Interferon-induced IL-10 drives systemic T-cell dysfunction during chronic liver injury.

BACKGROUND & AIMS: Patients with chronic liver disease (CLD), including cirrhosis, are at increased risk of intractable viral infections and are hyporesponsive to vaccination. Hallmarks of CLD and cirrhosis include microbial translocation and elevated levels of type I interferon (IFN-I). We aimed to investigate the relevance of microbiota-induced IFN-I in the impaired adaptive immune responses observed in CLD. METHODS: We combined bile duct ligation (BDL) and carbon tetrachloride (CCl4) models of liver injury with vaccination or lymphocytic choriomeningitis virus infection in transgenic mice lacking IFN-I in myeloid cells (LysM-Cre IFNARflox/flox), IFNAR-induced IL-10 (MX1-Cre IL10flox/flox) or IL-10R in T cells (CD4-DN IL-10R). Key pathways were blocked in vivo with specific antibodies (anti-IFNAR and anti-IL10R). We assessed T-cell responses and antibody titers after HBV and SARS-CoV-2 vaccinations in patients with CLD and healthy individuals in a proof-of-concept clinical study. RESULTS: We demonstrate that BDL- and CCL4-induced prolonged liver injury leads to impaired T-cell responses to vaccination and viral infection in mice, subsequently leading to persistent infection. We observed a similarly defective T-cell response to vaccination in patients with cirrhosis. Innate sensing of translocated gut microbiota induced IFN-I signaling in hepatic myeloid cells that triggered excessive IL-10 production upon viral infection. IL-10R signaling in antigen-specific T cells rendered them dysfunctional. Antibiotic treatment and inhibition of IFNAR or IL-10Ra restored antiviral immunity without detectable immune pathology in mice. Notably, IL-10Ra blockade restored the functional phenotype of T cells from vaccinated patients with cirrhosis. CONCLUSION: Innate sensing of translocated microbiota induces IFN-/IL-10 expression, which drives the loss of systemic T-cell immunity during prolonged liver injury. IMPACT AND IMPLICATIONS: Chronic liver injury and cirrhosis are associated with enhanced susceptibility to viral infections and vaccine hyporesponsiveness. Using different preclinical animal models and patient samples, we identified that impaired T-cell immunity in BDL- and CCL4-induced prolonged liver injury is driven by sequential events involving microbial translocation, IFN signaling leading to myeloid cell-induced IL-10 expression, and IL-10 signaling in antigen-specific T cells. Given the absence of immune pathology after interference with IL-10R, our study highlights a potential novel target to reconstitute T-cell immunity in patients with CLD that can be explored in future clinical studies.

Splenic Red Pulp Macrophages Cross-Prime Early Effector CTL That Provide Rapid Defense against Viral Infections.

Cross-presentation allows dendritic cells (DCs) to present peptides derived from endocytosed Ags on MHC class I molecules, which is important for activating CTL against viral infections and tumors. Type 1 classical DCs (cDC1), which depend on the transcription factor Batf3, are considered the main cross-presenting cells. In this study, we report that soluble Ags are efficiently cross-presented also by transcription factor SpiC-dependent red pulp macrophages (RPM) of the spleen. In contrast to cDC1, RPM used the mannose receptor for Ag uptake and employed the proteasome- and TAP-dependent cytosolic cross-presentation pathway, previously shown to be used in vitro by bone marrow-derived DCs. In an in vivo vaccination model, both cDC1 and RPM cross-primed CTL efficiently but with distinct kinetics. Within a few days, RPM induced very early effector CTL of a distinct phenotype (Ly6A/E+ Ly6C(+) KLRG1- CD127- CX3CR1- Grz-B+). In an adenoviral infection model, such CTL contained the early viral spread, whereas cDC1 induced short-lived effector CTL that eventually cleared the virus. RPM-induced early effector CTL also contributed to the endogenous antiviral response but not to CTL memory generation. In conclusion, RPM can contribute to antiviral immunity by generating a rapid CTL defense force that contains the virus until cDC1-induced CTL are available to eliminate it. This function can be harnessed for improving vaccination strategies aimed at inducing CTL.

Opposing roles of resident and infiltrating immune cells in the defense against Legionella longbeachae via IL-18R/IFN-γ/ROS axis in mice.

The immune response against Legionella longbeachae, a causative agent of the often-fatal Legionnaires' pneumonia, is poorly understood. Here, we investigated the specific roles of tissue-resident alveolar macrophages (AMs) and infiltrating phagocytes during infection with this pathogen. AMs were the predominant cell type that internalized bacteria 1 day after infection. A total of 3 and 5 days after infection, AM numbers were greatly reduced, whereas there was an influx of neutrophils and, later, monocyte-derived cells (MCs) into lung tissue. AMs carried greater numbers of viable L. longbeachae than neutrophils and MCs, which correlated with a higher capacity of L. longbeachae to translocate bacterial effector proteins required for bacterial replication into the AM cytosol. Cell ablation experiments demonstrated that AM promoted infection, whereas neutrophils and MC were required for efficient bacterial clearance. Interleukin (IL)-18 was important for interferon-γ production by IL-18R+ natural killer cells and T cells, which, in turn, stimulated reactive oxygen species-mediated bactericidal activity in neutrophils, resulting in the restriction of L. longbeachae infection. Ciliated bronchiolar epithelial cells also expressed IL-18R but did not play a role in IL-18-mediated L. longbeachae clearance. Our results have identified opposing innate functions of tissue-resident and infiltrating immune cells during L. longbeachae infection that may be manipulated to improve protective responses.

Monocyte-derived macrophages aggravate pulmonary vasculitis via cGAS/STING/IFN-mediated nucleic acid sensing.

Autoimmune vasculitis is a group of life-threatening diseases, whose underlying pathogenic mechanisms are incompletely understood, hampering development of targeted therapies. Here, we demonstrate that patients suffering from anti-neutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV) showed increased levels of cGAMP and enhanced IFN-I signature. To identify disease mechanisms and potential therapeutic targets, we developed a mouse model for pulmonary AAV that mimics severe disease in patients. Immunogenic DNA accumulated during disease onset, triggering cGAS/STING/IRF3-dependent IFN-I release that promoted endothelial damage, pulmonary hemorrhages, and lung dysfunction. Macrophage subsets played dichotomic roles in disease. While recruited monocyte-derived macrophages were major disease drivers by producing most IFN-ß, resident alveolar macrophages contributed to tissue homeostasis by clearing red blood cells and limiting infiltration of IFN-ß-producing macrophages. Moreover, pharmacological inhibition of STING, IFNAR-I, or its downstream JAK/STAT signaling reduced disease severity and accelerated recovery. Our study unveils the importance of STING/IFN-I axis in promoting pulmonary AAV progression and identifies cellular and molecular targets to ameliorate disease outcomes.

Interhemispheric interaction during the menstrual cycle.

Fluctuating levels of sex hormones and high levels of progesterone (P), in particular, have been suggested to reduce interhemispheric inhibition. The present study focuses on hormone-dependent modulation of interhemispheric integration. In two versions of the Banich-Belger task, participants were asked to match letters according to their physical (e.g., A vs. A) and semantic identity (e.g., A vs. a). Matches were presented either within or across visual half-fields. Moreover, a simple reaction-time task (Poffenberger task) that is assumed to estimate interhemispheric transfer time (IHTT) was used. Seventeen normally cycling women were tested during low P menses and high P midluteal phase. Saliva levels of P were analysed using chemiluminescence assays. Fifteen postmenopausal women performed the same tasks in corresponding time intervals. Additionally, 28 younger male controls were tested once. In agreement with previous results, the more demanding (semantic) interhemispheric-integration task revealed a typical across-field advantage (AFA) for all three groups. However, in normally cycling women, the AFA was significantly reduced during menses. IHTT did not change across the cycle phases. The results indicate that interhemispheric integration fluctuates across the menstrual cycle and is reduced during menses. During the luteal phase, however, the AFA is increased, suggesting that accompanying hormonal conditions favour an efficient interhemispheric integration. We conclude that transcallosal mechanisms involved in interhemispheric integration are profoundly altered when sex hormones are permanently reduced as in men and postmenopausal women. This difference enables an efficient interhemispheric integration without modulatory effects of P.

Induction of interferon synthesis by the PKR-inhibitory VA RNAs of adenoviruses.

In virus-infected cells, double-stranded RNA (dsRNA) activates the transcription factor interferon (IFN) regulatory factor-3 (IRF-3), which stimulates type I IFN (IFN-alpha/beta) gene expression. In addition, dsRNA activates the enzyme RNA-activated protein kinase (PKR), which phosphorylates the eukaryotic initiation factor 2alpha (eIF2beta), thereby inhibiting mRNA translation. Adenoviruses express highly structured RNA molecules termed VA RNAs (VA(I)/VA(II)) known to specifically inhibit PKR. As PKR impairs expression from transfected cDNA constructs, plasmids encoding VA RNAs are widely used as enhancers of transgene expression. Here, we describe induction of IFN synthesis as a novel feature of VA RNAs. Transfection of a VA(I)/VA(II)-expressing plasmid was found to induce type I IFN production, resulting in activation of IFN-dependent genes, such as IFN-stimulated gene 56 (ISG56) or MxA, and the establishment of an antiviral state in transfected cells. Curiously, VA RNAs did not activate IRF-3, suggesting an alternative pathway of IFN induction. These data may be considered when using genetically modified adenoviruses as therapeutic agents and suggest caution in choosing VA RNA constructs as a means to increase expression of a gene of interest.

Bunyamwera bunyavirus nonstructural protein NSs counteracts the induction of alpha/beta interferon.

Production of alpha/beta interferons (IFN-alpha/beta) in response to viral infection is one of the main defense mechanisms of the innate immune system. Many viruses therefore encode factors that subvert the IFN system to enhance their virulence. Bunyamwera virus (BUN) is the prototype of the Bunyaviridae family. By using reverse genetics, we previously produced a recombinant virus lacking the nonstructural protein NSs (BUNdelNSs) and showed that NSs is a nonessential gene product that contributes to viral pathogenesis. Here we demonstrate that BUNdelNSs is a strong inducer of IFN-alpha/beta, whereas in cells infected with the wild-type counterpart expressing NSs (wild-type BUN), neither IFN nor IFN mRNA could be detected. IFN induction by BUNdelNSs correlated with activation of NF-kappaB and was dependent on virally produced double-stranded RNA and on the IFN transcription factor IRF-3. Furthermore, both in cultured cells and in mice lacking a functional IFN-alpha/beta system, BUNdelNSs replicated to wild-type BUN levels, whereas in IFN-competent systems, wild-type BUN grew more efficiently. These results suggest that BUN NSs is an IFN induction antagonist that blocks the transcriptional activation of IFN-alpha/beta in order to increase the virulence of Bunyamwera virus.

Inhibition of RNA polymerase II phosphorylation by a viral interferon antagonist.

Many viruses subvert the cellular interferon (IFN) system with so-called IFN antagonists. Bunyamwera virus (BUNV) belongs to the family Bunyaviridae and is transmitted by arthropods. We have recently identified the nonstructural protein NSs of BUNV as a virulence factor that inhibits IFN-beta gene expression in the mammalian host. Here, we demonstrate that NSs targets the RNA polymerase II (RNAP II) complex. The C-terminal domain (CTD) of RNAP II consists of 52 repeats of the consensus sequence YSPTSPS. Phosphorylation at serine 5 is required for efficient initiation of transcription, and subsequent phosphorylation at serine 2 is required for mRNA elongation and 3'-end processing. In BUNV-infected mammalian cells, serine 5 phosphorylation occurred normally. Furthermore, RNAP II was able to bind to the IFN-beta gene promoter as revealed by chromatin immunoprecipitation analysis, indicating that the initiation of transcription was not disturbed by NSs. However, NSs prevented CTD phosphorylation at serine 2, suggesting a block in transition from initiation to elongation. Surprisingly, no interference with CTD phosphorylation was observed in insect cells. Our results indicate that BUNV uses an unconventional mechanism to block IFN synthesis in the mammalian host by directly dysregulating RNAP II. Moreover, by inducing a general transcriptional block, NSs may contribute to the lytic infection observed in mammalian cells as opposed to persistent infection in the insect host.