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International audience; As more cases of COVID-19 are studied and treated worldwide, it had become apparent that the lethal and most severe cases of pneumonia are due to an out-of-control inflammatory response to the SARS-CoV-2 virus. I explored the putative causes of this specific feature through a detailed genomic comparison with the closest SARS-CoV-2 relatives isolated from bats, as well as previous coronavirus strains responsible for the previous epidemics (SARS-CoV and MERS-CoV). The high variability region of the nsp3 protein was confirmed to exhibit the most variations between closest strains. It was then studied in the context of physiological and molecular data available in the literature. A number of convergent findings suggest de-mono-ADP-ribosylation (de-MARylation) of STAT1 by the SARS-CoV-2 nsp3 as a putative cause of the cytokine storm observed in the most severe cases of COVID-19. This may suggest new therapeutic approaches and help in designing assays to predict the virulence of naturally circulating SARS-like animal coronaviruses.

Type I IFN can exert pro- and anti-inflammatory activities in the immune system. Here, we have investigated the mechanism by which IFN-α enhances early expression of the anti-inflammatory cytokine IL-10 in human CD45RA+CD4+ T cells. With the use of transcriptomic and biochemical approaches, we found distinct and combined contributions of the IFN and the TCR signaling pathways to the induction of STAT1/2/3 and the basic leucine zipper activating transcription factor-like (BATF) family members. Moreover, IFN-induced STAT3 phosphorylation was prolonged by the TCR response, whereas IFN-induced STAT2 phosphorylation was of long duration. With the use of RNA interference (RNAi), we identified STAT3 as the major actor and STAT2 as a contributor of the IFN action on IL-10. Upon TCR/IFN costimulation, STAT3 directly bound at the IL-10 conserved noncoding sequence (CNS)- 9, an enhancer element known to recruit BATF in CD4 T cells. The cosilencing of the 3 BATFs resulted in an overall reduction of IL-10 expression, but the promoting activity of IFN-α was retained. These results support the notion that the IFN action is indexed on BATF function and provide evidence for a cooperation between BATFs and STAT3, the latter being activated via early IFN and delayed TCR effects.

The CDKN2A locus, which contains the tumor suppressor gene p16INK4a, is associated with an increased risk of age-related inflammatory diseases, such as cardiovascular disease and type 2 diabetes, in which macrophages play a crucial role. Monocytes can polarize toward classically (CAMφ) or alternatively (AAMφ) activated macrophages. However, the molecular mechanisms underlying the acquisition of these phenotypes are not well defined. Here, we show that p16INK4a deficiency (p16−/−) modulates the macrophage phenotype. Transcriptome analysis revealed that p16−/− BM-derived macrophages (BMDMs) exhibit a phenotype resembling IL-4–induced macrophage polarization. In line with this observation, p16−/− BMDMs displayed a decreased response to classically polarizing IFNγ and LPS and an increased sensitivity to alternative polarization by IL-4. Furthermore, mice transplanted with p16−/− BM displayed higher hepatic AAMφ marker expression levels on Schistosoma mansoni infection, an in vivo model of AAMφ phenotype skewing. Surprisingly, p16−/− BMDMs did not display increased IL-4–induced STAT6 signaling, but decreased IFNγ-induced STAT1 and lipopolysaccharide (LPS)–induced IKKα,β phosphorylation. This decrease correlated with decreased JAK2 phosphorylation and with higher levels of inhibitory acetylation of STAT1 and IKKα,β. These findings identify p16INK4a as a modulator of macrophage activation and polarization via the JAK2-STAT1 pathway with possible roles in inflammatory diseases.

International audience; Type I (α and β) interferons (IFNs) elicit antiproliferative and antiviral activities via the surface receptor IFNAR. Serendipitous observations in transgenic mice in 1988 strongly suggested that IFNα/β overexpression in the testis disrupts spermatogenesis. Here, we compare a new mouse strain transgenic for IFNβ (Tg10) and a sister strain lacking the IFNAR1 subunit of IFNAR (Tg10-Ifnar1(-/-)), both strains expressing the transgene in the testis. The main source of IFNβ RNA was the spermatid population. Importantly, the Tg10 mice, but not the double mutant Tg10-Ifnar1(-/-), showed altered spermatogenesis. The first IFNAR-dependent histological alteration was a higher apoptosis index in all germ cell categories apart from non-dividing spermatogonia. This occurred 3 weeks after the onset of IFNβ production at postnatal day 20 and in the absence of somatic cell defects in terms of cell number, expression of specific cell markers, and hormonal activities. Several known interferon-stimulated genes were up-regulated in Tg10 Sertoli cells and prepachytene germ cells but not in pachytene spermatocytes and spermatids. In concordance with this, pachytene spermatocytes and spermatids isolated from wild-type testes did not display measurable amounts of IFNAR1 and phosphorylated STAT1 upon IFNβ challenge in vitro, suggesting hyporesponsiveness of these cell types to IFN. At day 60, Tg10 males were sterile, and Sertoli cells showed increased amounts of anti-Mullerian hormone and decreased production of inhibin B, both probably attributable to the massive germ cell loss. Type I interferon signaling may lead to idiopathic infertilities by affecting the interplay between germ cells and Sertoli cells.

International audience; MPhi and DC are key elements in the control of tissue homeostasis and response to insult. In this work, we demonstrate that MPhi and DC are the major producers of the phenylalanine catabolizing enzyme IL-4-induced gene 1 (IL4I1) under inflammatory conditions. IL4I1 was first described in B cells, which indeed can produce IL4I1 in vitro, although at much lower levels. In vivo, IL4I1 is highly expressed by MPhi and DC of Th1 granulomas (sarcoidosis, tuberculosis) but poorly detected in Th2 granulomas (schistosomiasis). In vitro, expression of the enzyme is induced in mononuclear phagocytes by various pro-inflammatory stimuli through the activation of the transcription factors NF-kappaB and/or STAT1. B cells also express IL4I1 in response to NF-kappaB-activating stimuli such as CD40L; however, in contrast to myeloid cells, B cells are insensitive to IFN-gamma but respond to stimulation of the IL-4/STAT6 axis. As we show that the expression of IL4I1 by a monocytic cell line inhibits T-cell proliferation and production of IFN-gamma and inflammatory cytokines, we propose that IL4I1 participates in the downregulation of Th1 inflammation in vivo.

International audience; The V protein of measles virus (MV-V) is a potent inhibitor of IFN-alpha/beta signaling pathway. We previously reported that when physically dissociated, the N-terminal and C-terminal regions of MV-V (PNT and VCT, respectively) could independently impair signal transduction. The PNT region inhibited IFN-alpha/beta signaling by interacting with at least two components of this pathway: Jak1 and STAT1. Here we report a direct interaction between the VCT of MV-V and STAT2, a third component of IFN-alpha/beta transduction machinery. This interaction with STAT2 is carried by the cysteine-constrained peptide of 49 amino acids localized in the VCT region, and is essential to the inhibition of IFN-alpha/beta signaling. In parallel, we also mapped STAT1 binding site in the PNT region and identified a minimal peptide of only 11 amino acids. IFN-alpha/beta signaling was impaired in human cells treated with this MV-V peptide fused to a cell-penetrating sequence. Finally, we show that signaling downstream of IFN-lambda, a recently identified cytokine that also relies on STAT1, STAT2 and Jak1 to transduce, is blocked by MV-V. Altogether, our results illustrate how a single viral protein has evolved to achieve a robust inhibition of the antiviral response by interacting with several signaling molecules.

International audience; Interferon gamma (IFN-γ) and leukemia inhibitory factor (LIF) are key gestational factors that may differentially affect leukocyte function during gestation. Because IFN-γ induces a pro-inflammatory phenotype in macrophages and because trophoblast cells are principal targets of LIF in the placenta, we investigated whether and how soluble factors from trophoblast cells regulate the effects of IFN-γ on macrophage activation. IFN-γ reduces macrophage motility, but enhances Stat1 activation, pro-inflammatory gene expression and cytotoxic functions. Soluble factors from villous cytotrophoblasts (vCT+LIF cells) and BeWo cells (BW/ST+LIF cells) that were differentiated in the presence of LIF inhibit macrophage Stat1 activation but inversely sustain Stat3 activation in response to IFN-γ. vCT+LIF cells produce soluble factors that induce Stat3 activation; this effect is partially abrogated in the presence of neutralizing anti-interleukin 10 (IL-10) antibodies. Moreover, soluble factors from BW/ST+LIF cells reduce cell proliferation but enhance the migratory responses of monocytes. In addition, these factors reverse the inhibitory effect of IFN-γ on monocyte/macrophage motility. BW/ST+LIF cells also generate IFN-γ-activated macrophages with enhanced IL-10 expression, but reduced tumor-necrosis factor alpha (TNF-α), CD14 and CD40 expression as well as impaired cytotoxic function. Additional assays performed in the presence of neutralizing anti-IL-10 antibodies and exogenous IL-10 demonstrate that reduced macrophage cytotoxicity and proliferation, but increased cell motility result from the ability of trophoblast IL-10 to sustain Stat3 activation and suppress IFN-γ-induced Stat1 activation. These in vitro studies are the first to describe the regulatory role of the LIF-trophoblast-IL-10 axis in the process of macrophage activation in response to pro-inflammatory cytokines.

Constitutive activation of signal transducer and activator of transcription 1 (STAT1) is a distinctive feature of Epstein-Barr virus (EBV)-immortalized B cells (lymphoblastoid cell lines [LCLs]). The expression of STAT1 in these cells is modulated by the latent membrane protein 1 (LMP1), but the mechanism of STAT1 activation has remained unclear. We demonstrate that the tyrosine phosphorylation of STAT1 in LCLs results from an indirect pathway encompassing an NF-κB-dependent secretion of interferons (IFNs). The cell culture supernatant of LCLs induced tyrosine phosphorylation of STAT1 in cells with no constitutively activated STAT1. Moreover, removal of supernatant from LCLs was sufficient to decrease the phosphorylation of STAT1. Inhibition of NF-κB activity by different pharmacological inhibitors (i.e., parthenolide, MG132 and BAY 11-7082) and by overexpressed mutated IκBα prevented the activation of STAT1. To identify the factors involved, we performed macroarray cDNA profiling with or without inhibition of NF-κB. The expression of several cytokines was NF-κB dependent among those alpha and gamma IFNs (IFN-α and IFN-γ), known activators of STAT1. By real-time PCR and enzyme-linked immunosorbent assay we show that IFN-α and IFN-γ are expressed and released by LCLs in an NF-κB-dependent manner. Finally, the blocking of the IFN-α and IFN-γ by neutralizing antibodies led to the complete inhibition of tyrosine phosphorylation of STAT1. Taken together, our results clearly show that LMP1-induced tyrosine phosphorylation of STAT1 is almost exclusively due to the NF-κB-dependent secretion of IFNs. Whether this response, which is usually considered to be antiviral, is in fact required for the persistence of the virus remains to be elucidated.

International audience; As do cytokine receptors and receptor tyrosine kinases, G protein-coupled receptors (GPCRs) signal to Janus kinases (Jaks) and signal transducers and activators of transcription (STATs). However, the early biochemical events linking GPCRs to this signaling pathway have been unclear. Here we show that GPCR-stimulated Rac activity and the subsequent generation of reactive oxygen species are necessary for activating tyrosine phosphorylation of Jaks and STAT-dependent transcription. The requirement for Rac activity can be overcome by addition of hydrogen peroxide. Expression of activated mutants of Rac1 is sufficient to activate Jak2 and STAT-dependent transcription, and the activation of Jak2 correlates with the ability of Rac1 to bind to NADPH oxidase subunit p67(phox). We further show that GPCR agonists stimulate tyrosine phosphorylation of STAT1 and STAT3 proteins in a Rac-dependent manner. The tyrosine phosphorylation of STAT3 is biphasic; the first peak of phosphorylation is weak and correlates with rapid activation of Jaks by GPCRs, whereas the second peak is stronger and requires the synthesis of an autocrine factor. Rho also plays an essential role in the induction of STAT transcriptional activity. Our results highlight a novel role for Rho GTPases in mediating the regulatory effects of GPCRs on STAT-dependent gene expression.

A signal transduction pathway activated by many cytokines has recently been elaborated. The JAK kinases and the signal transducers and activators of transcription (STAT) factors have been found to be essential components. In this report, we describe the presence of constitutively activated STAT factors in peripheral blood cells from patients with acute leukemia. We used oligonucleotide probes from the beta-casein and IRF-1 gene promoters and the ISRE probe to detect STAT proteins in nuclear extracts from acute leukemia cells in bandshift assays. Specific DNA protein complex formation was observed with the probes from the beta-casein and IRF-1 gene promoters, but not with the ISRE oligonucleotide probe, when cell extracts from acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) were investigated. We used nonradioactive oligonucleotides as competitors to show the specificity of the complex formation. Specific antibodies directed against the individual STAT proteins were used in supershift experiments. STAT5- and STAT1-related factors were detected in ALL and STAT1-, STAT3-, and STAT5-related proteins were present in nuclear cell extracts from AML. Since the cells were not treated with cytokines before the nuclear proteins were extracted, we conclude that these factors are constitutively activated in vivo. It is likely that the constitutive activation of STAT proteins is a part of the events of leukemogenesis.

International audience; The CDKN2A locus, which contains the tumor suppressor gene p16(INK4a), is associated with an increased risk of age-related inflammatory diseases, such as cardiovascular disease and type 2 diabetes, in which macrophages play a crucial role. Monocytes can polarize toward classically (CAMϕ) or alternatively (AAMϕ) activated macrophages. However, the molecular mechanisms underlying the acquisition of these phenotypes are not well defined. Here, we show that p16(INK4a) deficiency (p16(-/-)) modulates the macrophage phenotype. Transcriptome analysis revealed that p16(-/-) BM-derived macrophages (BMDMs) exhibit a phenotype resembling IL-4-induced macrophage polarization. In line with this observation, p16(-/-) BMDMs displayed a decreased response to classically polarizing IFNγ and LPS and an increased sensitivity to alternative polarization by IL-4. Furthermore, mice transplanted with p16(-/-) BM displayed higher hepatic AAMϕ marker expression levels on Schistosoma mansoni infection, an in vivo model of AAMϕ phenotype skewing. Surprisingly, p16(-/-) BMDMs did not display increased IL-4-induced STAT6 signaling, but decreased IFNγ-induced STAT1 and lipopolysaccharide (LPS)-induced IKKα,β phosphorylation. This decrease correlated with decreased JAK2 phosphorylation and with higher levels of inhibitory acetylation of STAT1 and IKKα,β. These findings identify p16(INK4a) as a modulator of macrophage activation and polarization via the JAK2-STAT1 pathway with possible roles in inflammatory diseases.

International audience; STAT1 belongs to the STAT family of transcription factors, which comprises seven factors: STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B and STAT6. STAT1 is a 91 kDa protein originally identified as the mediator of the cellular response to interferon (IFN) alpha, and thereafter found to be a major component of the cellular response to IFNgamma. STAT1 is, in fact, involved in the response to several cytokines and to growth factors. It is activated by cytokine receptors via kinases of the JAK family. STAT1 becomes phosphorylated and forms a dimer which enters the nucleus and triggers the transcription of its targets. Although not lethal at birth, selective gene deletion of STAT1 in mice leads to rapid death from severe infections, demonstrating its major role in the response to pathogens. Similarly, in humans who do not express STAT1, there is a lack of resistance to pathogens leading to premature death. This indicates a key, non-redundant function of STAT1 in the defence against pathogens. Thus, to successfully infect organisms, bacterial, viral or parasitic pathogens must overcome the activity of STAT1, and almost all the steps of this pathway can be blocked or inhibited by proteins produced in infected cells. Interestingly, some pathogens, like the oncogenic Epstein-Barr virus, have evolved a strategy which uses STAT1 activation.

Macrophages are the first line of defense against pathogens. Upon infection macrophages usually produce high levels of proinflammatory mediators. However, macrophages can undergo an alternate polarization leading to a permissive state. In assessing global macrophage responses to the bacterial agent of Whipple's disease, Tropheryma whipplei, we found that T. whipplei induced M2 macrophage polarization which was compatible with bacterial replication. Surprisingly, this M2 polarization of infected macrophages was associated with apoptosis induction and a functional type I interferon (IFN) response, through IRF3 activation and STAT1 phosphorylation. Using macrophages from mice deficient for the type I IFN receptor, we found that this type I IFN response was required for T. whipplei-induced macrophage apoptosis in a JNK-dependent manner and was associated with the intracellular replication of T. whipplei independently of JNK. This study underscores the role of macrophage polarization in host responses and highlights the detrimental role of type I IFN during T. whipplei infection., Author Summary Innate immune cells are sentinels allowing the host to sense invading pathogens. Among them, macrophages are highly microbicidal and are able to kill microorganisms. However, several pathogens have evolved strategies to hijack macrophage responses in order to survive or replicate. Tropheryma whipplei is the agent of Whipple's disease, a systemic disease that associates arthropathy, weight loss and gastrointestinal symptoms. It has been known for several years that this bacterium has a tropism for macrophages, in which it replicates. In this study, we have shown that T. whipplei induces host cell apoptosis and a surprising macrophage activation, characterized by anti-inflammatory molecules and type I interferon (IFN) signaling, which is generally associated to viral infections. We demonstrate that this type I IFN response is critical for bacterial pathogenicity, as it is required for bacterial replication and provides the first step of the apoptotic program of infected macrophages. By identifying these signaling events induced in macrophage by T. whipplei, we can now better understand the molecular basis of the pathophysiology of Whipple's disease, of interest for clinical and therapeutic ends.

Intracellular parasites of the genus Leishmania generate severe diseases in humans, which are associated with a failure of the infected host to induce a protective interferon γ (IFNγ)-mediated immune response. We tested the role of the JAK/STAT1 signaling pathway in Leishmania pathogenesis by utilizing knockout mice lacking the signal transducer and activator of transcription 1 (Stat1) and derived macrophages. Unexpectedly, infection of Stat1-deficient macrophages in vitro with promastigotes from Leishmania major and attenuated LPG1 knockout mutants (lpg −) specifically lacking lipophosphoglycan (LPG) resulted in a twofold increased intracellular growth, which was independent of IFNγ and associated with a substantial increase in phagosomal pH. Phagosomes in Stat1−/− macrophages showed normal maturation as judged by the accumulation of the lysosomal marker protein rab7, and provided normal vATPase activity, but were defective in the anion conductive pathway required for full vesicular acidification. Our results suggest a role of acidic pH in the control of intracellular Leishmania growth early during infection and identify for the first time an unexpected role of Stat1 in natural anti-microbial resistance independent from its function as IFNγ-induced signal transducer. This novel Stat1 function may have important implications to studies of other pathogens, as the acidic phagolysosomal pH plays an important role in antigen processing and the uncoating process of many viruses., Author Summary Protozoan parasites of the genus Leishmania generate a variety of pathologies, collectively termed leishmaniasis, which afflict millions of people worldwide. Leishmania is transmitted during the blood meal of infested sand flies that inoculate highly infective metacyclic promastigotes into the mammalian host. Following uptake by host macrophages, metacyclics differentiate into the amastigote form that replicates inside the acidified phago-lysosome of the host cell. The cytokine interferon-γ activates infected macrophages to kill intracellular Leishmania through the production of nitric oxide. This process is mediated through Stat 1, a cytosolic transcription factor that translocates into the nucleus in response to the cytokine, where it induces a pleiotropic anti-microbial response. By utilizing Stat1-deficient macrophages we found evidence for a novel interferon-γ-independent physiological function of Stat1 in acidification of the host cell phago-lysosome. Stat1-deficient macrophages showed higher phago-lysosomal pH and increased susceptibility to Leishmania infection, which was linked to a defect in cellular chloride channel function. Vesicular pH and acidification are important factors affecting the infective cycle of bacterial and protozoan pathogens, and the uncoating process during viral entry. Thus, the role of cytokine-independent Stat1 functions in innate anti-microbial resistance may have a greater impact on host-pathogen interactions than previously appreciated.

International audience; Chemotherapeutic drugs such as fludarabine*, doxorubicin or cisplatin are very potent activators of the anti-oncogene p53. Convergent studies suggest that p53 and STAT1 (signal transducer and activator of transcription 1) cooperate in the induction of cell death. We show that these drugs are also activators of STAT1 in p53-expressing cells, but not in p53-null cells. STAT1 activation was obtained in the presence of both the secretion inhibitor brefeldine A and the inhibitor of RNA synthesis, actinomycin D. p53-dependent STAT1 activation was reversed by overexpression of MDM2 and siRNAs against p53. Genetic analysis of p53 showed that expression of transcriptionally inactive p53 punctual mutants markedly increased Y701-STAT1 phosphorylation, and suggests that the p53 DNA-binding domain was alternatively involved in STAT1 activation or p53 multimerization. Immunoprecipitation experiments showed that ataxia telangiectasia mutated, p53, STAT1 and c-Abl1 (Abelson murine leukaemia viral oncogene homologue 1) were associated together. Treatment of cells with the c-Abl1 tyrosine kinase inhibitor STI571 decreased STAT1 activation by genotoxic drugs. Finally, genotoxic agents sensitized cells in response to very low doses of both interferon alpha and gamma (IFNalpha and gamma). These results show that genotoxic drugs induce STAT1 activation, an effect that depends on p53 protein but not on p53 transcriptional activity, and point to a novel pathway of STAT1 activation by genotoxic drugs, with involvement of c-Abl1 tyrosine kinase in sensitizing cells to IFN response.

The interferon (IFN) response is one of the host response system's primary defense mechanisms against viral infection. Type I IFN (alpha/beta interferon [IFN-α/β]) is produced by most cells as a direct response to viral infection, while type II IFN (IFN-γ) is synthesized almost exclusively by activated NK cells and activated T cells in response to virus-infected cells. Both type I and II IFNs achieve antiviral effects by binding to their respective receptors (IFNAR for IFN-α/β or IFNGR for IFN-γ), resulting in the activation of a distinct but related “Janus” tyrosine kinase/signal transducer and activator of transcription (Jak/STAT) pathway (12). Briefly, the interaction of IFN-α/β with IFNAR leads to the activation of the Jak protein tyrosine kinases (Tyk 2 and Jak1) that phosphorylate STAT1 and STAT2. The phosphorylated STATs heterodimerize and bind to a DNA binding protein, IFN regulatory factor 9 (IRF9), to form a complex, IFN-stimulated growth factor 3 (ISGF3). ISGF3 translocates into the nucleus and binds to an IFN-stimulated response element (ISRE) to induce IFN-stimulated genes (ISGs). The binding of IFN-γ to its receptor, IFNGR, results in the phosphorylation of STAT1 by Jak1 and Jak2. STAT1 homodimers form, migrate to the nucleus, and bind to a DNA element termed GAS (for gamma-activated sequence) to induce specifically the transcription of IFN-γ target genes (12). All the IFN-induced biological responses are believed to be mediated by ISG products that have been shown to display intrinsic antiviral activities (8, 24). Viruses that require cellular machinery for their replication have evolved different strategies to counteract IFN action, particularly by altering IFN induction, IFN signaling, and IFN-induced mediators (1, 9, 15). Several viral proteins acting as IFN antagonists have been identified in Mononegavirales, such as members of the Paramyxoviridae families (10, 16). Very recently, interference with IFN production and signaling was described for rabies virus of the Lyssavirus genus that belongs to the Rhabdoviridae family (4, 5, 6, 28). Rabies virus has a linear, nonsegmented, single-strand RNA genome of negative polarity. The ribonucleoprotein contains the RNA genome tightly encapsidated by the viral nucleoprotein (N) and the RNA polymerase complex, which consists of the large protein (L) and its cofactor, the phosphoprotein (P). Both L and P are involved in transcription and replication. A positive-stranded leader RNA and five mRNAs are synthesized during transcription. The replication process yields nucleocapsids containing full-length antisense genome RNA, which in turn serves as a template for the synthesis of sense genome RNA. The rabies virus P protein is a noncatalytic cofactor and a regulatory protein that plays a role in viral transcription and replication: it stabilizes the RNA polymerase L to the N-RNA template and binds to the soluble N, preventing its aggregation and keeping it in a suitable form for specific encapsidation of viral RNA. P protein has other specific functions in the host cells (6). Interestingly, rabies virus P protein interacts directly with two proteins, STAT1 and promyelocytic leukemia protein (PML) (3, 28), playing an important role in the IFN-induced antiviral response. In addition, P protein impairs IRF-3 phosphorylation, leading to the inhibition of IFN production (4). This multifunctionality of P may be linked to the high polymorphism of protein expression. It is phosphorylated by two kinases, rabies virus protein kinase and protein kinase C, leading to the formation of different phosphorylated forms of the P protein (14). In addition, the P gene encodes not only P but also additional shorter P products (P2, P3, P4, and P5) whose translation is initiated from downstream and in-frame AUG codons by a leaky scanning mechanism (7). These small versions of P have different intracellular distributions. The nuclear localizations of P3, P4, and P5 are due to the presence of a nuclear localization signal (NLS) located in the C-terminal part of the protein, whereas the cytoplasmic distributions of P and P2 are the result of a CRM1 nuclear export signal (NES) located in the N-terminal part of the protein (20). We and others have previously shown that rabies virus P protein inhibits signaling by blocking the nuclear accumulation of STAT1 (5, 28). By analyzing the molecular mechanisms leading to the inhibition of IFN signaling by rabies virus P protein, we have shown that P protein and the nuclear P3 isoform inhibit an additional step that occurs in the nucleus: the binding of STAT1 or ISGF3 to the DNA promoters (i.e., to GAS and ISRE) of IFN-γ- or IFN-α-responsive genes, respectively.

Viruses have evolved various strategies to escape the antiviral activity of type I interferons (IFN-alpha/beta). For measles virus, this function is carried by the polycistronic gene P that encodes, by an unusual editing strategy, for the phosphoprotein P and the virulence factor V (MV-V). MV-V prevents STAT1 nuclear translocation by either sequestration or phosphorylation inhibition, thereby blocking IFN-alpha/beta pathway. We show that both the N- and C-terminal domains of MV-V (PNT and VCT) contribute to the inhibition of IFN-alpha/beta signaling. Using the two-hybrid system and co-affinity purification experiments, we identified STAT1 and Jak1 as interactors of MV-V and demonstrate that MV-V can block the direct phosphorylation of STAT1 by Jak1. A deleterious mutation within the PNT domain of MV-V (Y110H) impaired its ability to interact and block STAT1 phosphorylation. Thus, MV-V interacts with at least two components of IFN-alpha/beta receptor complex to block downstream signaling.

International audience; Deregulation of the proto-oncogene c-myc is a key event in the pathogenesis of many tumors. A paradigm is the activation of the c-myc gene by chromosomal translocations in Burkitt lymphoma (BL). Despite expression of a restricted set of Epstein-Barr viral (EBV) antigens, BL cells are not recognized by antigen-specific cytotoxic T cells (CTLs) because of their inability to process and present HLA class I-restricted antigens. In contrast, cells of EBV-driven posttransplant lymphoproliferative disease (PTLD) are recognized and rejected by EBV-specific CTLs. It is not known whether the poor immunogenicity of BL cells is due to nonexpression of viral antigens, overexpression of c-myc, or both. To understand the basis for immune recognition and escape, we have compared the mRNA expression profiles of BL and EBV-immortalized cells (as PTLD model). Among the genes expressed at low level in BL cells, we have identified many genes involved in the NF-kappaB and interferon response that play a pivotal role in antigen presentation and immune recognition. Using a cell line in which EBNA2 and c-myc can be regulated at will, we show that c-MYC negatively regulates STAT1, the central player linking the Type-I and Type-II interferon response. Switching off c-myc expression leads to STAT1 induction through a direct and indirect mechanism involving induction of Type-I interferons. c-MYC thus masks an interferon-inducing activity in these cells. Our findings imply that immune escape of tumor cells is not only a matter of in vivo selection but may be additionally promoted by activation of a cellular oncogene.

The broad and often contrasting effects of type I interferons (IFNs) in innate and adaptive immunity are belied by the signaling via a single receptor, IFN-α receptor (IFNAR). Here, we show that IFN-α/β induces opposing effects on the immunologic outcome of antigen cross-presentation depending on dendritic cell (DC) maturation status. Despite equivalent IFNAR expression, immature conventional DCs (cDCs) activate STAT1 in response to IFN-α/β, whereas exposure of mature DCs to IFN-α/β results in signaling via STAT4. Microarray analysis revealed numerous transcriptional changes resulting from the altered signaling. Importantly, STAT1 signaling resulted in significant inhibition of CD40L-induced IL-12 production, accounting for the inhibition of CD8+ T-cell activation. These data provide evidence for a molecular switch in signaling pathways concomitant with DC maturation that offers a novel mechanism by which DCs modulate the integration of signals from the surrounding environment.

International audience; BACKGROUND: The aim of this study was to investigate the molecular mechanisms involved in the production of Th1 cytokines, namely IL-12 and IL-27, when the intra-macrophage redox state was altered by different chemical entities such as GSH-C4, which is reduced glutathione carrying an aliphatic chain, or I-152, a pro-drug of N-acetyl-cysteine (NAC) and beta-mercaptoethylamine. We had already demonstrated that GSH-C4 and I-152 could shift the immune response towards Th1 in Ovalbumin-immunized mice as well as enhance Th1 response in HIV-1 Tat-immunized mice. METHODOLOGY/PRINCIPAL FINDINGS: By a new high performance liquid chromatography method, we found that 20 mM GSH-C4 provided a number of thiol species in the form of GSH, while 20 mM I-152 decreased GSH and increased the thiols in the form of NAC and I-152. Under these experimental conditions, GSH-C4 and I-152 enhanced and suppressed respectively the mRNA expression levels of IL-12 p40 induced by LPS/IFN-γ as assessed by Real-Time PCR. The protein production of IL-12 p40 was increased by GSH-C4 and decreased by I-152 as determined by Enzyme-linked immunosorbent assay. Western immunoblot and electrophoretic mobility shift assays revealed that Nuclear Factor -kB (NF-kB) activation was inhibited by I-152 and prolonged by GSH-C4. Twenty mM I-152 stimulated IL-27 p28 gene expression and sustained Signal Transducer and Activator of Transcription (STAT)-mediated interferon regulator factor 1 (IRF-1) de novo synthesis. By contrast, 20 mM GSH-C4 did not exert any effect on IL-27 p28 gene expression. CONCLUSIONS AND SIGNIFICANCE: an increase in the intra-macrophage redox state by GSH-C4 and I-152 enhances Th1 cytokine production although the chemical structure and the intra-cellular metabolism influence differently signalling pathways involved in IL-27 or IL-12 production. GSH-C4 and I-152 may be used as Th1 immunomodulators in some pathologies and in ageing where GSH depletion may contribute to the Th1/Th2 imbalance, and in new immunization strategies.