The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor whose activation induces the expression of numerous genes, with many effects on cells. However, AhR activation is not known to affect the replication of viruses. We show that AhR activation in macrophages causes a block to HIV-1 and HSV-1 replication. We find that AhR activation transcriptionally represses cyclin-dependent kinase (CDK)1/2 and their associated cyclins, thereby reducing SAMHD1 phosphorylation, cellular dNTP levels and both HIV-1 and HSV-1 replication. Remarkably, a different antiviral stimulus, interferon gamma (IFN-γ), that induces a largely non-overlapping set of genes, also transcriptionally represses CDK1, CDK2 and their associated cyclins, resulting in similar dNTP depletion and antiviral effects. Concordantly, the SIV Vpx protein provides complete and partial resistance to the antiviral effects of AhR and IFN-γ, respectively. Thus, distinct antiviral signaling pathways converge on CDK/cyclin repression, causing inhibition of viral DNA synthesis and replication.

Background: Anaemia is a major public health concern especially in African children living in malaria-endemic regions. Interferon-gamma (IFN-γ) is elevated during malaria infection and is thought to influence erythropoiesis and iron status. Genetic variants in the IFN-γ gene (IFNG) are associated with increased IFN-γ production. We investigated putative functional single nucleotide polymorphisms (SNPs) and haplotypes of IFNG in relation to nutritional iron status and anaemia in Gambian children over a malaria season. Methods: We used previously available data from Gambian family trios to determine informative SNPs and then used the Agena Bioscience MassArray platform to type five SNPs from the IFNG gene in a cohort of 780 Gambian children aged 2-6 years. We also measured haemoglobin and biomarkers of iron status and inflammation at the start and end of a malaria season. Results: We identified five IFNG haplotype-tagging SNPs ( IFNG-1616 [rs2069705], IFNG+874 [rs2430561], IFNG+2200 [rs1861493], IFNG+3234 [rs2069718] and IFNG+5612 [rs2069728]). The IFNG+2200C [rs1861493] allele was associated with reduced haemoglobin concentrations (adjusted β -0.44 [95% CI -0.75, -0.12]; Bonferroni adjusted P = 0.03) and a trend towards iron deficiency compared to wild-type at the end of the malaria season in multivariable models adjusted for potential confounders. A haplotype uniquely identified by IFNG+2200C was similarly associated with reduced haemoglobin levels and trends towards iron deficiency, anaemia and iron deficiency anaemia at the end of the malaria season in models adjusted for age, sex, village, inflammation and malaria parasitaemia. Conclusion: We found limited statistical evidence linking IFNG polymorphisms with a risk of developing iron deficiency and anaemia in Gambian children. More definitive studies are needed to investigate the effects of genetically influenced IFN-γ levels on the risk of iron deficiency and anaemia in children living in malaria-endemic areas.

Interferon gamma (IFN-γ) is best known for its ability to regulate host immune responses; however, its direct antiviral activity is less well studied. Transmissible gastroenteritis virus (TGEV) is an economically important swine enteric coronavirus and causes acute diarrhea in piglets. At present, little is known about the function of IFN-γ in the control of TGEV infection. In this study, we demonstrated that IFN-γ inhibited TGEV infection directly in ST cells and intestine epithelial IPEC-J2 cells and that the anti-TGEV activity of IFN-γ was independent of IFN-α/β. Moreover, IFN-γ suppressed TGEV infection in ST cells more efficiently than did IFN-α, and the combination of IFN-γ and IFN-α displayed a synergistic effect against TGEV. Mechanistically, using overexpression and functional knockdown experiments, we demonstrated that porcine interferon regulatory factor 1 (poIRF1) elicited by IFN-γ primarily mediated IFN-γ signaling cascades and the inhibition of TGEV infection by IFN-γ. Importantly, we found that TGEV elevated the expression of poIRF1 and IFN-γ in infected small intestines and peripheral blood mononuclear cells. Thus, IFN-γ plays a crucial role in curtailing enteric coronavirus infection and may serve as an effective prophylactic and/or therapeutic agent against TGEV infection.

The sole equine herpesvirus 1 (EHV-1) immediate-early protein (IEP) is essential for viral replication by transactivating viral immediate-early (IE), early (E), and late (L) genes. Here, we report that treatment of mouse MH-S, equine NBL6, and human MRC-5 cells with 20 ng/mL of IFN-γ reduced EHV-1 yield by 1122-, 631-, and 10,000-fold, respectively. However, IFN-γ reduced virus yield by only 2-4-fold in mouse MLE12, mouse L-M, and human MeWo cells compared to those of untreated cells. In luciferase assays with the promoter of the EHV-1 early regulatory EICP0 gene, IFN-γ abrogated trans-activation activity of the IEP by 96% in MH-S cells, but only by 21% in L-M cells. Similar results were obtained in assays with the early regulatory UL5 and IR4 promoter reporter plasmids. IFN-γ treatment reduced IEP protein expression by greater than 99% in MH-S cells, but only by 43% in L-M cells. The expression of IEP and UL5P suppressed by IFN-γ was restored by JAK inhibitor treatment, indicating that the inhibition of EHV-1 replication is mediated by JAK/STAT1 signaling. These results suggest that IFN-γ blocks EHV-1 replication by inhibiting the production of the IEP in a cell line-dependent manner. Affymetrix microarray analyses of IFN-γ-treated MH-S and L-M cells revealed that five antiviral ISGs (MX1, SAMHD1, IFIT2, NAMPT, TREX1, and DDX60) were upregulated 3.2-18.1-fold only in MH-S cells.

Systemic administration of interferon (IFN)-beta has been recently approved for the treatment of relapsing-remitting multiple sclerosis (RRMS). The immunological mechanism by which IFN-beta ameliorates MS is still partially unknown. We measured the number of blood circulating CD4(+), CD4(-), CD8(+), and CD8(-) T cells secreting IFN-gamma and IL-4 in 26 RRMS patients followed for up to 9 months of an alternate day s.c. treatment with 8x16 IU of IFN-beta1b. Compared to pre-treatment values, a significant (P<0.05) reduction of CD4(+), CD4(-), CD8(+) and CD8(-) cells producing IFN-gamma and of CD4(+) and CD4(-) cells producing IL-4 was observed in MS patients. The IFN-beta-associated effect was evident soon after the beginning of the treatment and persisted for the entire follow-up period. We did not observe any effect of IFN-beta treatment on the percentage of IL-4-producing CD8(+) and CD8(-) cells nor in that of natural killer (NK) cells producing IFN-gamma. Our results show that IFN-beta treatment in MS patients induces a profound and persistent down-regulation of the number of circulating T cells secreting IFN-gamma and IL-4 thus suggesting a broader rather than a specific immunomodulatory effect of IFN-beta in MS.

Two interferon gamma (IFN-gamma) genes are expressed in immune cells of teleost fish and are potentially implicated in B- and T-lymphocyte responses. IFN-gamma-2 shows structural and functional characteristics to other vertebrate IFN-gamma genes and is associated with T-lymphocyte function. Expression profiling shows IFN-gamma-2 upregulation in T-lymphocytes after phytohemagglutinin (PHA) stimulation in vitro. Unexpectedly, we found IFN-gamma-1, which is structurally different from IFN-gamma-2, to be expressed in lipopolysacharide (LPS)-stimulated IgM+ (B- lymphocyte enriched) fractions. Expression of T-box transcription factor T-bet, but not of GATA-binding protein 3 (GATA3), correlated with expression of both IFN-gamma genes. In-vivo parasite infection, but as predicted not zymosan-induced inflammation, resulted in concomitant upregulation of T-bet and IFN-gamma-2. This corroborates a genuine T-lymphocyte associated role for IFN-gamma-2.

IL-7 is a major regulator of lymphocyte homeostasis; however, little is known about the mechanisms that regulate IL-7 production. To study Il7 gene regulation in vivo, we generated a novel IL-7-reporter mouse, which allows the non-invasive quantification of Il7 gene activity in live mice and, additionally, the simultaneous activation/inactivation of target genes in IL-7-producing cells. With these IL-7-reporter mice, we identify thymus, skin and intestine as major sources of IL-7 in vivo. Importantly, we show that IFN-gamma and the commensal microflora promote steady-state IL-7 production in the intestine. Furthermore, we demonstrate that the blockade of IFN-gamma signaling in intestinal epithelial cells strongly reduces their IFN-gamma-driven IL-7 production. In summary, our data suggest a feedback loop in which commensal bacteria drive IFN-gamma production by lymphocytes, which in turn promotes epithelial cell IL-7 production and the survival of IL-7-dependent lymphocytes.

Interleukin-11 (IL-11) is an anti-apoptotic, anti-inflammatory cytokine with hematopoietic potential. The expression and protective actions of IL-11 have not been explored in the eye. The expression of IL-11 in primary cultures of human retinal pigment epithelial (HRPE) and human corneal fibroblast (HCRF) cells were evaluated in these studies. Constitutive secretion of IL-11 was not observed in either HRPE or HCRF. TNF-alpha+IL-1 induced IL-11 secretion and this production was inhibited by NFkappaB pathway inhibitors. IFN-gamma significantly inhibited TNF-alpha and IL-1 induced IL-11 secretion and inhibitors of JAK-STAT pathway reversed this inhibition. TGF-beta induced IL-11 secretion that was blocked by TGF-beta receptor 1 inhibitor but not by IFN-gamma. RT-PCR analysis confirmed the effects of IL-1, TNF-alpha, IFN-gamma and TGF-beta on IL-11 secretion at mRNA levels. Our results demonstrate that IL-11 is dramatically up regulated in retina and cornea cells and that IFN-gamma is a physiological inhibitor of IL-11 expression.

Infection of the central nervous system (CNS) by the neurotropic JHM strain of mouse hepatitis virus (JHMV) induces an acute encephalomyelitis associated with demyelination. To examine the anti-viral and/or regulatory role of interferon-gamma (IFN-gamma) signaling in the cell that synthesizes and maintains the myelin sheath, we analyzed JHMV pathogenesis in transgenic mice expressing a dominant-negative IFN-gamma receptor on oligodendroglia. Defective IFN-gamma signaling was associated with enhanced oligodendroglial tropism and delayed virus clearance. However, the CNS inflammatory cell composition and CD8(+) T-cell effector functions were similar between transgenic and wild-type mice, supporting unimpaired peripheral and CNS immune responses in transgenic mice. Surprisingly, increased viral load in oligodendroglia did not affect the extent of myelin loss, the frequency of oligodendroglial apoptosis, or CNS recruitment of macrophages. These data demonstrate that IFN-gamma receptor signaling is critical for the control of JHMV replication in oligodendroglia. In addition, the absence of a correlation between increased oligodendroglial infection and the extent of demyelination suggests a complex pathobiology of myelin loss in which infection of oligodendroglia is required but not sufficient.

As immune responses in the CNS are highly regulated, cell-specific differences in IFNgamma signaling may be integral in dictating the outcome of host cell responses. In comparing the response of IFNgamma-treated primary neurons to control MEF, we observed that neurons demonstrated lower basal expression of both STAT1 and STAT3, the primary signal transducers responsible for IFNgamma signaling. Following IFNgamma treatment of these cell populations, we noted muted and delayed STAT1 phosphorylation, no detectable STAT3 phosphorylation, and a 3-10-fold lower level of representative IFNgamma-responsive gene transcripts. Moreover, in response to a brief pulse of IFNgamma, a steady increase in STAT1 phosphorylation and IFNgamma gene expression over 48 h was observed in neurons, as compared to rapid attenuation in MEF. These distinct response kinetics in IFNgamma-stimulated neurons may reflect modifications in the IFNgamma negative feedback loop, which may provide a mechanism for the cell-specific heterogeneity of responses to IFNgamma.

Interferon γ (IFN-γ) induces an inflammatory response and apoptotic cell death. Rheumatoid arthritis (RA) is a systemic inflammatory disease associated with increased levels of inflammatory mediators, including tumour necrosis factor α (TNF-α) and T helper (Th) 17 cells, and downregulation of apoptosis of inflammatory cells. We hypothesized that IFN-γ would reduce inflammatory cell death in vitro and that loss of IFN-γ would aggravate inflammation in vivo. IFN-γ downregulated necroptosis and the expression of cellular FLICE-like inhibitory protein (cFLIPL) and mixed lineage kinase domain-like (MLKL). However, loss of IFN-γ promoted the production of cFLIPL and MLKL, and necroptosis. IFN-γ deficiency increased Th17 cell number and upregulated the expression of IL-17 and TNF-α. Expression of MLKL, receptor interacting protein kinase (RIPK)1, and RIPK3 was increased in the joints of mice with collagen-induced arthritis (CIA). Compared with wild-type mice with CIA, IFN-γ-/- CIA mice showed exacerbation of cartilage damage and joint inflammation, and acceleration of MLKL, RIPK1, and RIPK3 production in the joints. IFN-γ deficiency induced the activation of signal transducer and activator of transcription 3. These results suggest that IFN-γ regulates inflammatory cell death and may have potential for use in the treatment of RA.

IFI16, member of the IFN-inducible PYHIN-200 gene family, modulates proliferation, survival and differentiation of different cell lineages. In particular, IFI16 expression, which is regulated during the differentiation of B cells, was recently studied in B-CLL as well. Here, we compared IFI16 expression in several lymphomas including Burkitt lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, marginal zone lymphoma and mantle cell lymphoma with respect to normal cell counterparts. We observed that IFI16 expression was significantly deregulated only in mantle cell lymphoma (p < 0.05). Notably, IFI16 was associated with the expression of genes involved in interferon response, cell cycle, cell death and proliferation and, interestingly, lipid and glucose metabolism, suggesting that IFI16 deregulation might be associated with relevant changes in cell biology. In our group of mantle cell lymphoma samples a correlation between patient survival and IFI16 expression was not detected even though mantle cell lymphoma prognosis is known to be associated with cell proliferation. Altogether, these results suggest a complex relationship between IFI16 expression and MCL which needs to be analyzed in further studies.

Cathepsin S (catS) and cathepsin L (catL) mediate late stages of invariant chain (Ii) degradation in discrete antigen-presenting cell types. Macrophages (Mphis) are unique in that they express both proteases and here we sought to determine the relative contribution of each enzyme. We observe that catL plays no significant role in Ii cleavage in interferon (IFN)-gamma-stimulated Mphis. In addition, our studies show that the level of catL activity is significantly decreased in Mphis cultured in the presence of IFN-gamma whereas catS activity increases. The decrease in catL activity upon cytokine treatment occurs despite the persistence of high levels of mature catL protein, suggesting that a specific inhibitor of the enzyme is up-regulated in IFN-gamma-stimulated peritoneal Mphis. Similar inhibition of activity is observed in dendritic cells engineered to overexpress catL. Such enzymatic inhibition in Mphis exhibits only partial dependence upon Ii and therefore, other mechanisms of catL inhibition are regulated by IFN-gamma. Thus, during a T helper cell type 1 immune response catL inhibition in Mphis results in preferential usage of catS, such that major histocompatibility complex class II presentation by all bone marrow-derived antigen-presenting cell is regulated by catS.

Salmonella bacteria are a major cause of food-borne infectious diarrhoea and there is great interest in understanding the pathogenesis of Salmonella infection and in vaccine development. Potential vaccines include the aromatic mutants of S. typhimurium. Such non-lethal Aro mutants have also been useful for studying Salmonella infections in mouse models. Studies of systemic infection, using these Aro mutants, in both normal and cytokine gene knockout mice, indicate that interferon-gamma (IFN-gamma) plays a key role in the resolution of Salmonella infection. The present studies have investigated the outcome of oral infection in mice with attenuated Salmonella because this infection route mimics natural infection in humans. In IFN-gamma gene knockout (IFN-gamma-/-) mice, intestinal immunity was impaired and oral challenge resulted in disseminated septicaemia 2 weeks later. No dissemination of infection was seen in wild-type mice. In wild-type mice, both CD4 and CD8 cell numbers increased in the gut following Salmonella challenge, together with increased expression of major histocompatibility complex (MHC) II and vascular cell adhesion molecule-1 (VCAM-1). No such changes were seen in IFNgamma-/- mice. Following oral challenge, antilipopolysaccharide (LPS) and antiphosphoryl choline antibodies increased by more than 100-fold in both serum and faecal pellet extracts of IFNgamma-/- mice compared with wild-type mice. Our data show that IFN-gamma production is essential for resolution of enteric Salmonella infection and that antibody has little effect on this process.

The interferon signaling pathway is critical for host defense by serving diverse functions in both innate and adaptive immune responses. Here, we show that type I gamma phosphatidylinositol phosphate 5-kinase i5 (PIPKIγi5), an enzyme that synthesizes phosphatidylinositol-4,5-bisphosphate (PI4,5P2), controls the sensitivity to interferon in both human and mouse cells. PIPKIγi5 directly binds to the interferon-gamma (IFN-γ) downstream effector signal transducer and activator of transcription 1 (STAT1), which suppresses the STAT1 dimerization, IFN-γ-induced STAT1 nuclear translocation, and transcription of IFN-γ-responsive genes. Depletion of PIPKIγi5 significantly enhances IFN-γ signaling and strengthens an antiviral response. In addition, PIPKIγi5-synthesized PI4,5P2 can bind to STAT1 and promote the PIPKIγi5-STAT1 interaction. Similar to its interaction with STAT1, PIPKIγi5 is capable of interacting with other members of the STAT family, including STAT2 and STAT3, thereby suppressing the expression of genes mediated by these transcription factors. These findings identify the function of PIPKIγi5 in immune regulation.

The mucosal antiviral role of type I and III interferon in influenza virus infection is well established. However, much less is known about the antiviral mechanism of type II interferon (interferon-gamma). Here, we revealed an antiviral mechanism of interferon-gamma by inhibiting influenza A virus (IAV) attachment. By direct stochastic optical reconstruction microscopy, confocal microscopy, and flow cytometry, we have shown that interferon-gamma reduced the size of α-2,3 and α-2,6-linked sialic acid clusters, without changing the sialic acid or epidermal growth factor receptor expression levels, or the sialic acid density within cluster on the cell surface of A549 cells. Reversing the effect of interferon-gamma on sialic acid clustering by jasplakinolide reverted the cluster size, improved IAV attachment and replication. Our findings showed the importance of sialic acid clustering in IAV attachment and infection. We also demonstrated the interference of sialic acid clustering as an anti-IAV mechanism of IFN-gamma for IAV infection.

The protozoan parasite Trypanosoma cruzi is responsible for the zoonotic Chagas disease, a chronic and systemic infection in humans and warm-blooded animals typically leading to progressive dilated cardiomyopathy and gastrointestinal manifestations. In the present study, we report that the transcription factor STAT1 (signal transducer and activator of transcription 1) reduces the susceptibility of human cells to infection with T. cruzi. Our in vitro data demonstrate that interferon -γ (IFNγ) pre-treatment causes T. cruzi-infected cells to enter an anti-parasitic state through the activation of the transcription factor STAT1. Whereas stimulation of STAT1-expressing cells with IFNγ significantly impaired intracellular replication of parasites, no protective effect of IFNγ was observed in STAT1-deficient U3A cells. The gene encoding indoleamine 2, 3-dioxygenase (ido) was identified as a STAT1-regulated target gene engaged in parasite clearance. Exposure of cells to T. cruzi trypomastigotes in the absence of IFNγ resulted in both sustained tyrosine and serine phosphorylation of STAT1 and its increased DNA binding. Furthermore, we found that in response to T. cruzi the total amount of intracellular STAT1 increased in an infectious dose-dependent manner, both at the mRNA and protein level. While STAT1 activation is a potent strategy of the host in the fight against the invading pathogen, amastigotes replicating intracellularly antagonize this pathway by specifically promoting the dephosphorylation of STAT1 serine 727, thereby partially circumventing its protective effects. These findings point to the crucial role of the IFNγ/STAT1 signal pathway in the evolutionary combat between T. cruzi parasites and their host.

Activation of fibroblast growth factor receptor 3 (FGFR3) leads to attenuation of cartilage growth. The members of the STAT family of transcription factors are believed to participate in FGFR3 signaling in cartilage, however the molecular mechanism of this action is poorly understood. Here, we demonstrate that a chronic FGF stimulus leads to accumulation of STAT1, 3, 5 and 6, evident in both in vitro chondrocyte model and murine limb explant cultures. Despite the accumulation, both endogenous and cytokine-induced activation of STAT1 and STAT3 is impaired by FGF, as demonstrated by imaging of active STAT nuclear translocation and analyses of STAT activatory phosphorylation and transcriptional activation. Further, we demonstrate that FGF induces expression of CIS, SOCS1 and SOCS3 inhibitors of gp130, a common receptor for the IL6-family of cytokines. Since cytokine-gp130 signaling represents an important positive regulator of cartilage, its inhibition may contribute to the growth-inhibitory effect of FGFR3 in cartilage.

The proinflammatory cytokine interferon-gamma (IFN-gamma), which can be present in elevated levels in the central nervous system during pathological conditions, may be involved in the generation of persistent pain states by inducing neuronal hyperexcitability. The aim of the present study was to examine whether loss of dorsal horn GABAergic inhibition may underlie this IFN-gamma-mediated neuronal hyperexcitability. Repetitive intrathecal injections of recombinant rat IFN-gamma (1000 U) or control buffer were administered to rats every second day for eight days. Electrophysiological recordings from lumbar dorsal horn neurons (n=46) were performed under halothane anaesthesia. Cellular responses were recorded before, during and after microiontophoretic application of the GABA antagonist bicuculline. In control animals, all cellular responses studied were significantly enhanced in the presence of bicuculline, including increased spontaneous activity, enhanced responses to innocuous and noxious mechanical stimulation and reduced paired-pulse depression. In contrast, in IFN-gamma-treated animals, bicuculline ejection had little or no facilitating effect on neuronal responses and instead a significant proportion of neurons displayed reduced responses. Seventy-four percent of cells from IFN-gamma treated animals showed a reduction in the response to noxious stimulation and 47% of the cells showed increased rather than reduced paired-pulse depression in the presence of bicuculline, thus suggesting IFN-gamma-induced excitatory actions by GABA. These findings show that the prolonged presence of increased levels of IFN-gamma in the central nervous system may contribute to the generation of central sensitization and persistent pain by reducing inhibitory tone in the dorsal horn. This implies a potential link between disinhibition and cytokine action in the spinal cord.

Hematopoietic stem cell transplantation (HSCT) is a frequent therapeutic approach to restore hematopoiesis in patients with hematologic diseases. Patients receive a hematopoietic stem cell (HSC)-enriched donor cell infusion also containing immune cells, which may have a beneficial effect by eliminating residual neoplastic cells. However, the effect that donor innate immune cells may have on the donor HSCs has not been deeply explored. Here, we evaluate the influence of donor natural killer (NK) cells on HSC fate, concluded that NK cells negatively affect HSC frequency and function, and identified interferon-gamma (IFNγ) as a potential mediator. Interestingly, improved HSC fitness was achieved by NK cell depletion from murine and human donor infusions or by blocking IFNγ activity. Thus, our data suggest that suppression of inflammatory signals generated by donor innate immune cells can enhance engraftment and hematopoietic reconstitution during HSCT, which is particularly critical when limited HSC numbers are available and the risk of engraftment failure is high.