A TNF-IL-1 circuit controls Yersinia within intestinal pyogranulomas.

Tumor necrosis factor (TNF) is a pleiotropic inflammatory cytokine that mediates antimicrobial defense and granuloma formation in response to infection by numerous pathogens. We previously reported that Yersinia pseudotuberculosis colonizes the intestinal mucosa and induces the recruitment of neutrophils and inflammatory monocytes into organized immune structures termed pyogranulomas (PG) that control Yersinia infection. Inflammatory monocytes are essential for the control and clearance of Yersinia within intestinal PG, but how monocytes mediate Yersinia restriction is poorly understood. Here, we demonstrate that TNF signaling in monocytes is required for bacterial containment following enteric Yersinia infection. We further show that monocyte-intrinsic TNFR1 signaling drives the production of monocyte-derived interleukin-1 (IL-1), which signals through IL-1 receptors on non-hematopoietic cells to enable PG-mediated control of intestinal Yersinia infection. Altogether, our work reveals a monocyte-intrinsic TNF-IL-1 collaborative inflammatory circuit that restricts intestinal Yersinia infection.

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.

A TNF-IL-1 circuit controls Yersinia within intestinal granulomas.

Tumor necrosis factor (TNF) is a pleiotropic inflammatory cytokine that mediates antimicrobial defense and granuloma formation in response to infection by numerous pathogens. Yersinia pseudotuberculosis colonizes the intestinal mucosa and induces recruitment of neutrophils and inflammatory monocytes into organized immune structures termed pyogranulomas that control the bacterial infection. Inflammatory monocytes are essential for control and clearance of Yersinia within intestinal pyogranulomas, but how monocytes mediate Yersinia restriction is poorly understood. Here, we demonstrate that TNF signaling in monocytes is required for bacterial containment following enteric Yersinia infection. We further show that monocyte-intrinsic TNFR1 signaling drives production of monocyte-derived interleukin-1 (IL-1), which signals through IL-1 receptor on non-hematopoietic cells to enable pyogranuloma-mediated control of Yersinia infection. Altogether, our work reveals a monocyte-intrinsic TNF-IL-1 collaborative circuit as a crucial driver of intestinal granuloma function, and defines the cellular target of TNF signaling that restricts intestinal Yersinia infection.

Inflammatory monocytes promote granuloma control of Yersinia infection.

Granulomas are organized immune cell aggregates formed in response to chronic infection or antigen persistence. The bacterial pathogen Yersinia pseudotuberculosis (Yp) blocks innate inflammatory signalling and immune defence, inducing neutrophil-rich pyogranulomas (PGs) within lymphoid tissues. Here we uncover that Yp also triggers PG formation within the murine intestinal mucosa. Mice lacking circulating monocytes fail to form defined PGs, have defects in neutrophil activation and succumb to Yp infection. Yersinia lacking virulence factors that target actin polymerization to block phagocytosis and reactive oxygen burst do not induce PGs, indicating that intestinal PGs form in response to Yp disruption of cytoskeletal dynamics. Notably, mutation of the virulence factor YopH restores PG formation and control of Yp in mice lacking circulating monocytes, demonstrating that monocytes override YopH-dependent blockade of innate immune defence. This work reveals an unappreciated site of Yersinia intestinal invasion and defines host and pathogen drivers of intestinal granuloma formation.

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.

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.

Rotavirus susceptibility of antibiotic-treated mice ascribed to diminished expression of interleukin-22.

The commensal microbiota regulates susceptibility to enteric pathogens by fine-tuning mucosal innate immune responses, but how susceptibility to enteric viruses is shaped by the microbiota remains incompletely understood. Past reports have indicated that commensal bacteria may either promote or repress rotavirus replication in the small intestine of mice. We now report that rotavirus replicated more efficiently in the intestines of germ-free and antibiotic-treated mice compared to animals with an unmodified microbiota. Antibiotic treatment also facilitated rotavirus replication in type I and type III interferon (IFN) receptor-deficient mice, revealing IFN-independent proviral effects. Expression of interleukin-22 (IL-22) was strongly diminished in the intestine of antibiotic-treated mice. Treatment with exogenous IL-22 blocked rotavirus replication in microbiota-depleted wild-type and Stat1-/- mice, demonstrating that the antiviral effect of IL-22 in animals with altered microbiome is not dependent on IFN signaling. In antibiotic-treated animals, IL-22-induced a specific set of genes including Fut2, encoding fucosyl-transferase 2 that participates in the biosynthesis of fucosylated glycans which can mediate rotavirus binding. Interestingly, IL-22 also blocked rotavirus replication in antibiotic-treated Fut2-/- mice. Furthermore, IL-22 inhibited rotavirus replication in antibiotic-treated mice lacking key molecules of the necroptosis or pyroptosis pathways of programmed cell death. Taken together, our results demonstrate that IL-22 determines rotavirus susceptibility of antibiotic-treated mice, yet the IL-22-induced effector molecules conferring rotavirus resistance remain elusive.

Transferrable protection by gut microbes against STING-associated lung disease.

STING modulates immunity by responding to bacterial and endogenous cyclic dinucleotides (CDNs). Humans and mice with STING gain-of-function mutations develop a syndrome known as STING-associated vasculopathy with onset in infancy (SAVI), which is characterized by inflammatory or fibrosing lung disease. We hypothesized that hyperresponsiveness of gain-of-function STING to bacterial CDNs might explain autoinflammatory lung disease in SAVI mice. We report that depletion of gut microbes with oral antibiotics (vancomycin, neomycin, and ampicillin [VNA]) nearly eliminates lung disease in SAVI mice, implying that gut microbes might promote STING-associated autoinflammation. However, we show that germ-free SAVI mice still develop severe autoinflammatory disease and that transferring gut microbiota from antibiotics-treated mice to germ-free animals eliminates lung inflammation. Depletion of anaerobes with metronidazole abolishes the protective effect of the VNA antibiotics cocktail, and recolonization with the metronidazole-sensitive anaerobe Bacteroides thetaiotaomicron prevents disease, confirming a protective role of a metronidazole-sensitive microbe in a model of SAVI.

Norovirus evolution in immunodeficient mice reveals potentiated pathogenicity via a single nucleotide change in the viral capsid.

Interferons (IFNs) are key controllers of viral replication, with intact IFN responses suppressing virus growth and spread. Using the murine norovirus (MNoV) system, we show that IFNs exert selective pressure to limit the pathogenic evolutionary potential of this enteric virus. In animals lacking type I IFN signaling, the nonlethal MNoV strain CR6 rapidly acquired enhanced virulence via conversion of a single nucleotide. This nucleotide change resulted in amino acid substitution F514I in the viral capsid, which led to >10,000-fold higher replication in systemic organs including the brain. Pathogenicity was mediated by enhanced recruitment and infection of intestinal myeloid cells and increased extraintestinal dissemination of virus. Interestingly, the trade-off for this mutation was reduced fitness in an IFN-competent host, in which CR6 bearing F514I exhibited decreased intestinal replication and shedding. In an immunodeficient context, a spontaneous amino acid change can thus convert a relatively avirulent viral strain into a lethal pathogen.

The intestinal regionalization of acute norovirus infection is regulated by the microbiota via bile acid-mediated priming of type III interferon.

Evidence has accumulated to demonstrate that the intestinal microbiota enhances mammalian enteric virus infections1. For example, we and others previously reported that commensal bacteria stimulate acute and persistent murine norovirus infections2-4. However, in apparent contradiction of these results, the virulence of murine norovirus infection was unaffected by antibiotic treatment. This prompted us to perform a detailed investigation of murine norovirus infection in microbially deplete mice, revealing a more complex picture in which commensal bacteria inhibit viral infection of the proximal small intestine while simultaneously stimulating the infection of distal regions of the gut. Thus, commensal bacteria can regulate viral regionalization along the intestinal tract. We further show that the mechanism underlying bacteria-dependent inhibition of norovirus infection in the proximal gut involves bile acid priming of type III interferon. Finally, the regional effects of the microbiota on norovirus infection may result from distinct regional expression profiles of key bile acid receptors that regulate the type III interferon response. Overall, these findings reveal that the biotransformation of host metabolites by the intestinal microbiota directly and regionally impacts infection by a pathogenic enteric virus.

Disruption of Type III Interferon (IFN) Genes Ifnl2 and Ifnl3 Recapitulates Loss of the Type III IFN Receptor in the Mucosal Antiviral Response.

Type III interferon (IFN), or IFN lambda (IFN-λ), is an essential component of the innate immune response to mucosal viral infections. In both the intestine and the lung, signaling via the IFN-λ receptor (IFNLR) controls clinically important viral pathogens, including influenza virus, norovirus, and rotavirus. While it is thought that IFN-λ cytokines are the exclusive ligands for signaling through IFNLR, it is not known whether genetic ablation of these cytokines phenotypically recapitulates disruption of the receptor. Here, we report the serendipitous establishment of Ifnl2-/- Ifnl3-/- mice, which lack all known functional murine IFN-λ cytokines. We demonstrate that, like Ifnlr1-/- mice lacking IFNLR signaling, these mice display defective control of murine norovirus, reovirus, and influenza virus and therefore genocopy Ifnlr1-/- mice. Thus, for regulation of viral infections at mucosal sites of both the intestine and lung, signaling via IFNLR can be fully explained by the activity of known cytokines IFN-λ2 and IFN-λ3. Our results confirm the current understanding of ligand-receptor interactions for type III IFN signaling and highlight the importance of this pathway in regulation of mucosal viral pathogens.IMPORTANCE Type III interferons are potent antiviral cytokines important for regulation of viruses that infect at mucosal surfaces. Studies using mice lacking the Ifnlr1 gene encoding the type III interferon receptor have demonstrated that signaling through this receptor is critical for protection against influenza virus, norovirus, and reovirus. Using a genetic approach to disrupt murine type III interferon cytokine genes Ifnl2 and Ifnl3, we found that mice lacking these cytokines fully recapitulate the impaired control of viruses observed in mice lacking Ifnlr1 Our results support the idea of an exclusive role for known type III interferon cytokines in signaling via IFNLR to mediate antiviral effects at mucosal surfaces. These findings emphasize the importance of type III interferons in regulation of a variety of viral pathogens and provide important genetic evidence to support our understanding of the ligand-receptor interactions in this pathway.

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.

HOIL1 Is Essential for the Induction of Type I and III Interferons by MDA5 and Regulates Persistent Murine Norovirus Infection.

The linear ubiquitin chain assembly complex (LUBAC), composed of heme-oxidized IRP2 ubiquitin ligase 1 (HOIL1), HOIL1-interacting protein (HOIP), and SHANK-associated RH domain-interacting protein (SHARPIN), is a crucial regulator of multiple immune signaling pathways. In humans, HOIL1 or HOIP deficiency is associated with an immune disorder involving autoinflammation, immunodeficiency, and inflammatory bowel disease (IBD)-like symptoms. During viral infection, LUBAC is reported to inhibit the induction of interferon (IFN) by the cytosolic RNA sensor retinoic acid-inducible gene I (RIG-I). Surprisingly, we found that HOIL1 is essential for the induction of both type I and type III IFNs, as well as the phosphorylation of IFN regulatory factor 3 (IRF3), during murine norovirus (MNoV) infection in cultured dendritic cells. The RIG-I-like receptor, melanoma differentiation-associated protein 5 (MDA5), is also required for IFN induction and IRF3 phosphorylation during MNoV infection. Furthermore, HOIL1 and MDA5 were required for IFN induction after Theiler's murine encephalomyelitis virus infection and poly(I·C) transfection, but not Sendai virus or vesicular stomatitis virus infection, indicating that HOIL1 and LUBAC are required selectively for MDA5 signaling. Moreover, Hoil1-/- mice exhibited defective control of acute and persistent murine norovirus infection and defective regulation of MNoV persistence by the microbiome as also observed previously for mice deficient in interferon lambda (IFN-λ) receptor, signal transducer and activator of transcription factor 1 (STAT1), and IRF3. These data indicate that LUBAC plays a critical role in IFN induction to control RNA viruses sensed by MDA5.IMPORTANCE Human noroviruses are a leading cause of gastroenteritis throughout the world but are challenging to study in vivo and in vitro Murine norovirus (MNoV) provides a tractable genetic and small-animal model to study norovirus biology and immune responses. Interferons are critical mediators of antiviral immunity, but excessive expression can dysregulate the immune system. IFN-λ plays an important role at mucosal surfaces, including the gastrointestinal tract, and both IFN-λ and commensal enteric bacteria are important modulators of persistent MNoV infection. LUBAC, of which HOIL1 is a component, is reported to inhibit type I IFN induction after RIG-I stimulation. We show, in contrast, that HOIL1 is critical for type I and III IFN induction during infection with MNoV, a virus that preferentially activates MDA5. Moreover, HOIL1 regulates MNoV infection in vivo These data reveal distinct functions for LUBAC in these closely related signaling pathways and in modulation of IFN expression.

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.

Norovirus Cell Tropism Is Determined by Combinatorial Action of a Viral Non-structural Protein and Host Cytokine.

Cellular tropism during persistent viral infection is commonly conferred by the interaction of a viral surface protein with a host receptor complex. Norovirus, the leading global cause of gastroenteritis, can be persistently shed during infection, but its in vivo cellular tropism and tropism determinants remain unidentified. Using murine norovirus (MNoV), we determine that a small number of intestinal epithelial cells (IECs) serve as the reservoir for fecal shedding and persistence. The viral non-structural protein NS1, rather than a viral surface protein, determines IEC tropism. Expression of NS1 from a persistent MNoV strain is sufficient for an acute MNoV strain to target IECs and persist. In addition, interferon-lambda (IFN-λ) is a key host determinant blocking MNoV infection in IECs. The inability of acute MNoV to shed and persist is rescued in Ifnlr1-/- mice, suggesting that NS1 evades IFN-λ-mediated antiviral immunity. Thus, NS1 and IFN-λ interactions govern IEC tropism and persistence of MNoV.