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Heart disease link to inflammatory signalling A non-coding region on chromosome 9p21 was previously shown to associate with coronary artery disease and type 2 diabetes, and the region has been implicated in regulating neighbouring genes. Here the authors identify 33 distinct enhancers within this region and show that single nucleotide polymorphisms in one of the enhancers affect STAT1 binding. They further show that in human vascular endothelium cells, the enhancer interval physically interacts with a number of specific loci, and that interferon-γ activation strongly affects the chromatin structure and transcriptional regulation of the 9p21 locus, including STAT1 binding, long-range enhancer interactions and expression of neighbouring genes. A non-coding region on chromosome 9p21 was previously shown to associate with coronary artery disease and type 2 diabetes, and the region has been implicated in regulating neighbouring genes. Here, 33 distinct enhancers within this region are identified, showing that SNPs in one of the enhancers affect STAT1 binding. Furthermore, it is shown that in human vascular endothelial cells the enhancer interval physically interacts with a number of specific loci and that IFN-γ activation strongly affects the chromatin structure and transcriptional regulation of the 9p21 locus, including STAT1 binding, long-range enhancer interactions and expression of neighbouring genes. Genome-wide association studies have identified single nucleotide polymorphisms (SNPs) in the 9p21 gene desert associated with coronary artery disease (CAD) 1 , 2 , 3 , 4 and type 2 diabetes 5 , 6 , 7 . Despite evidence for a role of the associated interval in neighbouring gene regulation 8 , 9 , 10 , the biological underpinnings of these genetic associations with CAD or type 2 diabetes have not yet been explained. Here we identify 33 enhancers in 9p21; the interval is the second densest gene desert for predicted enhancers and six times denser than the whole genome ( P  < 6.55 × 10 −33 ). The CAD risk alleles of SNPs rs10811656 and rs10757278 are located in one of these enhancers and disrupt a binding site for STAT1. Lymphoblastoid cell lines homozygous for the CAD risk haplotype show no binding of STAT1, and in lymphoblastoid cell lines homozygous for the CAD non-risk haplotype, binding of STAT1 inhibits CDKN2BAS (also known as CDKN2B-AS1 ) expression, which is reversed by short interfering RNA knockdown of STAT1 . Using a new, open-ended approach to detect long-distance interactions, we find that in human vascular endothelial cells the enhancer interval containing the CAD locus physically interacts with the CDKN2A/B locus, the MTAP gene and an interval downstream of IFNA21 . In human vascular endothelial cells, interferon-γ activation strongly affects the structure of the chromatin and the transcriptional regulation in the 9p21 locus, including STAT1-binding, long-range enhancer interactions and altered expression of neighbouring genes. Our findings establish a link between CAD genetic susceptibility and the response to inflammatory signalling in a vascular cell type and thus demonstrate the utility of genome-wide association study findings in directing studies to novel genomic loci and biological processes important for disease aetiology.

The mechanisms by which the number of memory CD8 T cells is stably maintained remains incompletely understood. It has been postulated that maintaining them requires help from CD4 T cells, because adoptively transferred memory CD8 T cells persist poorly in MHC class II (MHCII)-deficient mice. Here we show that chronic interferon-γ signals, not CD4 T cell-deficiency, are responsible for their attrition in MHCII-deficient environments. Excess IFN-γ is produced primarily by endogenous colonic CD8 T cells in MHCII-deficient mice. IFN-γ neutralization restores the number of memory CD8 T cells in MHCII-deficient mice, whereas repeated IFN-γ administration or transduction of a gain-of-function STAT1 mutant reduces their number in wild-type mice. CD127 high memory cells proliferate actively in response to IFN-γ signals, but are more susceptible to attrition than CD127 low terminally differentiated effector memory cells. Furthermore, single-cell RNA-sequencing of memory CD8 T cells reveals proliferating cells that resemble short-lived, terminal effector cells and documents global downregulation of gene signatures of long-lived memory cells in MHCII-deficient environments. We propose that chronic IFN-γ signals deplete memory CD8 T cells by compromising their long-term survival and by diverting self-renewing CD127 high cells toward terminal differentiation. Memory CD8 + T cells persist poorly in MHCII-deficient mice. Here the authors show that this CD8 + T cell attrition is not caused by a lack of CD4 + T cell help, as previously proposed, but by chronic IFN-γ signals derived from endogenous colonic CD8 + T cells.

The local production of gamma interferon (IFN-γ) is important to control Toxoplasma gondii in the brain, but the basis for these protective effects is not fully understood. The studies presented here reveal that the ability of IFN-γ to inhibit parasite replication in astrocytes in vitro is dependent on signal transducer and activator of transcription 1 (STAT1) and that mice that specifically lack STAT1 in astrocytes are unable to limit parasite replication in the central nervous system (CNS). This susceptibility is associated with a loss of antimicrobial pathways and increased cyst formation in astrocytes. These results identify a critical role for astrocytes in limiting the replication of an important opportunistic pathogen. IMPORTANCE Astrocytes are the most numerous cell type in the brain, and they are activated in response to many types of neuroinflammation, but their function in the control of CNS-specific infection is unclear. The parasite Toxoplasma gondii is one of the few clinically relevant microorganisms that naturally infects astrocytes, and the studies presented here establish that the ability of astrocytes to inhibit parasite replication is essential for the local control of this opportunistic pathogen. Together, these studies establish a key role for astrocytes as effector cells and in the coordination of many aspects of the protective immune response that operates in the brain. Astrocytes are the most numerous cell type in the brain, and they are activated in response to many types of neuroinflammation, but their function in the control of CNS-specific infection is unclear. The parasite Toxoplasma gondii is one of the few clinically relevant microorganisms that naturally infects astrocytes, and the studies presented here establish that the ability of astrocytes to inhibit parasite replication is essential for the local control of this opportunistic pathogen. Together, these studies establish a key role for astrocytes as effector cells and in the coordination of many aspects of the protective immune response that operates in the brain.

The entire hepatitis C virus life cycle can be recapitulated in an inbred mouse model, allowing preclinical assessment of antiviral therapeutics and vaccines. Humanized mouse model for hepatitis C infection In a 2009 Nature paper, Alexander Ploss and colleagues showed that transient expression of the human genes CD81 and occludin ( OCLN ) constituted a minimal set of cellular factors required for uptake of hepatitis C virus (HCV) into immune-competent mouse cells. Now they report that transgenic immune-deficient mice stably expressing CD81 and OCLN can sustain the complete HCV replication cycle with measurable viraemia. The availability of this genetically humanized mouse model opens the way to closer study of HCV infection in vivo and should provide a valuable platform for testing potential therapeutics. More than 130 million people worldwide chronically infected with hepatitis C virus (HCV) are at risk of developing severe liver disease. Antiviral treatments are only partially effective against HCV infection, and a vaccine is not available. Development of more efficient therapies has been hampered by the lack of a small animal model. Building on the observation that CD81 and occludin (OCLN) comprise the minimal set of human factors required to render mouse cells permissive to HCV entry 1 , we previously showed that transient expression of these two human genes is sufficient to allow viral uptake into fully immunocompetent inbred mice 2 . Here we demonstrate that transgenic mice stably expressing human CD81 and OCLN also support HCV entry, but innate and adaptive immune responses restrict HCV infection in vivo . Blunting antiviral immunity in genetically humanized mice infected with HCV results in measurable viraemia over several weeks. In mice lacking the essential cellular co-factor cyclophilin A (CypA), HCV RNA replication is markedly diminished, providing genetic evidence that this process is faithfully recapitulated. Using a cell-based fluorescent reporter activated by the NS3-4A protease we visualize HCV infection in single hepatocytes in vivo . Persistently infected mice produce de novo infectious particles, which can be inhibited with directly acting antiviral drug treatment, thereby providing evidence for the completion of the entire HCV life cycle in inbred mice. This genetically humanized mouse model opens new opportunities to dissect genetically HCV infection in vivo and provides an important preclinical platform for testing and prioritizing drug candidates and may also have utility for evaluating vaccine efficacy.

The capacity of IL-10 and Tregs in the inflammatory tumor microenvironment to impair anticancer Th1 immunity makes them attractive targets for cancer immunotherapy. IL-10 and Tregs also suppress Th17 activity, which is associated with poor prognosis in several cancers. However, previous studies have overlooked their potential contribution to the regulation of pathogenic cancer-associated inflammation. In this study, we investigated the origin and function of IL-10–producing cells in the tumor microenvironment using transplantable tumor models in mice. The majority of tumor-associated IL-10 was produced by an activated Treg population. IL-10 production by Tregs was required to restrain Th17-type inflammation. Accumulation of activated IL-10+ Tregs in the tumor required type I IFN signaling but not inflammatory signaling pathways that depend on TLR adapter protein MyD88 or IL-12 family cytokines. IL-10 production limited Th17 cell numbers in both spleen and tumor. However, type I IFN was required to limit Th17 cells specifically in the tumor microenvironment, reflecting selective control of tumor-associated Tregs by type I IFN. Thus, the interplay of type I IFN, Tregs, and IL-10 is required to negatively regulate Th17 inflammation in the tumor microenvironment. Therapeutic interference of this network could therefore have the undesirable consequence of promoting Th17 inflammation and cancer growth.

Natural killer (NK) cells are important components of the innate immune defense against infections and cancers. Signal transducer and activator of transcription 1 (STAT1) is a transcription factor that is essential for NK cell maturation and NK cell-dependent tumor surveillance. Two alternatively spliced isoforms of STAT1 exist: a full-length STAT1α and a C-terminally truncated STAT1β isoform. Aberrant splicing is frequently observed in cancer cells and several anti-cancer drugs interfere with the cellular splicing machinery. To investigate whether NK cell-mediated tumor surveillance is affected by a switch in STAT1 splicing, we made use of knock-in mice expressing either only the STAT1α ( ) or the STAT1β ( ) isoform. NK cells from mice matured normally and controlled transplanted tumor cells as efficiently as NK cells from wild-type mice. In contrast, NK cells from mice showed impaired maturation and effector functions, albeit less severe than NK cells from mice that completely lack STAT1 ( ). Mechanistically, we show that NK cell maturation requires the presence of STAT1α in the niche rather than in NK cells themselves and that NK cell maturation depends on IFNγ signaling under homeostatic conditions. The impaired NK cell maturation in mice was paralleled by decreased IL-15 receptor alpha (IL-15Rα) surface levels on dendritic cells, macrophages and monocytes. Treatment of mice with exogenous IL-15/IL-15Rα complexes rescued NK cell maturation but not their effector functions. Collectively, our findings provide evidence that STAT1 isoforms are not functionally redundant in regulating NK cell activity and that the absence of STAT1α severely impairs, but does not abolish, NK cell-dependent tumor surveillance.

Background Signal transduction via the Signal Transducer and Activator of Transcription 1 (STAT1) pathway is indispensable for mediating the intracellular effects of interferon-α (IFN-α), interferon-γ (IFN-γ) and other cytokines in the brain and, thereby, crucial for antiviral and antibacterial responses during potential life-threatening CNS infections. However, the role of STAT1 signaling beyond the known IFN-α and IFN-γ effects in immediate antimicrobial defense is highly context-dependent, and studies in the existing literature using STAT1-targeted mouse models under normal physiological conditions remain scarce. Methods Here, we characterized a STAT1 targeted-disruption mouse model in the absence of infectious stimuli by employing established behavioral testing paradigms and immunohistochemical stainings, as well as bulk hippocampal transcriptomic and proteomic analyses. Results While we found neither overt behavioral alterations nor immunohistochemical changes with respect to microglial phagocytosis or proliferation, significant alterations were detected in gene and protein expression profiles implicated in neuroinflammatory processes and neuroprotection. Conclusion In summary, this study highlights the complex and context-dependent role of STAT1-mediated signaling even in the absence of any detectable behavioral and neuropathological changes.

Chapare virus (CHAPV) is an Arenaviridae family member and causative agent of Chapare hemorrhagic fever (CHHF). Endemic to Bolivia, CHAPV was found to be the cause of several outbreaks of CHHF in Bolivia in 2003 and 2019 with high case-fatality rates and instances of human-to-human transmission. The pathogenesis of CHAPV infection is poorly understood, and no vaccines or antivirals are available, in part due to a dearth of available animal models. Mice lacking signal transducer and activator of transcription 1 (STAT1-/-) have been shown to succumb to infection by related arenaviruses, including Machupo virus, and were investigated for their susceptibility to CHAPV infection. Challenge with CHAPV resulted in partial lethality in STAT1-/- mice with a biphasic disease course characterized by initial viral load and pathology in the spleen and liver followed by inflammation and high viral titers in the brain and spinal cord that immediately preceded mortality. Adaptation in the brains of STAT1-/- mice resulted in a fully lethal mouse-adapted CHAPV variant, with a similar biphasic disease course, but virus in tissues was detected more proximal to challenge. The result of this study is a lethal small-animal rodent model for CHAPV that recapitulates many aspects of human CHAPV disease.

Machupo virus (MACV), a member of the Arenaviridae family and causative agent of Bolivian hemorrhagic fever, results in lethality rates of 25–35% in humans. Mice lacking the signal transducer and activator of transcription 1 (STAT-1−/−) have previously been shown to succumb to MACV infection within 7–8 days via the intraperitoneal route. Despite these reports, we observed partial lethality in STAT-1−/− mice following challenge with wild-type MACV. Serial sampling studies to evaluate the temporal progression of infection and pathologic changes after challenge revealed a two-phase disease course. The first phase was characterized by viral load and pathological lesions in the spleen, liver, and kidney followed by a second, lethal phase, defined by high viral titers and inflammation in the brain and spinal cord resulting in neurological manifestations and subsequent mortality. Tissue adaptation in the brains of challenged STAT-1−/− mice resulted in a fully lethal model in STAT-1−/− mice (mouse-adapted; maMACV). A similar two-phase disease course was observed following maMACV challenge, but more rapid dissemination of the virus to the brain and overall pathology in this region was observed. The outcome of these studies is a lethal small rodent model of MACV that recapitulates many aspects of human disease.

Granulomatous diseases in childhood present a complex diagnostic landscape, particularly when histological and clinical findings overlap with those of systemic inflammatory or histiocytic disorders. A subset of these conditions may represent the clinical onset of inborn errors of immunity (IEI), such as Mendelian Susceptibility to Mycobacterial Disease (MSMD), where atypical or sterile granulomas may obscure the underlying infectious or genetic etiology. Recognition of IEI behind granulomatous diseases can radically alter patient’s prognosis and therapeutic management. This report describes the case of a 11-years-old with an initial diagnosis of Rosai-Dorfman disease based on clinical and and histological findings. Following relapse after steroid tapering the diagnosis was revised to sarcoidosis, supported by non-caseating granulomas and compatible laboratory findings. Only after cultures from biopsy specimens revealed Mycobacterium avium complex (MAC), immunological investigations were undertaken, revealing a STAT1 dominant negative deficiency, consistent with MSMD. This report underscores the need of considering IEI in pediatric patients presenting with granulomatous inflammation, especially when clinical course is atypical or refractory to standard immunosuppressive therapies. Early microbiological and immunogenetic assessment is essential to avoid diagnostic delay, prevent inappropriate treatment, and guide targeted antimicrobial therapy.