The transcription factor Aiolos restrains the activation of intestinal intraepithelial lymphocytes.

Intestinal intraepithelial lymphocytes (IELs) exhibit prompt innate-like responses to microenvironmental cues and require strict control of effector functions. Here we showed that Aiolos, an Ikaros zinc-finger family member encoded by Ikzf3, acted as a regulator of IEL activation. Ikzf3-/- CD8αα+ IELs had elevated expression of NK receptors, cytotoxic enzymes, cytokines and chemokines. Single-cell RNA sequencing of Ikzf3-/- and Ikzf3+/+ IELs showed an amplified effector machinery in Ikzf3-/- CD8αα+ IELs compared to Ikzf3+/+ counterparts. Ikzf3-/- CD8αα+ IELs had increased responsiveness to interleukin-15, which explained a substantial part, but not all, of the observed phenotypes. Aiolos binding sites were close to those for the transcription factors STAT5 and RUNX, which promote interleukin-15 signaling and cytolytic programs, and Ikzf3 deficiency partially increased chromatin accessibility and histone acetylation in these regions. Ikzf3 deficiency in mice enhanced susceptibility to colitis, underscoring the relevance of Aiolos in regulating the effector function in IELs.

Segmented Filamentous Bacteria Prevent and Cure Rotavirus Infection.

Rotavirus (RV) encounters intestinal epithelial cells amidst diverse microbiota, opening possibilities of microbes influencing RV infection. Although RV clearance typically requires adaptive immunity, we unintentionally generated RV-resistant immunodeficient mice, which, we hypothesized, reflected select microbes protecting against RV. Accordingly, such RV resistance was transferred by co-housing and fecal transplant. RV-protecting microbiota were interrogated by heat, filtration, and antimicrobial agents, followed by limiting dilution transplant to germ-free mice and microbiome analysis. This approach revealed that segmented filamentous bacteria (SFB) were sufficient to protect mice against RV infection and associated diarrhea. Such protection was independent of previously defined RV-impeding factors, including interferon, IL-17, and IL-22. Colonization of the ileum by SFB induced changes in host gene expression and accelerated epithelial cell turnover. Incubation of RV with SFB-containing feces reduced infectivity in vitro, suggesting direct neutralization of RV. Thus, independent of immune cells, SFB confer protection against certain enteric viral infections and associated diarrheal disease.

A Human STAT1 Gain-of-Function Mutation Impairs CD8+ T Cell Responses against Gammaherpesvirus 68.

Autosomal dominant STAT1 mutations in humans have been associated with chronic mucocutaneous candidiasis (CMC), as well as with increased susceptibility to herpesvirus infections. Prior studies have focused on mucosal and Th17-mediated immunity against Candida, but mechanisms of impaired antiviral immunity have not previously been examined. To begin to explore the mechanisms of STAT1-associated immunodeficiency against herpesviruses, we generated heterozygous STAT1 R274W knock-in mice that have a frequently reported STAT1 mutation associated in humans with susceptibility to herpesvirus infections. In primary macrophages and fibroblasts, we found that STAT1 R274W had no appreciable effect on cell-intrinsic immunity against herpes simplex virus 1 (HSV-1) or gammaherpesvirus 68 (γHV68) infection. However, intraperitoneal inoculation of mice with γHV68 was associated with impaired control of infection at day 14 in STAT1 R274W mice compared with that in wild-type (WT) littermate control animals. Infection of STAT1 R274W mice was associated with paradoxically decreased expression of IFN-stimulated genes (ISGs) and gamma interferon (IFN-γ), likely secondary to defective CD4+ and CD8+ T cell responses, including diminished numbers of antigen-specific CD8+ T cells. Viral pathogenesis studies in WT and STAT1 R274W mixed bone marrow chimeric mice revealed that the presence of WT leukocytes was sufficient to limit infection and that antigen-specific STAT1 R274W CD8+ T cell responses were impaired even in the presence of WT leukocytes. Thus, in addition to regulating Th17 responses against Candida, a STAT1 gain-of-function mutant impedes antigen-specific T cell responses against a common gammaherpesvirus in mice.IMPORTANCE Mechanisms of immunodeficiency related to STAT1 gain of function have not been previously studied in an animal model of viral pathogenesis. Using virological and immunological techniques, we examined the immune response to γHV68 in heterozygous mice that have an autosomal dominant mutation in the STAT1 coiled-coil domain (STAT1 R274W). We observed impaired control of infection, which was associated with diminished production of gamma interferon (IFN-γ), fewer effector CD4+ and CD8+ T cells, and a reduction in the number of antigen-specific CD8+ T cells. These findings indicate that a STAT1 gain-of-function mutation limits production of antiviral T cells, likely contributing to immunodeficiency against herpesviruses.

A Human Gain-of-Function STING Mutation Causes Immunodeficiency and Gammaherpesvirus-Induced Pulmonary Fibrosis in Mice.

We previously generated STING N153S knock-in mice that have a human disease-associated gain-of-function mutation in STING. Patients with this mutation (STING N154S in humans) develop STING-associated vasculopathy with onset in infancy (SAVI), a severe pediatric autoinflammatory disease characterized by pulmonary fibrosis. Since this mutation promotes the upregulation of antiviral type I interferon-stimulated genes (ISGs), we hypothesized that STING N153S knock-in mice may develop more severe autoinflammatory disease in response to a virus challenge. To test this hypothesis, we infected heterozygous STING N153S mice with murine gammaherpesvirus 68 (γHV68). STING N153S mice were highly vulnerable to infection and developed pulmonary fibrosis after infection. In addition to impairing CD8+ T cell responses and humoral immunity, STING N153S also promoted the replication of γHV68 in cultured macrophages. In further support of a combined innate and adaptive immunodeficiency, γHV68 infection was more severe in Rag1-/- STING N153S mice than in Rag1-/- littermate mice, which completely lack adaptive immunity. Thus, a gain-of-function STING mutation creates a combined innate and adaptive immunodeficiency that leads to virus-induced pulmonary fibrosis.IMPORTANCE A variety of human rheumatologic disease-causing mutations have recently been identified. Some of these mutations are found in viral nucleic acid-sensing proteins, but whether viruses can influence the onset or progression of these human diseases is less well understood. One such autoinflammatory disease, called STING-associated vasculopathy with onset in infancy (SAVI), affects children and leads to severe lung disease. We generated mice with a SAVI-associated STING mutation and infected them with γHV68, a common DNA virus that is related to human Epstein-Barr virus. Mice with the human disease-causing STING mutation were more vulnerable to infection than wild-type littermate control animals. Furthermore, the STING mutant mice developed lung fibrosis similar to that of patients with SAVI. These findings reveal that a human STING mutation creates severe immunodeficiency, leading to virus-induced lung disease in mice.

MicroRNA hsa-miR-324-5p Suppresses H5N1 Virus Replication by Targeting the Viral PB1 and Host CUEDC2.

MicroRNAs (miRNAs) are small noncoding RNAs that are crucial posttranscriptional regulators for host mRNAs. Recent studies indicate that miRNAs may modulate host response during RNA virus infection. However, the role of miRNAs in immune response against H5N1 infection is not clearly understood. In this study, we showed that expression of cellular miRNA miR-324-5p was downregulated in A549 cells in response to infection with RNA viruses H5N1, A/PR8/H1N1, and Newcastle disease virus (NDV) and transfection with poly(I·C). We found that miR-324-5p inhibited H5N1 replication by targeting the PB1 viral RNA of H5N1 in host cells. In addition, transcriptome analysis revealed that miR-324-5p enhanced the expression of type I interferon, type III interferon, and interferon-inducible genes (ISGs) by targeting CUEDC2, the negative regulator of the JAK1-STAT3 pathway. Together, these findings highlight that the miR-324-5p plays a crucial role in host defense against H5N1 by targeting viral PB1 and host CUEDC2 to inhibit H5N1 replication.IMPORTANCE Highly pathogenic influenza A virus (HPAIV) continues to pose a pandemic threat globally. From 2003 to 2017, H5N1 HPAIV caused 453 human deaths, giving it a high mortality rate (52.74%). This work shows that miR-324-5p suppresses H5N1 HPAIV replication by directly targeting the viral genome (thereby inhibiting viral gene expression) and cellular CUEDC2 gene, the negative regulator of the interferon pathway (thereby enhancing antiviral genes). Our study enhances the knowledge of the role of microRNAs in the cellular response to viral infection. Also, the study provides help in understanding how the host cells utilize small RNAs in controlling the viral burden.

The role of TLR9 polymorphism in susceptibility to pulmonary tuberculosis.

Mycobacterium tuberculosis (MTB) is the causative agent of pulmonary tuberculosis (PTB), a major health problem that leads to 1.5 million deaths annually. Host genetic factors play a significant role in disease resistance/susceptibility by altering immunity against MTB. Toll-like receptor (TLR) sensors such as TLR2, TLR4, TLR8, and TLR9 are known to play a pivotal role in PTB via modulating sensor expression and/or effector responses. Single-nucleotide polymorphism (SNP) rs187084 (T-1486C) of the TLR9 promoter is associated with various autoimmune disorders and cancers. A recent bioinformatic analysis predicted that the T-1486C SNP is involved in PTB, although its potential role is unclear. To investigate the role of T-1486C in PTB, we stimulated PBMCs with the H37Rv whole cell lysate. We found that the presence of the "C" allele increases the transcriptional activity of the TLR9, which in turn induces high levels of Interferon gamma-induced protein 10 (IP-10), a biomarker for PTB. However, the expression of protective cytokines such as IFNγ and TNFα was observed significantly less with "C" allele in comparison to "T" allele. We further selected three different tribe populations showing differential susceptibility to PTB and performed genotypic analyses for the TLR9 promoter. We found a significantly lower minor allele frequency (MAF) of T-1486C in the Baiga tribe, wherein fewer PTB cases were reported, than that in the Gond and Korku tribes. Collectively, these data suggest that the minor "C" allele at rs187084 locus may be associated with susceptibility to PTB, which may explain the relatively lower PTB rates observed in Baiga tribe members.

Recognition of bacterial infection by innate immune sensors.

Microbial challenges to the host initiate an array of defense processes through the activation of innate and adaptive immunity. Innate immunity consists of sensors or pattern-recognition receptors (PRRs) that are expressed on immune and non-immune cells and sense conserved pathogen-derived molecules or pathogen-associated molecular patterns (PAMPs) in various compartments of the host cells. Recognition of the PAMPs by PRRs triggers antimicrobial effector responses via the induction of proinflammatory cytokines and type I IFNs. Several families of PRRs, such as Toll-like receptors (TLRs), NOD-like receptors (NLRs), RIG-I-like receptors (RLRs), and DNA sensors and their respective PAMPs have been well studied in innate immunity and host defense. Here, we review the recent findings on bacterial recognition by TLRs and NLRs and the signaling pathways activated by these sensors.

Impact of the Microbiota on Viral Infections.

The mammalian gastrointestinal tract (GIT) hosts a diverse and highly active microbiota composed of bacteria, eukaryotes, archaea, and viruses. Studies of the GIT microbiota date back more than a century, although modern techniques, including mouse models, sequencing technology, and novel therapeutics in humans, have been foundational to our understanding of the roles of commensal microbes in health and disease. Here, we review the impacts of the GIT microbiota on viral infection, both within the GIT and systemically. GIT-associated microbes and their metabolites alter the course of viral infection through a variety of mechanisms, including direct interactions with virions, alteration of the GIT landscape, and extensive regulation of innate and adaptive immunity. Mechanistic understanding of the full breadth of interactions between the GIT microbiota and the host is still lacking in many ways but will be vital for the development of novel therapeutics for viral and nonviral diseases alike.

IFN-λ derived from nonsusceptible enterocytes acts on tuft cells to limit persistent norovirus.

Norovirus is a leading cause of epidemic viral gastroenteritis, with no currently approved vaccines or antivirals. Murine norovirus (MNoV) is a well-characterized model of norovirus pathogenesis in vivo, and persistent strains exhibit lifelong intestinal infection. Interferon-λ (IFN-λ) is a potent antiviral that rapidly cures MNoV. We previously demonstrated that IFN-λ signaling in intestinal epithelial cells (IECs) controls persistent MNoV, and here demonstrate that IFN-λ acts on tuft cells, the exclusive site of MNoV persistence, to limit infection. While interrogating the source of IFN-λ to regulate MNoV, we confirmed that MDA5-MAVS signaling, required for IFN-λ induction to MNoV in vitro, controls persistent MNoV in vivo. We demonstrate that MAVS in IECs and not immune cells controls MNoV. MAVS in nonsusceptible enterocytes, but not in tuft cells, restricts MNoV, implicating noninfected cells as the IFN-λ source. Our findings indicate that host sensing of MNoV is distinct from cellular tropism, suggesting intercellular communication between IECs for antiviral signaling induction in uninfected bystander cells.

Sequential early-life viral infections modulate the microbiota and adaptive immune responses to systemic and mucosal vaccination.

Increasing evidence points to the microbial exposome as a critical factor in maturing and shaping the host immune system, thereby influencing responses to immune challenges such as infections or vaccines. To investigate the effect of early-life viral exposures on immune development and vaccine responses, we inoculated mice with six distinct viral pathogens in sequence beginning in the neonatal period, and then evaluated their immune signatures before and after intramuscular or intranasal vaccination against SARS-CoV-2. Sequential viral infection drove profound changes in all aspects of the immune system, including increasing circulating leukocytes, altering innate and adaptive immune cell lineages in tissues, and markedly influencing serum cytokine and total antibody levels. Beyond these immune responses changes, these exposures also modulated the composition of the endogenous intestinal microbiota. Although sequentially-infected mice exhibited increased systemic immune activation and T cell responses after intramuscular and intranasal SARS-CoV-2 immunization, we observed decreased vaccine-induced antibody responses in these animals. These results suggest that early-life viral exposures are sufficient to diminish antibody responses to vaccination in mice, and highlight their potential importance of considering prior microbial exposures when investigating vaccine responses.

Intestinal antiviral signaling is controlled by autophagy gene Epg5 independent of the microbiota.

Mutations in the macroautophagy/autophagy gene EPG5 are responsible for Vici syndrome, a human genetic disease characterized by combined immunodeficiency. Previously, we found that epg5-/- mice exhibit hyperinflammation in the lungs mediated by IL1B/IL-1ß and TNF/TNFα, resulting in resistance to influenza. Here, we find that disruption of Epg5 results in protection against multiple enteric viruses including norovirus and rotavirus. Gene expression analysis reveals IFNL/IFN-λ responsive genes as a key alteration. Further, mice lacking Epg5 exhibit substantial alterations of the intestinal microbiota. Surprisingly, germ-free mouse studies indicate Epg5-associated inflammation of both the intestine and lung is microbiota-independent. Genetic studies support IFNL signaling as the primary mediator of resistance to enteric viruses, but not of microbial dysbiosis, in epg5-/- mice. This study unveils an important role, unexpectedly independent of the microbiota, for autophagy gene Epg5 in host organism protection by modulating intestinal IFNL responses.Abbreviations: CTNNB1: catenin (cadherin associated protein), beta 1; DAPI: 4',6-diamidino-2-phenylindole; EPG5: ectopic P-granules autophagy protein 5 homolog (C. elegans); FT: fecal transplant; IFI44: interferon-induced protein 44; IFIT1: interferon-induced protein with tetratricopeptide repeats 1; IFNG/IFN-γ: interferon gamma; IFNL/IFN-λ: interferon lambda; IFNLR1: interferon lambda receptor 1; IL1B/IL-1ß: interleukin 1 beta; ISG: interferon stimulated gene; GF: germ-free; LEfSe: linear discriminant analysis effect size; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MNoV: murine norovirus; MX2: MX dynamin-like GTPase 2; OAS1A: 2'-5' oligoadenylate synthetase 1A; RV: rotavirus; SPF: specific-pathogen free; SQSTM1/p62: sequestosome 1; STAT1: signal transducer and activator of transcription 1; STING1: stimulator of interferon response cGAMP interactor 1; TBK1: TANK-binding kinase 1; TNF/TNFα: tumor necrosis factor.

Homeostatic interferon-lambda response to bacterial microbiota stimulates preemptive antiviral defense within discrete pockets of intestinal epithelium.

Interferon-lambda (IFN-λ) protects intestinal epithelial cells (IECs) from enteric viruses by inducing expression of antiviral IFN-stimulated genes (ISGs). Here, we find that bacterial microbiota stimulate a homeostatic ISG signature in the intestine of specific pathogen-free mice. This homeostatic ISG expression is restricted to IECs, depends on IEC-intrinsic expression of IFN-λ receptor (Ifnlr1), and is associated with IFN-λ production by leukocytes. Strikingly, imaging of these homeostatic ISGs reveals localization to pockets of the epithelium and concentration in mature IECs. Correspondingly, a minority of mature IECs express these ISGs in public single-cell RNA sequencing datasets from mice and humans. Furthermore, we assessed the ability of orally administered bacterial components to restore localized ISGs in mice lacking bacterial microbiota. Lastly, we find that IECs lacking Ifnlr1 are hyper-susceptible to initiation of murine rotavirus infection. These observations indicate that bacterial microbiota stimulate ISGs in localized regions of the intestinal epithelium at homeostasis, thereby preemptively activating antiviral defenses in vulnerable IECs to improve host defense against enteric viruses.

Murine astrovirus tropism for goblet cells and enterocytes facilitates an IFN-λ response in vivo and in enteroid cultures.

Although they globally cause viral gastroenteritis in children, astroviruses are understudied due to the lack of well-defined animal models. While murine astroviruses (muAstVs) chronically infect immunodeficient mice, a culture system and understanding of their pathogenesis is lacking. Here, we describe a platform to cultivate muAstV using air-liquid interface (ALI) cultures derived from mouse enteroids, which support apical infection and release. Chronic muAstV infection occurs predominantly in the small intestine and correlates with higher interferon-lambda (IFN-λ) expression. MuAstV stimulates IFN-λ production in ALI, recapitulating our in vivo findings. We demonstrate that goblet cells and enterocytes are targets for chronic muAstV infection in vivo, and that infection is enhanced by parasite co-infection or type 2 cytokine signaling. Depletion of goblet cells from ALI limits muAstV infection in vitro. During chronic infection, muAstV stimulates IFN-λ production in infected cells and induces ISGs throughout the intestinal epithelium in an IFN-λ-receptor-dependent manner. Collectively, our study provides insights into the cellular tropism and innate immune responses to muAstV and establishes an enteroid-based culture system to propagate muAstV in vitro.

Enteric Viral Co-Infections: Pathogenesis and Perspective.

Enteric viral co-infections, infections involving more than one virus, have been reported for a diverse group of etiological agents, including rotavirus, norovirus, astrovirus, adenovirus, and enteroviruses. These pathogens are causative agents for acute gastroenteritis and diarrheal disease in immunocompetent and immunocompromised individuals of all ages globally. Despite virus-virus co-infection events in the intestine being increasingly detected, little is known about their impact on disease outcomes or human health. Here, we review what is currently known about the clinical prevalence of virus-virus co-infections and how co-infections may influence vaccine responses. While experimental investigations into enteric virus co-infections have been limited, we highlight in vivo and in vitro models with exciting potential to investigate viral co-infections. Many features of virus-virus co-infection mechanisms in the intestine remain unclear, and further research will be critical.

The Interplay Between Viral-Derived miRNAs and Host Immunity During Infection.

MicroRNAs are short non-coding RNAs that play a crucial role in the regulation of gene expression during cellular processes. The host-encoded miRNAs are known to modulate the antiviral defense during viral infection. In the last decade, multiple DNA and RNA viruses have been shown to produce miRNAs known as viral miRNAs (v-miRNAs) so as to evade the host immune response. In this review, we highlight the origin and biogenesis of viral miRNAs during the viral lifecycle. We also explore the role of viral miRNAs in immune evasion and hence in maintaining chronic infection and disease. Finally, we offer insights into the underexplored role of viral miRNAs as potential targets for developing therapeutics for treating complex viral diseases.

Viral complementation of immunodeficiency confers protection against enteric pathogens via interferon-λ.

Commensal microbes profoundly impact host immunity to enteric viral infections1. We have shown that the bacterial microbiota and host antiviral cytokine interferon-λ (IFN-λ) determine the persistence of murine norovirus in the gut2,3. However, the effects of the virome in modulating enteric infections remain unexplored. Here, we report that murine astrovirus can complement primary immunodeficiency to protect against murine norovirus and rotavirus infections. Protection against infection was horizontally transferable between immunocompromised mouse strains by co-housing and fecal transplantation. Furthermore, protection against enteric pathogens corresponded with the presence of a specific strain of murine astrovirus in the gut, and this complementation of immunodeficiency required IFN-λ signalling in gut epithelial cells. Our study demonstrates that elements of the virome can protect against enteric pathogens in an immunodeficient host.

Distinct Effects of Type I and III Interferons on Enteric Viruses.

Interferons (IFNs) are key host cytokines in the innate immune response to viral infection, and recent work has identified unique roles for IFN subtypes in regulating different aspects of infection. Currently emerging is a common theme that type III IFNs are critical in localized control of infection at mucosal barrier sites, while type I IFNs are important for broad systemic control of infections. The intestine is a particular site of interest for exploring these effects, as in addition to being the port of entry for a multitude of pathogens, it is a complex tissue with a variety of cell types as well as the presence of the intestinal microbiota. Here we focus on the roles of type I and III IFNs in control of enteric viruses, discussing what is known about signaling downstream from these cytokines, including induction of specific IFN-stimulated genes. We review viral strategies to evade IFN responses, effects of IFNs on the intestine, interactions between IFNs and the microbiota, and briefly discuss the role of IFNs in controlling viral infections at other barrier sites. Enhanced understanding of the coordinate roles of IFNs in control of viral infections may facilitate development of antiviral therapeutic strategies; here we highlight potential avenues for future exploration.

The microRNA miR-485 targets host and influenza virus transcripts to regulate antiviral immunity and restrict viral replication.

MicroRNAs (miRNAs) are small noncoding RNAs that are responsible for dynamic changes in gene expression, and some regulate innate antiviral responses. Retinoic acid-inducible gene I (RIG-I) is a cytosolic sensor of viral RNA; RIG-I activation induces an antiviral immune response. We found that miR-485 of the host was produced in response to viral infection and targeted RIG-I mRNA for degradation, which led to suppression of the antiviral response and enhanced viral replication. Thus, inhibition of the expression of mir-485 markedly reduced the replication of Newcastle disease virus (NDV) and the H5N1 strain of influenza virus in mammalian cells. Unexpectedly, miR-485 also bound to the H5N1 gene PB1 (which encodes an RNA polymerase required for viral replication) in a sequence-specific manner, thereby inhibiting replication of the H5N1 virus. Furthermore, miR-485 exhibited bispecificity, targeting RIG-I in cells with a low abundance of H5N1 virus and targeting PB1 in cells with increased amounts of the H5N1 virus. These findings highlight the dual role of miR-485 in preventing spurious activation of antiviral signaling and restricting influenza virus infection.

Low prevalence of CCR5-Δ32, CCR2-64I and SDF1-3'A alleles in the Baiga and Gond tribes of Central India.

Human immunodeficiency virus-1 (HIV-1) which causes acquired immune deficiency syndrome (AIDS), by infecting CD4(+) immune cells and hence weakening the host defense mechanism till death, is one of the major factor responsible for human demises worldwide. Both innate (monocytes and macrophages) and adaptive (T cells) immune cells expresses chemokines receptors (2 and 5) and stromal cell derived factor-1 (SDF-1) which play crucial role in HIV-1 virus entry and progression. Allele variants of genes CCR5 (CCR5-Δ32), CCR2 (CCR2-64I) and SDF1 (SDFA-3'A; the ligand of CXCR4) are known to slow down the HIV-1 progression in infected individual. In the present study, the frequency of CCR5-Δ32, CCR2-64I and SDF1-3'A alleles in primitive tribe (Baiga) and a non-primitive tribe (Gond) of central India were investigated. A total 200 seronegative samples for HIV from healthy individuals of tribes were analyzed and observed allele frequencies of CCR5-Δ32, CCR2-64I and SDF1-3'A were (0, 0.035, 0.080) and (0, 0.110, 0.100) in Baiga and Gond respectively. Minor allele frequency of these alleles of Gond and Baiga tribes were compared with different populations of the world for relative hazard (RH), which indicate the risk of progression after infection of HIV1. The RH values were calculated based on genotypic frequency, showed the high RH value (RH1-AIDS1993-0.98, RH2-AIDS1987-0.98 and death/RH3-0.97) in Baiga tribe, indicates the low level of resistance against HIV-1 progression after infection.