Inhibitory Effect Of IFN-γ Research Articles

Interferon-γ Induces Interleukin-6 Production and Alpha-smooth Muscle Actin Expression in Systemic Sclerosis Fibroblasts.

Systemic sclerosis (SSc) is an autoimmune systemic disease that is characterized by immune dysregulation, inflammation, vasculopathy, and fibrosis. Tissue fibrosis plays an important role in SSc and can affect several organs such as the dermis, lungs, and heart. Dysregulation of interferon (IFN) signaling contributes to the SSc pathogenesis and interferon regulatory factor 1 (IRF1) has been indicated as the main regulator of type I IFN. This study aimed to clarify the effect of IFN-gamma (-γ) and dexamethasone (DEX) on the IRF1, extracellular signal-regulated kinase 1/2 (ERK1/2), and the expression of alpha-smooth muscle actin (α-SMA) in myofibroblasts and genes involved in the inflammation and fibrosis processes in early diffuse cutaneous systemic sclerosis (dcSSc). A total of 10 early dcSSc patients (diffuse cutaneous form) and 10 unaffected control dermis biopsies were obtained to determine IFNγ and DEX effects on inflammation and fibrosis. Fibroblasts were treated with IFNγ and DEX at optimum time and dose. The expression level of genes and proteins involved in the fibrosis and inflammation processes have been quantified by quantitative real-time PCR (RT-qPCR) and western blot, respectively. IFNγ could up-regulate some of the inflammation-related genes (Interleukin-6; IL6) and down-regulate some of the fibrosis-related genes (COL1A1) in cultured fibroblasts of patients with early dcSSc compared to the untreated group. Besides, it has been revealed that IFNγ can induce fibroblast differentiation to the myofibroblast that expresses α-SMA. Concerning the inhibitory effect of IFNγ on some fibrotic genes and its positive effect on the inflammatory genes and myofibroblast differentiation, it seems that IFNγ may play a dual role in SSc.

The host transcriptome change involved in the inhibitory effect of exogenous interferon-γ on Getah virus replication.

Getah virus (GETV) has become a growing potential threat to the global livestock industry and public health. However, little is known about the viral pathogenesis and immune escape mechanisms, leading to ineffective control measures. In this study, the antiviral activity of exogenous interferons (IFNs) was assessed by using western blotting (WB), real-time quantitative PCR (RT-qPCR) and indirect immunofluorescence assay (IFA). The comparative transcriptomics among mock- and GETV-infected (MOI = 0.1) ST cells with or without IFN-γ was performed by RNA-seq, and then the transcriptome profiling of GETV-infected ST cells and key pathways and putative factors involved in inhibitory effect of IFN-γ on GETV replication were analyzed by bioinformatics methods and RT-qPCR. The results showed that treatment with IFN-γ could suppress GETV replication, and the inhibitory effect lasted for at least 48 h, while the exogenous IFN-α/ω and IFN-λ3 treatments failed to inhibit the viral infection and early replication in vitro. Furthermore, the blueprint of virus-host interaction was plotted by RNA-seq and RT-qPCR, showing systemic activation of inflammatory, apoptotic, and antiviral pathways in response to GETV infection, indicating viral hijacking and inhibition of innate host immunity such as IFN-I/III responses. Last and most importantly, activation of the JAK-STAT signaling pathway and complement and coagulation cascades may be a primary driver for IFN-γ-mediated inhibition of GETV replication. These findings revealed that GETV possessed the capability of viral immune escape and indicated that IFN-γ aided in the prevention and control of GETV, implying the potential molecular mechanism of suppression of GETV by IFN-γ, all of which warrant emphasis or further clarification.

The Paget's disease of bone risk gene PML is a negative regulator of osteoclast differentiation and bone resorption.

ABSTRACTPaget's disease of bone (PDB) is characterized by focal increases in bone remodelling. Genome-wide association studies identified a susceptibility locus for PDB tagged by rs5742915, which is located within the PML gene. Here, we have assessed the candidacy of PML as the predisposing gene for PDB at this locus. We found that the PDB-risk allele of rs5742915 was associated with lower PML expression and that PML expression in blood cells from individuals with PDB was lower than in controls. The differentiation, survival and resorptive activity of osteoclasts prepared from Pml−/− mice was increased compared with wild type. Furthermore, the inhibitory effect of IFN-γ on osteoclast formation from Pml−/− was significantly blunted compared with wild type. Bone nodule formation was also increased in osteoblasts from Pml−/− mice when compared with wild type. Although microCT analysis of trabecular bone showed no differences between Pml−/− mice and wild type, bone histomorphometry showed that Pml−/− mice had high bone turnover with increased indices of bone resorption and increased mineral apposition rate. These data indicate that reduced expression of PML predisposes an individual to PDB and identify PML as a novel regulator of bone metabolism. This article has an associated First Person interview with the first author of the paper.

Enhanced osteoclastogenesis in patients with MSMD due to impaired response to IFN-γ

Patients with Mendelian susceptibility to mycobacterial disease (MSMD) experience recurrent and/or persistent infectious diseases associated with poorly virulent mycobacteria. Multifocal osteomyelitis is among the representative manifestations of MSMD. The frequency of multifocal osteomyelitis is especially high in patients with MSMD etiologies that impair cellular response to IFN-γ, such as IFN-γR1, IFN-γR2, or STAT1 deficiency. This study sought to characterize the mechanism underlying multifocal osteomyelitis in MSMD. GM colonies prepared from bone marrow mononuclear cells from patients with autosomal dominant (AD) IFN-γR1 deficiency, AD STAT1 deficiency, or STAT1 gain of function (GOF) and from healthy controls were differentiated into osteoclasts in the presence or absence of IFN-γ. The inhibitory effect of IFN-γ on osteoclastogenesis was investigated by quantitative PCR, immunoblotting, tartrate-resistant acid phosphatase staining, and pit formation assays. Increased osteoclast numbers were identified by examining the histopathology of osteomyelitis in patients with AD IFN-γR1 deficiency or AD STAT1 deficiency. In the presence of receptor activator of nuclear factor kappa-B ligand and M-CSF, GM colonies from patients with AD IFN-γR1 deficiency, AD STAT1 deficiency, or STAT1 GOF differentiated into osteoclasts, similar to GM colonies from healthy volunteers. IFN-γ concentration-dependent inhibition of osteoclast formation was impaired in GM colonies from patients with AD IFN-γR1 deficiency or AD STAT1 deficiency, whereas it was enhanced in GM colonies from patients with STAT1 GOF. Osteoclast differentiation is increased in AD IFN-γR1 deficiency and AD STAT1 deficiency due to an impaired response to IFN-γ, leading to excessive osteoclast proliferation and, by inference, increased bone resorption in infected foci, which may underlie multifocal osteomyelitis.

IFN-γ inhibits acute allergic airway inflammation in mice

Objective To investigate the inhibitory effect of IFN-γ on acute allergic airway inflammation induced by IL-33 in mice. Methods Twenty-four female C57BL/6 mice (6-8 weeks) were randomly divided into four groups: IL-33 model group, IFN-γ treatment group, IL-33+ IFN-γ treatment group and PBS control group. A mouse model of acute allergic airway inflammation was induced by IL-33. Samples of bronchial alveolar lavage fluid (BALF) and lung tissues were collected. Group 2 innate lymphoid cells (ILC2s) and eosinophils were analyzed by flow cytometry. Levels of IL-5 and IL-13 in the supernatants of lung homogenate and BALF were measured by ELISA. Expression of IL-5, IL-13 and ST2 at mRNA level was detected by real-time PCR. Pathological changes in lung tissues were observed following hematoxylin and eosin (HE) and periodic acid-Schiff (PAS) staining. Results Compared with the PBS control group, no infiltration with inflammatory cells, goblet cell hyperplasia or mucus secretion was observed in the IFN-γ group; the numbers of ILC2s and eosinophils were not affected by IFN-γ; the levels of IL-5 and IL-13 in the supernatants of BALF and lung homogenate, and the expression of IL-5, IL-13 and ST2 at mRNA level in lung tissues were not significantly changed by IFN-γ (P>0.05). Compared with the PBS control group, massive infiltration with inflammatory cells, excessive mucus secretion, increased numbers of ILC2s and eosinophils, up-regulated levels of IL-5 and IL-13 in the supernatants of BALF and lung homogenate, and enhanced expression of IL-5, IL-13 and ST2 at mRNA level in lung tissues were detected in the IL-33 model group (P<0.05). Compared with the IL-33 model group, the combined treatment with IL-33 and IFN-γ significantly alleviated inflammatory cell infiltration, inhibited mucus secretion, reduced the numbers of ILC2s and eosinophils, down-regulated the levels of IL-5 and IL-13 in the supernatants of BALF and lung homogenate, and suppressed the expression of IL-5, IL-13 and ST2 at mRNA in lung tissues (P<0.05). Conclusion IFN-γ can inhibit the proliferation of eosinophils and ILC2s induced by IL-33, and reduce the secretion of IL-5 and IL-13, which indicates that IFN-γ has an inhibitory effect on acute allergic airway inflammation induced by IL-33 in mice. Key words: IFN-γ; Group 2 innate lymphoid cell; IL-33; Acute airway inflammation

Interferon-γ Perturbs Key Signaling Pathways Induced By Thrombopoietin, but Not Eltrombopag, in Human Hematopoietic Stem/Progenitor Cells

▪Thrombopoietin (TPO) is the main regulator of hematopoietic stem and progenitor cell (HSPC) self-renewal and survival. Upon binding to its receptor, c-MPL, TPO activates cell signaling, through JAK-STAT and other pathways, which is tightly balanced by negative regulatory signaling processes. Recent studies indicate that chronic exposure of HSPCs to IFNγ, as exemplified in subjects with severe aplastic anemia (SAA), impairs self-renewal by perturbing TPO signaling pathways. Despite elevated levels of TPO in subjects with SAA, the TPO receptor agonist Eltrombopag (Epag) improves trilineage hematopoiesis in refractory SAA, suggesting that it may activate signaling within HSPC in a way that is distinct from TPO under inflammatory conditions. To address the paradox of Epag efficacy despite high endogenous TPO levels in bone marrow failure, G-CSF mobilized human CD34+ cells from 6 healthy donors were cultured in the presence of SCF, FLT3 and either TPO 5 ng/ml (TPO5) or Epag 3 μg/ml (Epag), with or without IFNγ 100 ng/ml. After 7 days in culture, cells were characterized via flow cytometry, CFU assay and transplantation in immunodeficient (NSG) mice. The percentages of CD34+ cells in cultures containing TPO5 or Epag alone were similar (83.3 ± 9.7% and 87.6 ± 7.1%, respectively), but were better preserved with Epag than TPO5 in the presence of IFNγ (46.7 ± 16.1% and 24.6 ± 15.0% respectively, p<0.05). Accordingly, when comparing 7-day cultures with and without IFNγ, the absolute numbers of CD34+ cells were markedly reduced with TPO5 (average 7.6-fold, p<0.005) but only minimally decreased with Epag (average 1.6-fold, p = n.s.). The adjusted numbers of CFUs after 7 days in the presence of IFNγ similarly decreased 2.7-fold with TPO5 but remained unchanged with Epag compared to cultures without IFNγ. When the 7-day expanded progeny of an equal starting number of CD34+ cells was transplanted in NSG mice, human cell engraftment was superior with Epag (34 ± 3.8% human CD45+ cells) than with TPO5 (21 ± 1.8% human CD45+ cells, p<0.05) cultures in the presence of IFNγ, suggesting an impact of Epag on the most primitive long-term repopulating HSPCs. To investigate potential mechanisms by which Epag positively affects maintenance of HSPCs under inflammatory conditions, we examined cell signaling pathways induced upon binding of TPO, Epag and IFNγ to their respective receptors in human CD34+ cells. At a concentration of 5ng/mL, TPO induced a rapid (peak < 1 hour) and high potency rise in STAT5 phosphorylation followed by a rapid (< 2 hours) decay in signal. In contrast, Epag induced a slow (peak 4 hours) low potency rise in STAT5 phosphorylation, and the signal persisted for at least 10 hours. The difference in cell signaling potency and kinetics between TPO and Epag is likely related to their binding to distinct regions of c-MPL, resulting in alternate receptor conformational changes. We next investigated the impact of IFNγ on TPO and Epag-induced STAT5 phosphorylation at the signal peak (<1 and 4 hours, respectively). As previously shown in murine HSPCs, IFNγ impaired TPO signaling in human HSPCs (Figure, panels A, C). In contrast, Epag-induced STAT5 phosphorylation was preserved or increased in the presence of IFNγ (Figure, panels B, C). When Epag and TPO were combined, inhibition of TPO signaling by IFNγ was partially restored (Figure, panel D). By reducing the dose of TPO from 5 to 1ng/mL, and therefore reducing the potency of signaling to levels similar to Epag, the inhibitory effect of IFNγ on TPO signaling was abolished (Figure, panel E). Activation of IFNγ receptor by its ligand induces phosphorylation of STAT1 and subsequent expression of suppressor of cytokine signaling-1 (SOCS-1), a negative regulator of both IFNγ and c-MPL receptors via inhibition of STAT1 and STAT5 phosphorylation, respectively. We found that IFNγ-induced phosphorylation of STAT1 was increased in the presence of TPO 5ng/mL (1.5-fold increase, p<0.05) but unaffected by Epag (1.1-fold increase, p = n.s.) or TPO 1ng/mL (1.1-fold increase, p = n.s.). Our data indicate that Epag counters IFNγ-induced perturbation of TPO signaling in human HSPCs. Epag produces an unopposed low potency, slow kinetic positive signal and activates c-Mpl above a threshold level critical for HSPC self-renewal. Epag’s evasion of IFN blockade of a critical pathway of growth factor cell signaling may explain its efficacy in improving hematopoiesis in SAA. [Display omitted] DisclosuresCheng:Novartis: Research Funding. Cheruku:Novartis: Research Funding. Alvarado:Novartis: Research Funding. Cash:Novartis: Research Funding. Dunbar:Novartis: Research Funding. Young:Novartis: Research Funding. Larochelle:Novartis: Research Funding.

Study on IFN-γ/TGF-β level in a model of acute inflammation and melanoma

Objective:To build a mouse tumor model with a manufactured surgical wound representing acute inflammation,and to evaluate the relationship between acute inflammation or wound healing and the process of tumor growth.Then to observe the impact of IFN-γ/TGF-β on tumor growth.Methods:Male C57BL mice of six weeks were used and divided into the experiment group and the control group.The B16F10 melanoma cell suspension was injected into the left groin area of each mouse.A wound measured 1 cm in diameter was built on the opposite side of bodies in the experiment group when tumor volume was about 0.5 cm~3. The expression of IFN-γ/TGF-β in blood serum and tumor tissues were examined by ELISA.In order to further confirm the effect of TGF-β on tumor growth,another 16 mice models with melanoma were established and 8 of them received IFN-γ injection (the experiment group).Results:When acute inflammation had influences on tumor,a two-phase development was presented.In the early phase,the growth of tumor in the mice with wound was slower than that in the control group.In the early phase,the release of IFN-γ was higher and the release of TGF-β was lower in the experiment group.In the later phase,the growth of tumor in the mice with wound was similar to that in the controls and the release of TGF-β was higher.In vivo experiment confirmed the above results.In the early phase,the release of TGF-β was not significantly different between the experiment group and the control group (P0.05).In the later phase,the release of TGF-β in the experiment group was higher than that in the control group (P0.05).Conclusion:In the early phase of acute inflamma- tion,inhibitory effects of IFN-γ on tumor growth were presented.In the later phase,the inhibited tumor was resistant to IFN-γ through the release of TGF-β to balance the effect of inflammatory factors on tumor cells.

Experimental Colitis Is Associated with Transcriptional Inhibition of Na+/Ca2+ Exchanger Isoform 1 (NCX1) Expression by Interferon γ in the Renal Distal Convoluted Tubules

NCX1 is a Na(+)/Ca(2+) exchanger, which is believed to provide a key route for basolateral Ca(2+) efflux in the renal epithelia, thus contributing to renal Ca(2+) reabsorption. Altered mineral homeostasis, including intestinal and renal Ca(2+) transport may represent a significant component of the pathophysiology of the bone mineral density loss associated with Inflammatory Bowel Diseases (IBD). The objective of our research was to investigate the effects of TNBS and DSS colitis and related inflammatory mediators on renal Ncx1 expression. Colitis was associated with decreased renal Ncx1 expression, as examined by real-time RT-PCR, Western blotting, and immunofluorescence. In mIMCD3 cells, IFNγ significantly reduced Ncx1 mRNA and protein expression. Similar effects were observed in cells transiently transfected with a reporter construct bearing the promoter region of the kidney-specific Ncx1 gene. This inhibitory effect of IFNγ is mediated by STAT1 recruitment to the proximal promoter region of Ncx1. Further in vivo study with Stat1(-/-) mice confirmed that STAT1 is indeed required for the IFNγ mediated Ncx1 gene regulation. These results strongly support the hypothesis that impaired renal Ca(2+) handling occurs in experimental colitis. Negative regulation of NCX1- mediated renal Ca(2+) absorption by IFNγ may significantly contribute to the altered Ca(2+) homeostasis in IBD patients and to IBD-associated loss of bone mineral density.

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

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.

Reciprocal Negative Cross-Talk between Liver X Receptors (LXRs) and STAT1: Effects on IFN-γ–Induced Inflammatory Responses and LXR-Dependent Gene Expression

Liver X receptors (LXRs) exert key functions in lipid homeostasis and in control of inflammation. In this study we have explored the impact of LXR activation on the macrophage response to the endogenous inflammatory cytokine IFN-γ. Transcriptional profiling studies demonstrate that ∼38% of the IFN-γ-induced transcriptional response is repressed by LXR activation in macrophages. LXRs also mediated inhibitory effects on selected IFN-γ-induced genes in primary microglia and in a model of IFN-γ-induced neuroinflammation in vivo. LXR activation resulted in reduced STAT1 recruitment to the promoters tested in this study without affecting STAT1 phosphorylation. A closer look into the mechanism revealed that SUMOylation of LXRs, but not the presence of nuclear receptor corepressor 1, was required for repression of the NO synthase 2 promoter. We have also analyzed whether IFN-γ signaling exerts reciprocal effects on LXR targets. Treatment with IFN-γ inhibited, in a STAT1-dependent manner, the LXR-dependent upregulation of selective targets, including ATP-binding cassette A1 (ABCA1) and sterol response element binding protein 1c. Downregulation of ABCA1 expression correlated with decreased cholesterol efflux to apolipoprotein A1 in macrophages stimulated with IFN-γ. The inhibitory effects of IFN-γ on LXR signaling did not involve reduced binding of LXR/retinoid X receptor heterodimers to target gene promoters. However, overexpression of the coactivator CREB-binding protein/p300 reduced the inhibitory actions of IFN-γ on the Abca1 promoter, suggesting that competition for CREB-binding protein may contribute to STAT1-dependent downregulation of LXR targets. The results from this study suggest an important level of bidirectional negative cross-talk between IFN-γ/STAT1 and LXRs with implications both in the control of IFN-γ-mediated immune responses and in the regulation of lipid metabolism.

Target-Mediated Disposition Model Describing the Dynamics of IL12 and IFNγ after Administration of a Mifepristone-Inducible Adenoviral Vector for IL-12 Expression in Mice

Interleukin-12 (IL12) is a cytokine with potential applications in the treatment of cancer given the potent immune response that it triggers, in part due to its ability to stimulate expression of interferon-γ (IFNγ). To avoid the toxicity associated with systemic exposure to IL12, a high-capacity adenoviral vector carrying a liver-specific, mifepristone-inducible IL12 expression system (HC-Ad/RUmIL12) has been developed. However, the maintenance of IL12 expression at therapeutic levels is compromised by the inhibitory effect of IFNγ on inducible systems. The aim of this work is to develop a semi-mechanistic model to characterize the relationship between IL12 and IFNγ in wild-type and knock-out mice for the IFNγ receptor treated with HC-Ad/RUmIL12 under different dosing regimens in order to better understand the key mechanisms controlling the system. Rapid binding was considered to account for target-mediated disposition exhibited by both cytokines (equilibrium dissociation constant were 18 and 2.28pM for IL12 and IFNγ, respectively). The final model included: (1) IFNγ receptor turnover, (2) irreversible free cytokine elimination from the serum compartment, (3) internalization of the IL12 receptor complex, (4) IL12 expression upregulated by the co-administration of the adenoviral vector and mifepristone and downregulated by the IFNγ receptor, and (5) synthesis of IFNγ controlled by the relative increments in the bound IL12. In conclusion, a model simultaneously describing the kinetics of IL12 and IFNγ in the context of gene therapy was developed and validated with additional data. The model was applied to design an experimental dosing protocol intended to maintain sustained therapeutic IL12 levels.

IFN-γ and TNF-α Synergize to Inhibit CTGF Expression in Human Lung Endothelial Cells

Connective tissue growth factor (CTGF/CCN2) is an angiogenetic and profibrotic factor, acting downstream of TGF-β, involved in both airway- and vascular remodeling. While the T-helper 1 (Th1) cytokine interferon-gamma (IFN-γ) is well characterized as immune-modulatory and anti-fibrotic cytokine, the role of IFN-γ in lung endothelial cells (LEC) is less defined. Tumour necrosis factor alpha (TNF-α) is another mediator that drives vascular remodeling in inflammation by influencing CTGF expression. In the present study we investigated the influence of IFN-γ and TNF-α on CTGF expression in human LEC (HPMEC-ST1.6R) and the effect of CTGF knock down on human LEC. IFN-γ and TNF-α down-regulated CTGF in human LEC at the promoter-, transcriptional- and translational-level in a dose- and time-dependent manner. The inhibitory effect of IFN-γ on CTGF-expression could be almost completely compensated by the Jak inhibitor AG-490, showing the involvement of the Jak-Stat signaling pathway. Besides the inhibitory effect of IFN-γ and TNF-α alone on CTGF expression and LEC proliferation, these cytokines had an additive inhibitory effect on proliferation as well as on CTGF expression when administered together. To study the functional role of CTGF in LEC, endogenous CTGF expression was down-regulated by a lentiviral system. CTGF silencing in LEC by transduction of CTGF shRNA reduced cell proliferation, but did not influence the anti-proliferative effect of IFN-γ and TNF-α. In conclusion, our data demonstrated that CTGF was negatively regulated by IFN-γ in LEC in a Jak/Stat signaling pathway-dependent manner. In addition, an additive effect of IFN-γ and TNF-α on inhibition of CTGF expression and cell proliferation could be found. The inverse correlation between IFN-γ and CTGF expression in LEC could mean that screwing the Th2 response to a Th1 response with an additional IFN-γ production might be beneficial to avoid airway remodeling in asthma.

T-bet-independent effects of IL-12 family cytokines on regulation of Th17 responses to experimental T. cruzi infection

Tbx21 (i.e., T-bet) is an IFN-γ-inducible transcription factor that promotes Th1 differentiation. Previously, we reported that Tbx21(-/-) mice develop a robust Th17 response to the parasite Trypanosoma cruzi, including CD4(+) T cell subsets producing IL-17 and IFN-γ. Because of the known inhibitory effects of IFN-γ on Th17 cells, the purpose of this study was to determine the contribution of IFN-γ to regulation of Th17 differentiation during the course of T. cruzi infection. We observed that infection of IFN-γ(-/-) or Stat-1(-/-) mice generated increased numbers of IL-17-producing cells. In sharp contrast to infected Stat-1(-/-) or Tbx21(-/-) mice, however, IFN-γ(-/-) mice developed a lower overall Th17 response, suggesting that IFN-γ was not required for T-bet-dependent activity, including T-bet-dependent expression of CXCR3. To determine if IFN-γ could influence Th17 responses indirectly by acting on APCs, we neutralized IFN-γ in cultures containing APC and T. cruzi antigens. Although anti-IFN-γ increased IL-17 production modestly, anti-IFN-γ and anti-IL-12 led to a significant enhancement of T. cruzi-specific IL-17 (P<0.01). In contrast to the inhibitory effects of IL-12, IL-23 was able to stimulate Tbx21(-/-) T cells and cause a striking increase in T. cruzi-specific IL-17. These data show that the IL-12 family of cytokines can influence Th17 responses in a T-bet-independent manner and that the effects of IFN-γ are not necessarily related to its ability to induce T-bet expression in T cells.

Regulatory role of IFN-gamma on alveolar macrophage function during influenza infection (43.9)

Abstract Secondary bacterial infection often follows pulmonary virus infection. We previously reported that pulmonary IFN-γ produced during influenza infection in mice inhibits initial bacterial clearance by alveolar macrophages. As a result, there was a 100-fold increase in bacterial burden in viral-infected WT mice 48 hr after pneumococcal challenge compared to IFN-γ-/- mice. Although IFN-γ production by CD4 or CD8 T cells alone was sufficient to inhibit bacterial clearance, TCR transgenic OT-I and OT-II mice had no detectable IFN-γ following influenza infection. Thus, virus-specific T cells are responsible for significant IFN-γ production. We found that the inhibitory effect of IFN-γ was correlated with down regulation of the class A scavenger receptor MARCO. Indeed, prior influenza infection had no effect on initial bacterial clearance in MARCO-/- mice. Inhibition of MARCO expression following influenza infection could be prevented by IFN-γ neutralization. Preliminary data suggest that IFN-γ inhibits MARCO expression by signaling through STAT1 and there is significant induction of SOCS1 and SOCS3 in alveolar macrophages during influenza infection. Ongoing work is designed to determine whether influenza-induced IFN-γ facilitates activation of a SOCS negative-feedback loop in alveolar macrophages, thereby suppressing innate protection against extracellular bacteria. (NIH grant #AI41715)

IFN-gamma Impairs Release of IL-8 by IL-1beta-stimulated A549 Lung Carcinoma Cells

BackgroundProduction of interferon (IFN)-γ is key to efficient anti-tumor immunity. The present study was set out to investigate effects of IFNγ on the release of the potent pro-angiogenic mediator IL-8 by human A549 lung carcinoma cells.MethodsA549 cells were cultured and stimulated with interleukin (IL)-1β alone or in combination with IFNγ. IL-8 production by these cells was analyzed with enzyme linked immuno sorbent assay (ELISA). mRNA-expression was analyzed by real-time PCR and RNase protection assay (RPA), respectively. Expression of inhibitor-κ Bα, cellular IL-8, and cyclooxygenase-2 was analyzed by Western blot analysis.ResultsHere we demonstrate that IFNγ efficiently reduced IL-8 secretion under the influence of IL-1β. Surprisingly, real-time PCR analysis and RPA revealed that the inhibitory effect of IFNγ on IL-8 was not associated with significant changes in mRNA levels. These observations concurred with lack of a modulatory activity of IFNγ on IL-1β-induced NF-κB activation as assessed by cellular IκB levels. Moreover, analysis of intracellular IL-8 suggests that IFNγ modulated IL-8 secretion by action on the posttranslational level. In contrast to IL-8, IL-1β-induced cyclooxygenase-2 expression and release of IL-6 were not affected by IFNγ indicating that modulation of IL-1β action by this cytokine displays specificity.ConclusionData presented herein agree with an angiostatic role of IFNγ as seen in rodent models of solid tumors and suggest that increasing T helper type 1 (Th1)-like functions in lung cancer patients e.g. by local delivery of IFNγ may mediate therapeutic benefit via mechanisms that potentially include modulation of pro-angiogenic IL-8.

Interleukin-1 beta enhances and interferon-gamma suppresses activin A actions by reciprocally regulating activin A and follistatin secretion from bone marrow stromal fibroblasts.

Activin A is a multi-functional cytokine with a potent stimulation on erythroid cell differentiation in the bone marrow. The actions of activin A are determined by a balance of the levels of activin A and its inhibitor, follistatin (FS). However, the regulation of its actions in the bone marrow has been unclear. Here we show that bone marrow-derived stromal fibroblasts are the major source of activin A and FS in the bone marrow, and that the production of activin A is enhanced by interleukin-1beta (IL-1beta) and lipopolysaccharide (LPS), whereas interferon-gamma (IFN-gamma) inhibits the secretion of activin A by stromal fibroblasts. Concomitantly, IL-1beta as well as LPS inhibits and IFN-gamma stimulates FS secretion from stromal fibroblasts. Thus, these cytokines potently regulate activin A actions by reciprocal modulation of activin A and FS secretion from stromal fibroblasts. Because activin A exhibits anti-inflammatory effects in various tissues, up-regulation of activin A actions by IL-1beta and endotoxin in the bone marrow may play a protective role against inflammatory processes as well as anaemia. The present results also suggest that the inhibitory effect of IFN-gamma on erythropoiesis is mediated at least in part by a suppression of activin A actions in bone marrow.

Investigation of the Antifibrotic Effect of IFN-γ on Fibroblasts in a Cell Culture Model of Peyronie's Disease

ObjectiveA broad spectrum of options is available for treatment of Peyronie's disease; however, the effects of minimally invasive therapy are generally inadequate. Although useful, oral drugs must be administered at onset of the disease. Only a few patients request penile surgery. Therefore, new medical treatments for Peyronie's disease are needed. A better understanding of the pathogenesis of Peyronie's disease is required to facilitate development of these new medical treatments. Several studies have described an increased level of TGF-β in the fibrotic plaques of patients with Peyronie's disease, underscoring this important signalling pathway in the onset and/or development of Peyronie's disease. MethodsPlaque biopsies were taken from 16 patients with Peyronie's disease. Furthermore, 7 patients without Peyronie's disease were biopsied to provide control material. Fibroblasts were cultured from biopsy tissue, and cultured fibroblasts were stimulated with TGF-β1, BMP-2, IFN-γ, and IFN-γ combined with one of the other stimuli. Protein was extracted from treated fibroblasts and prepared for immunoblots. The membranes were probed for phosphorylated Smad and total Smad to indicate activation of TGF-β signalling. ResultsAn agonistic effect of IFN-γ on TGF-β signalling was observed. Stimulation with TGF-β1 increased levels of phospho-Smad2 and phospho-Smad3. After stimulation with TGF-β1 and IFN-γ combined, the levels of phospho-Smads were higher than those observed with stimulation withTGF-β1 alone. ConclusionsThe profibrotic effect of TGF-β1 is enhanced by IFN-γ in fibroblasts from patients with Peyronie's disease. The inhibitory effects of IFN-γ on the TGF-β pathway do not appear in Peyronie's disease. Therefore, IFN-γ cannot be taken as a useful tool in the therapy of Peyronie's disease.

IFN-γ Negatively Regulates CpG-Induced IL-10 in Bone Marrow-Derived Dendritic Cells

Dendritic cells (DCs) are important players in the regulation of Th1- and Th2-dominated immune responses. In these studies we showed that IFN-gamma, the key mediator of Th1 immunity, actively suppressed the production of IL-10 in murine DCs when activated with LPS or CpG. Our analysis revealed that both LPS and CpG induced IL-10 and IL-12 production but that the presence of IFN-gamma, in a dose-dependent manner, suppressed the production of IL-10 while enhancing that of IL-12. The observed inhibition of IL-10 production was independent of IL-12. Experiments performed with STAT-1 knockout mice demonstrated that the primary production of IL-12 induced by CpG was STAT-1 dependent, whereas the production of IL-10 was not. This finding was confirmed by the observation that CpG-induced IL-12 production could be inhibited by anti-IFN-beta Abs, whereas CpG-induced IL-10 production could not be inhibited. These data also demonstrated that the inhibitory effect of IFN-gamma on IL-10 expression was STAT-1 dependent and transcriptionally regulated. Thus, DCs respond to CpG by producing proinflammatory and anti-inflammatory cytokines such as IL-12 and IL-10, respectively, and IFN-gamma acts to not only enhance IL-12 but also to inhibit IL-10 production. The current data demonstrate a novel pathway for IFN-gamma-mediated immunoregulation and suggest that IFN-gamma-dependent suppression of IL-10 production by DCs may be involved in the antagonism between Th1 and Th2 patterns of immune reactivity.

Inhibitory Effect of Gamma Interferon on BK Virus Gene Expression and Replication

BK virus (BKV) is widely accepted to be the causative agent of polyomavirus nephropathy. In immunocompromised individuals, especially kidney transplant recipients, BKV can replicate in kidney epithelial cells, causing loss of renal function and eventual destruction of the graft. Advances in immunosuppressive therapies may be partially responsible for the increasing incidence of polyomavirus nephropathy among transplant recipients by more effectively eliminating components of the immune system, such as gamma interferon (IFN-gamma)-producing lymphocytes, that keep BKV infections at a subclinical level. In this study, we investigated the role of IFN-gamma in regulating lytic infection by BKV. Treatment with IFN-gamma inhibited the expression of the viral early protein large tumor antigen (TAg) and the late protein VP1 in a dose-dependent manner. We detected 1.6- and 12-fold reductions in TAg transcripts at 48 and 96 h postinfection, respectively, with 250 U/ml IFN-gamma, suggesting that IFN-gamma-mediated inhibition occurs at the level of transcription. Furthermore, IFN-gamma inhibited the level of viral progeny production as much as 50-fold at a multiplicity of infection (MOI) of 0.5 and 80-fold at an MOI of 0.1. The inhibitory effects of IFN-gamma were similar for three different strains of BKV examined. These results indicate an important role for IFN-gamma in regulating BKV lytic infection.

Inhibitory effects of interferon-γ on myocardial hypertrophy

Prostaglandin F 2α (PGF 2α) plays an important role in pathologic cardiac growth. After testing several immune cytokines, we found that interferon-γ (IFN-γ) inhibited responsiveness of adult myocytes to PGF 2α. The present study was designed to test the hypothesis that IFN-γ inhibits cardiac hypertrophy induced by PGF 2α. Incubation of cultured adult rat cardiac myocytes with PGF 2α caused cell spreading, which was inhibited by IFN-γ. The inhibitory effect was not affected by nitric oxide (NO) synthase inhibitors. In addition, administration of fluprostenol, a more selective agonist at the PGF 2α receptor, induced cardiac hypertrophy in rats. Chronic treatment with IFN-γ inhibited this myocardial growth, and the inhibitory effect of IFN-γ was not accompanied by an increase in myocardial NO synthase gene expression. Further, abdominal aortic constriction resulted in a substantial increase in heart, ventricular and left ventricular weights to BW ratio that was significantly attenuated by treatment with IFN-γ. The results demonstrate that IFN-γ inhibits the in vitro and in vivo effects of PGF 2α on cardiac hypertrophy, and that the mechanism of action is likely independent of NO production. IFN-γ also attenuated cardiac hypertrophy induced by pressure overload, suggesting that PGF 2α plays a role in the pathogeneses of this severe type of cardiac hypertrophy.