp3, Il12a, Il12b, Stat4) or function (Ifngr1, Irf1) were examined for mHgIA susceptibility. Absence of either Ifngr1 or Irf1 resulted in a striking reduction of disease, while deficiency of genes promoting IFN-γ expression had modest to no effect. Furthermore, both Irf1- and Ifng-deficiency only modestly reduced the expansion of CD44(hi) and CD44(hi)CD55(lo) CD4(+) T cells, indicating that they are not absolutely required for T cell activation. Thus, there is substantial redundancy in genes that regulate IFN-γ expression in contrast to those that mediate later signaling events. These findings have implications for the therapeutic targeting of IFN-γ pathways in systemic autoimmunity.

  3. Biosynthesis of the IFN-gamma binding protein of ectromelia virus, the causative agent of mousepox.

    PubMed

    Bai, Hongdong; Buller, R Mark L; Chen, Nanhai; Green, Michael; Nuara, Anthony A

    2005-03-30

    Ectromelia virus (ECTV), the causative agent of mousepox, expresses an extracellular interferon-gamma binding protein (IFN-gammaBP) with homology to the ligand binding domains of the IFN-gamma high affinity receptor (IFN-gammaR1). Unlike the cellular receptor, the IFN-gammaBP binds IFN-gamma from several species. The IFN-gammaBP is synthesized early after infection, accumulating in the extracellular milieu as dimers composed of two protein species with Mr of 34.6 or 33.0 kDa. Homodimers are covalently linked by an interchain disulphide bond at position 216. The IFN-gammaBP has complex N-linked oligosaccharides at positions 41 and 149 as determined by site-directed mutagenesis and glycosidase treatment. Glycosylation at position 41 is required for secretion from mammalian cells and may play a role in the activity of the IFN-gammaBP. Glycosylation at position 149 is not required for secretion, and the lack of glycosylation at this site does not diminish ligand binding as measured by surface plasmon resonance (SPR) and ELISA.

  4. Anti-inflammatory activity of IFN-beta in carrageenan-induced pleurisy in the mouse.

    PubMed Central

    Ghiara, P; Bartalini, M; Tagliabue, A; Boraschi, D

    1986-01-01

    The effect of IFN-beta on the development of the inflammatory reaction was studied in an experimental animal model, carrageenan-induced pleurisy in the mouse. Intrapleural inoculation of IFN-beta at the same time as carrageenan administration inhibited both migration of inflammatory cells and exudate formation in the pleural cavity in a dose-dependent fashion. Similarly, IFN-beta decreased the presence of the arachidonate metabolites PGI2, TXA2 and PGE2 (highly active molecules involved in the regulation of the inflammatory reaction) in inflammatory exudates. A marked inhibition of the inflammatory response to carrageenan was also evident when IFN-beta was administered several hours after the inflammatory challenge. In contrast, administration of IFN-gamma did not modify significantly any of the inflammatory parameters considered. PMID:3105936

  5. Bell's palsy associated with IFN-alpha and ribavirin therapy for hepatitis C virus infection.

    PubMed

    Hoare, Matthew; Woodall, Tracy; Alexander, Graeme J M

    2005-03-01

    First-line therapy for hepatitis C virus (HCV) infection comprises interferon-alpha (IFN-alpha) and ribavirin for 6 or 12 months. Mild complications of therapy are common, but more serious complications are rare. Three patients with chronic HCV infection, acquired through injecting drug use, developed idiopathic facial paralysis (Bell's palsy) during therapy, with spontaneous resolution after withdrawal of treatment. Large-scale cohort studies reveal that IFNs are associated rarely with neurologic complications, and only one previous report has linked IFN-alpha therapy and Bell's palsy. We postulate that IFN-alpha therapy led to a breakdown of peripheral tolerance to myelin sheath antigens, leading to neuropathy, just as IFN-alpha therapy can cause autoimmune thyroiditis through breakdown of tolerance to native thyroid antigens.

  6. Viral Evasion Strategies in Type I IFN Signaling – A Summary of Recent Developments

    PubMed Central

    Schulz, Katharina S.; Mossman, Karen L.

    2016-01-01

    The immune system protects the organism against infections and the damage associated with them. The first line of defense against pathogens is the innate immune response. In the case of a viral infection, it induces the interferon (IFN) signaling cascade and eventually the expression of type I IFN, which then causes an antiviral state in the cells. However, many viruses have developed strategies to counteract this mechanism and prevent the production of IFN. In order to modulate or inhibit the IFN signaling cascade in their favor, viruses have found ways to interfere at every single step of the cascade, for example, by inducing protein degradation or cleavage, or by mediate protein polyubiquitination. In this article, we will review examples of viruses that modulate the IFN response and describe the mechanisms they use. PMID:27891131

  7. Investigating the Role of TNF-α and IFN-γ Activation on the Dynamics of iNOS Gene Expression in LPS Stimulated Macrophages

    PubMed Central

    Salim, Taha; Sershen, Cheryl L.; May, Elebeoba E.

    2016-01-01

    Macrophage produced inducible nitric oxide synthase (iNOS) is known to play a critical role in the proinflammatory response against intracellular pathogens by promoting the generation of bactericidal reactive nitrogen species. Robust and timely production of nitric oxide (NO) by iNOS and analogous production of reactive oxygen species are critical components of an effective immune response. In addition to pathogen associated lipopolysaccharides (LPS), iNOS gene expression is dependent on numerous proinflammatory cytokines in the cellular microenvironment of the macrophage, two of which include interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α). To understand the synergistic effect of IFN-γ and TNF-α activation, and LPS stimulation on iNOS expression dynamics and NO production, we developed a systems biology based mathematical model. Using our model, we investigated the impact of pre-infection cytokine exposure, or priming, on the system. We explored the essentiality of IFN-γ priming to the robustness of initial proinflammatory response with respect to the ability of macrophages to produce reactive species needed for pathogen clearance. Results from our theoretical studies indicated that IFN-γ and subsequent activation of IRF1 are essential in consequential production of iNOS upon LPS stimulation. We showed that IFN-γ priming at low concentrations greatly increases the effector response of macrophages against intracellular pathogens. Ultimately the model demonstrated that although TNF-α contributed towards a more rapid response time, measured as time to reach maximum iNOS production, IFN-γ stimulation was significantly more significant in terms of the maximum expression of iNOS and the concentration of NO produced. PMID:27276061

  8. Neutrophil migration across cultured intestinal epithelial monolayers is modulated by epithelial exposure to IFN-gamma in a highly polarized fashion

    PubMed Central

    1993-01-01

    transmigration in the two directions. In contrast to apical-to-basolateral directed migration, IFN-gamma markedly downregulated transepithelial migration of PMN in the natural direction. This downregulation of PMN migration in the natural direction, however, was not due to failure of PMN to move across filters and into monolayers. Indeed, IFN-gamma exposure to epithelia increased the number of PMN which had moved into the basolateral space of the epithelium in naturally directed transmigration. These results represent the first detailed report of influences on PMN transepithelial migration by a cytokine, define conditions under which a qualitative difference in PMN transepithelial migration exists, and suggest that migration of PMN across epithelia in the natural direction may involve multiple steps which can be differentially regulated by cytokines.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:8093887

  9. A Model of DENV-3 Infection That Recapitulates Severe Disease and Highlights the Importance of IFN-γ in Host Resistance to Infection

    PubMed Central

    Valadão, Deborah F.; Cisalpino, Daniel; Dias, Ana Carolina F.; Silveira, Kátia D.; Kangussu, Lucas M.; Ávila, Thiago V.; Bonfim, Maria Rosa Q.; Bonaventura, Daniela; Silva, Tarcília A.; Sousa, Lirlândia P.; Rachid, Milene A.; Vieira, Leda Q.; Menezes, Gustavo B.; de Paula, Ana Maria; Atrasheuskaya, Alena; Ignatyev, George; Teixeira, Mauro M.; Souza, Danielle G.

    2012-01-01

    There are few animal models of dengue infection, especially in immunocompetent mice. Here, we describe alterations found in adult immunocompetent mice inoculated with an adapted Dengue virus (DENV-3) strain. Infection of mice with the adapted DENV-3 caused inoculum-dependent lethality that was preceded by several hematological and biochemical changes and increased virus dissemination, features consistent with severe disease manifestation in humans. IFN-γ expression increased after DENV-3 infection of WT mice and this was preceded by increase in expression of IL-12 and IL-18. In DENV-3-inoculated IFN-γ−/− mice, there was enhanced lethality, which was preceded by severe disease manifestation and virus replication. Lack of IFN-γ production was associated with diminished NO-synthase 2 (NOS2) expression and higher susceptibility of NOS2−/− mice to DENV-3 infection. Therefore, mechanisms of protection to DENV-3 infection rely on IFN-γ-NOS2-NO-dependent control of viral replication and of disease severity, a pathway showed to be relevant for resistance to DENV infection in other experimental and clinical settings. Thus, the model of DENV-3 infection in immunocompetent mice described here represents a significant advance in animal models of severe dengue disease and may provide an important tool to the elucidation of immunopathogenesis of disease and of protective mechanisms associated with infection. PMID:22666512

  10. Natural Killer Cell Sensing of Infected Cells Compensates for MyD88 Deficiency but Not IFN-I Activity in Resistance to Mouse Cytomegalovirus.

    PubMed

    Cocita, Clément; Guiton, Rachel; Bessou, Gilles; Chasson, Lionel; Boyron, Marilyn; Crozat, Karine; Dalod, Marc

    2015-05-01

    In mice, plasmacytoid dendritic cells (pDC) and natural killer (NK) cells both contribute to resistance to systemic infections with herpes viruses including mouse Cytomegalovirus (MCMV). pDCs are the major source of type I IFN (IFN-I) during MCMV infection. This response requires pDC-intrinsic MyD88-dependent signaling by Toll-Like Receptors 7 and 9. Provided that they express appropriate recognition receptors such as Ly49H, NK cells can directly sense and kill MCMV-infected cells. The loss of any one of these responses increases susceptibility to infection. However, the relative importance of these antiviral immune responses and how they are related remain unclear. In humans, while IFN-I responses are essential, MyD88 is dispensable for antiviral immunity. Hence, a higher redundancy has been proposed in the mechanisms promoting protective immune responses against systemic infections by herpes viruses during natural infections in humans. It has been assumed, but not proven, that mice fail to mount protective MyD88-independent IFN-I responses. In humans, the mechanism that compensates MyD88 deficiency has not been elucidated. To address these issues, we compared resistance to MCMV infection and immune responses between mouse strains deficient for MyD88, the IFN-I receptor and/or Ly49H. We show that selective depletion of pDC or genetic deficiencies for MyD88 or TLR9 drastically decreased production of IFN-I, but not the protective antiviral responses. Moreover, MyD88, but not IFN-I receptor, deficiency could largely be compensated by Ly49H-mediated antiviral NK cell responses. Thus, contrary to the current dogma but consistent with the situation in humans, we conclude that, in mice, in our experimental settings, MyD88 is redundant for IFN-I responses and overall defense against a systemic herpes virus infection. Moreover, we identified direct NK cell sensing of infected cells as one mechanism able to compensate for MyD88 deficiency in mice. Similar mechanisms likely

  11. CD127 Expression, Exhaustion Status and Antigen Specific Proliferation Predict Sustained Virologic Response to IFN in HCV/HIV Co-Infected Individuals

    PubMed Central

    Kared, Hassen; Saeed, Sahar; Klein, Marina B.; Shoukry, Naglaa H.

    2014-01-01

    Hepatitis C virus (HCV) infection is a major cause of morbidity and mortality in the HIV co-infected population. Interferon-alpha (IFN-α) remains a major component of anti-HCV therapy despite its deleterious effects on the immune system. Furthermore, IFN-α was recently shown to diminish the size of the latent HIV reservoir. The objectives of this study were to monitor the impact of IFN-α on T cell phenotype and proliferation of HIV and HCV-specific T cells during IFN therapy, and to identify immune markers that can predict the response to IFN in HICV/HIV co-infected patients. We performed longitudinal analyses of T cell numbers, phenotype and function in co-infected patients undergoing IFN-α therapy with different outcomes including IFN-α non-responders (NR) (n = 9) and patients who achieved sustained virologic response (SVR) (n = 19). We examined the expression of activation (CD38, HLA-DR), functional (CD127) and exhaustion markers (PD1, Tim-3, CD160 and CD244) on total CD4 and CD8 T cells before, during and after therapy. In addition, we examined the HIV- and HCV-specific proliferative responses against HIV-p24 and HCV-NS3 proteins. Frequencies of CD127+ CD4 T cells were higher in SVR than in NR patients at baseline. An increase in CD127 expression on CD8 T cells was observed after IFN-α therapy in all patients. In addition, CD8 T cells from NR patients expressed a higher exhaustion status at baseline. Finally, SVR patients exhibited higher proliferative response against both HIV and HCV antigens at baseline. Altogether, SVR correlated with higher expression of CD127, lower T cell exhaustion status and better HIV and HCV proliferative responses at baseline. Such factors might be used as non-invasive methods to predict the success of IFN–based therapies in co-infected individuals. PMID:25007250

  12. IFNs-signaling effects on lung cancer: an up-to-date pathways-specific review.

    PubMed

    Galani, Vasiliki; Kastamoulas, Michalis; Varouktsi, Anna; Lampri, Evangeli; Mitselou, Antigoni; Arvanitis, Dimitrios L

    2016-07-14

    IFNs have found important applications in clinical medicine, including the treatment of lung malignancies. The biological effect of the IFN-receptor signaling is regulated essentially by three factors: the expression profile of the IFN itself, the profile of the receptor, and the expression of target genes. IFNs initiate their signaling by binding to specific receptors. The activated IFNs can directly induce gene transcription and/or multiple downstream signaling that both induce diverse cellular responses including the cell cycle arrest and the apoptosis in tumor cells. We provided evidence that IFN-γ enhances the pro cell death effects of Fas/CD95 in human neoplastic alveolar epithelial cell line, A549. We also found that p27 protein plays a pivotal role in the inducing cell death of IFNγ-CH-11-treated A549 cells, since it is involved in the Ras/Raf signaling pathway. This article discusses recent insights into these possible additional functions of IFNs in lung cancer treatment.

  13. Lineage-Specific Expansion of IFIT Gene Family: An Insight into Coevolution with IFN Gene Family

    PubMed Central

    Liu, Ying; Zhang, Yi-Bing; Liu, Ting-Kai; Gui, Jian-Fang

    2013-01-01

    In mammals, IFIT (Interferon [IFN]-induced proteins with Tetratricopeptide Repeat [TPR] motifs) family genes are involved in many cellular and viral processes, which are tightly related to mammalian IFN response. However, little is known about non-mammalian IFIT genes. In the present study, IFIT genes are identified in the genome databases from the jawed vertebrates including the cartilaginous elephant shark but not from non-vertebrates such as lancelet, sea squirt and acorn worm, suggesting that IFIT gene family originates from a vertebrate ancestor about 450 million years ago. IFIT family genes show conserved gene structure and gene arrangements. Phylogenetic analyses reveal that this gene family has expanded through lineage-specific and species-specific gene duplication. Interestingly, IFN gene family seem to share a common ancestor and a similar evolutionary mechanism; the function link of IFIT genes to IFN response is present early since the origin of both gene families, as evidenced by the finding that zebrafish IFIT genes are upregulated by fish IFNs, poly(I:C) and two transcription factors IRF3/IRF7, likely via the IFN-stimulated response elements (ISRE) within the promoters of vertebrate IFIT family genes. These coevolution features creates functional association of both family genes to fulfill a common biological process, which is likely selected by viral infection during evolution of vertebrates. Our results are helpful for understanding of evolution of vertebrate IFN system. PMID:23818968

  14. Down modulation of IFN-{gamma} signaling in alveolar macrophages isolated from smokers

    SciTech Connect

    Dhillon, Navneet K.; Murphy, William J.; Filla, Michael B.; Crespo, Ana J.; Latham, Heath A.; O'Brien-Ladner, Amy

    2009-05-15

    The master cytokine, IFN-{gamma} possesses a wide spectrum of biological effects and is crucial for development of the highly activated macrophage phenotype characteristically found during inflammation. However, no data exists regarding the potential influence of cigarette smoke on the status of the expression of the cell surface receptor for IFN-{gamma} (IFN-{gamma}R) on alveolar macrophages (AM) of smokers. Here in, we report reduction in the expression of the IFN-{gamma}R {alpha}-chain on AM of cigarette smokers, when compared with non-smokers. Ensuing from the loss of receptor expression on the AM of smokers there was a decrease in IFN-{gamma}-mediated cell signaling. This included a decrease in the phosphorylation of signal transducer and activator of transcription (STAT)-1 and induction of interferon regulatory factor (IRF)-1. Further, diminished activation/induction of transcription factors did not appear to result from induction of known members of the 'suppressors of cytokine signaling (SOCS)' family. Decreased IFN-{gamma} signal transduction in AM from smokers may have an important implication regarding the use of therapeutic IFN-{gamma} in the lungs of patients that develop respiratory disorders as a result of tobacco use.

  15. IL-27 Induced by Select Candida spp. via TLR7/NOD2 Signaling and IFN-β Production Inhibits Fungal Clearance

    PubMed Central

    Patin, Emmanuel C.; Jones, Adam V.; Thompson, Aiysha; Clement, Mathew; Liao, Chia-Te; Griffiths, James S.; Wallace, Leah E.; Bryant, Clare E.; Lang, Roland; Rosenstiel, Philip; Humphreys, Ian R.; Taylor, Philip R.

    2016-01-01

    Candida spp. elicit cytokine production downstream of various pathogen recognition receptors, including C-type lectin-like receptors, TLRs, and nucleotide oligomerization domain (NOD)–like receptors. IL-12 family members IL-12p70 and IL-23 are important for host immunity against Candida spp. In this article, we show that IL-27, another IL-12 family member, is produced by myeloid cells in response to selected Candida spp. We demonstrate a novel mechanism for Candida parapsilosis–mediated induction of IL-27 in a TLR7-, MyD88-, and NOD2-dependent manner. Our data revealed that IFN-β is induced by C. parapsilosis, which in turn signals through the IFN-α/β receptor and STAT1/2 to induce IL-27. Moreover, IL-27R (WSX-1)–deficient mice systemically infected with C. parapsilosis displayed enhanced pathogen clearance compared with wild-type mice. This was associated with increased levels of proinflammatory cytokines in the serum and increased IFN-γ and IL-17 responses in the spleens of IL-27R–deficient mice. Thus, our data define a novel link between C. parapsilosis, TLR7, NOD2, IFN-β, and IL-27, and we have identified an important role for IL-27 in the immune response against C. parapsilosis. Overall, these findings demonstrate an important mechanism for the suppression of protective immune responses during infection with C. parapsilosis, which has potential relevance for infections with other fungal pathogens. PMID:27259855

  16. Respiratory Syncytial Virus Persistence in Murine Macrophages Impairs IFN-β Response but Not Synthesis

    PubMed Central

    Rivera-Toledo, Evelyn; Torres-González, Laura; Gómez, Beatriz

    2015-01-01

    Type-I interferon (IFN-I) production is an early response to viral infection and pathogenic viruses have evolved multiple strategies to evade this cellular defense. Some viruses can establish and maintain persistent infections by altering the IFN-I signaling pathway. Here, we studied IFN-I synthesis and response in an in vitro model of persistent infection by respiratory syncytial virus (RSV) in a murine macrophage-like cell line. In this model, interferon regulatory factor 3 was constitutively active and located at nuclei of persistently infected cells, inducing expression of IFN-beta mRNA and protein. However, persistently infected macrophages did not respond in an autocrine manner to the secreted-IFN-beta or to recombinant-IFN-beta, since phosphorylated-STAT1 was not detected by western blot and transcription of the interferon-stimulated genes (ISGs) Mx1 and ISG56 was not induced. Treatment of non-infected macrophages with supernatants from persistently infected cells induced STAT1 phosphorylation and ISGs expression, mediated by the IFN-I present in the supernatants, because blocking the IFN-I receptor inhibited STAT1 phosphorylation. Results suggest that the lack of autocrine response to IFN-I by the host cell may be one mechanism for maintenance of RSV persistence. Furthermore, STAT1 phosphorylation and ISGs expression induced in non-infected cells by supernatants from persistently infected macrophages suggest that RSV persistence may trigger a proinflammatory phenotype in non-infected cells as part of the pathogenesis of RSV infection. PMID:26501312

  17. ERIS, an endoplasmic reticulum IFN stimulator, activates innate immune signaling through dimerization.

    PubMed

    Sun, Wenxiang; Li, Yang; Chen, Lu; Chen, Huihui; You, Fuping; Zhou, Xiang; Zhou, Yi; Zhai, Zhonghe; Chen, Danying; Jiang, Zhengfan

    2009-05-26

    We report here the identification and characterization of a protein, ERIS, an endoplasmic reticulum (ER) IFN stimulator, which is a strong type I IFN stimulator and plays a pivotal role in response to both non-self-cytosolic RNA and dsDNA. ERIS (also known as STING or MITA) resided exclusively on ER membrane. The ER retention/retrieval sequence RIR was found to be critical to retain the protein on ER membrane and to maintain its integrity. ERIS was dimerized on innate immune challenges. Coumermycin-induced ERIS dimerization led to strong and fast IFN induction, suggesting that dimerization of ERIS was critical for self-activation and subsequent downstream signaling.

  18. Viral Inhibition of the IFN-Induced JAK/STAT Signalling Pathway: Development of Live Attenuated Vaccines by Mutation of Viral-Encoded IFN-Antagonists

    PubMed Central

    Fleming, Stephen B.

    2016-01-01

    The interferon (IFN) induced anti-viral response is amongst the earliest and most potent of the innate responses to fight viral infection. The induction of the Janus kinase/signal transducer and activation of transcription (JAK/STAT) signalling pathway by IFNs leads to the upregulation of hundreds of interferon stimulated genes (ISGs) for which, many have the ability to rapidly kill viruses within infected cells. During the long course of evolution, viruses have evolved an extraordinary range of strategies to counteract the host immune responses in particular by targeting the JAK/STAT signalling pathway. Understanding how the IFN system is inhibited has provided critical insights into viral virulence and pathogenesis. Moreover, identification of factors encoded by viruses that modulate the JAK/STAT pathway has opened up opportunities to create new anti-viral drugs and rationally attenuated new generation vaccines, particularly for RNA viruses, by reverse genetics. PMID:27367734

  19. NKT cells mediate pulmonary inflammation and dysfunction in murine sickle cell disease through production of IFN-γ and CXCR3 chemokines

    PubMed Central

    Wallace, Kori L.; Marshall, Melissa A.; Ramos, Susan I.; Lannigan, Joanne A.; Field, Joshua J.; Strieter, Robert M.

    2009-01-01

    Ischemia-reperfusion injury (IRI) triggers an inflammatory cascade that is initiated by the activation of CD1d-restricted iNKT cells. In sickle cell disease (SCD), misshapen erythrocytes evoke repeated transient bouts of microvascular IRI. Compared with C57BL/6 controls, NY1DD mice have more numerous and activated (CD69+, interferon-γ+ [IFN-γ+]) lung, liver, and spleen iNKT cells that are hyperresponsive to hypoxia/reoxygenation. NY1DD mice have increased pulmonary levels of IFN-γ, IFN-γ–inducible chemokines (CXCL9, CXCL10), and elevated numbers of lymphocytes expressing the chemokine receptor CXCR3. Treating NY1DD mice with anti-CD1d antibody to inhibit iNKT cell activation reverses baseline pulmonary dysfunction manifested as elevated vascular permeability, decreased arterial oxygen saturation, and increased numbers of activated leukocytes. Anti-CD1d antibodies decrease pulmonary levels of IFN-γ and CXCR3 chemokines. Neutralization of CXCR3 receptors ameliorates pulmonary dysfunction. Crossing NY1DD to lymphocyte-deficient Rag1−/− mice decreases pulmonary dysfunction. This is counteracted by the adoptive transfer of 1 million NKT cells. Like mice, people with SCD have increased numbers of activated circulating iNKT cells expressing CXCR3. Together, these data indicate that iNKT cells play a pivotal role in sustaining inflammation in SCD mice by a pathway involving IFN-γ and production of chemotactic CXCR3 chemokines and that this mechanism may translate to human disease. PMID:19433855

  20. Binding of /sup 125/I-labeled recombinant beta interferon (IFN-beta Ser17) to human cells

    SciTech Connect

    O'Rourke, E.C.; Drummond, R.J.; Creasey, A.A.

    1984-12-01

    The authors investigated the binding of /sup 125/I-labeled beta interferon (IFN-beta Ser17), a nonglycosylated recombinant human fibroblast interferon in which cysteine at position 17 is replaced by serine by site-specific mutagenesis. An optimized chloramine T radiolabeling method produced a highly labeled, fully active /sup 125/I-IFN suitable for these studies. Unlike the case with the chloramine T method, incorporation of a single mole of Bolton-Hunter reagent into a mole of IFN-beta Ser17 led to nearly complete loss of biological activity. /sup 125/I-IFN-beta Ser17, prepared by the chloramine T method, bound specifically to human lymphoblastoid cells (Daudi) with a dissociation constant of 0.24 nM. The number of binding sites per cell was 4,000. In competition assays, unlabeled beta interferons (native, recombinant IFN-beta Cys17, and various preparations of IFN-beta Ser17) equally displaced labeled IFN-beta Ser17 on Daudi cells. Recombinant IFN-alpha-1 displaced /sup 125/I-IFN-beta binding to Daudi cells less efficiently than did unlabeled native or recombinant beta interferon. However, at the concentrations tested, native gamma interferon showed no competition with /sup 125/I-IFN. The results indicate that IFN-beta Ser17 and native IFN-beta posses similar binding properties.

  1. THYROXINE (T4) CATABOLISM IN HUMAN AND RAT HEPATOCYTES INCREASES FOLLOWING EXPOSURE TO HEPATIC ENZYME INDUCERS

    EPA Science Inventory

    Nuclear receptor agonists phenobarbital (PB), 3-methylcholanthrene (3MC), pregnenolone-16a-carbonitrile (PCN), 2,2',4,4',5,5'-hexachlorobiphenyl (PCB 153), and 2,2' ,4,4'-tetrabromodiphenyl ether (BDE 47) decrease serum thyroxine (T4) in rats. This decrease is thought to occur th...

  2. Thyrocytes responding to IFN-gamma are essential for development of lymphocytic spontaneous autoimmune thyroiditis and inhibition of thyrocyte hyperplasia.

    PubMed

    Yu, Shiguang; Sharp, Gordon C; Braley-Mullen, Helen

    2006-01-15

    IFN-gamma promotes the development of lymphocytic spontaneous autoimmune thyroiditis (L-SAT) in NOD.H-2h4 mice and inhibits the development of thyrocyte hyperplasia and proliferation (TEC H/P). The precise mechanisms by which IFN-gamma promotes L-SAT and inhibits TEC H/P are unknown. To determine whether responsiveness of lymphocytes or thyrocytes to IFN-gamma is important for the development of these lesions, IFN-gammaR-/- mice, which develop TEC H/P similar to IFN-gamma-/- mice, were used as recipients for adoptive cell transfer. Wild-type (WT) splenocytes or bone marrow induced L-SAT and inhibited TEC H/P in IFN-gamma-/-, but not IFN-gammaR-/- recipients. IFN-gammaR-/- recipients of WT cells developed severe TEC H/P, but did not develop L-SAT, suggesting that thyrocytes responding to IFN-gamma are important for inhibition of TEC H/P. Unexpectedly, IFN-gammaR-/- splenocytes or bone marrow did not induce L-SAT in IFN-gamma-/- or WT mice even though IFN-gammaR-/- lymphocyte donors produced as much IFN-gamma as lymphocytes from WT donors, and thyrocytes could respond to IFN-gamma. Real-time PCR indicated that recipients of IFN-gammaR-/- bone marrow expressed less mRNA for IFN-gamma-inducible chemokines compared with recipients of WT bone marrow. This might limit the migration of IFN-gammaR-/- lymphocytes to thyroids. Few IFN-gammaR-/- lymphocytes infiltrated thyroids even in the presence of WT lymphocytes, suggesting that lymphocytes unable to respond to IFN-gamma are not induced to migrate to thyroids. These results suggest that thyrocytes must be able to respond to IFN-gamma for the development of L-SAT and inhibition of TEC H/P, and lymphocytes must be able to respond to IFN-gamma to induce L-SAT.

  3. Mesenchymal stromal cells expressing ErbB-2/neu elicit protective antibreast tumor immunity in vivo, which is paradoxically suppressed by IFN-gamma and tumor necrosis factor-alpha priming.

    PubMed

    Romieu-Mourez, Raphaëlle; François, Moïra; Abate, Amanda; Boivin, Marie-Noëlle; Birman, Elena; Bailey, Dana; Bramson, Jonathan L; Forner, Kathy; Young, Yoon-Kow; Medin, Jeffrey A; Galipeau, Jacques

    2010-10-15

    It is unknown whether mesenchymal stromal cells (MSC) can regulate immune responses targeting tumor autoantigens of low immunogenicity. We tested here whether immunization with MSC could break immune tolerance towards the ErbB-2/HER-2/neu tumor antigen and the effects of priming with IFN-γ and tumor necrosis factor-α (TNF-α) on this process. BALB/c- and C57BL/6-derived MSC were lentivirally transduced to express a kinase-inactive rat neu mutant (MSC/Neu). Immunization of BALB/c mice with nontreated or IFN-γ-primed allogeneic or syngeneic MSC/Neu induced similar levels of anti-neu antibody titers; however, only syngeneic MSC/Neu induced protective neu-specific CD8(+) T cell responses. Compared to immunization with nontreated or IFN-γ-primed syngeneic MSC/Neu, the number of circulating neu-specific CD8(+) T cells and titers of anti-neu antibodies were observed to be decreased after immunizations with IFN-γ- plus TNF-α-primed MSC/Neu. In addition, syngeneic MSC/Neu seemed more efficient than IFN-γ-primed MSC/Neu at inducing a protective therapeutic antitumor immune response resulting in the regression of transplanted neu-expressing mammary tumor cells. In vitro antigen-presenting cell assays performed with paraformaldehyde-fixed or live MSC showed that priming with IFN-γ plus TNF-α, compared to priming with IFN-γ alone, increased antigen presentation as well as the production of immunosuppressive factors. These data suggest that whereas MSC could effectively serve as antigen-presenting cells to induce immune responses aimed at tumor autoantigens, these functions are critically regulated by IFN-γ and TNF-α.

  4. IFN-γ, IL-21, and IL-10 co-expression in evolving autoimmune vitiligo lesions of Smyth line chickens.

    PubMed

    Shi, Fengying; Erf, Gisela F

    2012-03-01

    The Smyth line (SL) of chicken is an excellent animal model for human autoimmune vitiligo. In SL vitiligo (SLV), postnatal loss of melanocytes in feathers appears to be due to cell-mediated immunity. In this study, leukocyte infiltration and associated expression (RNA) of immune function-related cytokines in growing feathers were investigated throughout SLV development and progression. Both leukocyte infiltration and cytokine expression levels started to increase near visible SLV onset (early SLV), reached peak levels during active SLV, and decreased to near pre-vitiligo levels after complete loss of melanocytes. Specifically, significant increases were noticed in relative proportions of T cells, B cells, and major histocompatibility complex (MHC) II-expressing cells during active SLV. Levels of T-cell infiltration were higher than those of B cells, with more CD8+ than CD4+ cells throughout SLV. Elevated leukocyte infiltration in early and active SLV was accompanied by increased levels of cytokine expression, especially in IFN-γ, IL-10, and IL-21. Low expression of IL-4 and IL-17 did not suggest important roles of Th2 and Th17 cells in SLV pathogenesis. Taken together, SLV appears to be a Th1-polarized autoimmune disease, whereby IFN-γ expression is strongly associated with parallel increases in IL-10 and IL-21, particularly during early and active stages of SLV.

  5. A mouse model of vitiligo with focused epidermal depigmentation requires IFN-γ for autoreactive CD8⁺ T-cell accumulation in the skin.

    PubMed

    Harris, John E; Harris, Tajie H; Weninger, Wolfgang; Wherry, E John; Hunter, Christopher A; Turka, Laurence A

    2012-07-01

    Vitiligo is an autoimmune disease of the skin causing disfiguring patchy depigmentation of the epidermis and, less commonly, hair. Therapeutic options for vitiligo are limited, reflecting in part limited knowledge of disease pathogenesis. Existing mouse models of vitiligo consist of hair depigmentation but lack prominent epidermal involvement, which is the hallmark of human disease. They are thus unable to provide a platform to fully investigate disease mechanisms and treatment. CD8(+) T cells have been implicated in the pathogenesis of vitiligo, and expression of IFN-γ is increased in the lesional skin of patients, however, it is currently unknown what role IFN-γ has in disease. Here, we have developed an adoptive transfer mouse model of vitiligo using melanocyte-specific CD8(+) T cells, which recapitulates the human condition by inducing epidermal depigmentation while sparing the hair. Like active lesions in human vitiligo, histology of depigmenting skin reveals a patchy mononuclear infiltrate and single-cell infiltration of the epidermis. Depigmentation is accompanied by accumulation of autoreactive CD8(+) T cells in the skin, quantifiable loss of tyrosinase transcript, and local IFN-γ production. Neutralization of IFN-γ with antibody prevents CD8(+) T-cell accumulation and depigmentation, suggesting a therapeutic potential for this approach.

  6. Reversal of tumor acidosis by systemic buffering reactivates NK cells to express IFN-γ and induces NK cell-dependent lymphoma control without other immunotherapies.

    PubMed

    Pötzl, Johann; Roser, David; Bankel, Lorenz; Hömberg, Nadine; Geishauser, Albert; Brenner, Christoph D; Weigand, Michael; Röcken, Martin; Mocikat, Ralph

    2017-05-01

    Like other immune cells, natural killer (NK) cells show impaired effector functions in the microenvironment of tumors, but little is known on the underlying mechanisms. Since lactate acidosis, a hallmark of malignant tissue, was shown to contribute to suppression of effective antitumor immune responses, we investigated the impact of tissue pH and lactate concentration on NK-cell functions in an aggressive model of endogenously arising B-cell lymphoma. The progressive loss of IFN-γ production by NK cells observed during development of this disease could be ascribed to decreased pH values and lactate accumulation in the microenvironment of growing tumors. Interestingly, IFN-γ expression by lymphoma-derived NK cells could be restored by transfer of these cells into a normal micromilieu. Likewise, systemic alkalization by oral delivery of bicarbonate to lymphoma-developing mice was capable of enhancing IFN-γ expression in NK cells and increasing the NK-cell numbers in the lymphoid organs where tumors were growing. By contrast, NK-cell cytotoxicity was dampened in vivo by tumor-dependent mechanisms that seemed to be different from lactate acidosis and could not be restored in a normal milieu. Most importantly, alkalization and the concomitant IFN-γ upregulation in NK cells were sufficient to significantly delay tumor growth without any other immunotherapy. This effect was strictly dependent on NK cells.

  7. Quantifying the Antiviral Effect of IFN on HIV-1 Replication in Cell Culture

    NASA Astrophysics Data System (ADS)

    Ikeda, Hiroki; Godinho-Santos, Ana; Rato, Sylvie; Vanwalscappel, Bénédicte; Clavel, François; Aihara, Kazuyuki; Iwami, Shingo; Mammano, Fabrizio

    2015-06-01

    Type-I interferons (IFNs) induce the expression of hundreds of cellular genes, some of which have direct antiviral activities. Although IFNs restrict different steps of HIV replication cycle, their dominant antiviral effect remains unclear. We first quantified the inhibition of HIV replication by IFN in tissue culture, using viruses with different tropism and growth kinetics. By combining experimental and mathematical analyses, we determined quantitative estimates for key parameters of HIV replication and inhibition, and demonstrate that IFN mainly inhibits de novo infection (33% and 47% for a X4- and a R5-strain, respectively), rather than virus production (15% and 6% for the X4 and R5 strains, respectively). This finding is in agreement with patient-derived data analyses.

  8. IFN-γ and TNF-α are involved during Alzheimer disease progression and correlate with nitric oxide production: a study in Algerian patients.

    PubMed

    Belkhelfa, Mourad; Rafa, Hayet; Medjeber, Oussama; Arroul-Lammali, Amina; Behairi, Nassima; Abada-Bendib, Myriam; Makrelouf, Mohamed; Belarbi, Soreya; Masmoudi, Ahmed Nacer; Tazir, Meriem; Touil-Boukoffa, Chafia

    2014-11-01

    Alzheimer's disease (AD) is a neurodegenerative disease leading to a progressive and irreversible loss of mental functions. It is characterized by 3 stages according to the evolution and the severity of the symptoms. This disease is associated with an immune disorder, which appears with significant rise in the inflammatory cytokines and increased production of free radicals such as nitric oxide (NO). Our study aims to investigate interferon (IFN)-γ and tumor necrosis factor-α (TNF-α) involvement in NO production, in vivo and ex vivo, in peripheral blood mononuclear cells from Algerian patients (n=25), according to the different stages of the disease (mild Alzheimer's, moderate Alzheimer's, and severe Alzheimer's) in comparison to mild cognitive impairment (MCI) patients. Interestingly, we observed that in vivo IFN-γ and TNF-α levels assessed in patients with AD in mild and severe stages, respectively, are higher than those observed in patients with moderate stage and MCI. Our in vivo and ex vivo results show that NO production is related to the increased levels of IFN-γ and TNF-α, in mild and severe stages of AD. Remarkably, significant IFN-γ level is only detected in mild stage of AD. Our study suggests that NO production is IFN-γ dependent both in MCI and mild Alzheimer's patients. Further, high levels of NO are associated with an elevation of TNF-α levels in severe stage of AD. Collectively, our data indicate that the proinflammatory cytokine production seems, in part, to be involved in neurological deleterious effects observed during the development of AD through NO pathway.

  9. Prostacyclin inhibits IFN-γ-stimulated cytokine expression by reduced recruitment of p300/CBP to STAT1 in a SOCS-1 independent manner

    PubMed Central

    Strassheim, Derek; Riddle, Suzzette R.; Burke, Danielle L.; Geraci, Mark W; Stenmark, Kurt R.

    2009-01-01

    Increasing evidence indicates that Pulmonary Arterial Hypertension (PAH) is a vascular inflammatory disease. Prostacyclin (PGI2) is widely used to treat PAH and is believed to benefit patients largely through vasodilatory effects. PGI2 is also increasingly believed to have anti-inflammatory effects; including decreasing leukocyte cytokine production, yet few mechanistic details exist to explain how these effects are mediated at the transcriptional level. Since activated monocytes are critical sources of MCP-1 and other cytokines in cardiovascular inflammation, we examined the effects of iloprost on IFN-γ and IL-6 stimulated cytokine production in human monocytes. We found iloprost inhibited IFN-γ and IL-6-induced MCP-1, IL-8, RANTES, and TNF-α production in monocytes indicating wide-ranging anti-inflammatory action. We found that activation of STAT1 was critical for IFN-γ-induced MCP-1 production and demonstrated that iloprost inhibited STAT1 activation by several actions: 1) iloprost inhibited the phosphorylation of STAT1-S727 in the transactivation domain (TAD), thereby reducing recruitment of the histone acetylase and co-activator CBP/p300 to STAT1; 2) iloprost selectively inhibited activation of janus kinase 2 (JAK2), but not JAK1, both responsible for activation STAT1 via phosphorylation of STAT1-Y701, resulting in reduced nuclear recruitment and activation of STAT1; 3) SOCS-1, which normally terminates IFN-γ-signaling, was not involved in iloprost-mediated inhibition of STAT1, indicating divergence from the classical pathway for terminating IFN-γ-signaling. We conclude that PGI2 exerts anti-inflammatory action by inhibiting STAT1 induced cytokine production, in part by targeting the transactivation domain induced recruitment of the histone acetylase CBP/p300. PMID:19915063

  10. Antigenic modulation of metastatic breast and ovary carcinoma cells by intracavitary injection of IFN-alpha.

    PubMed Central

    Giacomini, P.; Mottolese, M.; Fraioli, R.; Benevolo, M.; Venturo, I.; Natali, P. G.

    1992-01-01

    Antigenic modulation of major histocompatibility and tumour associated antigens was observed in neoplastic cells obtained from patients with pleural and abdominal effusions of breast and ovary carcinomas following a single intracavitary dose of 18 x 10(6) U recombinant IFN-alpha. This regimen resulted in antigenic modulation in seven out of 11 tested cases, suggesting a potential, although limited, responsiveness of at least a fraction of breast and ovary carcinoma cells to in situ biomodification with IFN-alpha. PMID:1503908

  11. Induction of Type 1 Immune Responses to SIV by IFN-Gamma

    DTIC Science & Technology

    1997-06-01

    vaccination . Vaccinated macaques will be boosted with gag/env VLPs produced in recombinant baculoviruses and all macaques will be challenged with pathogenic...that express IFN-y and SIV virus-like particles ( VLPs ) containing SIVgag, env, and nef proteins. We hypothesize that vaccines expressing IFN-y will...1,000X lethal challenge with rinderpest virus (unpublished data). 3) Moreover, I/M vaccination of VV was used in some human vaccinee populations during the

  12. Mullerian Inhibiting Substance (MIS) Augments IFN-Gamma Mediated Inhibition of Breast Cancer Cell Growth

    DTIC Science & Technology

    2005-06-01

    demonstrated that MIS promotes IFN-y-induced apoptosis demonstrating a functional interaction between these two classes of signaling molecules in regulation...mammary tumors which do not express ER and have functional inactivation of Rb and p53. In agreement with results observed in the transgenic mice, MIS...Interferon regulatory factor-1 (IRF-1), a gene known for its growth inhibitory functions in breast cancer cells is induced by MIS and IFN-y through a NFkB

  13. Myocardial dysfunction in an experimental model of autoimmune myocarditis: role of IFN-gamma.

    PubMed

    Pérez Leirós, C; Goren, N; Sterin-Borda, L; Borda, E S

    1997-01-01

    Experimental autoimmune myocarditis obtained in mice by immunization with heart antigens is characterized by the presence of lymphomononuclear infiltrates in atria and ventricles. Here we show the ability of soluble factors released by immune cells from mice immunized with heart antigens to decrease heart contractility in a similar way to a muscarinic agonist. These effects appear to be mediated by IFN-gamma since all of them could be blocked by an anti-IFN-gamma monoclonal antibody. Moreover, the negative inotropic effect induced by immune cell-conditioned media was blocked by atropine, confirming previous findings that IFN acts as a muscarinic agonist on isolated atria. The role of locally released cytokines and especially of IFN-gamma was also evaluated in infiltrated autoimmune myocarditis hearts; thus, the addition of monoclonal anti-IFN-gamma antibody reversed the decreased contractility characteristics of this model. We conclude that IFN released both systemically and locally by autoreactive T cells may contribute to the impaired cardiac function in this experimental model of autoimmune myocarditis.

  14. T-Cell Activation under Hypoxic Conditions Enhances IFN-γ Secretion

    PubMed Central

    Roman, Jessica; Rangasamy, Tirumalai; Guo, Jia; Sugunan, Siva; Meednu, Nida; Packirisamy, Gopinath; Shimoda, Larissa A.; Golding, Amit; Semenza, Gregg; Georas, Steve N.

    2010-01-01

    Secondary lymphoid organs and peripheral tissues are characterized by hypoxic microenvironments, both in the steady state and during inflammation. Although hypoxia regulates T-cell metabolism and survival, very little is known about whether or how hypoxia influences T-cell activation. We stimulated mouse CD4+ T cells in vitro with antibodies directed against the T-cell receptor (CD3) and CD28 under normoxic (20% O2) and hypoxic (1% O2) conditions. Here we report that stimulation under hypoxic conditions augments the secretion of effector CD4+ T-cell cytokines, especially IFN-γ. The enhancing effects of hypoxia on IFN-γ secretion were independent of mouse strain, and were also unaffected using CD4+ T cells from mice lacking one copy of the gene encoding hypoxia-inducible factor-1α. Using T cells from IFN-γ receptor–deficient mice and promoter reporter studies in transiently transfected Jurkat T cells, we found that the enhancing effects of hypoxia on IFN-γ expression were not due to effects on IFN-γ consumption or proximal promoter activity. In contrast, deletion of the transcription factor, nuclear erythroid 2 p45–related factor 2 attenuated the enhancing effect of hypoxia on IFN-γ secretion and other cytokines. We conclude that hypoxia is a previously underappreciated modulator of effector cytokine secretion in CD4+ T cells. PMID:19372249

  15. The evolutionarily dynamic IFN-inducible GTPase proteins play conserved immune functions in vertebrates and cephalochordates.

    PubMed

    Li, Guang; Zhang, Juyong; Sun, Yi; Wang, Hua; Wang, Yiquan

    2009-07-01

    Interferon (IFN)-inducible GTPases currently include four families of proteins: myxovirus resistant proteins (Mxs), guanylate-binding proteins (GBPs), immunity-related GTPase proteins (IRGs), and very large inducible GTPase proteins (VLIGs). They are all under conserved regulation by IFNs in humans and mice and play a critical role in preventing microbial infections. However, differences between vertebrates are poorly characterized, and their evolutionary origins have not been studied in detail. In this study, we performed comparative genomic analysis of the four families in 18 representative animals that yielded several unexpected results. Firstly, we found that Mx, GBP, and IRG protein families arose before the divergence of chordate subphyla, but VLIG emerged solely in vertebrates. Secondly, IRG, GBP, and VLIG families have experienced a high rate of gene gain and loss during the evolution, with the GBP family being lost entirely in two pufferfish and VLIG family lost in primates and carnivores. Thirdly, the regulation of these genes by IFNs is highly conserved throughout vertebrates although the VLIG protein sequences in fish have lost the first 870 amino acid residues. Finally, amphioxus IFN-inducible GTPase genes are all highly expressed in immune-related organs such as gill, liver, and intestine and are upregulated after challenge with PolyI:C and pathogens, although no IFNs or their receptors were detected in the current amphioxus genome database. These results suggest that IFN-inducible GTPase genes play conserved immune functions both in vertebrates and in cephalochordates.

  16. Genetic and phenotypic analyses of sequential vpu alleles from HIV-infected IFN-treated patients.

    PubMed

    Vanwalscappel, Bénédicte; Rato, Sylvie; Perez-Olmeda, Mayte; Díez Fuertes, Francisco; Casartelli, Nicoletta; Alcami, José; Mammano, Fabrizio

    2017-01-01

    Treatment of HIV-infected patients with IFN-α results in significant, but clinically insufficient, reductions of viremia. IFN induces the expression of several antiviral proteins including BST-2, which inhibits HIV by multiple mechanisms. The viral protein Vpu counteracts different effects of BST-2. We thus asked if Vpu proteins from IFN-treated patients displayed improved anti-BST-2 activities as compared to Vpu from baseline. Deep-sequencing analyses revealed that in five of seven patients treated by IFN-α for a concomitant HCV infection in the absence of antiretroviral drugs, the dominant Vpu sequences differed before and during treatment. In three patients, vpu alleles that emerged during treatment improved virus replication in the presence of IFN-α, and two of them conferred improved virus budding from cells expressing BST-2. Differences were observed for the ability to down-regulate CD4, while all Vpu variants potently down-modulated BST-2 from the cell surface. This report discloses relevant consequences of IFN-treatment on HIV properties.

  17. Unique nonstructural proteins of Pneumonia Virus of Mice (PVM) promote degradation of interferon (IFN) pathway components and IFN-stimulated gene proteins.

    PubMed

    Dhar, Jayeeta; Barik, Sailen

    2016-12-01

    Pneumonia Virus of Mice (PVM) is the only virus that shares the Pneumovirus genus of the Paramyxoviridae family with Respiratory Syncytial Virus (RSV). A deadly mouse pathogen, PVM has the potential to serve as a robust animal model of RSV infection, since human RSV does not fully replicate the human pathology in mice. Like RSV, PVM also encodes two nonstructural proteins that have been implicated to suppress the IFN pathway, but surprisingly, they exhibit no sequence similarity with their RSV equivalents. The molecular mechanism of PVM NS function, therefore, remains unknown. Here, we show that recombinant PVM NS proteins degrade the mouse counterparts of the IFN pathway components. Proteasomal degradation appears to be mediated by ubiquitination promoted by PVM NS proteins. Interestingly, NS proteins of PVM lowered the levels of several ISG (IFN-stimulated gene) proteins as well. These results provide a molecular foundation for the mechanisms by which PVM efficiently subverts the IFN response of the murine cell. They also reveal that in spite of their high sequence dissimilarity, the two pneumoviral NS proteins are functionally and mechanistically similar.

  18. Unique nonstructural proteins of Pneumonia Virus of Mice (PVM) promote degradation of interferon (IFN) pathway components and IFN-stimulated gene proteins

    PubMed Central

    Dhar, Jayeeta; Barik, Sailen

    2016-01-01

    Pneumonia Virus of Mice (PVM) is the only virus that shares the Pneumovirus genus of the Paramyxoviridae family with Respiratory Syncytial Virus (RSV). A deadly mouse pathogen, PVM has the potential to serve as a robust animal model of RSV infection, since human RSV does not fully replicate the human pathology in mice. Like RSV, PVM also encodes two nonstructural proteins that have been implicated to suppress the IFN pathway, but surprisingly, they exhibit no sequence similarity with their RSV equivalents. The molecular mechanism of PVM NS function, therefore, remains unknown. Here, we show that recombinant PVM NS proteins degrade the mouse counterparts of the IFN pathway components. Proteasomal degradation appears to be mediated by ubiquitination promoted by PVM NS proteins. Interestingly, NS proteins of PVM lowered the levels of several ISG (IFN-stimulated gene) proteins as well. These results provide a molecular foundation for the mechanisms by which PVM efficiently subverts the IFN response of the murine cell. They also reveal that in spite of their high sequence dissimilarity, the two pneumoviral NS proteins are functionally and mechanistically similar. PMID:27905537

  19. NK-derived IFN-γ/IL-4 triggers the sexually disparate polarization of macrophages in CVB3-induced myocarditis.

    PubMed

    Liu, Li; Yue, Yan; Xiong, Sidong

    2014-11-01

    Coxsackievirus B3 (CVB3) is a common etiology of myocarditis with an increased morbidity and mortality in males. We previously reported that differential polarization of macrophages contributed to sexually dimorphic susceptibility of mice to CVB3-induced myocarditis. However, the underlying kinetics, impetus as well as the molecular mechanism remain unclear. Here, we demonstrated that myocardial macrophages started to polarize at as early as day 5 post CVB3 infection in both genders of BALB/c mice, with M1 phenotype detected in males and M2a phenotype in females, and this trend was further amplified at day 7 when myocarditis reached peak. In addition, we identified that prevailed IFN-γ in males and dominant IL-4 in females were critical myocardial cytokines for the disparate macrophage polarization, which respectively activated JAK1-STAT1 and JAK3-STAT6 pathways. Strikingly, we found that the main source of IFN-γ and IL-4 cytokines in both genders were myocardial infiltrating NK cells, which differentially secreted cytokines in various microenvironments manifested synergistically by sex hormones and CVB3 infection. Consistently, depletion of NK cells significantly impeded the myocardial macrophage polarization in both genders of CVB3-infected mice. Collectively, these data indicated that myocardial NK-derived IFN-γ/IL-4 was critical for the differential polarization of macrophages in CVB3-induced myocarditis via activating JAK1-STAT1 and JAK3-STAT6 pathways respectively. Our study may help understand the mechanism of sexually differential polarization of macrophages and provide clues for the gender bias in CVB3-induced myocarditis.

  20. Additive loss-of-function proteasome subunit mutations in CANDLE/PRAAS patients promote type I IFN production

    PubMed Central

    Brehm, Anja; Liu, Yin; Sheikh, Afzal; Marrero, Bernadette; Omoyinmi, Ebun; Zhou, Qing; Montealegre, Gina; Biancotto, Angelique; Reinhardt, Adam; Almeida de Jesus, Adriana; Pelletier, Martin; Tsai, Wanxia L.; Remmers, Elaine F.; Kardava, Lela; Hill, Suvimol; Kim, Hanna; Lachmann, Helen J.; Megarbane, Andre; Chae, Jae Jin; Brady, Jilian; Castillo, Rhina D.; Brown, Diane; Casano, Angel Vera; Gao, Ling; Chapelle, Dawn; Huang, Yan; Stone, Deborah; Chen, Yongqing; Sotzny, Franziska; Lee, Chyi-Chia Richard; Kastner, Daniel L.; Torrelo, Antonio; Zlotogorski, Abraham; Moir, Susan; Gadina, Massimo; McCoy, Phil; Wesley, Robert; Rother, Kristina; Hildebrand, Peter W.; Brogan, Paul; Krüger, Elke; Aksentijevich, Ivona; Goldbach-Mansky, Raphaela

    2015-01-01

    Autosomal recessive mutations in proteasome subunit β 8 (PSMB8), which encodes the inducible proteasome subunit β5i, cause the immune-dysregulatory disease chronic atypical neutrophilic dermatosis with lipodystrophy and elevated temperature (CANDLE), which is classified as a proteasome-associated autoinflammatory syndrome (PRAAS). Here, we identified 8 mutations in 4 proteasome genes, PSMA3 (encodes α7), PSMB4 (encodes β7), PSMB9 (encodes β1i), and proteasome maturation protein (POMP), that have not been previously associated with disease and 1 mutation in PSMB8 that has not been previously reported. One patient was compound heterozygous for PSMB4 mutations, 6 patients from 4 families were heterozygous for a missense mutation in 1 inducible proteasome subunit and a mutation in a constitutive proteasome subunit, and 1 patient was heterozygous for a POMP mutation, thus establishing a digenic and autosomal dominant inheritance pattern of PRAAS. Function evaluation revealed that these mutations variably affect transcription, protein expression, protein folding, proteasome assembly, and, ultimately, proteasome activity. Moreover, defects in proteasome formation and function were recapitulated by siRNA-mediated knockdown of the respective subunits in primary fibroblasts from healthy individuals. Patient-isolated hematopoietic and nonhematopoietic cells exhibited a strong IFN gene-expression signature, irrespective of genotype. Additionally, chemical proteasome inhibition or progressive depletion of proteasome subunit gene transcription with siRNA induced transcription of type I IFN genes in healthy control cells. Our results provide further insight into CANDLE genetics and link global proteasome dysfunction to increased type I IFN production. PMID:26524591

  1. The Gut Microbiota Reduces Colonization of the Mesenteric Lymph Nodes and IL-12-Independent IFN-γ Production During Salmonella Infection.

    PubMed

    Fernández-Santoscoy, María; Wenzel, Ulf A; Yrlid, Ulf; Cardell, Susanna; Bäckhed, Fredrik; Wick, Mary Jo

    2015-01-01

    The intestinal commensal microbiota is essential for many host physiological processes, but its impact on infectious diseases is poorly understood. Here we investigate the influence of the gut microbiota during oral Salmonella infection. We report a higher bacterial burden in mesenteric lymph nodes (MLN) of intragastrically infected germ-free (GF) mice compared to conventionally-raised (CONV-R) animals, despite similar inflammatory phagocyte recruitment. Salmonella penetration into the lamina propria of the small intestine and splenic bacterial burden were not altered in the absence of the microbiota. Intragastrically infected GF mice also displayed a higher frequency of IFN-γ-producing NK, NKT, CD4(+), and CD8(+) T cells in the MLN despite IL-12 levels similar to infected CONV-R mice. However, infecting mice intraperitoneally abrogated the difference in MLN bacterial load and IFN-γ-producing cells observed in intragastrically-infected animals. Moreover, mice treated with antibiotics (ABX) and intragastrically infected with Salmonella had a greater bacterial burden and frequency of IFN-γ-producing cells in the MLN. In ABX mice the number of Salmonella correlated with the frequency of IFN-γ-producing lymphocytes in the MLN, while no such correlation was observed in the MLN of infected GF mice. Overall, the data show that the lack of the microbiota influences pathogen colonization of the MLN, and the increased IFN-γ in the MLN of infected GF mice is not only due to the absence of commensals at the time of infection but the lack of immune signals provided by the microbiota from birth.

  2. The Gut Microbiota Reduces Colonization of the Mesenteric Lymph Nodes and IL-12-Independent IFN-γ Production During Salmonella Infection

    PubMed Central

    Fernández-Santoscoy, María; Wenzel, Ulf A.; Yrlid, Ulf; Cardell, Susanna; Bäckhed, Fredrik; Wick, Mary Jo

    2015-01-01

    The intestinal commensal microbiota is essential for many host physiological processes, but its impact on infectious diseases is poorly understood. Here we investigate the influence of the gut microbiota during oral Salmonella infection. We report a higher bacterial burden in mesenteric lymph nodes (MLN) of intragastrically infected germ-free (GF) mice compared to conventionally-raised (CONV-R) animals, despite similar inflammatory phagocyte recruitment. Salmonella penetration into the lamina propria of the small intestine and splenic bacterial burden were not altered in the absence of the microbiota. Intragastrically infected GF mice also displayed a higher frequency of IFN-γ-producing NK, NKT, CD4+, and CD8+ T cells in the MLN despite IL-12 levels similar to infected CONV-R mice. However, infecting mice intraperitoneally abrogated the difference in MLN bacterial load and IFN-γ-producing cells observed in intragastrically-infected animals. Moreover, mice treated with antibiotics (ABX) and intragastrically infected with Salmonella had a greater bacterial burden and frequency of IFN-γ-producing cells in the MLN. In ABX mice the number of Salmonella correlated with the frequency of IFN-γ-producing lymphocytes in the MLN, while no such correlation was observed in the MLN of infected GF mice. Overall, the data show that the lack of the microbiota influences pathogen colonization of the MLN, and the increased IFN-γ in the MLN of infected GF mice is not only due to the absence of commensals at the time of infection but the lack of immune signals provided by the microbiota from birth. PMID:26734581

  3. Impaired IFN-α production by plasmacytoid dendritic cells favors regulatory T-cell expansion that may contribute to breast cancer progression.

    PubMed

    Sisirak, Vanja; Faget, Julien; Gobert, Michael; Goutagny, Nadège; Vey, Nelly; Treilleux, Isabelle; Renaudineau, Sarah; Poyet, Gaelle; Labidi-Galy, Sana Intidhar; Goddard-Leon, Sophie; Durand, Isabelle; Le Mercier, Isabelle; Bajard, Agathe; Bachelot, Thomas; Puisieux, Alain; Puisieux, Isabelle; Blay, Jean-Yves; Ménétrier-Caux, Christine; Caux, Christophe; Bendriss-Vermare, Nathalie

    2012-10-15

    Infiltration and dysfunction of immune cells have been documented in many types of cancers. We previously reported that plasmacytoid dendritic cells (pDC) within primary breast tumors correlate with an unfavorable prognosis for patients. The role of pDC in cancer remains unclear but they have been shown to mediate immune tolerance in other pathophysiologic contexts. We postulated that pDC may interfere with antitumor immune response and favor tolerance in breast cancer. The present study was designed to decipher the mechanistic basis for the deleterious impact of pDC on the clinical outcome. Using fresh human breast tumor biopsies (N = 60 patients), we observed through multiparametric flow cytometry increased tumor-associated (TA) pDC (TApDC) rates in aggressive breast tumors, i.e., those with high mitotic index and the so-called triple-negative breast tumors (TNBT). Furthermore, TApDC expressed a partially activated phenotype and produced very low amounts of IFN-α following toll-like receptor activation in vitro compared with patients' blood pDC. Within breast tumors, TApDC colocalized and strongly correlated with TA regulatory T cells (TATreg), especially in TNBT. Of most importance, the selective suppression of IFN-α production endowed TApDC with the unique capacity to sustain FoxP3(+) Treg expansion, a capacity that was reverted by the addition of exogenous IFN-α. These findings indicate that IFN-α-deficient TApDC accumulating in aggressive tumors are involved in the expansion of TATreg in vivo, contributing to tumor immune tolerance and poor clinical outcome. Thus, targeting pDC to restore their IFN-α production may represent an attractive therapeutic strategy to overcome immune tolerance in breast cancer.

  4. Controlling nuclear JAKs and STATs for specific gene activation by IFN{gamma}

    SciTech Connect

    Noon-Song, Ezra N.; Ahmed, Chulbul M.; Dabelic, Rea; Canton, Johnathan; Johnson, Howard M.

    2011-07-08

    Highlights: {yields} Gamma interferon (IFN{gamma}) and its receptor subunit, IFNGR1, interact with the promoter region of IFN{gamma}-associated genes along with transcription factor STAT1{alpha}. {yields} We show that activated Janus kinases pJAK2 and pJAK1 also associate with IFNGR1 in the nucleus. {yields} The activated Janus kinases are responsible for phosphorylation of tyrosine 41 on histone H3, an important epigenetic event for specific gene activation. -- Abstract: We previously showed that gamma interferon (IFN{gamma}) and its receptor subunit, IFNGR1, interacted with the promoter region of IFN{gamma}-activated genes along with transcription factor STAT1{alpha}. Recent studies have suggested that activated Janus kinases pJAK2 and pJAK1 also played a role in gene activation by phosphorylation of histone H3 on tyrosine 41. This study addresses the question of the role of activated JAKs in specific gene activation by IFN{gamma}. We carried out chromatin immunoprecipitation (ChIP) followed by PCR in IFN{gamma} treated WISH cells and showed association of pJAK1, pJAK2, IFNGR1, and STAT1 on the same DNA sequence of the IRF-1 gene promoter. The {beta}-actin gene, which is not activated by IFN{gamma}, did not show this association. The movement of activated JAK to the nucleus and the IRF-1 promoter was confirmed by the combination of nuclear fractionation, confocal microscopy and DNA precipitation analysis using the biotinylated GAS promoter. Activated JAKs in the nucleus was associated with phosphorylated tyrosine 41 on histone H3 in the region of the GAS promoter. Unphosphorylated JAK2 was found to be constitutively present in the nucleus and was capable of undergoing activation in IFN{gamma} treated cells, most likely via nuclear IFNGR1. Association of pJAK2 and IFNGR1 with histone H3 in IFN{gamma} treated cells was demonstrated by histone H3 immunoprecipitation. Unphosphorylated STAT1 protein was associated with histone H3 of untreated cells. IFN

  5. A synergistic effect of oestradiol and prolactin influencing the incidence of 3-methylcholanthrene induced cerivical carcinomas in mice.

    PubMed

    Forsberg, J G; Breistein, L S

    1976-09-01

    Castrated NMRI mice were laparotomized and a thread impregnated with beeswax-methylcholanthrene was inserted into the uterine cervix. Beginning on the day of operation and for a further 5 days the animals were injected with oestradiol, prolactin, oestradiol-prolactin, oestradiol-prolactin-progesterone, or the solvents for the hormones only. One group of animals were injected with oestradiol-prolactin for 6 days and later with progesterone every third day until death. The animals were killed one or 4 weeks after the operation. Among the one-week animals the number of cervices presenting epithelial downgrowths ("buds") into the stroma was higher after treatment with a combination of oestradiol and prolactin than after treatment with each hormone separately or among the controls. Four weeks after operation, the incidence of squamous cervical carcinomas was seen to be significantly higher among animals injected with both oestradiol and prolactin than in controls or in those injected with oestradiol or prolactin alone. Progesterone had no definite effect on the oestradiol-prolactin induced incidence. The mechanism behind the synergistic effect of prolactin and oestradiol is discussed.

  6. IFN-γ and IL-17 production in Experimental Autoimmune Encephalomyelitis depends on local APC•T cell complement production*

    PubMed Central

    Liu, Jinbo; Lin, Feng; Strainic, Michael G.; An, Fengqi; Miller, Robert H.; Altuntas, Cengiz Z.; Heeger, Peter S.; Tuohy, Vincent K.; Medof, M. Edward

    2013-01-01

    Summary IFN-γ and IL-17 producing T cells autoreactive across myelin components are central to the pathogenesis of multiple sclerosis (MS). Using direct in vivo, adoptive transfer, and in vitro systems, here we show that the generation of these effectors in MOG35–55 induced EAE depends on interactions of locally produced C3a/C5a with APC and T cell C3aR/C5aR. In the absence of the cell surface C3/C5 convertase inhibitor decay accelerating factor (DAF) but not the combined absence of DAF and C5aR and/or C3aR on APC and T cells, a heightened local autoimmune response occurs in which myelin destruction is markedly augmented in concert with markedly more IFN-γ+ and IL-17+ T cell generation. The augmented T cell response is due to increased IL-12 and IL-23 elaboration by APCs together with increased T cell expression of the receptors for each cytokine. The results apply to initial generation of the IL-17 phenotype since naïve CD62Lhi Daf1−/− T cells produce 3-fold more IL-17 in response to TGF-β and IL-6 while CD62Lhi Daf1−/−C5aR−/−C3aR−/− T cells produce 4-fold less. PMID:18424707

  7. IL-2 phosphorylates STAT5 to drive IFN-γ production and activation of human dendritic cells.

    PubMed

    Herr, Florence; Lemoine, Roxane; Gouilleux, Fabrice; Meley, Daniel; Kazma, Ihab; Heraud, Audrey; Velge-Roussel, Florence; Baron, Christophe; Lebranchu, Yvon

    2014-06-15

    Human dendritic cells (hDCs) produce IL-2 and express IL-2R α-chain (CD25), but the role of IL-2 in DC functions is not well defined. A recent study suggested that the main function of CD25 on hDCs was to transpresent IL-2 to activate T lymphocytes. Our results demonstrate the expression of the three chains of the IL-2R on hDCs and that IL-2 induces STAT5 phosphorylation. Interestingly, use of inhibitors of p-STAT5 revealed that IL-2 increases LPS-induced IFN-γ through STAT5 phosphorylation. Finally, we report that IL-2 increases the ability of hDCs to activate helpless CD8(+) T cells, most likely because of IL-2-triggered IFN-γ synthesis, as we previously described. For the first time, to our knowledge, we disclose that IL-2 induces monocyte-derived hDC's functional maturation and activation through IL-2R binding. Interestingly, our study suggests a direct effect of anti-CD25 mAbs on hDCs that may contribute to their clinical efficacy.

  8. Activation of TLR4 is required for the synergistic induction of dual oxidase 2 and dual oxidase A2 by IFN-γ and lipopolysaccharide in human pancreatic cancer cell lines.

    PubMed

    Wu, Yongzhong; Lu, Jiamo; Antony, Smitha; Juhasz, Agnes; Liu, Han; Jiang, Guojian; Meitzler, Jennifer L; Hollingshead, Melinda; Haines, Diana C; Butcher, Donna; Roy, Krishnendu; Doroshow, James H

    2013-02-15

    Pancreatitis is associated with release of proinflammatory cytokines and reactive oxygen species and plays an important role in the development of pancreatic cancer. We recently demonstrated that dual oxidase (Duox)2, an NADPH oxidase essential for reactive oxygen species-related, gastrointestinal host defense, is regulated by IFN-γ-mediated Stat1 binding to the Duox2 promoter in pancreatic tumor lines. Because LPS enhances the development and invasiveness of pancreatic cancer in vivo following TLR4-related activation of NF-κB, we examined whether LPS, alone or combined with IFN-γ, regulated Duox2. We found that upregulation of TLR4 by IFN-γ in BxPC-3 and CFPAC-1 pancreatic cancer cells was augmented by LPS, resulting in activation of NF-κB, accumulation of NF-κB (p65) in the nucleus, and increased binding of p65 to the Duox2 promoter. TLR4 silencing with small interfering RNAs, as well as two independent NF-κB inhibitors, attenuated LPS- and IFN-γ-mediated Duox2 upregulation in BxPC-3 cells. Induction of Duox2 expression by IFN-γ and LPS may result from IFN-γ-related activation of Stat1 acting in concert with NF-κB-related upregulation of Duox2. Sustained extracellular accumulation of H(2)O(2) generated by exposure to both LPS and IFN-γ was responsible for an ∼50% decrease in BxPC-3 cell proliferation associated with a G(1) cell cycle block, apoptosis, and DNA damage. We also demonstrated upregulation of Duox expression in vivo in pancreatic cancer xenografts and in patients with chronic pancreatitis. These results suggest that inflammatory cytokines can interact to produce a Duox-dependent pro-oxidant milieu that could increase the pathologic potential of pancreatic inflammation and pancreatic cancer cells.

  9. Presence of HIV-1 Gag-specific IFN-gamma+IL-2+ and CD28+IL-2+ CD4 T cell responses is associated with nonprogression in HIV-1 infection.

    PubMed

    Boaz, Mark J; Waters, Anele; Murad, Shahed; Easterbrook, Philippa J; Vyakarnam, Annapurna

    2002-12-01

    HIV immunity is likely CD4 T cell dependent. HIV-specific CD4 T cell proliferative responses are reported to correlate inversely with virus load and directly with specific CD8 responses. However, the phenotype and cytokine profile of specific CD4 T cells that correlate with disease is unknown. We compared the number/function of Gag p24-specific CD4 T cells in 17 HIV-infected long-term nonprogressors (LTNPs) infected for a median of 14.6 years with those of 16 slow progressors (SPs), also HIV infected for a median of 14 years but whose CD4 count had declined to <500 cells/ micro l. Compared with SPs, LTNPs had higher numbers of specific CD4s that were double positive for IFN-gamma and IL-2 as well as CD28 and IL-2. However, CD4 T cells that produced IL-2 alone (IL-2(+)IFN-gamma(-)) or IFN-gamma alone (IFN-gamma(+)IL-2(-)) did not differ between LTNPs and SPs. The decrease in p24-specific CD28(+)IL-2(+) cells with a concomitant increase of p24-specific CD28(-)IL-2(+) cells occurred before those specific for a non-HIV Ag, CMV. p24-specific CD28(-)IL-2(+) cells were evident in LTNPs and SPs, whereas the CMV-specific CD28(-)IL-2(+) response was confined to SPs. The difference between LTNPs and SPs in the Gag p24 IFN-gamma(+)IL-2(+) response was maintained when responses to total Gag (p17 plus p24) were measured. The percentage and absolute number of Gag-specific IFN-gamma(+)IL-2(+) but not of IFN-gamma(+)IL-2(-) CD4s correlated inversely with virus load. The Gag-specific IFN-gamma(+)IL-2(+) CD4 response also correlated positively with the percentage of Gag-specific IFN-gamma(+) CD8 T cells in these subjects. Accumulation of specific CD28(-)IL-2(+) helpers and loss of IFN-gamma(+)IL-2(+) CD4 T cells may compromise specific CD8 responses and, in turn, immunity to HIV.

  10. The Complement Anaphylatoxins C5a and C3a Suppress IFN-β Production in Response to Listeria monocytogenes by Inhibition of the Cyclic Dinucleotide-Activated Cytosolic Surveillance Pathway.

    PubMed

    Mueller-Ortiz, Stacey L; Calame, Daniel G; Shenoi, Nancy; Li, Yi-Dong; Wetsel, Rick A

    2017-03-08

    Listeria monocytogenes is an intracellular Gram-positive bacterium that induces expression of type I IFNs (IFN-α/IFN-β) during infection. These cytokines are detrimental to the host during infection by priming leukocytes to undergo L. monocytogenes-mediated apoptosis. Our previous studies showed that C5aR1(-/-) and C3aR(-/-) mice are highly susceptible to L. monocytogenes infection as a result of increased IFN-β-mediated apoptosis of major leukocyte cell populations, including CD4(+) and CD8(+) T cells. However, the mechanisms by which C3a and C5a modulate IFN-β expression during L. monocytogenes infection were not examined in these initial investigations. Accordingly, we report in this article that C5a and C3a suppress IFN-β production in response to L. monocytogenes via cyclic di-AMP (c-di-AMP), a secondary messenger molecule of L. monocytogenes, in J774A.1 macrophage-like cells and in bone marrow-derived dendritic cells (BMDCs). Moreover, C5a and C3a suppress IFN-β production by acting through their respective receptors, because no inhibition was seen in C5aR1(-/-) or C3aR(-/-) BMDCs, respectively. C5a and C3a suppress IFN-β production in a manner that is dependent on Bruton's tyrosine kinase, p38 MAPK, and TANK-binding kinase 1 (TBK1), as demonstrated by the individual use of Bruton's tyrosine kinase, p38 MAPK, and TBK1 inhibitors. Pretreatment of cells with C5a and C3a reduced the expression of the IFN-β signaling molecules DDX41, STING, phosphorylated TBK1, and phosphorylated p38 MAPK in wild-type BMDCs following treatment with c-di-AMP. Collectively, these data demonstrate that C3a and C5a, via direct signaling through their specific receptors, suppress IFN-β expression by modulation of a distinct innate cytosolic surveillance pathway involving DDX41, STING, and other downstream molecular targets of L. monocytogenes-generated c-di-AMP.

  11. Adaptation of IFN-gamma ELISA and ELISPOT tests for feline tuberculosis.

    PubMed

    Rhodes, Shelley G; Gruffydd-Jones, Tim; Gunn-Moore, Danièlle; Jahans, Keith

    2008-08-15

    There are currently no reliable immunodiagnostic tests for feline tuberculosis. Infection of domestic cats in the UK is thought to occur via their contact with the relevant reservoir of infection, e.g. cattle and badgers for Mycobacterium bovis, and rodents for M. microti. In the African National Parks, where M. bovis infection of Bovidae is an increasing problem, transmission to big cats is occurring via their ingestion of infected carcasses. We have adapted feline ELISA and ELISPOT assays to potentially provide the first cell-based diagnostic test for the detection of tuberculosis in cats. We tested peripheral blood mononuclear cell antigen-specific IFN-gamma responses of 18 cats suspected of mycobacterial infection for which biopsy material was co-submitted to the Veterinary Laboratories Agency for mycobacterial culture and identification. Seventeen cats were tested by ELISA while seven cats were tested by ELISPOT (six cats were tested by both ELISA and ELISPOT). Six healthy control cats provided baseline data for these tests. Responses to bovine and avian tuberculins (PPDB and PPDA) and a protein cocktail of ESAT6 and CFP10 were measured, together with positive mitogen (PMA and calcium ionophore) and negative (medium) controls. Overall, both ELISPOT and ELISA tests were found to be suitable for generating rapid results (2 and 4 days, respectively), which provided good predictive information for M. bovis and M. microti infections, but were unable to reliably discern M. avium infection.

  12. Tc-99m labeled anti CEA F(ab{prime}){sub 2}: Augmenting tumor uptake with anitbody:Interferon (MAb:IFN-{alpha}-2b) conjugate

    SciTech Connect

    Thakur, M.L.; Donegan, M.; Li, J.

    1996-05-01

    Previously we have shown that IFN-{alpha}-2b, a potent biological response modifier (BRM) significantly enhanced tumor uptake of radiolabeled MAbs. Results were attributed to increased blood flow and upregulation of cell surface receptor glycoproteins. Probably because of the blood flow increase in all organs, radioactivity levels in other tissues, although to a lesser extent, were also enhanced. In order to validate our hypothesis that selective tumor targeting of IFN-{alpha}-2b (Schering-Plough, N.J.) may enhance tumor uptake and diminish uptake in normal organs, IFN-{alpha}-2b was conjugated with anti CEA MAb F-6 (IgG2a), by reacting with 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate (CMC) and N-hydroxysulfosuccinimide (NHS) in a 1:2:50:50 ratio by weight at pH 9.2 (22{degrees}C) for 24 hr. The conjugate (30{mu}g) was administered i.v. to nude mice bearing human colorectal carcinoma LS174T, 3hr prior to the i.v. administration of 300uCi / 20ug (1.5Ci / uM) F-6F(ab{prime}){sub 2}. Animals not receiving the conjugate served as controls. The conjugation efficiency as determined using I-125-IFN-{alpha}-2b was >70% and MAb:IFN-{alpha}-2b ratio was approximately 1:1. In animals receiving IFN conjugate tumor uptake enhanced by 180% and liver uptake decreased to 56%. Radioactivity also decreased in most other tissues. Furthermore with conjugate, the tumor/muscle ratio increased by 210% (10 {plus_minus} 1.2 Vs 4.7 {plus_minus} 0.5), tumor/blood ratio by 220% (5.4 {plus_minus} 0.6 Vs 2.5 {plus_minus} 0.3), and tumor/liver ratio by 317% (0.7 {plus_minus} 0.1 Vs 0.2 {plus_minus} 0.05).

  13. Association of IFN-γ and P2X7 Receptor Gene Polymorphisms in Susceptibility to Tuberculosis Among Iranian Patients.

    PubMed

    Shamsi, Mahdi; Zolfaghari, Mohammad Reza; Farnia, Parissa

    2016-03-01

    Interferon-gamma (IFN-γ) and P2X7 receptor are crucial for host defence against mycobacterial infections. Recent studies have indicated that IFN-γ, IFN-γ receptor 1 (IFN-γR1) andP2X7 gene polymorphisms are associated with susceptibility to pulmonary tuberculosis (TB). However, the relationship between IFN-γ and P2X7 polymorphism and TB susceptibility remains inconclusive in Iranian population. For this reason, single nucleotide polymorphisms (SNPs) in IFN-γ (G+2109A), IFN-γR1 (G-611A) and P2X7 genes (at -762, 1513 position) in patients (n = 100) were assessed using PCR-RFLP. Data were analysed with SPSS version 18. For the 2109 loci of IFN-γ gene, the frequency of mutant alleles between patients and controls were not statistically significant. However, there was a significant difference between the TB patient and controls for -611 alleles of IFN-γR1 (P = 0.01). Additionally, the frequency of P2X7 gene polymorphisms (SNP-762 and 1513) between patients and controls was statistically significant. In conclusions, our study revealed a significant association of IFN-γR1 and P2X7 genes polymorphisms with risk of developing TB in Iranian population.

  14. Intradermal cytosine-phosphate-guanosine treatment reduces lung inflammation but induces IFN-γ-mediated airway hyperreactivity in a murine model of natural rubber latex allergy.

    PubMed

    Haapakoski, Rita; Karisola, Piia; Fyhrquist, Nanna; Savinko, Terhi; Wolff, Henrik; Turjanmaa, Kristiina; Palosuo, Timo; Reunala, Timo; Lauerma, Antti; Alenius, Harri

    2011-05-01

    Asthma and other allergic diseases are continuously increasing, causing considerable economic and sociologic burden to society. The hygiene hypothesis proposes that lack of microbial T helper (Th) 1-like stimulation during early childhood leads to increased Th2-driven allergic disorders later in life. Immunostimulatory cytosine-phosphate-guanosine (CpG)-oligodeoxynucleotide motifs are candidate molecules for immunotherapeutic studies, as they have been shown to shift the Th2 response toward the Th1 direction and reduce allergic symptoms. Using natural rubber latex (NRL)-induced murine model of asthma, we demonstrated that intradermal CpG administration with allergen reduced pulmonary eosinophilia, mucus production, and Th2-type cytokines, but unexpectedly induced airway hyperreactivity (AHR) to inhaled methacholine, one of the hallmarks of asthma. We found that induction in AHR was dependent on STAT4, but independent of STAT6 signaling. CpG treatment increased production of IFN-γ in the airways and shifted the ratio of CD4(+):CD8(+) T cells toward CD8(+) dominance. By blocking soluble IFN-γ with neutralizing antibody, AHR diminished and the CD4(+):CD8(+) ratio returned to CD4(+) dominance. These results indicate that increased production of IFN-γ in the lungs may lead to severe side effects, such as enhancement of bronchial hyperreactivity to inhaled allergen. This finding should be taken into consideration when planning prophylaxis treatment of asthma with intradermal CpG injections.

  15. Identification of type I IFN in Chinese giant salamander (Andrias davidianus) and the response to an iridovirus infection.

    PubMed

    Chen, Qian; Ma, Jie; Fan, Yuding; Meng, Yan; Xu, Jin; Zhou, Yong; Liu, Wenzhi; Zeng, Xianhui; Zeng, Lingbing

    2015-06-01

    The type I IFNs play a major role in the first line of defense against virus infections. In this study, the type I IFN gene designated gsIFN was identified and characterized in the Chinese giant salamander (Andrias davidianus). The genomic DNA of gsIFN contains 5 exons and 4 introns and has a total length of 5622 bp. The full-length cDNA sequence of gsIFN is 1113 bp and encodes a putative protein of 186 amino acids that has a 43% identity to type I IFN of Xenopus tropicalis. The deduced amino acid sequence has the C-terminal CAWE motif, that is mostly conserved in the higher vertebrate type I IFNs. Real-time fluorescence quantitative RT-PCR analysis revealed broad expression of gsIFN in vivo and the highest level expression in blood, kidney and spleen. Additionally, the expression of gsIFN at the mRNA level was significantly induced in peripheral blood leucocytes after stimulation with poly I:C and after infection with the Chinese giant salamander iridovirus (GSIV). A plasmid expressing gsIFN was constructed and transfected into the Chinese giant salamander muscle cell line. Expression of the IFN-inducible gene Mx was up-regulated in the gsIFN-overexpressing cells after GSIV infection. The virus load and titer were significantly reduced compared with that in control cells. Additionally, a lower level of virus major capsid protein synthesis was confirmed by immunofluorescence assay compared to the control cells. These results suggest that the gsIFN gene plays an important role in the antiviral innate immune response.

  16. The impact of IFN-γ receptor on SLPI expression in active tuberculosis: association with disease severity.

    PubMed

    Tateosian, Nancy L; Pasquinelli, Virginia; Hernández Del Pino, Rodrigo E; Ambrosi, Nella; Guerrieri, Diego; Pedraza-Sánchez, Sigifredo; Santucci, Natalia; D'Attilio, Luciano; Pellegrini, Joaquín; Araujo-Solis, María A; Musella, Rosa M; Palmero, Domingo J; Hernandez-Pando, Rogelio; Garcia, Verónica E; Chuluyan, H Eduardo

    2014-05-01

    Interferon (IFN)-γ displays a critical role in tuberculosis (TB), modulating the innate and adaptive immune responses. Previously, we reported that secretory leukocyte protease inhibitor (SLPI) is a pattern recognition receptor with anti-mycobacterial activity against Mycobacterium tuberculosis (Mtb). Herein, we determined whether IFN-γ modulated the levels of SLPI in TB patients. Plasma levels of SLPI and IFN-γ were studied in healthy donors (HDs) and TB patients. Peripheral blood mononuclear cells from HDs and patients with TB or defective IFN-γ receptor 1* were stimulated with Mtb antigen and SLPI, and IFN-γR expression levels were measured. Both SLPI and IFN-γ were significantly enhanced in plasma from those with TB compared with HDs. A direct association between SLPI levels and the severity of TB was detected. In addition, Mtb antigen stimulation decreased the SLPI produced by peripheral blood mononuclear cells from HDs, but not from TB or IFN-γR patients. Neutralization of IFN-γ reversed the inhibition of SLPI induced by Mtb antigen in HDs, but not in TB patients. Furthermore, recombinant IFN-γ was unable to modify the expression of SLPI in TB patients. Finally, IFN-γR expression was lower in TB compared with HD peripheral blood mononuclear cells. These results show that Mtb-induced IFN-γ down-modulated SLPI levels by signaling through the IFN-γR in HDs. This inhibitory mechanism was not observed in TB, probably because of the low expression of IFN-γR detected in these individuals.

  17. Dengue Virus Control of Type I IFN Responses: A History of Manipulation and Control

    PubMed Central

    Castillo Ramirez, Jorge Andrés

    2015-01-01

    The arthropod-borne diseases caused by dengue virus (DENV) are a major and emerging problem of public health worldwide. Infection with DENV causes a series of clinical manifestations ranging from mild flu syndrome to severe diseases that include hemorrhage and shock. It has been demonstrated that the innate immune response plays a key role in DENV pathogenesis. However, in recent years, it was shown that DENV evades the innate immune response by blocking type I interferon (IFN-I). It has been demonstrated that DENV can inhibit both the production and the signaling of IFN-I. The viral proteins, NS2A and NS3, inhibit IFN-I production by degrading cellular signaling molecules. In addition, the viral proteins, NS2A, NS4A, NS4B, and NS5, can inhibit IFN-I signaling by blocking the phosphorylation of the STAT1 and STAT2 molecules. Finally, NS5 mediates the degradation of STAT2 using the proteasome machinery. In this study, we briefly review the most recent insights regarding the IFN-I response to DENV infection and its implication for pathogenesis. PMID:25629430

  18. The IFN-γ-induced Transcriptional Program of the CIITA Gene is Inhibited by Statins

    PubMed Central

    Lee, Sun Jung; Qin, Hongwei; Benveniste, Etty N.

    2009-01-01

    Summary Statins are 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors that exert anti-inflammatory effects. IFN-γ induction of class II MHC expression, which requires the class II transactivator (CIITA), is inhibited by statins, however, the molecular basis for suppression is undetermined. We describe that statins inhibit IFN-γ-induced class II MHC expression by suppressing CIITA gene expression, which is dependent on the HMG-CoA reductase pathway. In addition, CIITA expression is inhibited by GGTI-298 or Clostridium difficile Toxin A, specific inhibitors of Rho family protein prenylation, indicating the involvement of small GTPases. Rac1 is involved in IFN-γ inducible expression of CIITA, and statins inhibit IFN-γ-induced Rac1 activation, contributing to the inhibitory effect of statins. IFN-γ induction of the CIITA gene is regulated by the transcription factors STAT-1α, IRF-1 and USF-1. We previously reported that statins inhibit constitutive STAT-1α expression. IRF-1, a STAT-1 dependent gene, is also inhibited by statins. Therefore, statin treatment results in decreased recruitment of STAT-1α and IRF-1 to the endogenous CIITA pIV promoter. The recruitment of USF-1 to CIITA pIV is also reduced by statins, as is the recruitment of RNA Polymerase II, p300 and Brg-1. These data indicate that statins inhibit the transcriptional program of the CIITA gene. PMID:18601229

  19. Newcastle Disease Virus V Protein Targets Phosphorylated STAT1 to Block IFN-I Signaling

    PubMed Central

    Qiu, Xusheng; Fu, Qiang; Meng, Chunchun; Yu, Shengqing; Zhan, Yuan; Dong, Luna; Song, Cuiping; Sun, Yingjie; Tan, Lei; Hu, Shunlin; Wang, Xiaoquan; Liu, Xiaowen; Peng, Daxin; Liu, Xiufan; Ding, Chan

    2016-01-01

    Newcastle disease virus (NDV) V protein is considered as an effector for IFN antagonism, however, the mechanism remains unknown. In this study, the expression of STAT1 and phospho-STAT1 in cells infected with NDV or transfected with V protein-expressing plasmids were analyzed. Our results showed that NDV V protein targets phospho-STAT1 reduction in the cells depends on the stimulation of IFN-α. In addition, a V-deficient genotype VII recombinant NDV strain rZJ1-VS was constructed using reverse genetic technique to confirm the results. The rZJ1-VS lost the ability to reduce phospho-STAT1 and induced higher expression of IFN-responsive genes in infected cells. Furthermore, treatment with an ubiquitin E1 inhibitor PYR-41 demonstrated that phospho-STAT1 reduction was caused by degradation, but not de-phosphorylation. We conclude that NDV V protein targets phospho-STAT1 degradation to block IFN-α signaling, which adds novel knowledge to the strategies used by paramyxoviruses to evade IFN. PMID:26859759

  20. Bacterial Muramyl Dipeptide (MDP) Restricts Human Cytomegalovirus Replication via an IFN-β-Dependent Pathway

    PubMed Central

    Kapoor, Arun; Fan, Yi-Hsin; Arav-Boger, Ravit

    2016-01-01

    We recently reported that induction of NOD2 by human Cytomegalovirus (HCMV) resulted in virus inhibition and upregulation of antiviral and inflammatory cytokines. Here we investigated the effects of muramyl dipeptide (MDP), a bacterial cell wall component that activates NOD2, on HCMV replication and antiviral responses. HCMV infection of human foreskin fibroblasts induced NOD2, the downstream receptor-interacting serine/threonine-protein kinase 2 (RIPK2), resulting in phosphorylation of TANK-binding kinase 1 (TBK1) and interferon regulatory factor 3 (IRF3). MDP treatment following infection at low multiplicity (MOI = 0.1 PFU/cell) inhibited HCMV in a dose-dependent manner and further induced phosphorylation of TBK1, IRF3 and expression of IFN-β. None of these effects of MDP were observed following infection at multiplicity of 1. In infected NOD2 knocked-down cells MDP did not induce IFN-β, irrespective of MOI. Treatment with MDP before infection also inhibited HCMV, an effect augmented with treatment duration. Treatment with an IFN-β receptor blocking antibody or knockdown of IFN-β significantly attenuated the inhibitory effect of MDP on HCMV. MDP treatment before or after infection with herpesvirus 1 did not inhibit its replication. Summarized, NOD2 activation exerts anti-HCMV activities predominantly via IFN-β. Since MDP is a bacterial cell wall component, ongoing microbial exposure may influence HCMV replication. PMID:26830977

  1. The core protein of a pestivirus protects the incoming virus against IFN-induced effectors

    PubMed Central

    Riedel, Christiane; Lamp, Benjamin; Hagen, Benedikt; Indik, Stanislav; Rümenapf, Till

    2017-01-01

    A multitude of viral factors - either inhibiting the induction of the IFN-system or its effectors – have been described to date. However, little is known about the role of structural components of the incoming virus particle in protecting against IFN-induced antiviral factors during or immediately after entry. In this study, we take advantage of the previously reported property of Classical swine fever virus (family Flaviviridae, genus Pestivirus) to tolerate a deletion of the core protein if a compensatory mutation is present in the NS3-helicase-domain (Vp447∆c). In contrast to the parental virus (Vp447), which causes a hemorrhagic-fever-like disease in pigs, Vp447∆c is avirulent in vivo. In comparison to Vp447, growth of Vp447∆c in primary porcine cells and IFN-treated porcine cell lines was reduced >20-fold. Also, primary porcine endothelial cells and IFN-pretreated porcine cell lines were 8–24 times less susceptible to Vp447∆c. This reduction of susceptibility could be partially reversed by loading Vp447∆c particles with different levels of core protein. In contrast, expression of core protein in the recipient cell did not have any beneficial effect. Therefore, a protective effect of core protein in the incoming virus particle against the products of IFN-stimulated genes could be demonstrated. PMID:28290554

  2. Bacterial Muramyl Dipeptide (MDP) Restricts Human Cytomegalovirus Replication via an IFN-β-Dependent Pathway.

    PubMed

    Kapoor, Arun; Fan, Yi-Hsin; Arav-Boger, Ravit

    2016-02-02

    We recently reported that induction of NOD2 by human Cytomegalovirus (HCMV) resulted in virus inhibition and upregulation of antiviral and inflammatory cytokines. Here we investigated the effects of muramyl dipeptide (MDP), a bacterial cell wall component that activates NOD2, on HCMV replication and antiviral responses. HCMV infection of human foreskin fibroblasts induced NOD2, the downstream receptor-interacting serine/threonine-protein kinase 2 (RIPK2), resulting in phosphorylation of TANK-binding kinase 1 (TBK1) and interferon regulatory factor 3 (IRF3). MDP treatment following infection at low multiplicity (MOI = 0.1 PFU/cell) inhibited HCMV in a dose-dependent manner and further induced phosphorylation of TBK1, IRF3 and expression of IFN-β. None of these effects of MDP were observed following infection at multiplicity of 1. In infected NOD2 knocked-down cells MDP did not induce IFN-β, irrespective of MOI. Treatment with MDP before infection also inhibited HCMV, an effect augmented with treatment duration. Treatment with an IFN-β receptor blocking antibody or knockdown of IFN-β significantly attenuated the inhibitory effect of MDP on HCMV. MDP treatment before or after infection with herpesvirus 1 did not inhibit its replication. Summarized, NOD2 activation exerts anti-HCMV activities predominantly via IFN-β. Since MDP is a bacterial cell wall component, ongoing microbial exposure may influence HCMV replication.

  3. XBP-1 couples endoplasmic reticulum stress to augmented IFN-β induction via a cis-acting enhancer in macrophages

    PubMed Central

    Zeng, Ling; Liu, Yi-Ping; Sha, Haibo; Chen, Hui; Qi, Ling; Smith, Judith A.

    2010-01-01

    Perturbation of the endoplasmic reticulum (ER) results in a conserved stress response called the “Unfolded Protein Response” (UPR). Macrophages undergoing a UPR respond to LPS with log-fold increased production of IFN-β, a cytokine with diverse roles in innate and adaptive immunity. In this report, we found that thapsigargin-induced ER stress augmented recruitment of IRF-3, CBP/p300, and transcriptional machinery to the murine ifnb1 promoter during LPS stimulation. Although full synergistic IFN-β production requires XBP-1, this UPR-regulated transcription factor did not appreciably bind the ifnb1 promoter. However, XBP-1 bound a conserved site 6.1kb downstream of ifnb1, along with IRF-3 and CBP only during concomitant UPR and LPS stimulation. XBP-1 physically associates with p300, suggesting a mechanism of multi-molecular assembly at the +6.1kb site. Luciferase reporter assays provide evidence this +6kb region functions as an XBP-1-dependent enhancer of ifnb1 promoter activity. Thus, this study identifies a novel role for an UPR-dependent transcription factor in the regulation of an inflammatory cytokine. Our findings have broader mechanistic implications for the pathogenesis of diseases involving ER stress and type I interferon, including viral infection, ischemia-reperfusion injury, protein-misfolding and inflammatory diseases. PMID:20660350

  4. Natural killer cell intrinsic toll-like receptor MyD88 signaling contributes to IL-12-dependent IFN-γ production by mice during infection with Toxoplasma gondii.

    PubMed

    Ge, Yiyue; Chen, Jinling; Qiu, Xiaoyan; Zhang, Jie; Cui, Lunbiao; Qi, Yuhua; Liu, Xinjian; Qiu, Jingfan; Shi, Zhiyang; Lun, Zhaorong; Shen, Jilong; Wang, Yong

    2014-06-01

    Myeloid differentiation factor 88 (MyD88)-dependent IL-12 secretion by dendritic cells is critical for natural killer cell-mediated IFN-γ production and innate resistance to Toxoplasma gondii. Although MyD88(-/-) mice challenged with T. gondii have defective IL-12 responses and succumb to infection, administration of IL-12 to MyD88(-/-) mice fails to prevent acute mortality, suggesting that MyD88 may mediate signals within natural killer cells important for IL-12-dependent IFN-γ production and innate resistance to this parasite. In this study, we found that T. gondii antigens and IL-12 could synergistically trigger IFN-γ secretion by natural killer cells, which was dependent on toll-like receptor-MyD88 signaling. Further analysis showed that p38 mitogen-activated protein kinase, extracellular signal-regulated kinase, c-Jun N-terminal kinase and NF-κB multiple pathways downstream of MyD88 contributed to IFN-γ production by natural killer cells. Moreover, the well-established toll-like receptor agonists, T. gondii profilin (Tgprofilin) and T. gondii heat shock protein 70 (TgHSP70) could evoke a similar IFN-γ secretory response in natural killer cells to that evoked by T. gondii antigens. In vivo adoptive transfer experiments showed that, upon challenge with T. gondii, NOD/SCID-β2 microglobulin null (NOD/SCID-β2m(-/-)) mice injected i.v. with MyD88(-/-) natural killer cells had reduced serum IFN-γ levels and increased splenic tachyzoite burdens compared with those injected i.v. with wild-type natural killer cells. Taken together, these findings demonstrate a critical role for natural killer cell intrinsic toll-like receptor-MyD88 signaling in IL-12-dependent early IFN-γ production and innate resistance to T. gondii.

  5. Unique palindromic sequences in synthetic oligonucleotides are required to induce IFN [correction of INF] and augment IFN-mediated [correction of INF] natural killer activity.

    PubMed

    Yamamoto, S; Yamamoto, T; Kataoka, T; Kuramoto, E; Yano, O; Tokunaga, T

    1992-06-15

    Thirty-mer single-stranded oligonucleotides, with a sequence chosen from the known cDNA encoding the 64-kDa protein named Ag A or the MPB-70 protein of Mycobacterium bovis BCG and the human cellular proteins such as complement component 1 inhibitor and Ig rearranged lambda-chain, were used to dissect the capability to induce IFN and to augment NK cell activity of mouse spleen cells by coincubation in vitro. Three with the hexamer palindromic sequence as GACGTC were active, whereas two kinds of oligonucleotides with no palindrome were inactive. The oligonucleotides containing at least one of the different palindromic sequences showed no activity. When a portion of the sequence of the inactive oligonucleotides was substituted with either palindromic sequence of GACGTC, AGCGCT, or AACGTT, the oligonucleotide acquired the ability to augment NK activity. In contrast, the oligonucleotides substituted with another palindromic sequence such as ACCGGT was without effect. Furthermore, exchange of two neighboring mononucleotides within, but not outside, the active palindromic sequence destroyed the ability of the oligonucleotides to augment NK cell activity. Stimulation of spleen cells with the substituted oligonucleotide, A4a-AAC, induced production of significant amounts of IFN-alpha/beta and small amounts of IFN-gamma. Augmentation of NK activity of the cells by the oligonucleotide was ascribed to IFN-alpha/beta production. These results strongly suggest that the presence of the unique palindromic sequences, such as GACGTC, AGCGCT, and AACGTT, but not ACCGGT, is essential for the immunostimulatory activity of oligonucleotides.

  6. IFN-induced Guanylate Binding Proteins in Inflammasome Activation and Host Defense

    PubMed Central

    Kim, Bae-Hoon; Chee, Jonathan D.; Bradfield, Clinton J.; Park, Eui-Soon; Kumar, Pradeep; MacMicking, John D.

    2016-01-01

    Traditional views of the inflammasome highlight pre-existing core components being assembled under basal conditions shortly after infection or tissue damage. Recent work, however, suggests the inflammasome machinery is also subject to tunable or inducible signals that may accelerate its autocatalytic properties and dictate where inflammasome assembly takes place in the cell. Many of these immune signals operate downstream of interferon (IFN) receptors to elicit inflammasome regulators, including a new family of IFN-induced GTPases termed guanylate binding proteins (GBPs). Here, we examine the critical roles for IFN-induced GBPs in directing inflammasome subtype-specific responses and their consequences for cell-autonomous immunity against a wide variety of microbial pathogens. We discuss emerging mechanisms of action and the potential impact of these GBPs on predisposition to sepsis and other infectious or inflammatory diseases. PMID:27092805

  7. Mycobacterial disease and impaired IFN-γ immunity in humans with inherited ISG15 deficiency

    PubMed Central

    Bogunovic, Dusan; Byun, Minji; Durfee, Larissa A.; Abhyankar, Avinash; Sanal, Ozden; Mansouri, Davood; Salem, Sandra; Radovanovic, Irena; Grant, Audrey V.; Adimi, Parisa; Mansouri, Nahal; Okada, Satoshi; Bryant, Vanessa L.; Kong, Xiao-Fei; Kreins, Alexandra; Velez, Marcela Moncada; Boisson, Bertrand; Khalilzadeh, Soheila; Ozcelik, Ugur; Darazam, Ilad Alavi; Schoggins, John W.; Rice, Charles M.; Al-Muhsen, Saleh; Behr, Marcel; Vogt, Guillaume; Puel, Anne; Bustamante, Jacinta; Gros, Philippe; Huibregtse, Jon M.; Abel, Laurent; Boisson-Dupuis, Stéphanie; Casanova, Jean-Laurent

    2012-01-01

    ISG15 is an interferon (IFN)-α/β-inducible, ubiquitin-like intracellular protein. Its conjugation to various proteins (ISGylation) contributes to antiviral immunity in mice. We describe human patients with inherited ISG15 deficiency and mycobacterial, but not viral diseases. The lack of intracellular ISG15 production and protein ISGylation was not associated with cellular susceptibility to any viruses tested, consistent with the lack of viral diseases in these patients. By contrast, the lack of mycobacterium-induced ISG15 secretion by leukocytes — granulocytes in particular — reduced the production of IFN-γ by lymphocytes, including natural killer cells, probably accounting for the enhanced susceptibility to mycobacterial disease. This experiment of Nature shows that human ISGylation is largely redundant for antiviral immunity, but that ISG15 plays an essential role as an IFN-γ-inducing secreted molecule for optimal antimycobacterial immunity. PMID:22859821

  8. The External Quality Assurance Oversight Laboratory (EQAPOL) proficiency program for IFN-gamma enzyme-linked immunospot (IFN-γ ELISpot) assay.

    PubMed

    Sanchez, Ana M; Rountree, Wes; Berrong, Mark; Garcia, Ambrosia; Schuetz, Alexandra; Cox, Josephine; Frahm, Nicole; Manak, Mark; Sarzotti-Kelsoe, Marcella; D'Souza, M Patricia; Denny, Thomas; Ferrari, Guido

    2014-07-01

    The interferon-gamma enzyme-linked immunospot (IFN-γ ELISpot) assay has been developed and used as an end-point assay in clinical trials for infectious diseases and cancer to detect the magnitude of antigen-specific immune responses. The ability to compare data generated by different laboratories across organizations is pivotal to understand the relative potency of different therapeutic and vaccine strategies. We developed an external proficiency program for the IFN-γ ELISpot assay that evaluates laboratory performance based on five parameters: timeliness for data reporting; ability to handle cellular samples; detection of background (non-specific) responses; accuracy to consensus of the results; and precision of the measurements. Points are awarded for each criterion, and the sum of the points is used to determine a numeric and adjectival performance rating. Importantly, the evaluation of the accuracy to the consensus mean for the detection of antigen-specific responses using laboratory-specific procedures informs each laboratory and its sponsor on the degree of concordance of its results with those obtained by other laboratories. This study will ultimately provide the scientific community with information on how to organize and implement an external proficiency program to evaluate longitudinally the performance of the participating laboratories and, therefore, fulfill the requirements of the GCLP guidelines for laboratories performing end-point IFN-γ ELISpot assay for clinical trials.

  9. [Effects of IFN-γ treatment on biological characteristics and functions of dendritic cells].

    PubMed

    Liu, Yanling; Wang, Hongmei; Yu, Yanrong; Yuan, Keng; Zhang, Yujuan; Min, Weiping

    2016-08-01

    Objective To investigate the effect of IFN-γ treatment on the biological characteristics and functions of C57BL/6 murine dendritic cells (DCs). Methods In the process of DC culture, 20 ng/mL IFN-γ was added in the DCs at the early (day 2) or late (day 5) stage, and on day 7, LPS was added to stimulate DC maturation. The expressions of DC surface molecules CD11c, CD80 and CD86 were determined by flow cytometry. To analyze cell functions, DCs were co-cultured with BALB/c mouse-derived lymphocyte cells. The 5, 6-carboxyfluorescein diacetate N-succinimidyl ester (CFSE) labelling was used to detect their ability to stimulate allogeneic lymphocyte proliferation and flow cytometry was used to measure their ability to induce the production of regulatory T cells (Tregs). Results Compared with the control group, the early IFN-γ treatment group had decreased DC number and inhibited cell differentiation; though there was no difference in the expression of co-stimulatory molecules, early IFN-γ treatment resisted the stimulatory effect of LPS on DC maturation, weakened the ability to stimulate allogeneic lymphocyte proliferation and enhanced the ability to induce more Tregs. Compared with the control group, the late IFN-γ treatment group showed no change in DC number and differentiation; the expressions of co-stimulatory molecules CD86 and CD80 were upregulated; the results of DC maturation and mixed allogeneic lymphocyte reaction stimulated by LPS were similar to those in the control group, but its ability to induce Tregs was stronger. Conclusion DCs treated with IFN-γ at early stage and those at late stage showed obvious difference in biological characteristics and functions.

  10. Effect of anluohuaxian tablet combined with gamma-IFN on schistosomal liver fibrosis.

    PubMed

    Huang, Jiaquan; Huang, Haiyan; Jiao, Yuntao; Ai, Guo; Huang, Tiejun; Li, Lan; Yu, Haijing; Ma, Ke; Xiao, Fei

    2009-02-01

    The therapeutic effects of anluohuaxian tablet combined with gamma-IFN on schistosomal liver fibrosis and its mechanism were studied in a murine model and clinical cases of schistosomal liver fibrosis. Fifty Kunming mice were randomly divided into 5 groups: normal control group, infection control group, anluohuaxian tablet-treated group, gamma-IFN-treated group and combined treatment (anluohuaian tablet+gamma-IFN) group. Pathologic changes in liver, including hepatic pigmentation and the size of schistosomal egg granuloma, were observed by HE staining after treatment for 8 weeks. The expression of the type I and collagen III, and TIMP-1 was detected by immunohistochemistry. TGF-beta1 mRNA expression was examined by real-time fluorescent quantitative PCR. Sixty patients with schistosomal liver fibrosis were divided into treatment group and control group. The patients in treatment group were treated with anluohuaxian tablet in combination with gamma-IFN for 6 months. Before and after treatment, the changes of symptoms and signs, liver function, serum liver fibrosis indexes and imaging indexes were observed. The results showed that as compared with infection control group, all forms of treatments relieved the hepatic pathological injury with apparently diminished size of schistosomal egg nodules and decreased percentage of pigmentation (P<0.05). Furthermore, the expression of collagen I and III, TIMP-1, and TGF-beta1 mRNA in combined treatment group was significantly decreased as compared with anluohuaxian tablet-treated and gamma-IFN-treated groups (P<0.05). In the clinical observation, the serum liver fibrosis indexes, the portal vein width as well as the spleen thickness was significantly reduced in treatment group as compared with control group (P<0.05). It was concluded that the combined use of anluohuaxian tablet with gamma-IFN in schistosomal liver fibrosis could protect liver function, alleviate liver fibrosis, and could be used as a choice in treating

  11. Lion (Panthera leo) and cheetah (Acinonyx jubatus) IFN-gamma sequences.

    PubMed

    Maas, Miriam; Van Rhijn, Ildiko; Allsopp, Maria T E P; Rutten, Victor P M G

    2010-04-15

    Cloning and sequencing of the full length lion and cheetah interferon-gamma (IFN-gamma) transcript will enable the expression of the recombinant cytokine, to be used for production of monoclonal antibodies and to set up lion and cheetah-specific IFN-gamma ELISAs. These are relevant in blood-based diagnosis of bovine tuberculosis, an important threat to lions in the Kruger National Park. Alignment of nucleotide and amino acid sequences of lion and cheetah and that of domestic cats showed homologies of 97-100%.

  12. MLN4924, a First-in-Class NEDD8-Activating Enzyme Inhibitor, Attenuates IFN-β Production.

    PubMed

    Song, Hui; Huai, Wanwan; Yu, Zhongxia; Wang, Wenwen; Zhao, Jing; Zhang, Lining; Zhao, Wei

    2016-04-01

    Neddylation is a posttranslational protein modification that conjugates ubiquitin-like protein neural precursor cell-expressed developmentally downregulated protein 8 (NEDD8) to target proteins and regulates diverse cellular processes. MLN4924, a novel NEDD8 activating enzyme inhibitor, which has emerged as a promising anticancer drug, has a multifaceted function by inhibiting the process of neddylation. However, the potential roles of MLN4924 and neddylation in IFN-β production remain unknown. In this study, we show that MLN4924 inhibits TLR3/4- and retinoic acid-inducible gene-I-induced IFN-β expression in different cells, whereas NEDD8 knockdown had no effects on IFN-β expression. The ability of the MLN4924 to inhibit IFN-β production was confirmed in vivo, as mice treated with MLN4924 exhibited decreased levels of IFN-β upon LPS or polyinosinic-polycytidylic acid stimulation. Furthermore, we show that MLN4924 inhibits IFN regulatory factor 3 (IRF3) transcriptional activation and prevents IRF3 binding to IFN-β promoter. Our findings suggest that MLN4924 inhibits TLR3/4- and retinoic acid-inducible gene-I-induced IFN-β expression by preventing IRF3 binding to the IFN-β promoter, with a neddylation-independent manner. Therefore, our results provide new insight into the mechanism of MLN4924 and may have significant implications for the treatment of MLN4924.

  13. Identification of IFN-γ-producing T cells as the main mediators of the side effects associated to mouse interleukin-15 sustained exposure.

    PubMed

    Di Scala, Marianna; Gil-Fariña, Irene; Olagüe, Cristina; Vales, Africa; Sobrevals, Luciano; Fortes, Puri; Corbacho, David; González-Aseguinolaza, Gloria

    2016-08-02

    Interleukin-15 (IL-15) is a cell growth-factor that regulates lymphocyte function and homeostasis. Its strong immunostimulatory activity coupled with an apparent lack of toxicity makes IL-15 an exciting candidate for cancer therapy, somehow limited by its short half-life in circulation. To increase IL-15 bioavailability we constructed a recombinant adeno-associated vector expressing murine IL-15 (AAV-mIL15) in the liver. Mice injected with AAV-mIL15 showed sustained and vector dose-dependent levels of IL-15/IL-15Rα complexes in serum, production of IFN-γ and activation of CD8+ T-cells and macrophages. The antitumoral efficacy of AAV-mIL15 was tested in a mouse model of metastatic colorectal cancer established by injection of MC38 cells. AAV-mIL15 treatment slightly inhibits MC38 tumor-growth and significantly increases the survival of mice. However, mIL-15 sustained expression was associated with development of side effects like hepatosplenomegaly, liver damage and the development of haematological stress, which results in the expansion of hematopoietic precursors in the bone marrow. To elucidate the mechanism, we treated IFN-γ receptor-, RAG1-, CD1d- and µMT-deficient mice and performed adoptive transfer of bone marrow cells from WT mice to RAG1-defcient mice. We demonstrated that the side effects of murine IL-15 administration were mainly mediated by IFN-γ-producing T-cells.

  14. Suppression of miR-155 Expression in IFN-γ-Treated Astrocytes and Microglia by DJ-1: A Possible Mechanism for Maintaining SOCS1 Expression

    PubMed Central

    Kim, Jong-hyeon; Jou, Ilo

    2014-01-01

    Previously, we reported that DJ-1, encoded by a Parkinson's disease (PD)-associated gene, inhibits expression of proinflammatory mediators in interferon-gamma (IFN-γ)-treated astrocytes and microglia through inhibition of STAT1 activation. Here, using microglia and astrocytes cultured from wild-type (WT) and DJ-1-knockout (KO) mouse brains, we examined how DJ-1 regulates suppressor of cytokine signaling 1 (SOCS1), a negative feedback regulator of STAT1 (signal transducer and activator of transcription) that is also induced by STAT1. We found that IFN-γ significantly increased SOCS1 mRNA expression in WT microglia and astrocytes, but not in KO cells, although STAT1 was highly activated in these latter cells. We further found that SOCS mRNA stability was decreased in DJ-1-KO cells, an effect that appeared to be mediated by the microRNA, miR-155. IFNincreased the levels of miR-155 in DJ-1-KO cells but not in WT cells. In addition, an miR-155 inhibitor rescued SOCS1 expression and decreased STAT1 activation in DJ-1-KO cells. Taken together, these results suggest that DJ-1 efficiently regulates inflammation by maintaining SOCS1 expression through regulation of miR-155 levels, even under conditions in which STAT1 activation is decreased. PMID:24963279

  15. The correlations among serum tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma) and sialic acids with peripheral lymphocytes in bovine tropical theileriosis.

    PubMed

    Razavi, Seyed Mostafa; Nazifi, Saeed; Emadi, Mahboobeh; Rakhshandehroo, Ehsan

    2010-10-01

    The infection with protozoan parasite Theileria annulata induces changes triggering the activation and/or proliferation of the host lymphocytes. In order to find out the possible correlations among peripheral circulatory lymphocytes, cytokine activities and the level of sialic acids, 50 dairy Holstein cattle, naturally infected with T. annulata, were divided into 4 subgroups according to their parasitemia rates (<1%, 1-3%, 3-5% and >5%). Also, ten non-infected cattle were sampled as control group. Blood samples were taken from jugular vein into acid citrate dextrose-containing tubes for measuring hematological parameters and B and T (CD(4) and CD(8)) cell populations and without anticoagulant for TNF-alpha, IFN-gamma and sialic acid concentrations. Remarkable decreases observed in red blood cells (RBCs), white blood cells (WBCs) and packed cell volume (PCV) in infected cattle compared to healthy ones (P < 0.05). Also, with increase in parasitemia rate, total lymphocytes and monocytes alleviated in the diseased groups. By contrast, total neutrohpils and the concentrations of TNF-alpha, IFN-gamma and total sialic acids were significantly elevated (P < 0.05) in infected animals. Accordingly, the circulatory populations of CD(4) and CD(8) T cells and B cells showed a substantial decrease, while a significant increase was observed in T (CD(4) and CD(8)) cells in cattle infected with <1% parasitemia rates. Decreased circulatory T cell population shows the ineffective responses of T cells to the stimulatory cytokines such as IFN-gamma or TNF-alpha. On the other hand, the elevation of cytokines (particularly IFN-gamma) and sialic acids have presumably an inhibitory role on circulatory B cell population in infected cattle. In addition, a high level of sialic acid concentration indicates the probable role of sialic acid to regulate the parasite-host cell adhesion during sporozoites invasion.

  16. Sub-chronically exposing mice to a polycyclic aromatic hydrocarbon increases lipid accumulation in their livers.

    PubMed

    Jin, Yuanxiang; Miao, Wenyu; Lin, Xiaojian; Wu, Tao; Shen, Hangjie; Chen, Shan; Li, Yanhong; Pan, Qiaoqiao; Fu, Zhengwei

    2014-09-01

    The potential for exposing humans and wildlife to environmental polycyclic aromatic hydrocarbons (PAHs) has increased. Risk assessments describing how PAHs disturb lipid metabolism and induce hepatotoxicity have only received limited attention. In the present study, seven-week-old male ICR mice received intraperitoneal injections of 0, 0.01, 0.1 or 1mg/kg body weight 3-methylcholanthrene (3MC) per week for 10 weeks. A high-fat diet was provided during the exposure. Histopathological lipid accumulation and lipid metabolism-related genes were measured. We observed that sub-chronic 3MC exposure significantly increased lipid droplet and triacylglycerol (TG) levels in the livers. A low dose of 3MC activated the aryl hydrocarbon receptor, which negatively regulated lipid synthesis in the livers. The primary genes including acetyl-CoA carboxylase (Acc), fatty acid synthase (Fas) and stearoyl-CoA desaturase 1 (Scd1) decreased significantly when compared with those in the control group, indicating that de novo fatty acid synthesis in the hepatocytes was significantly inhibited by the sub-chronic 3MC exposure. However, the free fatty acid (FFA) synthesis in the adipose tissue was greatly enhanced by up-regulating the expression of peroxisome proliferator-activated receptor γ (PPARγ) and sterol regulatory element binding protein-1c (SREBP1C) and target genes including Acc, Fas and Scd1. The synthesized FFA was released into the blood and then transported into the liver by the up-regulation of Fat and Fatp2, which resulted in the gradual accumulation of lipids in the liver. In conclusion, histological examinations and molecular level analyses highlighted the development of lipid accumulation and confirmed that 3MC significantly impaired lipid metabolism in mice.

  17. VHSV G glycoprotein major determinants implicated in triggering the host type I IFN antiviral response as DNA vaccine molecular adjuvants.

    PubMed

    Martinez-Lopez, A; Garcia-Valtanen, P; Ortega-Villaizan, M; Chico, V; Gomez-Casado, E; Coll, J M; Estepa, A

    2014-10-14

    We have recently identified the two major determinants of the glycoprotein G of the viral hemorrhagic septicaemia rhabdovirus (gpGVHSV), peptides p31 and p33 implicated in triggering the host type I IFN antiviral response associated to these rhabdoviral antigens. With the aim to investigate the properties of these viral glycoprotein regions as DNA molecular adjuvants, their corresponding cDNA sequences were cloned into a plasmid (pMCV1.4) flanked by the signal peptide and transmembrane sequences of gpGVHSV. In addition, a plasmid construct encoding both sequences p31 and p33 (pMCV1.4-p31+p33) was also designed. In vitro transitory cell transfection assays showed that these VHSV gpG regions were able to induce the expression of type I IFN stimulated genes as well as to confer resistance to the infection with a different fish rhabdovirus, the spring viremia of carp virus (SVCV). In vivo, zebrafish intramuscular injection of only 1μg of the construct pMCV1.4-p31+p33 conferred fish protection against SVCV lethal challenge up to 45 days post-immunization. Moreover, pMCV1.4-p31+p33 construct was assayed for molecular adjuvantcity's for a DNA vaccine against SVCV based in the surface antigen of this virus (pAE6-GSVCV). The results showed that the co-injection of the SVCV DNA vaccine and the molecular adjuvant allowed (i) a ten-fold reduction in the dose of pAE6-Gsvcv without compromising its efficacy (ii) an increase in the duration of protection, and (iii) an increase in the survival rate. To our knowledge, this is the first report in which specific IFN-inducing regions from a viral gpG are used to design more-efficient and cost-effective viral vaccines, as well as to improve our knowledge on how to stimulate the innate immune system.

  18. IFN-γ secretion in gut of Ob/Ob mice after vertical sleeve gastrectomy and its function in weight loss mechanism.

    PubMed

    Du, Jin-Peng; Wang, Geng; Hu, Chao-Jie; Wang, Qing-Bo; Li, Hui-Qing; Xia, Wen-Fang; Shuai, Xiao-Ming; Tao, Kai-Xiong; Wang, Guo-Bin; Xia, Ze-Feng

    2016-06-01

    Vertical sleeve gastrectomy (VSG) is becoming more and more popular among the world. Despite its dramatic efficacy, however, the mechanism of VSG remains largely undetermined. This study aimed to test interferon (IFN)-γ secretion n of mesenteric lymph nodes in obese mice (ob/ob mice), a model of VSG, and its relationship with farnesoid X receptor (FXR) expression in the liver and small intestine, and to investigate the weight loss mechanism of VSG. The wild type (WT) mice and ob/ob mice were divided into four groups: A (WT+Sham), B (WT+VSG), C (ob/ob+Sham), and D (ob/ob+VSG). Body weight values were monitored. The IFN-γ expression in mesenteric lymph nodes of ob/ob mice pre- and post-operation was detected by flow cytometry (FCM). The FXR expression in the liver and small intestine was detected by Western blotting. The mouse AML-12 liver cells were stimulated with IFN-γ at different concentrations in vitro. The changes of FXR expression were also examined. The results showed that the body weight of ob/ob mice was significantly declined from (40.6±2.7) g to (27.5±3.8) g on the 30th day after VSG (P<0.05). At the same time, VSG induced a higher level secretion of IFN-γ in mesenteric lymph nodes of ob/ob mice than that pre-operation (P<0.05). The FXR expression levels in the liver and small intestine after VSG were respectively 0.97±0.07 and 0.84±0.07 fold of GAPDH, which were significantly higher than pre-operative levels of 0.50±0.06 and 0.48±0.06 respectively (P<0.05). After the stimulation of AML-12 liver cells in vitro by different concentrations of IFN-γ (0, 10, 25, 50, 100, and 200 ng/mL), the relative FXR expression levels were 0.22±0.04, 0.31±0.04, 0.39±0.05, 0.38±0.05, 0.56±0.06, and 0.35±0.05, respectively, suggesting IFN-γ could distinctly promote the FXR expression in a dose-dependent manner in comparison to those cells without IFN-γ stimulation (P<0.05). It was concluded that VSG induces a weight loss in ob/ob mice by increasing IFN

  19. Phenotypic and functional characteristics of active suppressor cells against IFN-gamma production in PHA-stimulated cord blood lymphocytes

    SciTech Connect

    Seki, H.; Taga, K.; Matsuda, A.; Uwadana, N.; Hasui, M.; Miyawaki, T.; Taniguchi, N.

    1986-11-15

    Cord blood mononuclear cells (MNC) were defective in their ability to produce interferon-gamma (IFN-gamma) on stimulation with phytohemagglutinin (PHA) or recombinant interleukin 2, whereas cord MNC could induce comparable amounts of IFN-gamma with adult controls on stimulation with a streptococcal preparation, OK-432. Moreover, irradiation of cord MNC with 1500 rad before PHA stimulation could restore the IFN-gamma production. Kinetic studies indicated that such augmentation of IFN-gamma production by irradiation was evident when cord MNC were irradiated before or by 12 hr of PHA-stimulated culture. But irradiation after 18 hr or more of PHA stimulation did not exert any significant augmentation on IFN-gamma production by cord MNC. It seemed most likely that the ability of IFN-gamma production is already mature at birth, but radiosensitive suppressor effectors on IFN-gamma production are activated within cord MNC at an early stage of PHA stimulation, resulting in poor IFN-gamma production by cord MNC. PHA-induced IFN-gamma production by OKT3+, OKT4+, and OKT8- cord cells were markedly enhanced by irradiation with 1,500 rad before the culture. Coculture experiments disclosed that cord OKT4+ cells, but not OKT4- cells, when prestimulated with PHA for 24 hr, exerted active suppression on PHA-induced IFN-gamma production by adult MNC in a dose-dependent manner. These results suggested that radiosensitive suppressor effectors on IFN-gamma production were induced within the OKT4+ T cell subset of cord MNC on PHA stimulation.

  20. IκBζ augments IL-12- and IL-18-mediated IFN-γ production in human NK cells.

    PubMed

    Kannan, Yashaswini; Yu, Jianhua; Raices, Raquel M; Seshadri, Sudarshan; Wei, Min; Caligiuri, Michael A; Wewers, Mark D

    2011-03-10

    Interferon-γ (IFN-γ) production by natural killer (NK) cells and cytotoxic lymphocytes is a key component of innate and adaptive immune responses. Because inhibitor of κB-ζ (IκBζ), a Toll-like receptor (TLR)/interleukin-1 receptor (IL-1R) inducible transcription factor, regulates IFN-γ production in KG-1 cells, we tested IκBζ's role in the classic lymphocyte pathway of IL-12/IL-18-induced IFN-γ. Upon stimulation with IL-12/IL-18, monocyte-depleted human peripheral blood lymphocytes expressed the 79-kDa form of IκBζ and released IFN-γ. CD56(+) NK cells were shown to be the IκBζ-producing lymphocyte subpopulation, which also released abundant IFN-γ in response to IL-12/IL-18. Importantly, IκBζ was undetectable in CD56(-) lymphocytes where IFN-γ release was 10-fold lower. In addition, small interfering RNA knockdown of IκBζ suppressed IFN-γ expression in CD56(+) cells. The association of IκBζ with the IFN-γ promoter was documented by chromatin immunoprecipitation. IFN-γ promoter activity from IκBζ overexpression was confirmed by luciferase reporter assay. Finally, IκBζ coprecipitated with p65 and p50 NF-κB in NK cells in response to IL-12/IL-18, suggesting that IκBζ's effects on IFN-γ promoter activity are coregulated by NF-κB. These results suggest that IκBζ functions as an important regulator of IFN-γ in human NK cells, further expanding the class of IκBζ-modulated genes.

  1. Interferon (IFN)-λ is a potential mediator in lupus nephritis

    PubMed Central

    Zickert, Agneta; Oke, Vilija; Svenungsson, Elisabet; Sundström, Yvonne; Gunnarsson, Iva

    2016-01-01

    Objectives Interferon (IFN)-α is thought to be central in the pathogenesis for lupus nephritis (LN) and recent studies also indicate a role for IFNλ. Little is known about these cytokines in the context of treatment response. We studied levels of IFNα and IFNλ in patients with LN in association with clinical and histological response (HR) to treatment. Methods Fifty-six patients with active LN were included. Renal biopsies were performed at baseline and after immunosuppressive therapy. Serum levels of IFNα and IFNλ were analysed at both biopsy occasions and in 163 controls. The biopsies were evaluated according to the International Society of Nephrology/Renal Pathology Society classification. Clinical response was defined according to recent definitions. HR was defined as class I, II or III/IV-C on repeat biopsies. The expression of IFNλ in renal tissue was assessed by immunohistochemistry. Results At baseline, serum levels of both IFNα and IFNλ were higher in patients versus controls (p=0.01 and p=0.03, respectively). There was no correlation between IFNα and IFNλ. Overall, IFNα decreased after treatment (p=0.003) but IFNλ remained unchanged. However in patients with HR, IFNλ decreased (p=0.01). The highest levels of IFNλ were seen in patients with poor HR. Immunostaining of renal tissue revealed expression of IFNλ, particularly in crescent formations, inflammatory infiltrates and tubular cells. Conclusions The study supports a role for IFNλ in LN, both in circulation and at a tissue level. Levels of IFNα and IFNλ did not correlate and were affected differently by immunosuppression, indicating that they are differently involved in subgroups of LN. Persistent increased levels of IFNλ were associated to an unfavourable HR to treatment. PMID:27933198

  2. HER-2/neu raises SHP-2, stops IFN-{gamma} anti-proliferation in bladder cancer

    SciTech Connect

    Su, W.-P.; Tu, I-H.; Hu, S.-W.; Yeh, H.-H.; Shieh, D.-B.; Chen, T.-Y.; Su, W.-C. . E-mail: sunnysu@mail.ncku.edu.tw

    2007-04-27

    Gene amplification or HER-2/neu protein overexpression signals a poor outcome for bladder cancer patients. We investigated the anti-proliferative effect of IFN-{gamma} in HER-2/neu-transfected human bladder cancer cells (TCC-N5 and TCC-N10). The cells continued growing after IFN-{gamma} stimulation but did not activate the Janus kinase (Jak)/Stat pathway. We found Jak/Stat protein phosphatase in TCC-N5 and TCC-N10 cells with upregulated Src homology 2-containing protein tyrosine phosphatase-2 (SHP-2). After the cells had been treated with AG825, a HER-2/neu-specific inhibitor, SHP-2 expression declined, and Jak2/Stat1 reactivated. Similar results were reported in a mouse bladder cancer cell line, MBT2, with constitutive HER-2/neu overexpression. Further, AG825 pretreatment restored the anti-proliferation activity of IFN-{gamma} in TCC-N5 and TCC-N10 cells. Therefore, the suppression of IFN-{gamma} signaling in HER-2/neu-overexpressing bladder cancer cells might be due to SHP-2 upregulation. The regulation of SHP-2 by HER-2/neu provides a new target for blocking the HER-2/neu oncogenic pathwa000.

  3. Apoptotic Caspases Suppress mtDNA-Induced STING-Mediated Type I IFN Production

    PubMed Central

    McArthur, Kate; Metcalf, Donald; Lane, Rachael M.; Cambier, John C.; Herold, Marco J.; van Delft, Mark F.; Bedoui, Sammy; Lessene, Guillaume; Ritchie, Matthew E.; Huang, David C.S.

    2015-01-01

    SUMMARY Activated caspases are a hallmark of apoptosis induced by the intrinsic pathway, but they are dispensable for cell death and the apoptotic clearance of cells in vivo. This has led to the suggestion that caspases are activated not just to kill but to prevent dying cells from triggering a host immune response. Here, we show that the caspase cascade suppresses type I interferon (IFN) production by cells undergoing Bak/Bax-mediated apoptosis. Bak and Bax trigger the release of mitochondrial DNA. This is recognized by the cGAS/STING-dependent DNA sensing pathway, which initiates IFN production. Activated caspases attenuate this response. Pharmacological caspase inhibition or genetic deletion of caspase-9, Apaf-1, or caspase-3/7 causes dying cells to secrete IFN-β. In vivo, this precipitates an elevation in IFN-β levels and consequent hematopoietic stem cell dysfunction, which is corrected by loss of Bak and Bax. Thus, the apoptotic caspase cascade functions to render mitochondrial apoptosis immunologically silent. PMID:25525874

  4. Apoptotic caspases suppress mtDNA-induced STING-mediated type I IFN production.

    PubMed

    White, Michael J; McArthur, Kate; Metcalf, Donald; Lane, Rachael M; Cambier, John C; Herold, Marco J; van Delft, Mark F; Bedoui, Sammy; Lessene, Guillaume; Ritchie, Matthew E; Huang, David C S; Kile, Benjamin T

    2014-12-18

    Activated caspases are a hallmark of apoptosis induced by the intrinsic pathway, but they are dispensable for cell death and the apoptotic clearance of cells in vivo. This has led to the suggestion that caspases are activated not just to kill but to prevent dying cells from triggering a host immune response. Here, we show that the caspase cascade suppresses type I interferon (IFN) production by cells undergoing Bak/Bax-mediated apoptosis. Bak and Bax trigger the release of mitochondrial DNA. This is recognized by the cGAS/STING-dependent DNA sensing pathway, which initiates IFN production. Activated caspases attenuate this response. Pharmacological caspase inhibition or genetic deletion of caspase-9, Apaf-1, or caspase-3/7 causes dying cells to secrete IFN-β. In vivo, this precipitates an elevation in IFN-β levels and consequent hematopoietic stem cell dysfunction, which is corrected by loss of Bak and Bax. Thus, the apoptotic caspase cascade functions to render mitochondrial apoptosis immunologically silent.

  5. IFN-γ is required for cytotoxic T cell-dependent cancer genome immunoediting

    PubMed Central

    Takeda, Kazuyoshi; Nakayama, Masafumi; Hayakawa, Yoshihiro; Kojima, Yuko; Ikeda, Hiroaki; Imai, Naoko; Ogasawara, Kouetsu; Okumura, Ko; Thomas, David M.; Smyth, Mark J.

    2017-01-01

    Genetic evolution that occurs during cancer progression enables tumour heterogeneity, thereby fostering tumour adaptation, therapeutic resistance and metastatic potential. Immune responses are known to select (immunoedit) tumour cells displaying immunoevasive properties. Here we address the role of IFN-γ in mediating the immunoediting process. We observe that, in several mouse tumour models such as HA-expressing 4T1 mammary carcinoma cells, OVA-expressing EG7 lymphoma cells and CMS5 MCA-induced fibrosarcoma cells naturally expressing mutated extracellular signal-regulated kinase (ERK) antigen, the action of antigen-specific cytotoxic T cell (CTL) in vivo results in the emergence of resistant cancer cell clones only in the presence of IFN-γ within the tumour microenvironment. Moreover, we show that exposure of tumours to IFN-γ-producing antigen-specific CTLs in vivo results in copy-number alterations (CNAs) associated with DNA damage response and modulation of DNA editing/repair gene expression. These results suggest that enhanced genetic instability might be one of the mechanisms by which CTLs and IFN-γ immunoedits tumours, altering their immune resistance as a result of genetic evolution. PMID:28233863

  6. IFN-γ is required for cytotoxic T cell-dependent cancer genome immunoediting.

    PubMed

    Takeda, Kazuyoshi; Nakayama, Masafumi; Hayakawa, Yoshihiro; Kojima, Yuko; Ikeda, Hiroaki; Imai, Naoko; Ogasawara, Kouetsu; Okumura, Ko; Thomas, David M; Smyth, Mark J

    2017-02-24

    Genetic evolution that occurs during cancer progression enables tumour heterogeneity, thereby fostering tumour adaptation, therapeutic resistance and metastatic potential. Immune responses are known to select (immunoedit) tumour cells displaying immunoevasive properties. Here we address the role of IFN-γ in mediating the immunoediting process. We observe that, in several mouse tumour models such as HA-expressing 4T1 mammary carcinoma cells, OVA-expressing EG7 lymphoma cells and CMS5 MCA-induced fibrosarcoma cells naturally expressing mutated extracellular signal-regulated kinase (ERK) antigen, the action of antigen-specific cytotoxic T cell (CTL) in vivo results in the emergence of resistant cancer cell clones only in the presence of IFN-γ within the tumour microenvironment. Moreover, we show that exposure of tumours to IFN-γ-producing antigen-specific CTLs in vivo results in copy-number alterations (CNAs) associated with DNA damage response and modulation of DNA editing/repair gene expression. These results suggest that enhanced genetic instability might be one of the mechanisms by which CTLs and IFN-γ immunoedits tumours, altering their immune resistance as a result of genetic evolution.

  7. Ubiquilin 1 Promotes IFN-γ-Induced Xenophagy of Mycobacterium tuberculosis.

    PubMed

    Sakowski, Erik T; Koster, Stefan; Portal Celhay, Cynthia; Park, Heidi S; Shrestha, Elina; Hetzenecker, Stefanie E; Maurer, Katie; Cadwell, Ken; Philips, Jennifer A

    2015-07-01

    The success of Mycobacterium tuberculosis (Mtb) as a pathogen rests upon its ability to grow intracellularly in macrophages. Interferon-gamma (IFN-γ) is critical in host defense against Mtb and stimulates macrophage clearance of Mtb through an autophagy pathway. Here we show that the host protein ubiquilin 1 (UBQLN1) promotes IFN-γ-mediated autophagic clearance of Mtb. Ubiquilin family members have previously been shown to recognize proteins that aggregate in neurodegenerative disorders. We find that UBQLN1 can interact with Mtb surface proteins and associates with the bacilli in vitro. In IFN-γ activated macrophages, UBQLN1 co-localizes with Mtb and promotes the anti-mycobacterial activity of IFN-γ. The association of UBQLN1 with Mtb depends upon the secreted bacterial protein, EsxA, which is involved in permeabilizing host phagosomes. In autophagy-deficient macrophages, UBQLN1 accumulates around Mtb, consistent with the idea that it marks bacilli that traffic through the autophagy pathway. Moreover, UBQLN1 promotes ubiquitin, p62, and LC3 accumulation around Mtb, acting independently of the E3 ligase parkin. In summary, we propose a model in which UBQLN1 recognizes Mtb and in turn recruits the autophagy machinery thereby promoting intracellular control of Mtb. Thus, polymorphisms in ubiquilins, which are known to influence susceptibility to neurodegenerative illnesses, might also play a role in host defense against Mtb.

  8. IFN-γ Hinders Recovery from Mucosal Inflammation during Antibiotic Therapy for Salmonella Gut Infection.

    PubMed

    Dolowschiak, Tamas; Mueller, Anna Angelika; Pisan, Lynn Joanna; Feigelman, Rounak; Felmy, Boas; Sellin, Mikael Erik; Namineni, Sukumar; Nguyen, Bidong Dinh; Wotzka, Sandra Yvonne; Heikenwalder, Mathias; von Mering, Christian; Mueller, Christoph; Hardt, Wolf-Dietrich

    2016-08-10

    Salmonella Typhimurium (S.Tm) causes acute enteropathy resolving after 4-7 days. Strikingly, antibiotic therapy does not accelerate disease resolution. We screened for factors blocking remission using a S.Tm enterocolitis model. The antibiotic ciprofloxacin clears pathogen stool loads within 3-24 hr, while gut pathology resolves more slowly (ψ50: ∼48 hr, remission: 6-9 days). This delayed resolution is mediated by an interferon-γ (IFN-γ)-dependent response that is triggered during acute infection and continues throughout therapy. Specifically, IFN-γ production by mucosal T and NK cells retards disease resolution by maintaining signaling through the transcriptional regulator STAT1 and boosting expression of inflammatory mediators like IL-1β, TNF, and iNOS. Additionally, sustained IFN-γ fosters phagocyte accumulation and hampers antimicrobial defense mediated by IL-22 and the lectin REGIIIβ. These findings reveal a role for IFN-γ in delaying resolution of intestinal inflammation and may inform therapies for acute Salmonella enteropathy, chronic inflammatory bowel diseases, or disease resolution during antibiotic treatment.

  9. Effects of Saussurea lappa roots extract in ethanol on leukocyte phagocytic activity, lymphocyte proliferation and interferon-gamma (IFN-gamma).

    PubMed

    Sarwar, Anas; Enbergs, H

    2007-07-01

    Effects of Saussurea lappa root extracts prepared in ethanol according to the homeopathic principles were assessed on leukocyte phagocytic activity, lymphocyte transformation and mitogen-induced interferon-gamma (IFN-gamma) in the cultures of peripheral blood mononuclear cells of goats (PBMC) in vitro. Leukocyte phagocytic activity was measured by flow cytometry, lymphocyte proliferation by MTT and IFN-gamma level in cell culture supernatants was determined by ELISA. The results obtained demonstrated that all test dilutions (D4, D6, D8) of Saussurea lappa in ethanol have exerted a stimulating effect on leukocyte phagocytic activity in dose-dependent manner. A 10 microl dose of Saussurea lappa of each dilution markedly enhanced phagocytic activity, while other doses tested made only a feeble stimulating effect. The increases with 10 microl dose were found significantly (P<0.01) different between each dilution, maximal stimulation was observed by D8 dilution. Different doses (10 microl, 2 microl, 1 microl, 0.5 microl) of all test dilutions (D4, D6, D8) of Saussurea lappa in sterile 0.9% NaCl solution inhibited lymphocyte proliferation. Maximal inhibitory effect was observed with the 2 microl dose. Similarly, Saussurea lappa suppressed the secretion of IFN-gamma by mitogen-activated (PHA; 2.5 microg/ml) of peripheral mononuclear cells in dose-dependent manner. In conclusion these findings suggest that enhanced leukocyte phagocytic activity may be helpful to clear the soluble immune complexes produced during a sustained immune response against self antigens which causes chronic inflammatory injury of tissue. On the other hand, inhibition of lymphocyte proliferation and IFN-gamma by Saussurea lappa may contribute to suppress immune-mediated inflammatory reactions possibly through a cell-mediated cytokine pathway. Thus it is concievable that ethanolic extracts of Saussurea lappa roots in homeopathetic dilutions may be considered as a potential candidate for therapeutic

  10. Activated human mesenchymal stem/stromal cells suppress metastatic features of MDA-MB-231 cells by secreting IFN-β.

    PubMed

    Yoon, N; Park, M S; Shigemoto, T; Peltier, G; Lee, R H

    2016-04-14

    Our recent study showed that human mesenchymal stem/stromal cells (hMSCs) are activated to express tumor necrosis factor (TNF)-α-related apoptosis-inducing ligand (TRAIL) by exposure to TNF-α and these activated hMSCs effectively induce apoptosis in triple-negative breast cancer MDA-MB-231 (MDA) cells in vitro and in vivo. Here, we further demonstrated that activated hMSCs not only induced apoptosis of MDA cells but also reduced metastatic features in MDA cells. These activated hMSC-exposed MDA cells showed reduced tumorigenicity and suppressed formation of lung metastasis when implanted in the mammary fat pad. Surprisingly, the activated hMSC-exposed MDA cells increased TRAIL expression, resulting in apoptosis in MDA cells. Interestingly, upregulation of TRAIL in MDA cells was mediated by interferon-beta (IFN-β) secreted from activated hMSCs. Furthermore, IFN-β in activated hMSCs was induced by RNA and DNA released from apoptotic MDA cells in absent in melanoma 2 (AIM2) and IFN induced with helicase C domain 1 (IFIH1)-dependent manners. These observations were only seen in the TRAIL-sensitive breast cancer cell lines but not in the TRAIL-resistant breast cancer cell lines. Consistent with these results, Kaplan-Meier survival analysis also showed that lack of innate sensors detecting DNA or RNA is strongly associated with poor survival in estrogen receptor-negative breast cancer patients. In addition, cancer-associated fibroblasts (CAF) isolated from a breast cancer patient were also able to express TRAIL and IFN-β upon DNA and RNA stimulation. Therefore, our results suggest that the crosstalk between TRAIL-sensitive cancer cells and stromal cells creates a tumor-suppressive microenvironment and further provide a novel therapeutic approach to target stromal cells within cancer microenvironment for TRAIL sensitive cancer treatment.

  11. IFN-γ regulates xanthine oxidase-mediated iNOS-independent oxidative stress in maneb- and paraquat-treated rat polymorphonuclear leukocytes.

    PubMed

    Singh, Deepali; Kumar, Vinod; Singh, Chetna

    2017-03-01

    Maneb (MB) and paraquat (PQ) provoke oxidative stress-mediated cell damage. Role of xanthine oxidase (XO) in oxidative stress and its association with nitric oxide (NO)/NO synthase (NOS) have been widely reported. While inducible NOS (iNOS) is implicated in MB+PQ-induced toxicity in rat polymorphonuclear leukocytes (PMNs), role of XO and its alliance with iNOS have not yet been established. The study investigated the role of XO in MB+PQ-induced oxidative stress in rat PMNs and its regulation by iNOS and inflammatory cytokines. MB+PQ-augmented reactive oxygen species (ROS), superoxide, nitro-tyrosine, lipid peroxidation (LPO), and nitrite levels along with the catalytic activity of iNOS, superoxide dismutase (SOD), and XO. XO inhibitor, allopurinol (AP), alleviated MB+PQ-induced changes except nitrite content and iNOS activity. Conversely, an iNOS inhibitor, aminoguanidine, mitigated MB+PQ-induced LPO, nitrite, iNOS, and nitro-tyrosine levels; however, no change was observed in ROS, SOD, and XO. Nuclear factor-κB inhibitor, pyrrolidine dithiocarbamate (PDTC), tumor necrosis factor-alpha (TNF-α) inhibitor, pentoxyfylline, and an anti-inflammatory agent, dexamethasone, attenuated MB+PQ-induced increase in XO, superoxide, and ROS with parallel reduction in the expression of interferon-gamma (IFN-γ), TNF-α, and interleukin-1β (IL-1β) in rat PMNs. Exogenous IFN-γ, TNF-α, and IL-1β enhanced superoxide, ROS, and XO in the PMNs of control and MB+PQ-treated rats; however, IFN- γ was found to be the most potent inducer. Moreover, AP ameliorated cytokine-induced free radical generation and restored XO activity towards normalcy. The results thus demonstrate that XO mediates oxidative stress in MB+PQ-treated rat PMNs via iNOS-independent but cytokine (predominantly IFN-γ)-dependent mechanism.

  12. Hepatitis C Virus-Induced Myeloid-Derived Suppressor Cells Suppress NK Cell IFN-γ Production by Altering Cellular Metabolism via Arginase-1.

    PubMed

    Goh, Celeste C; Roggerson, Krystal M; Lee, Hai-Chon; Golden-Mason, Lucy; Rosen, Hugo R; Hahn, Young S

    2016-03-01

    The hepatitis C virus (HCV) infects ∼ 200 million people worldwide. The majority of infected individuals develop persistent infection, resulting in chronic inflammation and liver disease, including cirrhosis and hepatocellular carcinoma. The ability of HCV to establish persistent infection is partly due to its ability to evade the immune response through multiple mechanisms, including suppression of NK cells. NK cells control HCV replication during the early phase of infection and regulate the progression to chronic disease. In particular, IFN-γ produced by NK cells limits viral replication in hepatocytes and is important for the initiation of adaptive immune responses. However, NK cell function is significantly impaired in chronic HCV patients. The cellular and molecular mechanisms responsible for impaired NK cell function in HCV infection are not well defined. In this study, we analyzed the interaction of human NK cells with CD33(+) PBMCs that were exposed to HCV. We found that NK cells cocultured with HCV-conditioned CD33(+) PBMCs produced lower amounts of IFN-γ, with no effect on granzyme B production or cell viability. Importantly, this suppression of NK cell-derived IFN-γ production was mediated by CD33(+)CD11b(lo)HLA-DR(lo) myeloid-derived suppressor cells (MDSCs) via an arginase-1-dependent inhibition of mammalian target of rapamycin activation. Suppression of IFN-γ production was reversed by l-arginine supplementation, consistent with increased MDSC arginase-1 activity. These novel results identify the induction of MDSCs in HCV infection as a potent immune evasion strategy that suppresses antiviral NK cell responses, further indicating that blockade of MDSCs may be a potential therapeutic approach to ameliorate chronic viral infections in the liver.

  13. Antigen-Specific Mammary Inflammation Depends on the Production of IL-17A and IFN-γ by Bovine CD4+ T Lymphocytes

    PubMed Central

    Rainard, Pascal; Cunha, Patricia; Ledresseur, Marion; Staub, Christophe; Touzé, Jean-Luc; Kempf, Florent; Gilbert, Florence B.; Foucras, Gilles

    2015-01-01

    Intramammary infusion of the antigen used to sensitize cows by the systemic route induces a local inflammation associated with neutrophil recruitment. We hypothesize that this form of delayed type hypersensitivity, which may occur naturally during infections or could be induced intentionally by vaccination, can impact the outcome of mammary gland infections. We immunized cows with ovalbumin to identify immunological correlates of antigen-specific mammary inflammation. Intraluminal injection of ovalbumin induced a mastitis characterized by a prompt tissue reaction (increase in teat wall thickness) and an intense influx of leukocytes into milk of 10 responder cows out of 14 immunized animals. The magnitude of the local inflammatory reaction, assessed through milk leukocytosis, correlated with antibody titers, skin thickness test, and production of IL-17A and IFN-γ in a whole-blood antigen stimulation assay (WBA). The production of these two cytokines significantly correlated with the magnitude of the milk leukocytosis following the ovalbumin intramammary challenge. The IL-17A and IFN-γ production in the WBA was dependent on the presence of CD4+ cells in blood samples. In vitro stimulation of peripheral blood lymphocytes with ovalbumin followed by stimulation with PMA/ionomycin allowed the identification by flow cytometry of CD4+ T cells producing either IL-17A, IFN-γ, or both cytokines. The results indicate that the antigen-specific WBA, and specifically IL-17A and IFN-γ production by circulating CD4+ cells, can be used as a predictor of mammary hypersensitivity to protein antigens. This prompts further studies aiming at determining how Th17 and/or Th1 lymphocytes modulate the immune response of the mammary gland to infection. PMID:26375594

  14. Cloning and expression analyses of interferon regulatory factor (IRF) 3 and 7 genes in European eel, Anguilla anguilla with the identification of genes involved in IFN production.

    PubMed

    Huang, Bei; Huang, Wen Shu; Nie, P

    2014-04-01

    Interferon regulatory factor (IRF) 3 and IRF7 have been identified as regulators of type I interferon (IFN) gene expression in mammals. In the present study, the two genes were cloned and characterized in the European eel, Anguilla anguilla. The full-length cDNA sequence of IRF3 and IRF7 in the European eel, named as AaIRF3 and AaIRF7 consists of 2879 and 2419 bp respectively. Multiple alignments showed that the two IRFs have a highly conserved DNA binding domain (DBD) in the N terminus, with the characteristic motif containing five tryptophan residues, which is a feature present in their mammalian homologues. But, IRF7 has only four of the five residues in other species of fish. The expression of AaIRF3 and AaIRF7 both displayed an obvious dose-dependent manner following polyinosinic:polycytidylic acid (PolyI:C) challenge. In vivo expression analysis showed that the mRNA level of AaIRF3 and AaIRF7 was significantly up-regulated in response to PolyI:C stimulation in all examined tissues/organs except in muscle, with a lower level of increase observed in response to lipopolysaccharide (LPS) challenge and Edwardsiella tarda infection, indicating that AaIRF3 and AaIRF7 may be more likely involved in antiviral immune response. In addition, some pattern recognition receptors genes related with the production of type I IFNs and those genes in response to type I IFNs were identified in the European eel genome database, indicating a relatively conserved system in the production of type I IFN and its signalling in the European eel.

  15. Increased expression of nonmuscle myosin IIs is associated with 3MC-induced mouse tumor.

    PubMed

    Saha, Shekhar; Dey, Sumit K; Das, Provas; Jana, Siddhartha S

    2011-11-01

    Administration of the chemical carcinogen, 3-methylcholanthrene (3MC), in the hind leg induces the progressive formation of tumors in mice within 110 days. Previous reports suggest that transformation of muscle cells to atypical cells is one of the causes of tumor formation. Molecular events that lead to transformation of normal cells to atypical cells are not well understood. Here, we investigate the effect of 3MC on the expression of nonmuscle myosin IIs (NM IIs) which are known to be involved in cell migration, division and adhesion. Mass spectroscopy analysis reveals that tumor tissue contains 64.5% NM II-A, 34% II-B and only 1.5% II-C of total NM IIs, whereas these three isoforms of NM IIs are undetectable by mass spectroscopy in normal tissue associated with the tumor (NTAT) from the hind leg. Quantification of heavy chain mRNAs of NM II suggests that tumor tissue contains 25.7-fold and 19.03-fold more of NM II-A and II-B, respectively, compared with NTAT. Unlike NM II-B, which is detected only after tumor formation, II-A is detectable as early as day 7 after a second dose of 3MC. Immunofluorescence confocal microscopy reveals that fibroblast cells which are sparsely distributed in normal tissue are densely populated but of atypical shape in the tumor. These findings suggest that transformation of fibroblasts or non-fibroblast cells to atypical, cancerous cells is associated with increased levels of NM II-A and NM II-B expression in the 3MC-induced tumor mouse model. 3MC-induced transformation is further demonstrated in C2C12 myotubes.

  16. The links between chronic obstructive pulmonary disease and comorbid depressive symptoms: role of IL-2 and IFN-γ.

    PubMed

    Rybka, Joanna; Korte, S Mechiel; Czajkowska-Malinowska, Małgorzata; Wiese, Małgorzata; Kędziora-Kornatowska, Kornelia; Kędziora, Józef

    2016-11-01

    Depression is highly prevalent in COPD patients, and both diseases are believed to be associated with inflammation. The aim of this study was to elucidate the role of the immune system alterations in pathogenesis of depression in COPD patients. Blood was collected from patients diagnosed with chronic obstructive pulmonary disease and comorbid depressive symptoms [COPD + DS, (N = 13)], from individuals with either COPD (N = 16) or recurrent depressive disorder (rDD) alone (N = 15), and from healthy controls (N = 19). Surface phenotype expression of T regulatory and T effector cells was analyzed with a flow cytometry, and IL-2, IL-6, IL-8, IFN-γ, IL-17, and neopterin were detected with ELISA. We demonstrated that COPD, depression, and COPD with comorbid depression are associated with increased IL-6 levels when compared with healthy controls 42.2 ± 1.87, 40.9 ± 2.12, 41.7 ± 1.31, and 33.2 ± 1.23 pg/ml, respectively (p < 0.05). A significant increase in neopterin levels was observed both in rDD and COPD patients when compared with controls (15.69 ± 0.095, 13.98 ± 0.887 vs. 9.22 ± 0.466 nmol/l, p < 0.001 and p < 0.05, respectively). Concentrations of IFN-γ were significantly increased in COPD + DS patients when compared with controls (24.3 ± 1.49 and 17.8 ± 0.70 pg/ml, respectively, p < 0.05). IL-2 levels were highest in COPD + DS (3.20 ± 0.389 pg/ml) and differed significantly when this group was compared with controls (2.20 ± 0.184 pg/ml), p ≤ 0.05). In this study, we demonstrated for the first time that depressive symptoms in COPD patients may be related to inflammatory state as confirmed by increased levels of IL-6 both in COPD and depression and also in COPD with comorbid depressive symptoms, despite the fact that the patients were treated with anti-inflammatory drugs and/or antidepressants. We also identified IFN-γ and IL-2 as putative inflammatory agents associated with depressive symptoms in COPD

  17. Production of IL-8, IL-17, IFN-gamma and IP-10 in human astrocytes correlates with alphavirus attenuation.

    PubMed

    Peng, Bi-Hung; Borisevich, Viktoriya; Popov, Vsevolod L; Zacks, Michele A; Estes, D Mark; Campbell, Gerald A; Paessler, Slobodan

    2013-05-03

    Venezuelan equine encephalitis virus (VEEV) is an important, naturally emerging zoonotic pathogen. Recent outbreaks in Venezuela and Colombia in 1995 indicate that VEEV still poses a serious public health threat. Astrocytes may be target cells in human and mouse infection and they play an important role in repair through gliosis. In this study, we report that virulent VEEV efficiently infects cultured normal human astrocytes, three different murine astrocyte cell lines and astrocytes in the mouse brain. The attenuation of virus replication positively correlates with the increased levels of production of IL-8, IL-17, IFN-gamma and IP-10. In addition, VEEV infection induces release of basic fibroblast growth factor and production of potent chemokines such as RANTES and MIP-1-beta from cultured human astrocytes. This growth factor and cytokine profile modeled by astrocytes in vitro may contribute to both neuroprotection and repair and may play a role in leukocyte recruitment in vivo.

  18. Regulatory effects of SKAR in interferon α signaling and its role in the generation of type I IFN responses.

    PubMed

    Kroczynska, Barbara; Mehrotra, Swarna; Majchrzak-Kita, Beata; Arslan, Ahmet Dirim; Altman, Jessica K; Stein, Brady L; McMahon, Brandon; Kozlowski, Piotr; Kahle, Philipp J; Eklund, Elizabeth A; Fish, Eleanor N; Platanias, Leonidas C

    2014-08-05

    We provide evidence that S6 kinase 1 (S6K1) Aly/REF-like target (SKAR) is engaged in IFN-α signaling and plays a key role in the generation of IFN responses. Our data demonstrate that IFN-α induces phosphorylation of SKAR, which is mediated by either the p90 ribosomal protein S6 kinase (RSK) or p70 S6 kinase (S6K1), in a cell type-specific manner. This type I IFN-inducible phosphorylation of SKAR results in enhanced interaction with the eukaryotic initiation factor (eIF)4G and recruitment of activated RSK1 to 5' cap mRNA. Our studies also establish that SKAR is present in cap-binding CBP80 immune complexes and that this interaction is mediated by eIF4G. We demonstrate that inducible protein expression of key IFN-α-regulated protein products such as ISG15 and p21(WAF1/CIP1) requires SKAR activity. Importantly, our studies define a requirement for SKAR in the generation of IFN-α-dependent inhibitory effects on malignant hematopoietic progenitors from patients with chronic myeloid leukemia or myeloproliferative neoplasms. Taken altogether, these findings establish critical and essential roles for SKAR in the regulation of mRNA translation of IFN-sensitive genes and induction of IFN-α biological responses.

  19. Enhancing Specific-Antibody Production to the ragB Vaccine with GITRL That Expand Tfh, IFN-γ+ T Cells and Attenuates Porphyromonas gingivalis Infection in Mice

    PubMed Central

    Su, Zhaoliang; Kong, Fanzhi; Shi, Xiaoju; Tong, Jia; Shen, Pei; Peng, Tianqing; Wang, Shengjun; Xu, Huaxi

    2013-01-01

    The outer membrane protein RagB is one of the major virulence factors of the periodontal pathogen Porphyromonas gingivalis (P. gingivalis). In order to induce protective immune response against P. gingivalis infection, an mGITRL gene-linked ragB DNA vaccine (pIRES-ragB-mGITRL ) was constructed. Six-week-old female BALB/c mice were immunized with pIRES-ragB-mGITRL through intramuscular injection and then challenged by subcutaneous injection in the abdomen with P. gingivalis. RagB-specific antibody-forming cells were evaluated by an Enzyme-linked immunosorbent spot, and specific antibody was determined by enzyme-linked immunosorbent assay. In addition, the frequencies of Tfh and IFN-γ+ T cells in spleen were measured using flow cytometer, and the levels of IL-21 and IFN-γ mRNA or proteins were detected by real time RT-PCR or ELISA. The data showed that the mGITRL-linked ragB DNA vaccine induced higher levels of RagB-specific IgG in serum and RagB-specific antibody-forming cells in spleen. The frequencies of Tfh and IFN-γ+ T cells were obviously expanded in mice immunized by pIRES-ragB-mGITRL compared with other groups (pIRES or pIRES-ragB ). The levels of Tfh and IFN-γ+ T cells associated cytokines were also significantly increased in pIRES-ragB-mGITRL group. Therefore, the mice immunized with ragB plus mGITRL showed the stronger resistant to P. gingivalis infection and a significant reduction of the lesion size caused by P. gingivalis infection comparing with other groups. Taken together, our findings demonstrated that intramuscular injection of DNA vaccine ragB together with mGITRL induced protective immune response dramatically by increasing Tfh and IFN-γ+ T cells and antibody production to P. gingivalis. PMID:23560053

  20. Microarray analysis on germfree mice elucidates the primary target of a traditional Japanese medicine juzentaihoto: acceleration of IFN-α response via affecting the ISGF3-IRF7 signaling cascade

    PubMed Central

    2012-01-01

    Background The traditional Japanese medicine juzentaihoto (JTX) is a pharmaceutical grade multi-herbal medicine widely used for the prevention of cancer metastasis and infection in immuno-compromized patients in Japan. The effect of JTX has been supposed to be intimately affected by the immunological properties of host and enteric microflora. The influence of JTX on the gene expression profile in the large and small intestines was investigated by microarray analyses using mice of different strains with or without enteric microflora. Results In all types of mice, including germfree (GF) animals, the genes most affected by two-week oral JTX treatment were the type 1 interferon (IFN)-related genes including Stat1, Isgf3g and Irf7, which play a critical role in the feedback loop of IFN-α production cascade. In IQI specific pathogen free (SPF) mice JTX increased the steady state level of the expression of IFN-related genes, but had the opposite effect in IQI GF and BALB/c SPF mice. Promoter analysis suggests that tandem repeated $IRFF (the promoter sequences for interferon regulatory factors) may be a primary target for JTX action. Pre-treatment of JTX accelerated the effects of an oral IFN "inducer" 2-amino-5-bromo-6-methyl-4-pyrimidinol (ABMP) (up-regulation of IFN-α production in IQI strain and down-regulation in BALB/c mice), which is in good accordance with the effect of JTX on gene expression of type 1 IFN-related genes. Conclusions Microarray analysis revealed that the target of JTX might be the transcription machinery regulating the steady-state level of genes involved in the ISGF3-IRF7 cascade, whose effect is bi-directional in a strain- and microbiota-dependent manner. PMID:22257721

  1. TGF-β Promotes Proliferation of Thyroid Epithelial Cells in IFN-γ−/− Mice by Down-Regulation of p21 and p27 via AKT Pathway

    PubMed Central

    Fang, Yujiang; Yu, Shiguang; Braley-Mullen, Helen

    2012-01-01

    IFN-γ−/− NOD.H-2h4 mice develop an autoimmune disease characterized by hyperplasia and proliferation of thyroid epithelial cells (TEC H/P). Proliferating TECs produce TGF-β, and IFN-γ inhibits TEC H/P. In the present study, cultured TECs were used to directly determine the mechanisms by which these cytokines act on TECs to result in proliferation or inhibition of proliferation. With TECs from IFN-γ−/− NOD.H-2h4 mice or mice expressing the dominant negative TGF-β type II receptor on TECs, TGF-β was shown to promote TEC proliferation and IFN-γ was shown to inhibit TEC proliferation in vitro. TGF-β may promote TEC proliferation by down-regulating antiproliferative molecules p21 and p27, whereas IFN-γ may inhibit proliferation by up-regulating antiproliferative molecules p18 and p21 and down-regulating the pro-proliferative molecule cyclin D. Inhibition of AKT abolished the effect of TGF-β on p21 and p27, resulting in similar proliferation of TGF-β-treated and control TECs. Increased expression of proliferating cell nuclear antigen (PCNA), TGF-β, and p-AKT and decreased expression of p21 and p27 by proliferating TECs correlated with the proliferative state of TEC H/P. Taken together, the results suggest that TGF-β promotes TEC proliferation by down-regulating p21 and p27 via the AKT pathway in IFN-γ−/− NOD.H-2h4 mice, which may have significant implications for development of effective therapeutic strategies targeting the TGF-β and AKT pathways for treatment of hyperplasia and/or neoplasia. PMID:22119715

  2. High Pulmonary Levels of IL-6 and IL-1β in Children with Chronic Suppurative Lung Disease Are Associated with Low Systemic IFN-γ Production in Response to Non-Typeable Haemophilus influenzae.

    PubMed

    Pizzutto, Susan J; Upham, John W; Yerkovich, Stephanie T; Chang, Anne B

    2015-01-01

    Non-typeable Haemophilus influenzae (NTHi) is commonly associated with chronic suppurative lung disease in children. We have previously shown that children with chronic suppurative lung disease have a reduced capacity to produce IFN-γ in response to NTHi compared with healthy control children. The aim of this study was to determine if deficient NTHi-specific IFN-γ production is associated with heightened systemic or airway inflammation. We measured a panel of cytokines (IFN-γ, IL-1β, IL-6, IL-8, IL-12 p70), antimicrobial proteins (LL-37, IP-10) as well as cellular and clinical factors associated with airway and systemic inflammation in 70 children with chronic suppurative lung disease. IFN-γ was measured in peripheral blood mononuclear cells challenged in vitro with live NTHi. Regression analysis was used to assess the association between the systemic and airway inflammation and the capacity to produce IFN-γ. On multivariate regression, NTHi-specific IFN-γ production was significantly negatively associated with the BAL concentrations of the inflammatory cytokines IL-6 (β=-0.316; 95%CI -0.49, -0.14; p=0.001) and IL-1β (β=-0.023; 95%CI -0.04, -0.01; p=0.001). This association was independent of bacterial or viral infection, BAL cellularity and the severity of bronchiectasis (using modified Bhalla score on chest CT scans). We found limited evidence of systemic inflammation in children with chronic suppurative lung disease. In summary, increased local airway inflammation is associated with a poorer systemic cell-mediated immune response to NTHi in children with chronic suppurative lung disease. These data support the emerging body of evidence that impaired cell-mediated immune responses and dysregulated airway inflammation may be linked and contribute to the pathobiology of chronic suppurative lung disease.

  3. The Yaa locus and IFN-α fine-tune germinal center B cell selection in murine systemic lupus erythematosus.

    PubMed

    Moisini, Ioana; Huang, Weiqing; Bethunaickan, Ramalingam; Sahu, Ranjit; Ricketts, Peta-Gay; Akerman, Meredith; Marion, Tony; Lesser, Martin; Davidson, Anne

    2012-11-01

    Male NZW/BXSB.Yaa (W/B) mice express two copies of TLR7 and develop pathogenic autoantibodies, whereas females with only one copy of TLR7 have attenuated disease. Our goal was to analyze the regulation of the autoantibody response in male and female W/B mice bearing the autoreactive site-directed H chain transgene 3H9. Serum anti-dsDNA Abs appeared in males at 12 wk, and most had high-titer IgG anti-dsDNA and anti-cardiolipin Abs and developed >300 mg/dl proteinuria by 8 mo. Females had only low-titer IgG anti-cardiolipin Abs, and none developed proteinuria by 1 y. Males had a smaller marginal zone than females with a repertoire that was distinct from the follicular repertoire, indicating that the loss of marginal zone B cells was not due to diversion to the follicular compartment. Vk5-43 and Vk5-48, which were rare in the naive repertoire, were markedly overrepresented in the germinal center repertoire of both males and females, but the VJ junctions differed between males and females with higher-affinity autoreactive B cells being selected into the germinal centers of males. Administration of IFN-α to females induced anti-cardiolipin and anti-DNA autoantibodies and proteinuria and was associated with a male pattern of junctional diversity in Vk5-43 and Vk5-48. Our studies are consistent with the hypothesis that presence of the Yaa locus, which includes an extra copy of Tlr7, or administration of exogenous IFN-α relaxes the stringency for selection in the germinal centers resulting in increased autoreactivity of the Ag-driven B cell repertoire.

  4. Eomes expression reports the progressive differentiation of IFN-γ-producing Th1-like γδ T cells.

    PubMed

    Lino, Ciro N R; Barros-Martins, Joana; Oberdörfer, Linda; Walzer, Thierry; Prinz, Immo

    2017-04-06

    The transcription factor Eomesodermin (Eomes) plays a crucial role in regulating cytotoxic function, development and survival of immune cells. γδ T cells can express Eomes, but its contribution to their differentiation is unknown. Using Eomes-IRES-GFP mice, we show that Eomes(+) γδ T cells are unequally distributed among organs, with the highest proportion in spleen. While the majority of Eomes(+) γδ T cells expressed Vγ1(+) and Vγ4(+) TCRs, Eomes was absent in Vγ5(+) , Vγ6(+) and Vγ7(+) subsets. Moreover, Eomes was co-expressed in γδ T cells with Th1 lineage-related factors such as CD27, T-bet and Ly6C, but not with Th17 lineage-related genes. Eomes(+) and Eomes(-) γδ T-cell populations showed distinct gene expression profiles, with an increase of cytotoxic-related genes in Eomes(+) γδ T cells. Furthermore, Eomes could be induced in peripheral γδ T cells by IL-12 and IL-4, and Eomes(+) γδ T cells presented a higher proliferation rate and IFN-γ production when stimulated in vitro with IL-12 and IL-18. However, γδ T cells with very high Eomes levels displayed an exhausted phenotype with high levels of PD-1, and were less capable of IFN-γ production. Together, this study highlights Eomes as a marker for the differentiation of Th1-like effector γδ T cells. This article is protected by copyright. All rights reserved.

  5. OAS/PKR Pathways and α/β TCR+ T Cells are Required for Ad: IFN-γ Inhibition of HSV-1 in Cornea1

    PubMed Central

    Austin, Bobbie Ann; Halford, William P.; Williams, Bryan R. G.; Carr, Daniel J. J.

    2007-01-01

    An adenoviral vector containing the muIFN-γ transgene (Ad:IFN-γ) was evaluated for its capacity to inhibit HSV-1. To measure effectiveness, viral titers were analyzed in cornea and trigeminal ganglia (TG) during acute ocular HSV-1 infection. Ad: IFN-γ potently suppressed HSV-1 replication in a dose-dependent fashion, requiring IFN-γ R. Moreover, Ad:IFN-γ was effective when delivered -72 and -24 h prior to infection as well as 24 h post infection. Associated with anti-viral opposition, TG from Ad: IFN-γ transduced mice harbored fewer T cells. Also related to T cell involvement, Ad:IFN-γ was effective but attenuated in TG from α/β TCR deficient mice. In corneas, α/β TCR+ T cells were obligatory for protection against viral multiplication. Type I IFN involvement amid anti-viral efficacy of Ad: IFN-γ was further investigated because type I and II IFN pathways have synergistic anti-HSV-1 activity. Ad:IFN-γ inhibited viral reproduction in corneas and TG from IFN-α/β R deficient (CD118 −/−) mice, although viral titers were 2–3 fold higher in cornea and TG, compared to wild type. The absence of IFN-stimulated anti-viral proteins, 2’-5’ oligoadenylate synthetase/RNase L and ds RNA dependent protein kinase R, completely eliminated the anti-viral effectiveness of Ad:IFN-γ. Collectively, the results demonstrate: (1) nonexistence of type I IFN R does not abolish defense of Ad:IFN-γ against HSV-1; (2) anti-viral pathways, OAS/RNase L and PKR are mandatory; and (3) α/β TCR+ T cells are compulsory for Ad: IFN-γ effectiveness against HSV-1 in cornea but not in TG. PMID:17404299

  6. TNF-alpha, but not IFN-gamma, regulates CCN2 (CTGF), collagen type I, and proliferation in mesangial cells: possible roles in the progression of renal fibrosis.

    PubMed

    Cooker, Laurinda A; Peterson, Darryl; Rambow, Joann; Riser, Melisa L; Riser, Rebecca E; Najmabadi, Feridoon; Brigstock, David; Riser, Bruce L

    2007-07-01

    Connective tissue growth factor (CCN2) is a profibrotic factor acting downstream and independently of TGF-beta to mediate renal fibrosis. Although inflammation is often involved in the initiation and/or progression of fibrosis, the role of inflammatory cytokines in regulation of glomerular CCN2 expression, cellular proliferation, and extracellular matrix accumulation is unknown. We studied two such cytokines, TNF-alpha and IFN-gamma, for their effects on cultured mesangial cells in the presence or absence of TGF-beta, as a model for progressive renal fibrosis. Short-term treatment with TNF-alpha, like TGF-beta, significantly increased secreted CCN2 per cell, but unlike TGF-beta inhibited cellular replication. TNF-alpha combined with TGF-beta further increased CCN2 secretion and mRNA levels and reduced proliferation. Surprisingly, however, TNF-alpha treatment decreased baseline collagen type I protein and mRNA levels and largely blocked their stimulation by TGF-beta. Long-term treatment with TGF-beta or TNF-alpha alone no longer increased CCN2 protein levels. However, the combination synergistically increased CCN2. IFN-gamma had no effect on either CCN2 or collagen activity and produced a mild inhibition of TGF-beta-induced collagen only at a high concentration (500 U/ml). In summary, we report a strong positive regulatory role for TNF-alpha, but not IFN-gamma, in CCN2 production and secretion, including that driven by TGF-beta. The stimulation of CCN2 release by TNF-alpha, unlike TGF-beta, is independent of cellular proliferation and not linked to increased collagen type I accumulation. This suggests that the paradigm of TGF-beta-driven CCN2 with subsequent collagen production may be overridden by an as yet undefined inhibitory mechanism acting either directly or indirectly on matrix metabolism.

  7. IFN-γ Directly Controls IL-33 Protein Level through a STAT1- and LMP2-dependent Mechanism*

    PubMed Central

    Kopach, Pavel; Lockatell, Virginia; Pickering, Edward M.; Haskell, Ronald E.; Anderson, Richard D.; Hasday, Jeffrey D.; Todd, Nevins W.; Luzina, Irina G.; Atamas, Sergei P.

    2014-01-01

    IL-33 contributes to disease processes in association with Th1 and Th2 phenotypes. IL-33 mRNA is rapidly regulated, but the fate of synthesized IL-33 protein is unknown. To understand the interplay among IL-33, IFN-γ, and IL-4 proteins, recombinant replication-deficient adenoviruses were produced and used for dual expression of IL-33 and IFN-γ or IL-33 and IL-4. The effects of such dual gene delivery were compared with the effects of similar expression of each of these cytokines alone. In lung fibroblast culture, co-expression of IL-33 and IFN-γ resulted in suppression of the levels of both proteins, whereas co-expression of IL-33 and IL-4 led to mutual elevation. In vivo, co-expression of IL-33 and IFN-γ in the lungs led to attenuation of IL-33 protein levels. Purified IFN-γ also attenuated IL-33 protein in fibroblast culture, suggesting that IFN-γ controls IL-33 protein degradation. Specific inhibition of caspase-1, -3, and -8 had minimal effect on IFN-γ-driven IL-33 protein down-regulation. Pharmacological inhibition, siRNA-mediated silencing, or gene deficiency of STAT1 potently up-regulated IL-33 protein expression levels and attenuated the down-regulating effect of IFN-γ on IL-33. Stimulation with IFN-γ strongly elevated the levels of the LMP2 proteasome subunit, known for its role in IFN-γ-regulated antigen processing. siRNA-mediated silencing of LMP2 expression abrogated the effect of IFN-γ on IL-33. Thus, IFN-γ, IL-4, and IL-33 are engaged in a complex interplay. The down-regulation of IL-33 protein levels by IFN-γ in pulmonary fibroblasts and in the lungs in vivo occurs through STAT1 and non-canonical use of the LMP2 proteasome subunit in a caspase-independent fashion. PMID:24619410

  8. Chlamydia trachomatis induces expression of IFN-gamma-inducible protein 10 and IFN-beta independent of TLR2 and TLR4, but largely dependent on MyD88.

    PubMed

    Nagarajan, Uma M; Ojcius, David M; Stahl, Lynn; Rank, Roger G; Darville, Toni

    2005-07-01

    IFN-gamma-inducible protein 10 (IP-10) is a chemokine important in the attraction of T cells, which are essential for resolution of chlamydial genital tract infection. During infections with Gram-negative bacteria, the IP-10 response mediated through type I IFNs usually occurs as a result of TLR4 stimulation by bacterial LPS. However, we found that levels of IP-10 in genital tract secretions of Chlamydia trachomatis-infected female wild-type mice were similar to those of infected TLR2- and TLR4-deficient mice but significantly greater than those of infected MyD88-deficient mice. We investigated the mechanism of IP-10 and IFN-beta induction during chlamydial infection using mouse macrophages and fibroblasts infected ex vivo. The induction of IP-10 and IFN-beta was unchanged in Chlamydia-infected TLR2- and TLR4-deficient cells compared with wild-type cells. However, infection of MyD88-deficient cells resulted in significantly decreased responses. These results suggest a role for MyD88-dependent pathways in induction of IP-10 and IFN-beta during chlamydial infection. Furthermore, treatment of infected macrophages with an endosomal maturation inhibitor significantly reduced chlamydial-induced IFN-beta. Because endosomal maturation is required for MyD88-dependent intracellular pathogen recognition receptors to function, our data suggest a role for the intracellular pathogen recognition receptor(s) in induction of IFN-beta and IP-10 during chlamydial infection. Furthermore, the intracellular pathways that lead to chlamydial-induced IFN-beta function through TANK-binding kinase mediated phosphorylation and nuclear translocation of IFN regulatory factor-3.

  9. Lycopene, quercetin and tyrosol prevent macrophage activation induced by gliadin and IFN-gamma.

    PubMed

    De Stefano, Daniela; Maiuri, Maria Chiara; Simeon, Vittorio; Grassia, Gianluca; Soscia, Antonio; Cinelli, Maria Pia; Carnuccio, Rosa

    2007-07-02

    Oxidative stress plays an important role in inflammatory process of celiac disease. We have studied the effect of the lycopene, quercetin and tyrosol natural antioxidants on the inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) gene expression in RAW 264.7 macrophages stimulated by gliadin in association with IFN-gamma. The IFN-gamma plus gliadin combination treatment was capable of enhancing iNOS and COX-2 gene expression and nuclear factor-kappaB (NF-kappaB), interferon regulatory factor-1 (IRF-1) and signal transducer and activator of transcription-1alpha (STAT-1alpha) activation induced by reactive oxygen species generation at 24 h. Lycopene, quercetin and tyrosol inhibited all these effects. The results here reported suggest that these compounds may represent non toxic agents for the control of pro-inflammatory genes involved in celiac disease.

  10. Naturally Occurring Fc-Dependent Antibody From HIV-Seronegative Individuals Promotes HIV-Induced IFN-α Production

    PubMed Central

    Lum, Thomas; Green, Jon A.

    2016-01-01

    A majority of adults without HIV infection and with a low risk of HIV-exposure have plasma IgG antibodies that enhance the rate and magnitude of HIV-induced interferon alpha (IFN-α) production. Fc-dependent IgG-HIV complexes induce IFN-α rapidly and in high titers in response to HIV concentrations that are too low to otherwise stimulate an effective IFN-α response. IFN-α promoting antibody (IPA) counters HIV-specific inhibition of IFN-α production, and compensates for the inherent delay in IFN-α production common to HIV infection and other viruses. Naturally occurring IPA has the potential to initiate a potent IFN-α response early in the course of HIV mucosal invasion in time to terminate infection prior to the creation of a pool of persistently infected cells. The current study adds IPA as a mediator of an Fc-dependent antiviral state capable of preventing HIV infection. PMID:27881846

  11. IFN-γ regulates human dental pulp stem cells behavior via NF-κB and MAPK signaling

    PubMed Central

    He, Xinyao; Jiang, Wenkai; Luo, Zhirong; Qu, Tiejun; Wang, Zhihua; Liu, Ningning; Zhang, Yaqing; Cooper, Paul R.; He, Wenxi

    2017-01-01

    During caries, dental pulp expresses a range of pro-inflammatory cytokines in response to the infectious challenge. Interferon gamma (IFN-γ) is a dimerized soluble cytokine, which is critical for immune responses. Previous study has demonstrated that IFN-γ at relative high concentration (100 ng/mL) treatment improved the impaired dentinogenic and immunosuppressive regulatory functions of disease-derived dental pulp stem cells (DPSCs). However, little is known about the regulatory effects of IFN-γ at relative low concentration on healthy DPSC behavior (including proliferation, migration, and multiple-potential differentiation). Here we demonstrate that IFN-γ at relatively low concentrations (0.5 ng/mL) promoted the proliferation and migration of DPSCs, but abrogated odonto/osteogenic differentiation. Additionally, we identified that NF-κB and MAPK signaling pathways are both involved in the process of IFN-γ-regulated odonto/osteogenic differentiation of DPSCs. DPSCs treated with IFN-γ and supplemented with pyrrolidine dithiocarbamate (PDTC, an NF-κB inhibitor) or SB203580 (a MAPK inhibitor) showed significantly improved potential for odonto/osteogenic differentiation of DPSCs both in vivo and in vitro. These data provide important insight into the regulatory effects of IFN-γ on the biological behavior of DPSCs and indicate a promising therapeutic strategy for dentin/pulp tissue engineering in future endodontic treatment. PMID:28098169

  12. Role of the IFN I system against the VHSV infection in juvenile Senegalese sole (Solea senegalensis).

    PubMed

    Alvarez-Torres, Daniel; Podadera, Ana M; Bejar, Julia; Bandin, Isabel; Alonso, M Carmen; Garcia-Rosado, Esther

    2016-01-08

    Senegalese sole is susceptible to marine VHSV isolates but is not affected by freshwater isolates, which may indicate differences regarding virus-host immune system interaction. IFN I induces an antiviral state in fish, stimulating the expression of genes encoding antiviral proteins (ISG). In this study, the stimulation of the Senegalese sole IFN I by VHSV infections has been evaluated by the relative quantification of the transcription of several ISG (Mx, Isg15 and Pkr) after inoculation with marine (pathogenic) and freshwater (non-pathogenic) VHSV isolates. Compared to marine VHSV, lower levels of RNA of the freshwater VHSV induced transcription of ISG to similar levels, with the Isg15 showing the highest fold induction. The protective role of the IFN I system was evaluated in poly I:C-inoculated animals subsequently challenged with VHSV isolates. The cumulative mortality caused by the marine isolate in the control group was 68%, whereas in the poly I:C-stimulated group was 5%. The freshwater VHSV isolate did not cause any mortality. Furthermore, viral RNA fold change and viral titers were lower in animals from the poly I:C + VHSV groups than in the controls. The implication of the IFN I system in the protection observed was confirmed by the transcription of the ISG in animals from the poly I:C + VHSV groups. However, the marine VHSV isolate exerts a negative effect on the ISG transcription at 3 and 6 h post-inoculation (hpi), which is not observed for the freshwater isolate. This difference might be partly responsible for the virulence shown by the marine isolate.

  13. Use of recombinant adenovirus vectored consensus IFN-α to avert severe arenavirus infection.

    PubMed

    Gowen, Brian B; Ennis, Jane; Russell, Andrew; Sefing, Eric J; Wong, Min-Hui; Turner, Jeffrey

    2011-01-01

    Several arenaviruses can cause viral hemorrhagic fever, a severe disease with case-fatality rates in hospitalized individuals ranging from 15-30%. Because of limited prophylaxis and treatment options, new medical countermeasures are needed for these viruses classified by the National Institutes of Allergy and Infectious Diseases (NIAID) as top priority biodefense Category A pathogens. Recombinant consensus interferon alpha (cIFN-α) is a licensed protein with broad clinical appeal. However, while cIFN-α has great therapeutic value, its utility for biodefense applications is hindered by its short in vivo half-life, mode and frequency of administration, and costly production. To address these limitations, we describe the use of DEF201, a replication-deficient adenovirus vector that drives the expression of cIFN-α, for pre- and post-exposure prophylaxis of acute arenaviral infection modeled in hamsters. Intranasal administration of DEF201 24 h prior to challenge with Pichindé virus (PICV) was highly effective at protecting animals from mortality and preventing viral replication and liver-associated disease. A significant protective effect was still observed with a single dosing of DEF201 given two weeks prior to PICV challenge. DEF201 was also efficacious when administered as a treatment 24 to 48 h post-virus exposure. The protective effect of DEF201 was largely attributed to the expression of cIFN-α, as dosing with a control empty vector adenovirus did not protect hamsters from lethal PICV challenge. Effective countermeasures that are highly stable, easily administered, and elicit long lasting protective immunity are much needed for arena and other viral infections. The DEF201 technology has the potential to address all of these issues and may serve as a broad-spectrum antiviral to enhance host defense against a number of viral pathogens.

  14. Enhancing pIFN-α production and process stability in fed-batch culture of Pichia pastoris by controlling the methanol concentration and monitoring the responses of OUR/DO levels.

    PubMed

    Gao, Min-Jie; Zhan, Xiao-Bei; Zheng, Zhi-Yong; Wu, Jian-Rong; Dong, Shi-Juan; Li, Zhen; Shi, Zhong-Ping; Lin, Chi-Chung

    2013-11-01

    Effective expression of porcine interferon-α (pIFN-α) with recombinant Pichia pastoris was conducted in a bench-scale fermentor using an in situ methanol electrode-based feeding process with the control level of methanol concentration linearly increased to 10 g l⁻¹ for the first 20 h and maintained at 10 g l⁻¹ for the rest of expression phase. With this two-stage control process, the highest pIFN-α concentration reached a level of 1.81 g l⁻¹, which was 1.5-fold of that in the previous constant 10 g l⁻¹ induction experiments. There is an improvement of the pIFN-α productivity from more distribution of carbon flux to protein expression. The pIFN-α expression stability could be further enhanced by a simple on-line fault diagnosis method for methanol overfeeding based on oxygen uptake rate changing patterns. By implementing corrective action of feeding glycerol after fault detection, the production yield increased to twice the amount it would have been without the diagnosis.

  15. Serum levels of IFN-β are associated with days of evolution but not with severity of dengue.

    PubMed

    Pech Torres, Ramón Enrique; Cedillo Rivera, Roberto Manuel; Loroño Pino, Maria Alba; Sánchez Burgos, Gilma Guadalupe

    2016-03-01

    The serum levels of beta interferon (IFN-β) were evaluated in clinical samples taken in the acute phase of dengue fever for 107 patients during the 2011 dengue outbreak in Yucatán, México. Dengue diagnoses were confirmed by NS1 or IgM/IgG serology in all patients. Average serum IFN-β levels in patients with dengue fever without warning signs (n = 53) were 140 pg/ml, and 105 pg/ml for patients with warning signs (n = 54). There was no statistically significant difference between IFN-β levels for the two groups (Mann-Whitney U Test P > 0.05) and no association with warning or mild signs (OR: 0.57; 95%CI: 0.26-1.22) was indicated. Thrombocytopenia was the most prevalent warning sign (91%) in one group of patients (49) with mean 113 pg/ml IFN-β levels. In contrast, the patients without thrombocytopenia (50) had 126 pg/ml of IFN-β, but this level was not statistically significant (Mann-Whitney U Test P > 0.05). The average levels of IFN-β were also found to have statistically similar results, using the 1997 WHO classification system. The amount of IFN-β at 1-3, 4-6, and 7-9 days after onset of illness, however, did show significant differences (ANOVA P = 0.038) between patients for the 1-3 and 4-6 days pair (Scheffe post-hoc P = 0.043). These results suggest that serum levels of IFN-β do not correlate well with the severity of dengue illness, but there is a clear association between changes in IFN-β levels and the days of evolution during the acute phase of the disease.

  16. Natural Killer Cells-Produced IFN-γ Improves Bone Marrow-Derived Hepatocytes Regeneration in Murine Liver Failure Model

    PubMed Central

    Li, Lu; Zeng, Zhutian; Qi, Ziping; Wang, Xin; Gao, Xiang; Wei, Haiming; Sun, Rui; Tian, Zhigang

    2015-01-01

    Bone-marrow transplantation (BMT) can repopulate the liver through BM-derived hepatocyte (BMDH) generation, although the underlying mechanism remains unclear. Using fumarylacetoacetate hydrolase–deficient (Fah−/−) mice as a liver-failure model, we confirmed that BMDHs were generated by fusion of BM-derived CD11b+F4/80+myelomonocytes with resident Fah−/− hepatocytes. Hepatic NK cells became activated during BMDH generation and were the major IFN-γ producers. Indeed, both NK cells and IFN-γ were required for BMDH generation since WT, but not NK-, IFN-γ–, or IFN-γR1–deficient BM transplantation successfully generated BMDHs and rescued survival in Fah−/− hosts. BM-derived myelomonocytes were determined to be the IFN-γ–responding cells. The IFN-γ–IFN-γR interaction contributed to the myelomonocyte–hepatocyte fusion process, as most of the CD11b+ BMDHs in mixed BM chimeric Fah−/− hosts transplanted with a 1:1 ratio of CD45.1+ WT and CD45.2+ Ifngr1−/− BM cells were of CD45.1+ WT origin. Confirming these findings in vitro, IFN-γ dose-dependently promoted the fusion of GFP+ myelomonocytes with Fah−/− hepatocytes due to a direct effect on myelomonocytes; similar results were observed using activated NK cells. In conclusion, BMDH generation requires NK cells to facilitate myelomonocyte–hepatocyte fusion in an IFN-γ–dependent manner, providing new insights for treating severe liver failure. PMID:26345133

  17. Sialoadhesin Promotes Rapid Proinflammatory and Type I IFN Responses to a Sialylated Pathogen, Campylobacter jejuni

    PubMed Central

    Klaas, Mariliis; Oetke, Cornelia; Lewis, Leanne E.; Erwig, Lars P.; Heikema, Astrid P.; Easton, Alistair; Willison, Hugh J.

    2012-01-01

    Sialoadhesin (Sn) is a macrophage (Mϕ)-restricted receptor that recognizes sialylated ligands on host cells and pathogens. Although Sn is thought to be important in cellular interactions of Mϕs with cells of the immune system, the functional consequences of pathogen engagement by Sn are unclear. As a model system, we have investigated the role of Sn in Mϕ interactions with heat-killed Campylobacter jejuni expressing a GD1a-like, sialylated glycan. Compared to Sn-expressing bone marrow-derived macrophages (BMDM) from wild-type mice, BMDM from mice either deficient in Sn or expressing a non-glycan–binding form of Sn showed greatly reduced phagocytosis of sialylated C. jejuni. This was accompanied by a strong reduction in MyD88-dependent secretion of TNF-α, IL-6, IL-12, and IL-10. In vivo studies demonstrated that functional Sn was required for rapid TNF-α and IFN-β responses to i.v.-injected sialylated C. jejuni. Bacteria were captured within minutes after i.v. injection and were associated with Mϕs in both liver and spleen. In the spleen, IFN-β–reactive cells were localized to Sn+ Mϕs and other cells in the red pulp and marginal zone. Together, these studies demonstrate that Sn plays a key role in capturing sialylated pathogens and promoting rapid proinflammatory cytokine and type I IFN responses. PMID:22851711

  18. Genetic polymorphisms, their allele combinations and IFN-β treatment response in Irish multiple sclerosis patients

    PubMed Central

    O’Doherty, Catherine; Favorov, Alexander; Heggarty, Shirley; Graham, Colin; Favorova, Olga; Ochs, Michael; Hawkins, Stanley; Hutchinson, Michael; O’Rourke, Killian; Vandenbroeck, Koen

    2009-01-01

    Introduction IFN-β is widely used as first-line immunomodulatory treatment for multiple sclerosis. Response to treatment is variable (30–50% of patients are nonresponders) and requires a long treatment duration for accurate assessment to be possible. Information about genetic variations that predict responsiveness would allow appropriate treatment selection early after diagnosis, improve patient care, with time saving consequences and more efficient use of resources. Materials & methods We analyzed 61 SNPs in 34 candidate genes as possible determinants of IFN-β response in Irish multiple sclerosis patients. Particular emphasis was placed on the exploration of combinations of allelic variants associated with response to therapy by means of a Markov chain Monte Carlo-based approach (APSampler). Results The most significant allelic combinations, which differed in frequency between responders and nonresponders, included JAK2–IL10RB–GBP1–PIAS1 (permutation p-value was pperm = 0.0008), followed by JAK2–IL10–CASP3 (pperm = 0.001). Discussion The genetic mechanism of response to IFN-β is complex and as yet poorly understood. Data mining algorithms may help in uncovering hidden allele combinations involved in drug response versus nonresponse. PMID:19604093

  19. Morphological features of IFN-γ–stimulated mesenchymal stromal cells predict overall immunosuppressive capacity

    PubMed Central

    Klinker, Matthew W.; Marklein, Ross A.; Lo Surdo, Jessica L.; Wei, Cheng-Hong

    2017-01-01

    Human mesenchymal stromal cell (MSC) lines can vary significantly in their functional characteristics, and the effectiveness of MSC-based therapeutics may be realized by finding predictive features associated with MSC function. To identify features associated with immunosuppressive capacity in MSCs, we developed a robust in vitro assay that uses principal-component analysis to integrate multidimensional flow cytometry data into a single measurement of MSC-mediated inhibition of T-cell activation. We used this assay to correlate single-cell morphological data with overall immunosuppressive capacity in a cohort of MSC lines derived from different donors and manufacturing conditions. MSC morphology after IFN-γ stimulation significantly correlated with immunosuppressive capacity and accurately predicted the immunosuppressive capacity of MSC lines in a validation cohort. IFN-γ enhanced the immunosuppressive capacity of all MSC lines, and morphology predicted the magnitude of IFN-γ–enhanced immunosuppressive activity. Together, these data identify MSC morphology as a predictive feature of MSC immunosuppressive function. PMID:28283659

  20. IFN type I and II induce BAFF secretion from human decidual stromal cells.

    PubMed

    Lundell, Anna-Carin; Nordström, Inger; Andersson, Kerstin; Lundqvist, Christina; Telemo, Esbjörn; Nava, Silvia; Kaipe, Helen; Rudin, Anna

    2017-01-06

    B cell activating factor (BAFF) is a critical cytokine for maturation of immature B cells. In murine lymph nodes, BAFF is mainly produced by podoplanin-expressing stromal cells. We have previously shown that circulating BAFF levels are maximal at birth, and that farmers' children exhibit higher BAFF levels in cord blood than non-farmers' children. Here, we sought to investigate whether maternal-derived decidual stromal cells from placenta secrete BAFF and examine what factors could stimulate this production. We found that podoplanin is expressed in decidua basalis and in the underlying villous tissue as well as on isolated maternal-derived decidual stromal cells. Decidual stromal cells produced BAFF when stimulated with IFN-γ and IFN-α, and NK cells and NK-T-like cells competent of IFN-γ production were isolated from the decidua. Finally, B cells at different maturational stages are present in decidua and all expressed BAFF-R, while stromal cells did not. These findings suggest that decidual stromal cells are a cellular source of BAFF for B cells present in decidua during pregnancy.

  1. IFN type I and II induce BAFF secretion from human decidual stromal cells

    PubMed Central

    Lundell, Anna-Carin; Nordström, Inger; Andersson, Kerstin; Lundqvist, Christina; Telemo, Esbjörn; Nava, Silvia; Kaipe, Helen; Rudin, Anna

    2017-01-01

    B cell activating factor (BAFF) is a critical cytokine for maturation of immature B cells. In murine lymph nodes, BAFF is mainly produced by podoplanin-expressing stromal cells. We have previously shown that circulating BAFF levels are maximal at birth, and that farmers’ children exhibit higher BAFF levels in cord blood than non-farmers’ children. Here, we sought to investigate whether maternal-derived decidual stromal cells from placenta secrete BAFF and examine what factors could stimulate this production. We found that podoplanin is expressed in decidua basalis and in the underlying villous tissue as well as on isolated maternal-derived decidual stromal cells. Decidual stromal cells produced BAFF when stimulated with IFN-γ and IFN-α, and NK cells and NK-T-like cells competent of IFN-γ production were isolated from the decidua. Finally, B cells at different maturational stages are present in decidua and all expressed BAFF-R, while stromal cells did not. These findings suggest that decidual stromal cells are a cellular source of BAFF for B cells present in decidua during pregnancy. PMID:28057926

  2. Characterization of CD4 and CD8 T cells producing IFN-γ in human latent and active tuberculosis.

    PubMed

    Rueda, Cesar M; Marín, Nancy D; García, Luis F; Rojas, Mauricio

    2010-11-01

    Patients with pulmonary tuberculosis (PTB) frequently have reduced IFN-γ production in response to mycobacterial antigens, compared to individuals with latent Mycobacterium tuberculosis infection (LTBi). However, it is not clear whether this reduced responsiveness is restricted to a particular T cell subset. Herein, PBMCs from 26 PTB patients, 30 household contacts (HHCs) of PTB, and 30 tuberculin positive (TST+) healthy subjects not recently exposed to PTB, were stained with CFSE and stimulated non-specific (PPD) for 120 h, and specific (CFP-10/ESAT-6) and latency (HSpX) mycobacterial antigens for 144 h and the percentage of CD4(+) and CD8(+)IFN-γ(+) T cells responding determined by flow cytometry, in addition to their memory phenotype by the CD45RO and CD27 expression. PTB had decreased frequency of both CD4(+) and CD8(+) precursor cells, as well as decreased number of CD4(+)IFN-γ(+) cells in response to all antigens, whereas CD8(+)IFN-γ(+) cells were decreased in response to PPD and ESAT-6, but not to CFP-10 and HSpX. HHCs exhibited the highest precursor frequencies and IFN-γ responses, irrespective of the antigen employed. The CD4(+)/CD8(+) cell ratios showed that in response to PPD CD4(+) precursor and IFN-γ-producer cells are more frequent than their CD8(+) counterparts, and that PTB have a decreased CD4(+)IFN-γ(+)/CD8(+)IFN-γ(+) ratio in response to PPD, CFP-10, and ESAT-6. CD4(+)IFN-γ(+) and CD8(+)IFN-γ(+) cells exhibited a central memory phenotype (CD45RO(+)CD27(+)), irrespective of the group of subjects and the antigen used for stimulation. In conclusion, PTB patients had a decreased percentage of CD4(+) and CD8(+) precursor cells and CD4(+)IFN-γ(+). HHCs exhibited the highest frequency of CD4(+) and CD8(+) precursors and CD4(+)IFN-γ(+)-producing cells.

  3. IL-7 Promotes CD95-Induced Apoptosis in B Cells via the IFN-γ/STAT1 Pathway

    PubMed Central

    Sammicheli, Stefano; Dang Vu Phuong, Linh; Ruffin, Nicolas; Pham Hong, Thang; Lantto, Rebecka; Vivar, Nancy; Chiodi, Francesca; Rethi, Bence

    2011-01-01

    Interleukin-7 (IL-7) concentrations are increased in the blood of CD4+ T cell depleted individuals, including HIV-1 infected patients. High IL-7 levels might stimulate T cell activation and, as we have shown earlier, IL-7 can prime resting T cell to CD95 induced apoptosis as well. HIV-1 infection leads to B cell abnormalities including increased apoptosis via the CD95 (Fas) death receptor pathway and loss of memory B cells. Peripheral B cells are not sensitive for IL-7, due to the lack of IL-7Ra expression on their surface; however, here we demonstrate that high IL-7 concentration can prime resting B cells to CD95-mediated apoptosis via an indirect mechanism. T cells cultured with IL-7 induced high CD95 expression on resting B cells together with an increased sensitivity to CD95 mediated apoptosis. As the mediator molecule responsible for B cell priming to CD95 mediated apoptosis we identified the cytokine IFN-γ that T cells secreted in high amounts in response to IL-7. These results suggest that the lymphopenia induced cytokine IL-7 can contribute to the increased B cell apoptosis observed in HIV-1 infected individuals. PMID:22194871

  4. Increase in bile flow and biliary excretion of glutathione-derived sulfhydryls in rats by drug-metabolizing enzyme inducers is mediated by multidrug resistance protein 2.

    PubMed

    Johnson, David R; Habeebu, Sultan S M; Klaassen, Curtis D

    2002-03-01

    Glutathione (GSH) is an important cellular constituent for normal liver homeostasis. Certain drug-metabolizing enzyme inducers (i.e., phenobarbital [PB] and pregnenolone-16alpha-carbonitrile [PCN]) increase biliary excretion of GSH-derived sulfhydryls (SH) as well as bile flow, whereas other drug-metabolizing enzyme inducers (i.e., 3-methylcholanthrene [3MC] and benzo(a)pyrene [BaP]), do not. The purpose of the study was to determine whether rat multidrug resistance protein 2 (Mrp2) is the inducible transporter responsible for increasing biliary SH excretion and bile flow. Sprague-Dawley (SD) rats were injected ip daily for 4 days with PB, PCN, 3MC, BaP, or vehicle; Mrp2-null Eisai hyperbilirubinemic (EHBR) rats were injected ip daily for 4 days with PCN or vehicle. Although no drug-metabolizing enzyme inducer altered hepatic GSH in SD rats, PB and PCN significantly increased the rate of biliary SH excretion and bile flow. Neither 3MC nor BaP affected the biliary SH excretion rate or bile flow. In control EHBR rats, despite elevated hepatic GSH, the rate of biliary SH excretion was almost completely eliminated and bile flow was dramatically reduced compared with SD rats. Furthermore, PCN treatment did not affect bile flow or the biliary SH excretion rate in EHBR rats. PB and PCN also increased Mrp2 protein levels, but 3MC and BaP did not. None of the drug-metabolizing enzyme inducers tested significantly increased Mrp2 mRNA levels. PCN increased Mrp2 protein, but not Mrp2 mRNA, in a time-dependent manner. In conclusion, Mrp2 is the inducible efflux transporter responsible for increased biliary SH excretion and bile flow after administration of some drug-metabolizing enzyme inducers.

  5. Study of immunological aspects of aspergillosis in mice and effect of polyene macrolide antibiotic (SJA-95) and IFN-γ: a possible role of IFN-γ as an adjunct in antifungal therapy.

    PubMed

    Naik, Suresh R; Thakare, Vishnu N; Desai, Sandhya K; Rahalkar, Prabhakar R

    2011-12-30

    New polyene macrolide antibiotic SJA-95 in free as well as liposomal (lip.) forms, with and without interferon-γ (IFN-γ) was studied in mice model of aspergillosis using biological and biochemical parameters viz. colony forming units (CFU) in liver, spleen, kidney, lung and brain, and serum IgG, and interleukin-4 (IL-4). Treatment with free and lip SJA-95 along with IFN-γ prolonged the survival time, reduced CFU in vital organs, decreased serum IgG and IL-4 levels. SJA-95 lip form showed greater antifungal activity as compared to free form. The combined treatment of lip SJA-95 with IFN-γ showed further enhancement in antifungal activity of SJA-95 (lip). The present experimental findings demonstrated IFN-γ might act as a potent modulator in immune reaction during fungal infection and can be a useful adjunctive in antifungal therapy in the management of deep seated systemic mycoses.

  6. Recombinant fowlpox viruses coexpressing chicken type I IFN and Newcastle disease virus HN and F genes: influence of IFN on protective efficacy and humoral responses of chickens following in ovo or post-hatch administration of recombinant viruses.

    PubMed

    Karaca, K; Sharma, J M; Winslow, B J; Junker, D E; Reddy, S; Cochran, M; McMillen, J

    1998-10-01

    We have constructed recombinant (r) fowl pox viruses (FPVs) coexpressing chicken type I interferon (IFN) and/or hemagglutinin-neuraminidase (HN) and fusion (F) proteins of Newcastle disease virus (NDV). We administered rFPVs and FPV into embryonated chicken eggs at 17 days of embryonation or in chickens after hatch. Administration of FPV or rFPVs did not influence hatchability and survival of hatched chicks. In ovo or after hatch vaccination of chickens with the recombinant viruses resulted in protection against challenge with virulent FPV and NDV. Chickens vaccinated with FPV or FPV-NDV recombinant had significantly lower body weight 2 weeks following vaccination. This loss in body weight was not detected in chickens receiving FPV-IFN and FPV-NDV-IFN recombinants. Chickens vaccinated with FPV coexpressing IFN and NDV genes produced less antibodies against NDV in comparison with chickens vaccinated with FPV expressing NDV genes.

  7. Dysregulated expression of IFN-γ and IL-10 and impaired IFN-γ-mediated responses at different disease stages in patients with genital herpes simplex virus-2 infection

    PubMed Central

    SINGH, R; KUMAR, A; CREERY, W D; RUBEN, M; GIULIVI, A; DIAZ-MITOMA, F

    2003-01-01

    Cell-mediated T-helper type-1 (Th1) responses play a vital role in the immunopathogenesis of genital infections caused by herpes simplex virus 2 (HSV-2). We investigated the role of Th responses in HSV-2 infection at different disease stages by analysing the production of Th cytokines in HSV-stimulated peripheral blood mononuclear cells (PBMCs). IFN-γ production decreased over time following a recurrence, whereas levels of IL-10, and to a lesser extent IL-2, remained elevated during this period. In addition, PBMCs from asymptomatic seropositive individuals produced high levels of IFN-γ and low levels of IL-10, in contrast to individuals with a history of genital ulcers. Following a recurrence, virus copy number in the genital lesions decreased progressively over time, in a manner similar to IFN-γ production by HSV-2-stimulated PBMCs. Enhanced production of IFN-γ may modulate HSV replication and B7 expression on monocytic cells of HSV-infected individuals. In contrast to seronegative controls, IFN-γ failed to enhance B7 expression on monocytic cells of HSV-infected individuals. In addition, monocytic cells from HSV-2-infected individuals with recurrent disease supported greater HSV replication than did those of HSV-infected asymptomatic individuals or seronegative controls. Furthermore, addition of IFN-γ resulted in enhanced HSV replication in monocytic cells of HSV-infected individuals with recurrent disease, in contrast to the inhibition observed in HSV-seropositive asymptomatic individuals and seronegative controls. Taken together, our results suggest that dysregulated production of IFN-γ at different disease stages and the impaired ability of monocytic cells to respond to IFN-γ may play a role in the pathogenesis of recurrent genital herpes disease. PMID:12823283

  8. Interferon-γ is increased in the gut of patients with irritable bowel syndrome and modulates serotonin metabolism.

    PubMed

    Barbaro, Maria Raffaella; Di Sabatino, Antonio; Cremon, Cesare; Giuffrida, Paolo; Fiorentino, Michelangelo; Altimari, Annalisa; Bellacosa, Lara; Stanghellini, Vincenzo; Barbara, Giovanni

    2016-03-15

    Mucosal immune activation and altered serotonin metabolism participate in the pathophysiology of irritable bowel syndrome (IBS). However, the reciprocal interplay between these two systems remains unknown. We evaluated the expression and release of interferon (IFN)-γ from the colonic mucosa of patients with IBS and its impact on serotonin reuptake transporter (SERT) gene expression in Caco-2 cells. qPCR was used to evaluate IFN-γ gene expression in colonic mucosal biopsies, whereas IFN-γ protein amount was assessed by ELISA. Colonic T box expressed in T cells (T-bet) and phosphorylated signal transducer and activator of transcription 4 protein amount were evaluated by Western blot. The impact of colonic mucosal mediators on SERT gene expression was evaluated in Caco-2 cells using qPCR. IFN-γ receptor was silenced in Caco-2 cells to determine the effect of IFN-γ released by mucosal biopsies. Compared with asymptomatic controls (ACs), the expression of IFN-γ gene and its transcription factor T-bet were markedly increased in the colonic mucosa of patients with IBS. Compared with ACs, IFN-γ protein tissue levels and its release by mucosal biopsies were significantly increased in IBS. The exposure of Caco-2 cells to IBS supernatants induced a significant decrease in SERT gene expression, independently of IBS subtypes, compared with AC mucosal supernatants. In Caco-2 cells, IFN-γ receptor silencing reversed the reduction of SERT expression evoked by IBS supernatants vs. nonsilenced cell lines. IFN-γ gene, its transcription factor T-bet, IFN-γ protein expression, and its release are increased in the colonic mucosa of patients with IBS and downregulate SERT gene expression in vitro. These results suggest that IFN-γ downregulates SERT expression, hence likely playing a role in altered serotonin metabolism of patients with IBS.

  9. Dimethylnitrosamine-demethylase: absence of increased enzyme catabolism and multiplicity of effector sites in repression. Hemoprotein involvement.

    PubMed

    Argus, M F; Arcos, J C; Pastor, K M; Wu, B C; Venkatesan, N

    1976-05-01

    Evidence is presented that the previously observed decrease of the Vmax of hepatic microsomal demethylation of dimethylnitrosamine (DMN), following pretreatment of rats with 3-methylcholanthrene (MC), is not due to increase in the rate of breakdown but to decrease of de novo synthesis. Determinations of Vmax at time intervals in the transition from the high steady-state level induced by a carbohydrate-devoid casein diet, down to the low steady-state level of carbohydrate-containing basal diet, yielded two consecutive slopes; descent from the basal diet level to the lower steady-state level following pretreatment with MC yielded one slope. Plotting these slopes against the initial Vmax values gave a typical exponential curve (or straight line if the logs of slopes are used) indicating that the rate of enzyme decay in the MC-treated animals is not greater than that expected from normal enzyme catabolism. A multiplicity of effector sites appears to be involved in the repressor action of different structural types; for example, repression by MC (46.6%) and by phenobarbital (23.9%) in combination are approximately additive (62.0%), rather than competitive, indicating that the two agents act at different sites. A P-450 type cytochrome is involved in the demethylation of DMN. DMN-demethylase is inhibited by carbon monoxide, but the susceptibility to CO is far greater than that observed previously with 3,4-benzopyrene hydroxylation; inhibition of DMN-demethylase as a function of CO concentration follows typical enzyme kinetics. However, while both phenobarbital and MC powerfully repress the DMN-demethylase, we have confirmed that they are strong inducers of the synthesis of P-450 and P-448, respectively, as estimated from the difference spectra.

  10. Harnessing Mechanistic Knowledge on Beneficial Versus Deleterious IFN-I Effects to Design Innovative Immunotherapies Targeting Cytokine Activity to Specific Cell Types

    PubMed Central

    Tomasello, Elena; Pollet, Emeline; Vu Manh, Thien-Phong; Uzé, Gilles; Dalod, Marc

    2014-01-01

    Type I interferons (IFN-I) were identified over 50 years ago as cytokines critical for host defense against viral infections. IFN-I promote anti-viral defense through two main mechanisms. First, IFN-I directly reinforce or induce de novo in potentially all cells the expression of effector molecules of intrinsic anti-viral immunity. Second, IFN-I orchestrate innate and adaptive anti-viral immunity. However, IFN-I responses can be deleterious for the host in a number of circumstances, including secondary bacterial or fungal infections, several autoimmune diseases, and, paradoxically, certain chronic viral infections. We will review the proposed nature of protective versus deleterious IFN-I responses in selected diseases. Emphasis will be put on the potentially deleterious functions of IFN-I in human immunodeficiency virus type 1 (HIV-1) infection, and on the respective roles of IFN-I and IFN-III in promoting resolution of hepatitis C virus (HCV) infection. We will then discuss how the balance between beneficial versus deleterious IFN-I responses is modulated by several key parameters including (i) the subtypes and dose of IFN-I produced, (ii) the cell types affected by IFN-I, and (iii) the source and timing of IFN-I production. Finally, we will speculate how integration of this knowledge combined with advanced biochemical manipulation of the activity of the cytokines should allow designing innovative immunotherapeutic treatments in patients. Specifically, we will discuss how induction or blockade of specific IFN-I responses in targeted cell types could promote the beneficial functions of IFN-I and/or dampen their deleterious effects, in a manner adapted to each disease. PMID:25400632

  11. Interferon-resistant Daudi cells are deficient in interferon-alpha-induced ISGF3 alpha activation, but remain sensitive to the interferon-alpha-induced increase in ISGF3 gamma content.

    PubMed

    Dron, M; Tovey, M G

    1993-10-01

    Low levels of the transcription factor ISGF3 alpha were detected in the cytoplasm and nucleus of untreated Daudi cells, which increased markedly following interferon (IFN) treatment. In contrast no ISGF3 alpha was detected in an IFN-resistant clone of Daudi cells, DIF8, and only low levels were detected in these cells after IFN-alpha treatment. High levels of ISGF3 were produced in vitro, however, by the addition of ISGF3 alpha to extracts of IFN-treated DIF8 cells, indicating that IFN is unable to produce substantial amounts of functional ISGF3 alpha in DIF8 cells. A second clone of IFN-resistant Daudi cells, DIF3, also exhibited defective ISGF3 alpha production, which was restored to normal in the subclone DIF3REV5 that had reverted to high IFN sensitivity. Thus, the antiproliferative effect of IFN on Daudi cells and derived clones is closely related to the level of ISGF3 present in the nucleus of these cells. IFN-alpha, however, also enhances the content of ISGF3 gamma in IFN-resistant cells as well as certain proteins of unknown function, raising the possibility that a second pathway of IFN-alpha signal transduction, distinct from the ISGF3 pathway, remains functional in both DIF8 and DIF3 cells.

  12. Alantolactone from Saussurea lappa Exerts Antiinflammatory Effects by Inhibiting Chemokine Production and STAT1 Phosphorylation in TNF-α and IFN-γ-induced in HaCaT cells.

    PubMed

    Lim, Hye-Sun; Jin, Sung-Eun; Kim, Ohn-Soon; Shin, Hyeun-Kyoo; Jeong, Soo-Jin

    2015-07-01

    Skin inflammation is the most common condition seen in dermatology practice and can be caused by various allergic reactions and certain toxins or chemicals. In the present study, we investigated the antiinflammatory effects of Saussurea lappa, a medicinal herb, and its marker compounds alantolactone, caryophyllene, costic acid, costunolide, and dehydrocostuslactone in the HaCaT human keratinocyte cell line. HaCaT cells were stimulated with tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ), and treated with S. lappa or each of five marker compounds. Chemokine production and expression were analyzed by enzyme-linked immunosorbent assay and reverse transcription-polymerase chain reaction, respectively. Phosphorylation of signal transducer and activator of transcription (STAT) 1 was determined by immunoblotting. Stimulation with TNF-α and IFN-γ significantly increased the production of the following chemokines: thymus-regulated and activation-regulated chemokine (TARC): regulated on activation, normal T-cell expressed and secreted (RANTES): macrophage-derived chemokine (MDC): and interleukin-8 (IL-8). By contrast, S. lappa and the five marker compounds significantly reduced the production of these chemokines by TNF-α and IFN-γ-treated cells. S. lappa and alantolactone suppressed the TNF-α and IFN-γ-stimulated increase in the phosphorylation of STAT1. Our results demonstrate that alantolactone from S. lappa suppresses TNF-α and IFN-γ-induced production of RANTES and IL-8 by blocking STAT1 phosphorylation in HaCaT cells.

  13. Role of mouse and human autophagy proteins in IFN-γ-induced cell-autonomous responses against Toxoplasma gondii.

    PubMed

    Ohshima, Jun; Lee, Youngae; Sasai, Miwa; Saitoh, Tatsuya; Su Ma, Ji; Kamiyama, Naganori; Matsuura, Yoshiharu; Pann-Ghill, Suh; Hayashi, Mikako; Ebisu, Shigeyuki; Takeda, Kiyoshi; Akira, Shizuo; Yamamoto, Masahiro

    2014-04-01

    IFN-γ mediates cellular innate immunity against an intracellular parasite, Toxoplasma gondii, by inducing immunity-related GTPases such as p47 IFN-γ-regulated GTPases (IRGs) and p65 guanylate-binding proteins (GBPs), which also participate in antibacterial responses via autophagy. An essential autophagy protein, Atg5, was previously shown to play a critical role in anti-T. gondii cell-autonomous immunity. However, the involvement of other autophagy proteins remains unknown. In this study, we show that essential autophagy proteins differentially participate in anti-T. gondii cellular immunity by recruiting IFN-γ-inducible GTPases. IFN-γ-induced suppression of T. gondii proliferation and recruitment of an IRG Irgb6 and GBPs are profoundly impaired in Atg7- or Atg16L1-deficient cells. In contrast, cells lacking other essential autophagy proteins, Atg9a and Atg14, are capable of mediating the anti-T. gondii response and recruiting Irgb6 and GBPs to the parasites. Although IFN-γ also stimulates anti-T. gondii cellular immunity in humans, whether this response requires GBPs and human autophagy proteins remains to be seen. To analyze the role of human ATG16L1 and GBPs in IFN-γ-mediated anti-T. gondii responses, human cells lacking ATG16L1 or GBPs were generated by the Cas9/CRISPR genome-editing technique. Although both ATG16L1 and GBPs are dispensable for IFN-γ-induced inhibition of T. gondii proliferation in the human cells, human ATG16L1 is also required for the recruitment of GBPs. Taken together, human ATG16L1 and mouse autophagy components Atg7 and Atg16L1, but not Atg9a and Atg14, participate in the IFN-γ-induced recruitment of the immunity-related GTPases to the intracellular pathogen.

  14. Development of a subunit vaccine containing recombinant chicken anemia virus VP1 and pigeon IFN-γ.

    PubMed

    Shen, Sin Ying; Chang, Wei Chun; Yi, Hsiang Heng; Tsai, Shinn-Shong; Liu, Hung Jen; Liao, Pei-Chun; Chuang, Kuo Pin

    2015-10-15

    Chicken anemia virus (CAV) is a severe threat to the chicken industry and causes heavy economic losses worldwide. In this study, we evaluated the immune response and protective efficacy provided by a subunit vaccine containing recombinant VP1 (rVP1) and pigeon interferon-γ (rPiIFN-γ). Results indicated that rPiIFN-γ enhanced humoral immunity elicited by rVP1 as early as 10 day after primary immunization and reach the high titer after secondary immunization. When compared to chickens immunized with rVP1, inactivated vaccine, chickens immunized with rVP1+rPiIFN-γ showed faster and higher levels (p<0.05) of antibody titer. The CAV challenge result showed that the rVP1+rPiIFN-γ vaccine prevent the reducing of hematocrit values in comparison with the rVP1 or inactivated groups. The relative fold inductions of mRNA expression of Th1-type (IFN-γ), but not Th2-type (IL-4) cytokines in splenocytes isolated from chickens immunized with rVP1+rPiIFN-γ were significantly higher than those of the rVP1 or inactivated vaccine groups. In conclusion, our study found that rPiIFN-γ can enhance both humoral and cellular immunity elicited by an rVP1 vaccine. The rVP1+rPiIFN-γ vaccine may provide a new strategy vaccine against CAV in chicken.

  15. Mycobacterium simiae infection in two unrelated patients with different forms of inherited IFN-γR2 deficiency

    PubMed Central

    Martínez-Barricarte, Rubén; Megged, Orli; Stepensky, Polina; Casimir, Pierre; Moncada-Velez, Marcela; Averbuch, Diana; Assous, Marc Victor; Abuzaitoun, Omar; Kong, Xiao-Fei; Pedergnana, Vincent; Deswarte, Caroline; Migaud, Mélanie; Rose-John, Stefan; Itan, Yuval; Boisson, Bertrand; Belkadi, Aziz; Conti, Francesca; Abel, Laurent; Vogt, Guillaume; Boisson-Dupuis, Stephanie; Casanova, Jean-Laurent; Bustamante, Jacinta

    2014-01-01

    Interferon-γ receptor 2 (IFN-γR2) deficiency is a rare primary immunodeficiency characterized by predisposition to infections with weakly virulent mycobacteria, such as environmental mycobacteria and BCG vaccines. We describe here two children with IFN-γR2 deficiency, from unrelated, consanguineous kindreds of Arab and Israeli descent. The first patient was a boy who died at the age of 4.5 years, from recurrent, disseminated disease caused by Mycobacterium simiae. His IFN-γR2 defect was autosomal recessive and complete. The second patient was a girl with multiple disseminated mycobacterial infections, including infection with M. simiae. She died at the age of five years, a short time after the transplantation of umbilical cord blood cells from an unrelated donor. Her IFN-γR2 defect was autosomal recessive and partial. Autosomal recessive IFN-γR2 deficiency is life-threatening, even in its partial form, and genetic diagnosis and familial counseling are therefore particularly important for this condition. These two cases are the first of IFN-γR2 deficiency associated with M simiae infection to be described. PMID:25135595

  16. A cyclometalated iridium(III) complex used as a conductor for the electrochemical sensing of IFN

    NASA Astrophysics Data System (ADS)

    Miao, Xiangmin; Ko, Chung-Nga; Vellaisamy, Kasipandi; Li, Zongbing; Yang, Guanjun; Leung, Chung-Hang; Ma, Dik-Lung

    2017-02-01

    A novel iridium(III) complex was prepared and used as a conductor for sensitive and enzyme-free electrochemical detection of interferon gamma (IFN-γ). This assay is based on a dual signal amplification mechanism involving positively charged gold nanoparticles ((+)AuNPs) and hybridization chain reaction (HCR). To construct the sensor, nafion (Nf) and (+)AuNPs composite membrane was first immobilized onto the electrode surface. Subsequently, a loop-stem structured capture probe (CP) containing a special IFN-γ interact strand was modified onto the (+)AuNP surface via the formation of Au-S bonds. Upon addition of IFN-γ, the loop-stem structure of CP was opened, and the newly exposed “sticky” region of CP then hybridized with DNA hairpin-1 (H1), which in turn opened its hairpin structure for hybridizing with DNA hairpin-2 (H2). Happen of HCR between H1 and H2 thus generated a polymeric duplex DNA (dsDNA) chain. Meanwhile, the iridium(III) complex could interact with the grooves of the dsDNA polymer, producing a strong current signal that was proportional to IFN-γ concentration. Thus, sensitive detection of IFN-γ could be realized with a detection limit down to 16.3 fM. Moreover, satisfied results were achieved by using this method for the detection of IFN-γ in human serum samples.

  17. Successful Treatment of Human Visceral Leishmaniasis Restores Antigen-Specific IFN-γ, but not IL-10 Production

    PubMed Central

    Adem, Emebet; Tajebe, Fitsumbirhan; Getahun, Mulusew; Kiflie, Amare; Diro, Ermias; Hailu, Asrat; Shkedy, Ziv; Mengesha, Bewketu; Mulaw, Tadele; Atnafu, Saba; Deressa, Tekalign; Mathewos, Biniam; Abate, Ebba; Modolell, Manuel; Munder, Markus; Müller, Ingrid; Takele, Yegnasew; Kropf, Pascale

    2016-01-01

    One of the key immunological characteristics of active visceral leishmaniasis (VL) is a profound immunosuppression and impaired production of Interferon-γ (IFN-γ). However, recent studies from Bihar in India showed using a whole blood assay, that whole blood cells have maintained the capacity to produce IFN-γ. Here we tested the hypothesis that a population of low-density granulocytes (LDG) might contribute to T cell responses hyporesponsiveness via the release of arginase. Our results show that this population is affected by the anticoagulant used to collect blood: the frequency of LDGs is significantly lower when the blood is collected with heparin as compared to EDTA; however, the anticoagulant does not impact on the levels of arginase released. Next, we assessed the capacity of whole blood cells from patients with active VL to produce IFN-γ and IL-10 in response to antigen-specific and polyclonal activation. Our results show that whole blood cells produce low or levels below detection limit of IFN-γ and IL-10, however, after successful treatment of VL patients, these cells gradually regain their capacity to produce IFN-γ, but not IL-10, in response to activation. These results suggest that in contrast to VL patients from Bihar, India, whole blood cells from VL patients from Gondar, Ethiopia, have lost their ability to produce IFN-γ during active VL and that active disease is not associated with sustained levels of IL-10 production following stimulation. PMID:26962865

  18. A cyclometalated iridium(III) complex used as a conductor for the electrochemical sensing of IFN

    PubMed Central

    Miao, Xiangmin; Ko, Chung-Nga; Vellaisamy, Kasipandi; Li, Zongbing; Yang, Guanjun; Leung, Chung-Hang; Ma, Dik-Lung

    2017-01-01

    A novel iridium(III) complex was prepared and used as a conductor for sensitive and enzyme-free electrochemical detection of interferon gamma (IFN-γ). This assay is based on a dual signal amplification mechanism involving positively charged gold nanoparticles ((+)AuNPs) and hybridization chain reaction (HCR). To construct the sensor, nafion (Nf) and (+)AuNPs composite membrane was first immobilized onto the electrode surface. Subsequently, a loop-stem structured capture probe (CP) containing a special IFN-γ interact strand was modified onto the (+)AuNP surface via the formation of Au-S bonds. Upon addition of IFN-γ, the loop-stem structure of CP was opened, and the newly exposed “sticky” region of CP then hybridized with DNA hairpin-1 (H1), which in turn opened its hairpin structure for hybridizing with DNA hairpin-2 (H2). Happen of HCR between H1 and H2 thus generated a polymeric duplex DNA (dsDNA) chain. Meanwhile, the iridium(III) complex could interact with the grooves of the dsDNA polymer, producing a strong current signal that was proportional to IFN-γ concentration. Thus, sensitive detection of IFN-γ could be realized with a detection limit down to 16.3 fM. Moreover, satisfied results were achieved by using this method for the detection of IFN-γ in human serum samples. PMID:28198433

  19. Successful Treatment of Human Visceral Leishmaniasis Restores Antigen-Specific IFN-γ, but not IL-10 Production.

    PubMed

    Adem, Emebet; Tajebe, Fitsumbirhan; Getahun, Mulusew; Kiflie, Amare; Diro, Ermias; Hailu, Asrat; Shkedy, Ziv; Mengesha, Bewketu; Mulaw, Tadele; Atnafu, Saba; Deressa, Tekalign; Mathewos, Biniam; Abate, Ebba; Modolell, Manuel; Munder, Markus; Müller, Ingrid; Takele, Yegnasew; Kropf, Pascale

    2016-03-01

    One of the key immunological characteristics of active visceral leishmaniasis (VL) is a profound immunosuppression and impaired production of Interferon-γ (IFN-γ). However, recent studies from Bihar in India showed using a whole blood assay, that whole blood cells have maintained the capacity to produce IFN-γ. Here we tested the hypothesis that a population of low-density granulocytes (LDG) might contribute to T cell responses hyporesponsiveness via the release of arginase. Our results show that this population is affected by the anticoagulant used to collect blood: the frequency of LDGs is significantly lower when the blood is collected with heparin as compared to EDTA; however, the anticoagulant does not impact on the levels of arginase released. Next, we assessed the capacity of whole blood cells from patients with active VL to produce IFN-γ and IL-10 in response to antigen-specific and polyclonal activation. Our results show that whole blood cells produce low or levels below detection limit of IFN-γ and IL-10, however, after successful treatment of VL patients, these cells gradually regain their capacity to produce IFN-γ, but not IL-10, in response to activation. These results suggest that in contrast to VL patients from Bihar, India, whole blood cells from VL patients from Gondar, Ethiopia, have lost their ability to produce IFN-γ during active VL and that active disease is not associated with sustained levels of IL-10 production following stimulation.

  20. IFN-γ prevents adenosine receptor (A2bR) upregulation to sustain the macrophage activation response

    PubMed Central

    Cohen, Heather B.; Ward, Amanda; Hamidzadeh, Kajal; Ravid, Katya; Mosser, David M.

    2015-01-01

    The priming of macrophages with IFN-γ prior to TLR stimulation results in enhanced and prolonged inflammatory cytokine production. Here, we demonstrate that following TLR stimulation, macrophages up regulate the adenosine 2b receptor (A2bR) to enhance their sensitivity to immunosuppressive extracellular adenosine. This up-regulation of A2bR leads to the induction of a macrophage with an immunoregulatory phenotype and the down regulation of inflammation. IFN-γ priming of macrophages, selectively prevents the induction of the A2bR in macrophages to mitigate sensitivity to adenosine and prevent this regulatory transition. IFN-γ-mediated A2bR blockade leads to a prolonged production of TNFα and IL-12 in response to TLR ligation. The pharmacological inhibition or the genetic deletion of the A2bR results in a hyper-inflammatory response to TLR ligation, similar to IFN-γ treatment of macrophages. Conversely, the overexpression of A2bR on macrophages blunts the IFN-γ effects and promotes the development of immunoregulatory macrophages. Thus, we propose a novel mechanism whereby IFN-γ contributes to host defense, by desensitizing macrophages to the immunoregulatory effects of adenosine. This mechanism overcomes the transient nature of TLR activation, and prolongs the anti-microbial state of the classically activated macrophage. This study may offer promising new targets to improve the clinical outcome of inflammatory diseases in which macrophage activation is dysregulated. PMID:26355158

  1. IFN-τ Alleviates Lipopolysaccharide-Induced Inflammation by Suppressing NF-κB and MAPKs Pathway Activation in Mice.

    PubMed

    Wu, Haichong; Zhao, Gan; Jiang, Kangfeng; Chen, Xiuying; Rui, Guangze; Qiu, Changwei; Guo, Mengyao; Deng, Ganzhen

    2016-06-01

    IFN-τ, which is a type I interferon with low cytotoxicity, is defined as a pregnancy recognition signal in ruminants. Type I interferons have been used as anti-inflammatory agents, but their side effects limit their clinical application. The present study aimed to determine the anti-inflammatory effects of IFN-τ in a lipopolysaccharide-stimulated acute lung injury (ALI) model and in RAW264.7 cells and to confirm the mechanism of action involved. The methods used included histopathology, measuring the lung wet/dry ratio, determining the myeloperoxidase activity, ELISA, qPCR, and western blot. The results revealed that IFN-τ greatly ameliorated the infiltration of inflammatory cells and the expression of TNF-α, IL-1β, and IL-6. Further analysis revealed that IFN-τ down-regulated the expression of TLR-2 and TLR-4 mRNA and the activity of the NF-κB and MAPK pathways both in a lipopolysaccharide-induced ALI model and in RAW264.7 cells. The results demonstrated that IFN-τ suppressed the levels of pro-inflammatory cytokines by inhibiting the phosphorylation of the NF-κB and MAPK pathways. Thus, IFN-τ may be an optimal target for the treatment of inflammatory diseases.

  2. Ligand-induced IFN gamma receptor tyrosine phosphorylation couples the receptor to its signal transduction system (p91).

    PubMed Central

    Greenlund, A C; Farrar, M A; Viviano, B L; Schreiber, R D

    1994-01-01

    Herein we report that interferon-gamma (IFN gamma) induces the rapid and reversible tyrosine phosphorylation of the IFN gamma receptor. Using a panel of receptor intracellular domain mutants, we show that a membrane-proximal LPKS sequence (residues 266-269) is required for ligand-induced tyrosine kinase activation and/or kinase-receptor association and biological responsiveness, and a functionally critical membrane-distal tyrosine residue (Y440) is a target of the activated enzyme. The biological significance of Y440 phosphorylation was demonstrated by showing that a receptor-derived nonapeptide corresponding to receptor residues 436-444 and containing phosphorylated Y440 bound specifically to p91, blocked p91 phosphorylation and inhibited the generation of an active p91-containing transcription factor complex. In contrast, nonphosphorylated wild-type, phosphorylated mutant, or phosphorylated irrelevant peptides did not. Moreover, the phosphorylated Y440-containing peptide did not interact with a related but distinct latent transcription factor (p113) which is activatible by IFN alpha but not IFN gamma. These results thus document the specific and inducible association of p91 with the phosphorylated IFN gamma receptor and thereby elucidate the mechanism by which ligand couples the IFN gamma receptor to its signal transduction system. Images PMID:8156998

  3. Complement Component 3 Regulates IFN-α Production by Plasmacytoid Dendritic Cells following TLR7 Activation by a Plant Virus-like Nanoparticle.

    PubMed

    Lebel, Marie-Ève; Langlois, Marie-Pierre; Daudelin, Jean-François; Tarrab, Esther; Savard, Pierre; Leclerc, Denis; Lamarre, Alain

    2017-01-01

    The increasing use of plant viruses for the development of new vaccines and immunotherapy approaches poses questions regarding the mechanism by which the mammalian immune system recognizes these viruses. For example, although natural Abs (NA) and complement are key components of the innate immune system involved in the opsonization, phagocytosis, and destruction of microorganisms infecting mammals, their implication in plant virus recognition and immunogenicity is not well defined. In this study, we address the involvement of NA and the complement system in the activation of innate immunity through engagement of TLR7 with papaya mosaic virus (PapMV)-like nanoparticles. We demonstrate that NA, although binding to PapMV, are not involved in its recognition by the immune system. On the other hand, C3 strongly binds to PapMV nanoparticles and its depletion significantly reduces PapMV's interaction with immune cells. Unexpectedly, however, we observed increased immune cell activation following administration of PapMV to complement-depleted mice. TLR7 activation by PapMV in the absence of C3 induced higher IFN-α production, resulting in superior immune cell activation and increased immunotherapeutic properties. In conclusion, in this study we established the involvement of the complement system in the recognition and the phagocytosis of PapMV nanoparticles and identified an unsuspected role for C3 in regulating the production of IFN-α following TLR7 activation.

  4. Alpha-interferon does not increase the efficacy of 5-fluorouracil in advanced colorectal cancer.

    PubMed

    Thirion, P; Piedbois, P; Buyse, M; O'Dwyer, P J; Cunningham, D; Man, A; Greco, F A; Colucci, G; Köhne, C H; Di Constanzo, F; Piga, A; Palmeri, S; Dufour, P; Cassano, A; Pajkos, G; Pensel, R A; Aykan, N F; Marsh, J; Seymour, M T

    2001-03-02

    Two meta-analyses were conducted to quantify the benefit of combining alpha-IFN to 5FU in advanced colorectal cancer in terms of tumour response and survival. Analyses were based on a total of 3254 individual patient data provided by principal investigators of each trial. The meta-analysis of 5FU +/- LV vs. 5FU +/- LV + alpha-IFN combined 12 trials and 1766 patients. The meta-analysis failed to show any statistically significant difference between the two treatment groups in terms of tumour response or survival. Overall tumour response rates were 25% for patients receiving no alpha-IFN vs. 24% for patients receiving alpha-IFN (relative risk, RR = 1.02), and median survivals were 11.4 months for patients receiving no alpha-IFN vs. 11.5 months for patients receiving alpha-IFN (hazard ratio, HR = 0.95). The meta-analysis of 5FU + LV vs. 5FU + alpha-IFN combined 7 trials, and 1488 patients. This meta-analysis showed an advantage for 5FU + LV over 5FU + alpha-IFN which was statistically significant in terms of tumour response (23% vs. 18%; RR = 1.26;P = 0.042), and of a borderline significance for overall survival (HR = 1.11;P = 0.066). Metastases confined to the liver and primary rectal tumours were independent favourable prognostic factors for tumour response, whereas good performance status, metastases confined to the liver or confined to the lung, and primary tumour in the rectum were independent favourable prognostic factors for survival. We conclude that alpha-IFN does not increase the efficacy of 5FU or of 5FU + LV, and that 5FU + alpha-IFN is significantly inferior to 5FU + LV, for patients with advanced colorectal cancer.

  5. Direct Type I IFN but Not MDA5/TLR3 Activation of Dendritic Cells Is Required for Maturation and Metabolic Shift to Glycolysis after Poly IC Stimulation

    PubMed Central

    Haddad, Elias; Wood, Elizabeth G.; Longhi, M. Paula

    2014-01-01

    Type I interferons (IFNs) play an important role in direct antiviral defense as well as linking the innate and adaptive immune responses. On dendritic cells (DCs), IFNs facilitate their activation and contribute to CD8+ and CD4+ T cell priming. However, the precise molecular mechanism by which IFNs regulate maturation and immunogenicity of DCs in vivo has not been studied in depth. Here we show that, after in vivo stimulation with the TLR ligand poly IC, IFNs dominate transcriptional changes in DCs. In contrast to direct TLR3/mda5 signaling, IFNs are required for upregulation of all pathways associated with DC immunogenicity. In addition, metabolic pathways, particularly the switch from oxidative phosphorylation to glycolysis, are also regulated by IFNs and required for DC maturation. These data provide evidence for a metabolic reprogramming concomitant with DC maturation and offer a novel mechanism by which IFNs modulate DC maturation. PMID:24409099

  6. Characterization of Amphioxus IFN Regulatory Factor Family Reveals an Archaic Signaling Framework for Innate Immune Response.

    PubMed

    Yuan, Shaochun; Zheng, Tingting; Li, Peiyi; Yang, Rirong; Ruan, Jie; Huang, Shengfeng; Wu, Zhenxin; Xu, Anlong

    2015-12-15

    The IFN regulatory factor (IRF) family encodes transcription factors that play important roles in immune defense, stress response, reproduction, development, and carcinogenesis. Although the origin of the IRF family has been dated back to multicellular organisms, invertebrate IRFs differ from vertebrate IRFs in genomic structure and gene synteny, and little is known about their functions. Through comparison of multiple amphioxus genomes, in this study we suggested that amphioxus contains nine IRF members, whose orthologs are supposed to be shared among three amphioxus species. As the orthologs to the vertebrate IRF1 and IRF4 subgroups, Branchiostoma belcheri tsingtauense (bbt)IRF1 and bbtIRF8 bind the IFN-stimulated response element (ISRE) and were upregulated when amphioxus intestinal cells were stimulated with poly(I:C). As amphioxus-specific IRFs, both bbtIRF3 and bbtIRF7 bind ISRE. When activated, they can be phosphorylated by bbtTBK1 and then translocate into nucleus for target gene transcription. As transcriptional repressors, bbtIRF2 and bbtIRF4 can inhibit the transcriptional activities of bbtIRF1, 3, 7, and 8 by competing for the binding of ISRE. Interestingly, amphioxus IRF2, IRF8, and Rel were identified as target genes of bbtIRF1, bbtIRF7, and bbtIRF3, respectively, suggesting a dynamic feedback regulation among amphioxus IRF and NF-κB. Collectively, to our knowledge we present for the first time an archaic IRF signaling framework in a basal chordate, shedding new insights into the origin and evolution of vertebrate IFN-based antiviral networks.

  7. The role of macrophage IL-10/innate IFN interplay during virus-induced asthma

    PubMed Central

    Zdrenghea, Mihnea T; Makrinioti, Heidi; Muresan, Adriana; Johnston, Sebastian L; Stanciu, Luminita A

    2015-01-01

    Activation through different signaling pathways results in two functionally different types of macrophages, the pro-inflammatory (M1) and the anti-inflammatory (M2). The polarization of macrophages toward the pro-inflammatory M1 phenotype is considered to be critical for efficient antiviral immune responses in the lung. Among the various cell types that are present in the asthmatic airways, macrophages have emerged as significant participants in disease pathogenesis, because of their activation during both the inflammatory and resolution phases, with an impact on disease progression. Polarized M1 and M2 macrophages are able to reversibly undergo functional redifferentiation into anti-inflammatory or pro-inflammatory macrophages, respectively, and therefore, macrophages mediate both processes. Recent studies have indicated a predominance of M2 macrophages in asthmatic airways. During a virus infection, it is likely that M2 macrophages would secrete higher amounts of the suppressor cytokine IL-10, and less innate IFNs. However, the interactions between IL-10 and innate IFNs during virus-induced exacerbations of asthma have not been well studied. The possible role of IL-10 as a therapy in allergic asthma has already been suggested, but the divergent roles of this suppressor molecule in the antiviral immune response raise concerns. This review attempts to shed light on macrophage IL-10–IFNs interactions and discusses the role of IL-10 in virus-induced asthma exacerbations. Whereas IL-10 is important in terminating pro-inflammatory and antiviral immune responses, the presence of this immune regulatory cytokine at the beginning of virus infection could impair the response to viruses and play a role in virus-induced asthma exacerbations. PMID:25430775

  8. Sphingosine Kinase 2 Deficiency Attenuates Kidney Fibrosis via IFN-γ.

    PubMed

    Bajwa, Amandeep; Huang, Liping; Kurmaeva, Elvira; Ye, Hong; Dondeti, Krishna R; Chroscicki, Piotr; Foley, Leah S; Balogun, Z Ayoade; Alexander, Kyle J; Park, Hojung; Lynch, Kevin R; Rosin, Diane L; Okusa, Mark D

    2017-04-01

    Maladaptive repair after AKI may lead to progressive fibrosis and decline in kidney function. Sphingosine 1-phosphate has an important role in kidney injury and pleiotropic effects in fibrosis. We investigated the involvement of sphingosine kinase 1 and 2 (SphK1 and SphK2), which phosphorylate sphingosine to produce sphingosine 1-phosphate, in kidney fibrosis induced by folic acid (FA) or unilateral ischemia-reperfusion injury. Analysis of Masson trichrome staining and fibrotic marker protein and mRNA expression 14 days after AKI revealed that wild-type (WT) and Sphk1(-/-) mice exhibited more kidney fibrosis than Sphk2(-/-) mice. Furthermore, kidneys of FA-treated WT and Sphk1(-/-) mice had greater immune cell infiltration and expression of fibrotic and inflammatory markers than kidneys of FA-treated Sphk2(-/-) mice. In contrast, kidneys of Sphk2(-/-) mice exhibited greater expression of Ifng and IFN-γ-responsive genes (Cxcl9 and Cxcl10) than kidneys of WT or Sphk1(-/-) mice did at this time point. Splenic T cells from untreated Sphk2(-/-) mice were hyperproliferative and produced more IFN-γ than did those of WT or Sphk1(-/-) mice. IFN-γ blocking antibody administered to Sphk2(-/-) mice or deletion of Ifng (Sphk2(-/-)Ifng(-/-) mice) blocked the protective effect of SphK2 deficiency in fibrosis. Moreover, adoptive transfer of Sphk2(-/-) (but not Sphk2(-/-)Ifng(-/-) ) CD4 T cells into WT mice blocked FA-induced fibrosis. Finally, a selective SphK2 inhibitor blocked FA-induced kidney fibrosis in WT mice. These studies demonstrate that SphK2 inhibition may serve as a novel therapeutic approach for attenuating kidney fibrosis.

  9. Effect of a four-week exercise program on the secretion of IFN-γ, TNF-α, IL-2 and IL-6 cytokines in elite Taekwondo athletes

    PubMed Central

    Kaya, Oktay

    2016-01-01

    The aim of the present study was to examine how a 4-week exercise program affects the serum levels of certain cytokines in Taekwondo athletes. The study involved 10 elite male Taekwondo athletes (mean age, 20.67±0.24 years; mean weight, 65.45±1.69 kg) who were studying at the Physical Education and Sports High School of Selçuk University (Konya, Turkey) in June 2014. The subjects were involved in a Taekwondo exercise program on every weekday for 4 weeks. The subjects were also engaged in an exercise to exhaustion session twice; once before starting the 4-week exercise program and once upon completion of the program. Blood samples were collected from the subjects in four rounds: During rest, upon fatigue, and before and after the 4-week exercise program. These samples were analyzed to establish the serum levels of interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), interleukin (IL)-2 and IL-6 using enzyme-linked immunosorbent assay test kits. Pre- and post-exercise program, the IFN-γ and TNF-α levels did not show any significant difference. When compared with the pre-exercise levels, serum IL-2 levels of the subjects were found to be elevated after the 4-week exercise program. The highest serum IL-6 values were established after the subjects were exercised to fatigue before the exercise program was initiated (P<0.05). The 4-week exercise program resulted in a decrease in IL-6 levels (P<0.05). The findings of the study indicate that a 4-week exercise program did not result in significant changes in IFN-γ and TNF-α levels, but led to an increase in IL-2 levels. The notable finding of the present study is that a 4-week exercise program reduces cellular immune functions and, thus, the levels of IL-6, which negatively influences performance. PMID:27588179

  10. NKG2D⁺ IFN-γ⁺ CD8⁺ T cells are responsible for palladium allergy.

    PubMed

    Kawano, Mitsuko; Nakayama, Masafumi; Aoshima, Yusuke; Nakamura, Kyohei; Ono, Mizuho; Nishiya, Tadashi; Nakamura, Syou; Takeda, Yuri; Dobashi, Akira; Takahashi, Akiko; Endo, Misato; Ito, Akiyo; Ueda, Kyosuke; Sato, Naoki; Higuchi, Shigehito; Kondo, Takeru; Hashimoto, Suguru; Watanabe, Masamichi; Watanabe, Makoto; Takahashi, Tetsu; Sasaki, Keiichi; Nakamura, Masanori; Sasazuki, Takehiko; Narushima, Takayuki; Suzuki, Ryuji; Ogasawara, Kouetsu

    2014-01-01

    Nickel, cobalt, and chromium are well known to be causal agents of allergic contact dermatitis. Palladium (Pd) can also cause allergic disease and exposure results from wide use of this metal in dental restorations and jewelry. Metal allergy is categorized as a delayed-type hypersensitivity, and metal-responsive T cell clones have been isolated from allergic patients. However, compared to nickel, little is known about the pathology of allergic disease mediated by Pd, and pathogenic T cells are poorly understood. To identify the pathogenic T cells that are responsible for onset of Pd allergy, we enriched metal-responsive lymphocytes by sequential adoptive transfer of involved lymph node cells. Here we show that sequential adoptive transfer gradually increased the incidence and the intensity of Pd allergy, and CD8⁺ T cells are responsible for the disease as CD8⁺ T cell-depleted mice and β2-microglobulin-deficient mice did not develop Pd allergy. In addition, we found that draining lymph node cells skewed toward CD8⁺ T cells in response to Pd challenge in 8th adoptive transferred recipient mice. The CD8⁺ T cells expressed NKG2D, a costimulatory molecule involved in the production of IFN-γ. NKG2D ligand was also induced in Pd-injected tissues. Furthermore, both NKG2D ligand-transgenic mice, where NKG2D is downmodulated, and IFN-γ-deficient mice showed impaired Pd allergy. Taken together, these results indicate that IFN-γ-producing NKG2D⁺ CD8⁺ T cells are responsible for Pd allergy and suggest that NKG2D is a potential therapeutic target for treatment of metal allergy.

  11. Both CXCR3 and CXCL10/IFN-inducible protein 10 are required for resistance to primary infection by dengue virus.

    PubMed

    Hsieh, Ming-Fang; Lai, Szu-Liang; Chen, Jia-Perng; Sung, Jui-Ming; Lin, Yi-Ling; Wu-Hsieh, Betty A; Gerard, Craig; Luster, Andrew; Liao, Fang

    2006-08-01

    We examined the extent to which CXCR3 mediates resistance to dengue infection. Following intracerebral infection with dengue virus, CXCR3-deficient (CXCR3(-/-)) mice showed significantly higher mortality rates than wild-type (WT) mice; moreover, surviving CXCR3(-/-) mice, but not WT mice, often developed severe hind-limb paralysis. The brains of CXCR3(-/-) mice showed higher viral loads than those of WT mice, and quantitative analysis using real-time PCR, flow cytometry, and immunohistochemistry revealed fewer T cells, CD8(+) T cells in particular, in the brains of CXCR3(-/-) mice. This suggests that recruitment of effector T cells to sites of dengue infection was diminished in CXCR3(-/-) mice, which impaired elimination of the virus from the brain and thus increased the likelihood of paralysis and/or death. These results indicate that CXCR3 plays a protective rather than an immunopathological role in dengue virus infection. In studies to identify critical CXCR3 ligands, CXCL10/IFN-inducible protein 10-deficient (CXCL10/IP-10(-/-)) mice infected with dengue virus showed a higher mortality rate than that of the CXCR3(-/-) mice. Although CXCL10/IP-10, CXCL9/monokine induced by IFN-gamma, and CXCL11/IFN-inducible T cell alpha chemoattractant share a single receptor and all three of these chemokines are induced by dengue virus infection, the latter two could not compensate for the absence of CXCL10/IP-10 in this in vivo model. Our results suggest that both CXCR3 and CXCL10/IP-10 contribute to resistance against primary dengue virus infection and that chemokines that are indistinguishable in in vitro assays differ in their activities in vivo.

  12. Effect of a four-week exercise program on the secretion of IFN-γ, TNF-α, IL-2 and IL-6 cytokines in elite Taekwondo athletes.

    PubMed

    Kaya, Oktay

    2016-09-01

    The aim of the present study was to examine how a 4-week exercise program affects the serum levels of certain cytokines in Taekwondo athletes. The study involved 10 elite male Taekwondo athletes (mean age, 20.67±0.24 years; mean weight, 65.45±1.69 kg) who were studying at the Physical Education and Sports High School of Selçuk University (Konya, Turkey) in June 2014. The subjects were involved in a Taekwondo exercise program on every weekday for 4 weeks. The subjects were also engaged in an exercise to exhaustion session twice; once before starting the 4-week exercise program and once upon completion of the program. Blood samples were collected from the subjects in four rounds: During rest, upon fatigue, and before and after the 4-week exercise program. These samples were analyzed to establish the serum levels of interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), interleukin (IL)-2 and IL-6 using enzyme-linked immunosorbent assay test kits. Pre- and post-exercise program, the IFN-γ and TNF-α levels did not show any significant difference. When compared with the pre-exercise levels, serum IL-2 levels of the subjects were found to be elevated after the 4-week exercise program. The highest serum IL-6 values were established after the subjects were exercised to fatigue before the exercise program was initiated (P<0.05). The 4-week exercise program resulted in a decrease in IL-6 levels (P<0.05). The findings of the study indicate that a 4-week exercise program did not result in significant changes in IFN-γ and TNF-α levels, but led to an increase in IL-2 levels. The notable finding of the present study is that a 4-week exercise program reduces cellular immune functions and, thus, the levels of IL-6, which negatively influences performance.

  13. Mycobacterium tuberculosis promotes Th17 expansion via regulation of human dendritic cells toward a high CD14 and low IL-12p70 phenotype that reprograms upon exogenous IFN-γ.

    PubMed

    Søndergaard, Jonas Nørskov; Laursen, Janne Marie; Rosholm, Lisbeth Buus; Brix, Susanne

    2014-12-01

    The capacity to develop protective immunity against mycobacteria is heterogeneously distributed among human beings, and it is currently unknown why the initial immune response induced against Mycobacterium tuberculosis (Mtb) does not provide proper clearance of this pathogen. Dendritic cells (DCs) are some of the first cells to interact with Mtb and they play an essential role in development of protective immunity against Mtb. Given that Mtb-infected macrophages have difficulties in degrading Mtb, they need help from IFN-γ-producing CD4+ T cells propagated via IL-12p70-producing DCs. Here we report that Mtb modifies human DC plasticity by expanding a CD14+ DC subset with weak IL-12p70-producing capacity. The CD14+ Mtb-promoted subset was furthermore poor inducers of IFN-γ by naive CD4+ T cells, but instead prompted IL-17A-producing RORγT+ CD4+ T cells. Mtb-derived peptidoglycan and mannosylated lipoarabinomannan partly recapitulated the subset partition induced by Mtb. Addition of IFN-γ, but neither IL-17A nor IL-22, which are potentially produced by Mtb-exposed γ/δ-T cells in mucosal linings, inhibited the differentiation toward CD14+ DCs and promoted high-level IL-12p70 in Mtb-challenged DCs. We conclude that Mtb exploits DC plasticity to reduce production of IL-12p70, and that this process is entirely divertible by exogenous IFN-γ. These data suggest that strategies to increase local IFN-γ production in the lungs of tuberculosis patients may boost host immunity toward Mtb.

  14. IFN-α-Induced Downregulation of miR-221 in Dendritic Cells: Implications for HCV Pathogenesis and Treatment

    PubMed Central

    Sehgal, Mohit; Zeremski, Marija; Talal, Andrew H.; Ginwala, Rashida; Elrod, Elizabeth; Grakoui, Arash; Li, Qi-Ging; Philip, Ramila; Khan, Zafar K.

    2015-01-01

    Although interferon (IFN)-α is known to exert immunomodulatory and antiproliferative effects on dendritic cells (DCs) through induction of protein-coding IFN-stimulated genes (ISGs), little is known about IFN-α-regulated miRNAs in DCs. Since several miRNAs are involved in regulating DC functions, it is important to investigate whether IFN-α's effects on DCs are mediated through miRNAs as well. In this study, we examined miRNA expression patterns in myeloid DCs (mDCs) and plasmacytoid DCs after exposing them to IFN-α. We report that IFN-α downregulates miR-221 in both DC subsets via inhibition of STAT3. We validated proapoptotic proteins BCL2L11 and CDKN1C as miR-221 targets suggesting that IFN-α can induce DC apoptosis via miR-221 downregulation. In addition, we validated another miR-221 target, SOCS1, which is known to be a negative regulator of JAK/STAT signaling. Consistent with this, miR-221 overexpression in mDCs enhanced the secretion of proinflammatory cytokines IL-6 and TNF-α. In peripheral blood mononuclear cells (PBMCs) of HIV-1/HCV co-infected individuals undergoing IFN-α-based treatment the baseline miR-221 expression was lower in non-responders compared with responders; and miR-221 expression directly correlated with DC frequency and IL-6/TNF-α secretion. In addition to PBMCs, we isolated total liver cells and kupffer cells from HCV-infected individuals and individuals with alcoholic cirrhosis. We found that both total liver cells and kupffer cells from HCV-infected individuals had significantly higher miR-221 levels compared with individuals with cirrhosis. Overall, we demonstrate that IFN-α exerts both antiproliferative and immunomodulatory effects on mDCs via miR-221 downregulation; and IFN-miR-221 axis can play important role in HCV pathogenesis and treatment. PMID:26090579

  15. Combination of TLR1/2 and TLR3 ligands enhances CD4+ T cell longevity and antibody responses by modulating type I IFN production

    PubMed Central

    Lee, Bo Ryeong; Jeong, Soo Kyung; Ahn, Byung Cheol; Lee, Byeong-Jae; Shin, Sung Jae; Yum, Jung Sun; Ha, Sang-Jun

    2016-01-01

    Despite the possibility of combining Toll-like receptor (TLR) ligands as adjuvants to improve vaccine efficacy, it remains unclear which combinations of TLR ligands are effective or what their underlying mechanisms may be. Here, we investigated the mechanism of action of L-pampo, a proprietary adjuvant composed of TLR1/2 and TLR3 ligands. L-pampo dramatically increased humoral immune responses against the tested target antigens, which was correlated with an increase in follicular helper T cells and the maintenance of antigen-specific CD4+ T cells. During the initial priming phase, in contrast to the induction of type I interferon (IFN) and pro-inflammatory cytokines stimulated by polyI:C, L-pampo showed a greatly diminished induction of type I IFN, but not of other cytokines, and remarkably attenuated IRF3 signaling, which appeared to be critical to L-pampo-mediated adjuvanticity. Collectively, our results demonstrate that the adjuvant L-pampo contributes to the promotion of antigen-specific antibodies and CD4+ T cell responses via a fine regulation of the TLR1/2 and TLR3 signaling pathways, which may be helpful in the design of improved vaccines. PMID:27580796

  16. Junín Virus Infection of Human Hematopoietic Progenitors Impairs In Vitro Proplatelet Formation and Platelet Release via a Bystander Effect Involving Type I IFN Signaling

    PubMed Central

    Pozner, Roberto G.; Ure, Agustín E.; Jaquenod de Giusti, Carolina; D'Atri, Lina P.; Italiano, Joseph E.; Torres, Oscar; Romanowski, Victor; Schattner, Mirta; Gómez, Ricardo M.

    2010-01-01

    phenotype. Our study introduces a potential mechanism for thrombocytopenia in VHF and other diseases associated with increased bone marrow type I IFN levels. PMID:20419155

  17. T cells detect intracellular DNA but fail to induce type I IFN responses: implications for restriction of HIV replication.

    PubMed

    Berg, Randi K; Rahbek, Stine H; Kofod-Olsen, Emil; Holm, Christian K; Melchjorsen, Jesper; Jensen, David G; Hansen, Anne Louise; Jørgensen, Louise B; Ostergaard, Lars; Tolstrup, Martin; Larsen, Carsten S; Paludan, Søren R; Jakobsen, Martin R; Mogensen, Trine H

    2014-01-01

    HIV infects key cell types of the immune system, most notably macrophages and CD4+ T cells. Whereas macrophages represent an important viral reservoir, activated CD4+ T cells are the most permissive cell types supporting high levels of viral replication. In recent years, it has been appreciated that the innate immune system plays an important role in controlling HIV replication, e.g. via interferon (IFN)-inducible restriction factors. Moreover, innate immune responses are involved in driving chronic immune activation and the pathogenesis of progressive immunodeficiency. Several pattern recognition receptors detecting HIV have been reported, including Toll-like receptor 7 and Retinoic-inducible gene-I, which detects viral RNA. Here we report that human primary T cells fail to induce strong IFN responses, despite the fact that this cell type does express key molecules involved in DNA signaling pathways. We demonstrate that the DNA sensor IFI16 migrates to sites of foreign DNA localization in the cytoplasm and recruits the signaling molecules stimulator of IFN genes and Tank-binding kinase, but this does not result in expression of IFN and IFN-stimulated genes. Importantly, we show that cytosolic DNA fails to affect HIV replication. However, exogenous treatment of activated T cells with type I IFN has the capacity to induce expression of IFN-stimulated genes and suppress HIV replication. Our data suggest the existence of an impaired DNA signaling machinery in T cells, which may prevent this cell type from activating cell-autonomous anti-HIV responses. This phenomenon could contribute to the high permissiveness of CD4+ T cells for HIV-1.

  18. Mechanisms of transcriptional modulation of the human anion exchanger SLC26A3 gene expression by IFN

    PubMed Central

    Saksena, Seema; Singla, Amika; Goyal, Sonia; Katyal, Shivani; Bansal, Nikhil; Gill, Ravinder K.; Alrefai, Waddah A.; Ramaswamy, Krishnamurthy; Dudeja, Pradeep K.

    2010-01-01

    Two members of the SLC26 gene family, SLC26A3 or DRA (downregulated in adenoma) and SLC26A6 (putative anion transporter 1, PAT1), are known to play a major role in apical Cl−/OH− (HCO3−) exchange process in the human intestine. We have previously shown the inhibitory effects of IFN-γ (30 ng/ml, 24 h) on both SLC26A3 and A6 expression and promoter activity. We also demonstrated that the effects of IFN-γ on SLC26A6 gene expression were mediated via IRF-1 transcription factor. However, the molecular mechanisms underlying the transcriptional modulation of SLC26A3 gene expression by IFN-γ in the intestine are not known. The present studies were, therefore, designed to elucidate the signaling mechanisms and transcription factor(s) involved in mediating the inhibitory effects of IFN-γ on DRA promoter (p-−1183/+114) activity. Deletion analysis indicated that the IFN-γ response element is located within the −1183 to −790 region, and sequence analysis of this region revealed the presence of potential γ-activated site (GAS), a binding site (−933/−925 bp) for signal transducer and activator of transcription factor 1 (STAT1). Mutations in the potential GAS element abrogated the inhibitory effects of IFN-γ. These studies provide evidence for the involvement of STAT1 in the inhibition of SLC26A3 gene expression by IFN-γ in the human intestine. PMID:19940027

  19. The IFN-gamma +874T/A gene polymorphism is associated with retinochoroiditis toxoplasmosis susceptibility.

    PubMed

    Albuquerque, Maíra Cavalcanti de; Aleixo, Ana Luisa Quintella do Couto; Benchimol, Eliezer Israel; Leandro, Ana Cristina Câmara S; das Neves, Leandro Batista; Vicente, Regiane Trigueiro; Bonecini-Almeida, Maria da Glória; Amendoeira, Maria Regina Reis

    2009-05-01

    Toxoplasmosis is a worldwide zoonosis that generally produces an asymptomatic infection. In some cases, however, toxoplasmosis infection can lead to ocular damage. The immune system has a crucial role in both the course of the infection and in the evolution of toxoplasmosis disease. In particular, IFN-gamma plays an important role in resistance to toxoplasmosis. Polymorphisms in genes encoding cytokines have been shown to have an association with susceptibility to parasitic diseases. The aim of this work was to analyse the occurrence of polymorphisms in the gene encoding IFN-gamma (+874T/A) among Toxoplasma gondii seropositive individuals, including those with ocular lesions caused by the parasite, from a rural population of Santa Rita de Cássia, Barra Mansa, state of Rio de Janeiro, Brazil. Further, we verified which of these polymorphisms could be related to susceptibility to the development of ocular toxoplasmosis. This study included 34 individuals with ocular toxoplasmosis (ocular group) and 134 without ocular lesions (control group). The differences between A and T allele distributions were not statistically significant between the two groups. However, we observed that a higher frequency of individuals from the ocular group possessed the A/A genotype, when compared with the control group, suggesting that homozygocity for the A allele could enhance susceptibility to ocular toxoplasmosis in T. gondii infection.

  20. The Role of Response Elements Organization in Transcription Factor Selectivity: The IFN-β Enhanceosome Example

    PubMed Central

    Pan, Yongping; Nussinov, Ruth

    2011-01-01

    What is the mechanism through which transcription factors (TFs) assemble specifically along the enhancer DNA? The IFN-β enhanceosome provides a good model system: it is small; its components' crystal structures are available; and there are biochemical and cellular data. In the IFN-β enhanceosome, there are few protein-protein interactions even though consecutive DNA response elements (REs) overlap. Our molecular dynamics (MD) simulations on different motif combinations from the enhanceosome illustrate that cooperativity is achieved via unique organization of the REs: specific binding of one TF can enhance the binding of another TF to a neighboring RE and restrict others, through overlap of REs; the order of the REs can determine which complexes will form; and the alternation of consensus and non-consensus REs can regulate binding specificity by optimizing the interactions among partners. Our observations offer an explanation of how specificity and cooperativity can be attained despite the limited interactions between neighboring TFs on the enhancer DNA. To date, when addressing selective TF binding, attention has largely focused on RE sequences. Yet, the order of the REs on the DNA and the length of the spacers between them can be a key factor in specific combinatorial assembly of the TFs on the enhancer and thus in function. Our results emphasize cooperativity via RE binding sites organization. PMID:21698143

  1. A Novel Function of F-Box Protein FBXO17 in Negative Regulation of Type I IFN Signaling by Recruiting PP2A for IFN Regulatory Factor 3 Deactivation.

    PubMed

    Peng, Di; Wang, Zining; Huang, Anfei; Zhao, Yong; Qin, F Xiao-Feng

    2017-01-15

    The F-box proteins were originally identified as the key component of SKP1-Cullin1-F-box E3 ligase complexes that control the stability of their specific downstream substrates essential for cell growth and survival. However, the involvement of these proteins in type I IFN (IFN-I) signaling during innate immunity has not been investigated. In this study we report that the F-box protein FBXO17 negatively regulates IFN-I signaling triggered by double-strand DNA, RNA, or viral infection. We found that FBXO17 specifically interacts with IFN regulatory factor 3 (IRF3) and decreases its dimerization and nuclear translocation. The decrease of IRF3 dimerization and nuclear translocation is due to the recruitment of protein phosphatase 2 (PP2A) mediated by FBXO17, resulting in IRF3 dephosphorylation. Interestingly, PP2A recruitment does not require the F-box domain but instead the F-box associated region of the protein; thus, the recruitment is independent of the canonical function of the SKP1-Cullin1-F-box family of E3 ligase. Together, our studies identify a previously unreported role of FBXO17 in regulating IFN-I signaling and further demonstrate a novel mechanism for IRF3 deactivation by F-box protein-mediated recruitment of PP2A.

  2. A Novel Function of F-Box Protein FBXO17 in Negative Regulation of Type I IFN Signaling by Recruiting PP2A for IFN Regulatory Factor 3 Deactivation

    PubMed Central

    Peng, Di; Wang, Zining; Huang, Anfei

    2017-01-01

    The F-box proteins were originally identified as the key component of SKP1-Cullin1-F-box E3 ligase complexes that control the stability of their specific downstream substrates essential for cell growth and survival. However, the involvement of these proteins in type I IFN (IFN-I) signaling during innate immunity has not been investigated. In this study we report that the F-box protein FBXO17 negatively regulates IFN-I signaling triggered by double-strand DNA, RNA, or viral infection. We found that FBXO17 specifically interacts with IFN regulatory factor 3 (IRF3) and decreases its dimerization and nuclear translocation. The decrease of IRF3 dimerization and nuclear translocation is due to the recruitment of protein phosphatase 2 (PP2A) mediated by FBXO17, resulting in IRF3 dephosphorylation. Interestingly, PP2A recruitment does not require the F-box domain but instead the F-box associated region of the protein; thus, the recruitment is independent of the canonical function of the SKP1-Cullin1-F-box family of E3 ligase. Together, our studies identify a previously unreported role of FBXO17 in regulating IFN-I signaling and further demonstrate a novel mechanism for IRF3 deactivation by F-box protein-mediated recruitment of PP2A. PMID:27956528

  3. Conventional but Not Plasmacytoid Dendritic Cells Foster the Systemic Virus–Induced Type I IFN Response Needed for Efficient CD8 T Cell Priming

    PubMed Central

    Riezu-Boj, Jose-Ignacio; Mancheño, Uxua; Rueda, Paloma; Lopez, Lissette; Alignani, Diego; Rodríguez-García, Estefanía; Thieblemont, Nathalie

    2014-01-01

    Plasmacytoid dendritic cells (pDCs) are considered to be the principal type-I IFN (IFN-I) source in response to viruses, whereas the contribution of conventional DCs (cDCs) has been underestimated because, on a per-cell basis, they are not considered professional IFN-I–producing cells. We have investigated their respective roles in the IFN-I response required for CTL activation. Using a nonreplicative virus, baculovirus, we show that despite the high IFN-I–producing abilities of pDCs, in vivo cDCs but not pDCs are the pivotal IFN-I producers upon viral injection, as demonstrated by selective pDC or cDC depletion. The pathway involved in the virus-triggered IFN-I response is dependent on TLR9/MyD88 in pDCs and on stimulator of IFN genes (STING) in cDCs. Importantly, STING is the key molecule for the systemic baculovirus-induced IFN-I response required for CTL priming. The supremacy of cDCs over pDCs in fostering the IFN-I response required for CTL activation was also verified in the lymphocytic choriomeningitis virus model, in which IFN-β promoter stimulator 1 plays the role of STING. However, when the TLR-independent virus-triggered IFN-I production is impaired, the pDC-induced IFNs-I have a primary impact on CTL activation, as shown by the detrimental effect of pDC depletion and IFN-I signaling blockade on the residual lymphocytic choriomeningitis virus–triggered CTL response detected in IFN-β promoter stimulator 1−/− mice. Our findings reveal that cDCs play a major role in the TLR-independent virus-triggered IFN-I production required for CTL priming, whereas pDC-induced IFNs-I are dispensable but become relevant when the TLR-independent IFN-I response is impaired. PMID:24973449

  4. Dual oxidase 2 and pancreatic adenocarcinoma: IFN-γ-mediated dual oxidase 2 overexpression results in H2O2-induced, ERK-associated up-regulation of HIF-1α and VEGF-A

    PubMed Central

    Wu, Yongzhong; Meitzler, Jennifer L.; Antony, Smitha; Juhasz, Agnes; Lu, Jiamo; Jiang, Guojian; Liu, Han; Hollingshead, Melinda; Haines, Diana C.; Butcher, Donna; Panter, Michaela S.; Roy, Krishnendu; Doroshow, James H.

    2016-01-01

    Several NADPH oxidase family members, including dual oxidase 2 [DUOX2], are expressed in human tumors, particularly gastrointestinal cancers associated with long-standing chronic inflammation. We found previously that exposure of pancreatic ductal adenocarcinoma cells to the pro-inflammatory cytokine IFNincreased DUOX2 expression (but not other NADPH oxidases) leading to long-lived H2O2 production. To elucidate the pathophysiology of DUOX2-mediated H2O2 formation in the pancreas further, we demonstrate here that IFN-γ-treated BxPC-3 and CFPAC-1 pancreatic cancer cells (known to increase DUOX2 expression) produce significant levels of intracellular oxidants and extracellular H2O2 which correlate with concomitant up-regulation of VEGF-A and HIF-1α transcription. These changes are not observed in the PANC-1 line that does not increase DUOX2 expression following IFN-γ treatment. DUOX2 knockdown with short interfering RNA significantly decreased IFN-γ-induced VEGF-A or HIF-1α up-regulation, as did treatment of pancreatic cancer cells with the NADPH oxidase inhibitor diphenylene iodonium, the multifunctional reduced thiol N-acetylcysteine, and the polyethylene glycol-modified form of the hydrogen peroxide detoxifying enzyme catalase. Increased DUOX2-related VEGF-A expression appears to result from reactive oxygen-mediated activation of ERK signaling that is responsible for AP-1-related transcriptional effects on the VEGF-A promoter. To clarify the relevance of these observations in vivo, we demonstrate that many human pre-malignant pancreatic intraepithelial neoplasms and frank pancreatic cancers express substantial levels of DUOX protein compared to histologically normal pancreatic tissues, and that expression of both DUOX2 and VEGF-A mRNAs is significantly increased in surgically-resected pancreatic cancers compared to the adjacent normal pancreas. PMID:27637085

  5. Three functional variants of IFN regulatory factor 5 (IRF5) define risk and protective haplotypes for human lupus

    PubMed Central

    Graham, Robert R.; Kyogoku, Chieko; Sigurdsson, Snaevar; Vlasova, Irina A.; Davies, Leela R. L.; Baechler, Emily C.; Plenge, Robert M.; Koeuth, Thearith; Ortmann, Ward A.; Hom, Geoffrey; Bauer, Jason W.; Gillett, Clarence; Burtt, Noel; Cunninghame Graham, Deborah S.; Onofrio, Robert; Petri, Michelle; Gunnarsson, Iva; Svenungsson, Elisabet; Rönnblom, Lars; Nordmark, Gunnel; Gregersen, Peter K.; Moser, Kathy; Gaffney, Patrick M.; Criswell, Lindsey A.; Vyse, Timothy J.; Syvänen, Ann-Christine; Bohjanen, Paul R.; Daly, Mark J.; Behrens, Timothy W.; Altshuler, David

    2007-01-01

    Systematic genome-wide studies to map genomic regions associated with human diseases are becoming more practical. Increasingly, efforts will be focused on the identification of the specific functional variants responsible for the disease. The challenges of identifying causal variants include the need for complete ascertainment of genetic variants and the need to consider the possibility of multiple causal alleles. We recently reported that risk of systemic lupus erythematosus (SLE) is strongly associated with a common SNP in IFN regulatory factor 5 (IRF5), and that this variant altered spicing in a way that might provide a functional explanation for the reproducible association to SLE risk. Here, by resequencing and genotyping in patients with SLE, we find evidence for three functional alleles of IRF5: the previously described exon 1B splice site variant, a 30-bp in-frame insertion/deletion variant of exon 6 that alters a proline-, glutamic acid-, serine- and threonine-rich domain region, and a variant in a conserved polyA+ signal sequence that alters the length of the 3′ UTR and stability of IRF5 mRNAs. Haplotypes of these three variants define at least three distinct levels of risk to SLE. Understanding how combinations of variants influence IRF5 function may offer etiological and therapeutic insights in SLE; more generally, IRF5 and SLE illustrates how multiple common variants of the same gene can together influence risk of common disease. PMID:17412832

  6. MAP kinase p38α regulates type III interferon (IFN-λ1) gene expression in human monocyte-derived dendritic cells in response to RNA stimulation.

    PubMed

    Jiang, Miao; Österlund, Pamela; Fagerlund, Riku; Rios, Diana N; Hoffmann, Alexander; Poranen, Minna M; Bamford, Dennis H; Julkunen, Ilkka

    2015-02-01

    Recognition of viral nucleic acids leads to type I and type III IFN gene expression and activation of host antiviral responses. At present, type III IFN genes are the least well-characterized IFN types. Here, we demonstrate that the p38 MAPK signaling pathway is involved in regulating IFN-λ1 gene expression in response to various types of RNA molecules in human moDCs. Inhibition of p38 MAPK strongly reduced IFN gene expression, and overexpression of p38α MAPK enhanced IFN-λ1 gene expression in RNA-stimulated moDCs. The regulation of IFN gene expression by p38 MAPK signaling was independent of protein synthesis and thus, a direct result of RNA stimulation. Moreover, the RIG-I/MDA5-MAVS-IRF3 pathway was required for p38α MAPK to up-regulate IFN-λ1 promoter activation, whereas the MyD88-IRF7 pathway was not needed, and the regulation was not involved directly in IRF7-dependent IFN-α1 gene expression. The stimulatory effect of p38α MAPK on IFN-λ1 mRNA expression in human moDCs did not take place directly via the activating TBK1/IKKε complex, but rather, it occurred through some other parallel pathways. Furthermore, mutations in ISRE and NF-κB binding sites in the promoter region of the IFN-λ1 gene led to a significant reduction in p38α MAPK-mediated IFN responses after RNA stimulation. Altogether, our data suggest that the p38α MAPK pathway is linked with RLR signaling pathways and regulates the expression of early IFN genes after RNA stimulation cooperatively with IRF3 and NF-κB to induce antiviral responses further.

  7. Molecular cloning and functional characterization of porcine DNA-dependent activator of IFN-regulatory factors (DAI).

    PubMed

    Xie, Lilan; Fang, Liurong; Wang, Dang; Luo, Rui; Cai, Kaimei; Chen, Huanchun; Xiao, Shaobo

    2010-03-01

    The DNA-dependent activator of IFN-regulatory factors (DAI) is a recently identified DNA sensor for intracellular DNA that triggers a signal for the production of type I IFN. Here we report the cloning and characterization of porcine DAI (poDAI). The full-length of poDAI encodes 439 amino acids, contains two N-terminal DNA-binding domains and shows similarity to mouse, rat, dog, monkey, human, horse and cattle counterparts ranging from 44% to 67%. poDAI mRNA expression was mainly detected in spleen, lung, kidney and small intestine. Over-expression of poDAI activated transcription factors IRF3 and NF-kappaB and induced IFN-beta in different porcine cell lines, but to varying degrees. Deletion mutant analysis revealed that both the DNA-binding domains and the C-terminus are required for full activation of IFN-beta. siRNA targeting poDAI significantly decreased poly(dAT:dAT)- or Pseudorabies virus (PRV)-induced IFN-beta activation. These results indicate that DAI is an important immuno-regulator of the porcine innate immune system.

  8. Lack of Neuronal IFN-β-IFNAR Causes Lewy Body- and Parkinson’s Disease-like Dementia

    PubMed Central

    Ejlerskov, Patrick; Hultberg, Jeanette Göransdotter; Wang, JunYang; Carlsson, Robert; Ambjørn, Malene; Kuss, Martin; Liu, Yawei; Porcu, Giovanna; Kolkova, Kateryna; Friis Rundsten, Carsten; Ruscher, Karsten; Pakkenberg, Bente; Goldmann, Tobias; Loreth, Desiree; Prinz, Marco; Rubinsztein, David C.; Issazadeh-Navikas, Shohreh

    2015-01-01

    Summary Neurodegenerative diseases have been linked to inflammation, but whether altered immunomodulation plays a causative role in neurodegeneration is not clear. We show that lack of cytokine interferon-β (IFN-β) signaling causes spontaneous neurodegeneration in the absence of neurodegenerative disease-causing mutant proteins. Mice lacking Ifnb function exhibited motor and cognitive learning impairments with accompanying α-synuclein-containing Lewy bodies in the brain, as well as a reduction in dopaminergic neurons and defective dopamine signaling in the nigrostriatal region. Lack of IFN-β signaling caused defects in neuronal autophagy prior to α-synucleinopathy, which was associated with accumulation of senescent mitochondria. Recombinant IFN-β promoted neurite growth and branching, autophagy flux, and α-synuclein degradation in neurons. In addition, lentiviral IFN-β overexpression prevented dopaminergic neuron loss in a familial Parkinson’s disease model. These results indicate a protective role for IFN-β in neuronal homeostasis and validate Ifnb mutant mice as a model for sporadic Lewy body and Parkinson’s disease dementia. PMID:26451483

  9. Excessive production and extreme editing of human metapneumovirus defective interfering RNA is associated with type I IFN induction.

    PubMed

    van den Hoogen, Bernadette G; van Boheemen, Sander; de Rijck, Jonneke; van Nieuwkoop, Stefan; Smith, Derek J; Laksono, Brigitta; Gultyaev, Alexander; Osterhaus, Albert D M E; Fouchier, Ron A M

    2014-08-01

    Type I IFN production is one of the hallmarks of host innate immune responses upon virus infection. Whilst most respiratory viruses carry IFN antagonists, reports on human metapneumovirus (HMPV) have been conflicting. Using deep sequencing, we have demonstrated that HMPV particles accumulate excessive amounts of defective interfering RNA (DIs) rapidly upon in vitro passage, and that these are associated with IFN induction. Importantly, the DIs were edited extensively; up to 70% of the original A and T residues had mutated to G or C, respectively. Such high editing rates of viral RNA have not, to our knowledge, been reported before. Bioinformatics and PCR assays indicated that adenosine deaminase acting on RNA (ADAR) was the most likely editing enzyme. HMPV thus has an unusually high propensity to generate DIs, which are edited at an unprecedented high frequency. The conflicting published data on HMPV IFN induction and antagonism are probably explained by DIs in virus stocks. The interaction of HMPV DIs with the RNA-editing machinery and IFN responses warrants further investigation.

  10. Human neural stem cells transduced with IFN-beta and cytosine deaminase genes intensify bystander effect in experimental glioma.

    PubMed

    Ito, S; Natsume, A; Shimato, S; Ohno, M; Kato, T; Chansakul, P; Wakabayashi, T; Kim, S U

    2010-05-01

    Previously, we have shown that the genetically modified human neural stem cells (NSCs) show remarkable migratory and tumor-tropic capability to track down brain tumor cells and deliver therapeutic agents with significant therapeutic benefit. Human NSCs that were retrovirally transduced with cytosine deaminase (CD) gene showed remarkable 'bystander killer effect' on the glioma cells after application of the prodrug, 5-fluorocytosine (5-FC). Interferon-beta (IFN-beta) is known for its antiproliferative effects in a variety of cancers. In our pilot clinical trial in glioma, the IFN-beta gene has shown potent antitumor activity in patients with malignant glioma. In the present study, we sought to examine whether human NSCs genetically modified to express both CD and IFN-beta genes intensified antitumor effect on experimental glioma. In vitro studies showed that CD/IFN-beta-expressing NSCs exerted a remarkable bystander effect on human glioma cells after the application of 5-FC, as compared with parental NSCs and CD-expressing NSCs. In animal models with human glioma orthotopic xenograft, intravenously infused CD/IFN-beta-expressing NSCs produced striking antitumor effect after administration of the prodrug 5-FC. Furthermore, the same gene therapy regimen prolonged survival periods significantly in the experimental animals. The results of the present study indicate that the multimodal NSC-based treatment strategy might have therapeutic potential against gliomas.

  11. IFNs Modify the Proteome of Legionella-Containing Vacuoles and Restrict Infection Via IRG1-Derived Itaconic Acid.

    PubMed

    Naujoks, Jan; Tabeling, Christoph; Dill, Brian D; Hoffmann, Christine; Brown, Andrew S; Kunze, Mareike; Kempa, Stefan; Peter, Andrea; Mollenkopf, Hans-Joachim; Dorhoi, Anca; Kershaw, Olivia; Gruber, Achim D; Sander, Leif E; Witzenrath, Martin; Herold, Susanne; Nerlich, Andreas; Hocke, Andreas C; van Driel, Ian; Suttorp, Norbert; Bedoui, Sammy; Hilbi, Hubert; Trost, Matthias; Opitz, Bastian

    2016-02-01

    Macrophages can be niches for bacterial pathogens or antibacterial effector cells depending on the pathogen and signals from the immune system. Here we show that type I and II IFNs are master regulators of gene expression during Legionella pneumophila infection, and activators of an alveolar macrophage-intrinsic immune response that restricts bacterial growth during pneumonia. Quantitative mass spectrometry revealed that both IFNs substantially modify Legionella-containing vacuoles, and comparative analyses reveal distinct subsets of transcriptionally and spatially IFN-regulated proteins. Immune-responsive gene (IRG)1 is induced by IFNs in mitochondria that closely associate with Legionella-containing vacuoles, and mediates production of itaconic acid. This metabolite is bactericidal against intravacuolar L. pneumophila as well as extracellular multidrug-resistant Gram-positive and -negative bacteria. Our study explores the overall role IFNs play in inducing substantial remodeling of bacterial vacuoles and in stimulating production of IRG1-derived itaconic acid which targets intravacuolar pathogens. IRG1 or its product itaconic acid might be therapeutically targetable to fight intracellular and drug-resistant bacteria.

  12. A Critical Role of Bacterioferritin in Salmonella pullorum-Induced IFN-β Expression in DF-1 Cells

    PubMed Central

    Xu, Zhichao; Qin, Yao; Wang, Yongqiang; Li, Xiaoqi; Cao, Hong; Zheng, Shijun J.

    2016-01-01

    Salmonella enterica serovar Pullorum (S. pullorum) causes pullorum disease in poultry and results in great economic losses to the poultry industry. Although an eradication program has been successfully performed in some countries, it remains a major threat to countries with poor poultry disease surveillance. Currently there are no effective control measures for pullorum disease except eradication. In particular, the pathogenesis of S. pullorum infection is still largely unknown. Here we identified bacterioferritin (Bfr) as a major antigen of S. pullorum to elicit a humoral immune response. Furthermore, we demonstrate that Bfr induces activation of IFN-β promoter and mRNA expression in DF-1 cells, and that the amino acids 1–50 form a critical domain involved in IFN-β expression. Moreover, we found that the p38 MAPK signaling pathway was essential for Bfr-induced IFN-β expression. Importantly, S. pullorum-induced IFN-β expression was totally abolished by deficiency of Bfr in the bacteria, indicating that Bfr plays a critical role in S. pullorum induced IFN-β expression in DF-1 cells. Our findings provide new insights into the molecular mechanisms of the host response to S. pullorum infection. PMID:26870001

  13. IFN-{gamma}+ CD8+ T Lymphocytes: Possible Link Between Immune and Radiation Responses in Tumor-Relevant Hypoxia

    SciTech Connect

    De Ridder, Mark Jiang Heng; Esch, Gretel van; Law, Kalun; Monsaert, Christinne; Berge, Dirk L. van den; Verellen, Dirk; Verovski, Valeri N.; Storme, Guy A.

    2008-07-01

    Activated T lymphocytes are known to kill tumor cells by triggering cytolytic mechanisms; however, their ability to enhance radiation responses remains unclear. This study examined the radiosensitizing potential of mouse CD8+ T cells, obtained by T-cell-tailored expansion and immunomagnetic purification. Activated CD8+ T cells displayed an interferon (IFN)-{gamma}+ phenotype and enhanced by 1.8-fold the radiosensitivity of EMT-6 tumor cells in 1% oxygen, which modeled tumor-relevant hypoxia. Radiosensitization was counteracted by neutralizing IFN-{gamma} or by blocking the inducible isoform of nitric oxide synthase, thus delineating the immune-tumor cell interaction through the IFN-{gamma} secretion pathway. Reverse transcriptase-polymerase chain reaction, enzyme-linked immunosorbent assay, and fluorescence-activated cell sorter data in agreement detected downregulation of the IFN-{gamma} gene by hypoxia, which caused IFN-{gamma} deficiency next to radioresistance. Therefore, immune and radiation responses are likely to be allied in the hypoxic tumor microenvironment, and CD8+ T cells may bridge immunostimulatory and radiosensitizing strategies.

  14. Heterogeneity of subordination of the IL-18/IFN-γ axis to caspase-1 among patients with Crohn's disease.

    PubMed

    Jarry, Anne; Bossard, Céline; Droy-Dupré, Laure; Volteau, Christelle; Bourreille, Arnaud; Meurette, Guillaume; Mosnier, Jean-François; Laboisse, Christian L

    2015-10-01

    In Crohn's disease (CD), hierarchical architecture of the inflammatory network, including subordination of IL-18, an IFN-γ-inducing cytokine, to the inflammasome, have remained undeciphered. Heterogeneity among patients of such a subordination cannot be evaluated by animal models, monofactorial in their etiology and homogenous in disease progression. To address these issues, we set up an ex vivo model of inflamed mucosa explant cultures from patients with active long-standing CD. Th1 cytokine production, especially IFN-γ and IL-18, was assessed in relation with inflammation intensity. Subordination of the Th1 response to caspase-1, effector of the inflammasome, was determined in explant cultures subjected to pharmacological inhibition of caspase-1 by YVAD. We showed a correlation between secreted IFN-γ/IL-18 levels, and caspase-1 activation, with inflammation intensity of intestinal CD mucosa explants. Inhibition of caspase-1 activation using the specific inhibitor YVAD identified a homogenous non responder group featuring a caspase-1-independent IL-18/IFN-γ response, and a heterogenous responder group, in which both IL-18 and IFN-γ responses were caspase-1-dependent, with a 40-70% range of inhibition by YVAD. These findings bring out the concept of heterogeneity of subordination of the Th1 response to inflammasome activation among CD patients. This ex vivo model should have therapeutic relevance in allowing to determine eligibility of CD patients for new targeted therapies.

  15. Interferon (IFN) and Cellular Immune Response Evoked in RNA-Pattern Sensing During Infection with Hepatitis C Virus (HCV).

    PubMed

    Nakai, Masato; Oshiumi, Hiroyuki; Funami, Kenji; Okamoto, Masaaki; Matsumoto, Misako; Seya, Tsukasa; Sakamoto, Naoya

    2015-10-23

    Hepatitis C virus (HCV) infects hepatocytes but not dendritic cells (DCs), but DCs effectively mature in response to HCV-infected hepatocytes. Using gene-disrupted mice and hydrodynamic injection strategy, we found the MAVS pathway to be crucial for induction of type III interferons (IFNs) in response to HCV in mouse. Human hepatocytes barely express TLR3 under non-infectious states, but frequently express it in HCV infection. Type I and III IFNs are induced upon stimulation with polyI:C, an analog of double-stranded (ds)RNA. Activation of TLR3 and the TICAM-1 pathway, followed by DC-mediated activation of cellular immunity, is augmented during exposure to viral RNA. Although type III IFNs are released from replication-competent human hepatocytes, DC-mediated CTL proliferation and NK cell activation hardly occur in response to the released type III IFNs. Yet, type I IFNs and HCV-infected hepatocytes can induce maturation of DCs in either human or mouse origin. In addition, mouse CD8+ DCs mature in response to HCV-infected hepatocytes unless the TLR3/TICAM-1 pathway is blocked. We found the exosomes containing HCV RNA in the supernatant of the HCV-infected hepatocytes act as a source of TLR3-mediated DC maturation. Here we summarize our view on the mechanism by which DCs mature to induce NK and CTL in a status of HCV infection.

  16. A soluble nonglycosylated recombinant infectious hematopoietic necrosis virus (IHNV) G-protein induces IFNs in rainbow trout (Oncorhynchus mykiss).

    PubMed

    Verjan, Noel; Ooi, Ei Lin; Nochi, Tomonori; Kondo, Hidehiro; Hirono, Ikuo; Aoki, Takashi; Kiyono, Hiroshi; Yuki, Yoshikazu

    2008-07-01

    Viral glycoproteins interact with cell-surface receptors to mediate virus entry and innate immune system activation. We found that a soluble recombinant infectious hematopoietic necrosis virus G-protein (rIHNV-G) stimulated an early innate immune response mediated by proinflammatory cytokines, IFN1 and IFN-gamma in rainbow trout (Oncorhynchus mykiss) fry. Expression of both IFN1 and IFN-gamma mRNA transcripts was an early event and was rIHNV-G dose-dependent. In addition, preliminary evidence revealed that the innate immune response induced by rIHNV-G protein could protect rainbow trout fry from a subsequent IHNV virus challenge. Finally, the binding and distribution of FITC-rIHNV-G protein on rainbow trout spleen and head kidney leukocytes resemble morphological changes which occur on the cell membrane during antigen-receptor interaction including membrane reorganization, patching, polarization and capping. Thus a soluble nonglycosylated rIHNV-G protein could mediate the activation of rainbow trout leukocytes, with concomitant production of proinflammatory cytokines and IFNs.

  17. Detection of IFN-γ for latent tuberculosis diagnosis using an anodized aluminum oxide-based capacitive sensor.

    PubMed

    Kim, Joo Hyoung; Chang, Young Wook; Bok, Eun; Kim, Hyun-Jeong; Lee, Hyejon; Cho, Sang-Nae; Shin, Jeon-Soo; Yoo, Kyung-Hwa

    2014-01-15

    We describe a rapid, sensitive, and label-free method to detect interferon-gamma (IFN-γ), a biomarker of latent tuberculosis infection (LTBI). IFN-γ is detected by measuring the capacitance change caused by its binding to an anti-IFN-γ antibody. The antibody is immobilized on the surface of an anodized aluminum oxide (AAO)-based capacitive sensor. With this technique, IFN-γ can be detected in the range of ~0.1 pg/ml to ~10 ng/ml, with a detection limit of 0.2 pg/ml. We have also measured the concentration of IFN-γ in clinical samples using the AAO-based capacitive sensor and compared this concentration with the results of the commercial QuantiFERON-TB Gold (QFT-G) ELISA kit to determine whether the two sets of data are consistent. Comparable results were obtained with the two measurement strategies, demonstrating the applicability of the AAO-based capacitive sensor to the diagnosis of LTBI.

  18. IFNs Modify the Proteome of Legionella-Containing Vacuoles and Restrict Infection Via IRG1-Derived Itaconic Acid

    PubMed Central

    Naujoks, Jan; Kunze, Mareike; Kempa, Stefan; Peter, Andrea; Mollenkopf, Hans-Joachim; Dorhoi, Anca; Kershaw, Olivia; Gruber, Achim D.; Sander, Leif E.; Witzenrath, Martin; Herold, Susanne; Nerlich, Andreas; Hocke, Andreas C.; van Driel, Ian; Suttorp, Norbert; Bedoui, Sammy; Hilbi, Hubert; Trost, Matthias; Opitz, Bastian

    2016-01-01

    Macrophages can be niches for bacterial pathogens or antibacterial effector cells depending on the pathogen and signals from the immune system. Here we show that type I and II IFNs are master regulators of gene expression during Legionella pneumophila infection, and activators of an alveolar macrophage-intrinsic immune response that restricts bacterial growth during pneumonia. Quantitative mass spectrometry revealed that both IFNs substantially modify Legionella-containing vacuoles, and comparative analyses reveal distinct subsets of transcriptionally and spatially IFN-regulated proteins. Immune-responsive gene (IRG)1 is induced by IFNs in mitochondria that closely associate with Legionella-containing vacuoles, and mediates production of itaconic acid. This metabolite is bactericidal against intravacuolar L. pneumophila as well as extracellular multidrug-resistant Gram-positive and -negative bacteria. Our study explores the overall role IFNs play in inducing substantial remodeling of bacterial vacuoles and in stimulating production of IRG1-derived itaconic acid which targets intravacuolar pathogens. IRG1 or its product itaconic acid might be therapeutically targetable to fight intracellular and drug-resistant bacteria. PMID:26829557

  19. Suppression of human anti-inflammatory plasma cytokines IL-10 and IL-1RA with elevation of proinflammatory cytokine IFN-gamma during the isolation of the Antarctic winter

    NASA Technical Reports Server (NTRS)

    Shearer, William T.; Lee, Bang-Ning; Cron, Stanley G.; Rosenblatt, Howard M.; Smith, E. O'Brian; Lugg, Desmond J.; Nickolls, Peter M.; Sharp, Robert M.; Rollings, Karl; Reuben, James M.

    2002-01-01

    Cellular immune function has been shown to be decreased and latent virus shedding to be increased in human beings isolated during the Antarctic winter, a model used for assessing some effects of space flight. However, the balance of proinflammatory (IFN-gamma) and anti-inflammatory (IL-10 and IL-1RA) cytokines has not previously been evaluated. We therefore sought to determine whether isolation during the Antarctic winter would alter the proinflammatory and anti-inflammatory cytokine balance. Cytokine levels were measured with ELISA in monthly plasma samples from January through September 1999 in 21 study subjects in the Antarctic and 7 control subjects on Macquarie Island. There was a significant time-dependent increase in plasma IFN-gamma (P =.039) as well as decreases in IL-10 (P =.042) and IL-1RA (P =.053) in the study subjects compared with the control subjects. The study subjects also had significantly increased plasma IFN-gamma levels (P < or =.045) but decreased IL-10 and IL-1RA levels (P < or =.036) at individual time points of isolation. Isolation of human beings in the Antarctic appears to shift the plasma cytokine balance toward a proinflammatory profile. These observations are consistent with T-cell activation that might be due to activation of latent viruses, and they could hold importance for determining the risks of space flight.

  20. Plasmacytoid Dendritic Cells Respond Directly to Apoptotic Cells by Secreting Immune Regulatory IL-10 or IFN

    PubMed Central

    Simpson, Joanne; Miles, Katherine; Trüb, Marta; MacMahon, Roisin; Gray, Mohini

    2016-01-01

    Plasmacytoid dendritic cells (pDCs) play a pivotal role in driving the autoimmune disease systemic lupus erythematosus, via the secretion of IFN-α in response to nuclear self-antigens complexed with autoantibodies. Apoptotic cells, generated at sites of inflammation or secondary lymphoid organs, are exposed to activated pDCs and also express the same nuclear antigens on their cell surface. Here, we show that in the absence of autoantibodies, activated pDCs directly respond to apoptotic cell-expressed chromatin complexes by secreting IL-10 and IL-6, which also induces T cells to secrete IL-10. Conversely, when activated by the viral mimetic CpG-A, apoptotic cells enhance their secretion of IFN-α. This study demonstrates that activated pDCs respond directly to apoptotic cells and may maintain tolerance via IL-10, or promote inflammation through secretion of IFN-α, depending on the inflammatory context. PMID:28018356

  1. Cloning and sequence analysis of IL-2, IL-4 and IFN-γ from Indian Dromedary camels (Camelus dromedarius).

    PubMed

    Nagarajan, G; Swami, Shelesh Kumar; Ghorui, S K; Pathak, K M L; Singh, R K; Patil, N V

    2012-06-01

    The cDNAs of three cytokines, viz., IL-2, IL-4 and IFN-γ from Dromedary camels were amplified by PCR using Bactrian camel sequences and subsequently cloned for sequence analysis. Relationship based on amino acid sequences revealed that Dromedary camel IL-2 shared 99.5% and 99.3% identity at the nucleotide and amino acid levels with Bactrian camel IL-2. In the case of IL-4, the identity of Dromedary camel was 99.7% and 99.2% at the nucleotide and amino acid levels, respectively with that of Bactrian camel. The Dromedary camel IFN-γ shared 100% identity both at nucleotide and amino acid levels with Bactrian camel IFN-γ. Phylogenetic analysis based on amino acid sequences indicated the close relationship in these cytokine genes between the Dromedary camel and other camelids.

  2. Glycyrrhiza flavescens subsp. antalyensis exerts antiproliferative effects on melanoma cells via altering TNF-α and IFN-α levels.

    PubMed

    Aydemir, Esra Arsl