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.

Selective Polyprotein Processing Determines Norovirus Sensitivity to Trim7.

Noroviruses are a leading cause of gastroenteritis worldwide, yet the molecular mechanisms of how host antiviral factors restrict norovirus infection are poorly understood. Here, we present a CRISPR activation screen that identifies mouse genes which inhibit murine norovirus (MNV) replication. Detailed analysis of the major hit Trim7 demonstrates a potent inhibition of the early stages of MNV replication. Leveraging in vitro evolution, we identified MNV mutants that escape Trim7 restriction by altering the cleavage of the viral NS6-7 polyprotein precursor. NS6, but not the NS6-7 precursor, directly binds the substrate-binding domain of Trim7. Surprisingly, the selective polyprotein processing that enables Trim7 evasion inflicts a significant evolutionary burden, as viruses with decreased NS6-7 cleavage are strongly attenuated in viral replication and pathogenesis. Our data provide an unappreciated mechanism of viral evasion of cellular antiviral factors through selective polyprotein processing and highlight the evolutionary tradeoffs in acquiring resistance to host restriction factors. IMPORTANCE To maximize a limited genetic capacity, viruses encode polyproteins that can be subsequently separated into individual components by viral proteases. While classically viewed as a means of economy, recent findings have indicated that polyprotein processing can spatially and temporally coordinate the distinct phases of the viral life cycle. Here, we present a function for alternative polyprotein processing centered on immune defense. We discovered that selective polyprotein processing of the murine norovirus polyprotein shields MNV from restriction by the host antiviral protein Trim7. Trim7 can bind the viral protein NS6 but not the viral precursor protein NS6-7. Our findings provide insight into the evolutionary pressures that define patterns of viral polyprotein processing and uncover a trade-off between viral replication and immune evasion.

The bacterial microbiota regulates normal hematopoiesis via metabolite-induced type 1 interferon signaling.

Antibiotic therapy, especially when administered long term, is associated with adverse hematologic effects such as cytopenia. Signals from the intestinal microbiota are critical to maintain normal hematopoiesis, and antibiotics can cause bone marrow suppression through depletion of the microbiota. We reported previously that STAT1 signaling is necessary for microbiota-dependent hematopoiesis, but the precise mechanisms by which the gut microbiota signals to the host bone marrow to regulate hematopoiesis remain undefined. We sought to identify the cell type(s) through which STAT1 promotes microbiota-mediated hematopoiesis and to elucidate which upstream signaling pathways trigger STAT1 signaling. Using conditional knockout and chimeric mice, we found that the microbiota induced STAT1 signaling in non-myeloid hematopoietic cells to support hematopoiesis and that STAT1 signaling was specifically dependent on type I interferons (IFNs). Indeed, basal type I IFN signaling was reduced in hematopoietic progenitor cells with antibiotic treatment. In addition, we discovered that oral administration of a commensal-derived product, NOD1 ligand, rescues the hematopoietic defects induced by antibiotics in mice. Using metabolomics, we identified additional microbially produced candidates that can stimulate type I IFN signaling to potentially rescue the hematopoietic defects induced by antibiotics, including phosphatidylcholine and γ-glutamylalanine. Overall, our studies define a signaling pathway through which microbiota promotes normal hematopoiesis and identify microbial metabolites that may serve as therapeutic agents to ameliorate antibiotic-induced bone marrow suppression and cytopenia.

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.

CD300lf Conditional Knockout Mouse Reveals Strain-Specific Cellular Tropism of Murine Norovirus.

Noroviruses are a leading cause of gastrointestinal infection in humans and mice. Understanding human norovirus (HuNoV) cell tropism has important implications for our understanding of viral pathogenesis. Murine norovirus (MNoV) is extensively used as a surrogate model for HuNoV. We previously identified CD300lf as the receptor for MNoV. Here, we generated a Cd300lf conditional knockout (CD300lfF/F ) mouse to elucidate the cell tropism of persistent and nonpersistent strains of murine norovirus. Using this mouse model, we demonstrated that CD300lf expression on intestinal epithelial cells (IECs), and on tuft cells in particular, is essential for transmission of the persistent MNoV strain CR6 (MNoVCR6) in vivo In contrast, the nonpersistent MNoV strain CW3 (MNoVCW3) does not require CD300lf expression on IECs for infection. However, deletion of CD300lf in myelomonocytic cells (LysM Cre+) partially reduces CW3 viral load in lymphoid and intestinal tissues. Disruption of CD300lf expression on B cells (CD19 Cre), neutrophils (Mrp8 Cre), and dendritic cells (CD11c Cre) did not affect MNoVCW3 viral RNA levels. Finally, we show that the transcription factor STAT1, which is critical for the innate immune response, partially restricts the cell tropism of MNoVCW3 to LysM+ cells. Taken together, these data demonstrate that CD300lf expression on tuft cells is essential for MNoVCR6; that myelomonocytic cells are a major, but not exclusive, target cell of MNoVCW3; and that STAT1 signaling restricts the cellular tropism of MNoVCW3 This study provides the first genetic system for studying the cell type-specific role of CD300lf in norovirus pathogenesis.IMPORTANCE Human noroviruses (HuNoVs) are a leading cause of gastroenteritis resulting in up to 200,000 deaths each year. The receptor and cell tropism of HuNoV in immunocompetent humans are unclear. We use murine norovirus (MNoV) as a model for HuNoV. We recently identified CD300lf as the sole physiologic receptor for MNoV. Here, we leverage this finding to generate a Cd300lf conditional knockout mouse to decipher the contributions of specific cell types to MNoV infection. We demonstrate that persistent MNoVCR6 requires CD300lf expression on tuft cells. In contrast, multiple CD300lf+ cell types, dominated by myelomonocytic cells, are sufficient for nonpersistent MNoVCW3 infection. CD300lf expression on epithelial cells, B cells, neutrophils, and dendritic cells is not critical for MNoVCW3 infection. Mortality associated with the MNoVCW3 strain in Stat1-/- mice does not require CD300lf expression on LysM+ cells, highlighting that both CD300lf receptor expression and innate immunity regulate MNoV cell tropism in vivo.

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.

CD300LF Polymorphisms of Inbred Mouse Strains Confer Resistance to Murine Norovirus Infection in a Cell Type-Dependent Manner.

Human norovirus is the leading cause of gastroenteritis worldwide, yet basic questions about its life cycle remain unanswered due to an historical lack of robust experimental systems. Recent studies on the closely related murine norovirus (MNV) have identified CD300LF as an indispensable entry factor for MNV. We compared the MNV susceptibilities of cells from different mouse strains and identified polymorphisms in murine CD300LF which are critical for its function as an MNV receptor. Bone marrow-derived macrophages (BMDMs) from I/LnJ mice were resistant to infection from multiple MNV strains which readily infect BMDMs from C57BL/6J mice. The resistance of I/LnJ BMDMs was specific to MNV, since the cells supported infection of other viruses comparably to C57BL/6J BMDMs. Transduction of I/LnJ BMDMs with C57BL/6J CD300LF made the cells permissible to MNV infection, suggesting that the cause of resistance lies in the entry step of MNV infection. In fact, we mapped this phenotype to a 4-amino-acid difference at the CC' loop of CD300LF; swapping of these amino acids between C57BL/6J and I/LnJ CD300LF proteins made the mutant C57BL/6J CD300LF functionally impaired and the corresponding mutant of I/LnJ CD300LF functional as an MNV entry factor. Surprisingly, expression of the I/LnJ CD300LF in other cell types made the cells infectible by MNV, even though the I/LnJ allele did not function as an MNV receptor in macrophage-like cells. Correspondingly, I/LnJ CD300LF bound MNV virions in permissive cells but not in nonpermissive cells. Collectively, our data suggest the existence of a cell type-specific modifier of MNV entry.IMPORTANCE MNV is a prevalent model system for studying human norovirus, which is the leading cause of gastroenteritis worldwide and thus a sizeable public health burden. Elucidating mechanisms underlying susceptibility of host cells to MNV infection can lead to insights on the roles that specific cell types play during norovirus pathogenesis. Here, we show that different alleles of the proteinaceous receptor for MNV, CD300LF, function in a cell type-dependent manner. In contrast to the C57BL/6J allele, which functions as an MNV entry factor in all tested cell types, including human cells, I/LnJ CD300LF does not function as an MNV entry factor in macrophage-like cells but does allow MNV entry in other cell types. Together, these observations indicate the existence of cell type-specific modifiers of CD300LF-dependent MNV entry.

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 Stem-Cell-Derived Platform Enables Complete Cryptosporidium Development In Vitro and Genetic Tractability.

Despite being a frequent cause of severe diarrheal disease in infants and an opportunistic infection in immunocompromised patients, Cryptosporidium research has lagged due to a lack of facile experimental methods. Here, we describe a platform for complete life cycle development and long-term growth of C. parvum in vitro using "air-liquid interface" (ALI) cultures derived from intestinal epithelial stem cells. Transcriptomic profiling revealed that differentiating epithelial cells grown under ALI conditions undergo profound changes in metabolism and development that enable completion of the parasite life cycle in vitro. ALI cultures support parasite expansion > 100-fold and generate viable oocysts that are transmissible in vitro and to mice, causing infection and animal death. Transgenic parasite lines created using CRISPR/Cas9 were used to complete a genetic cross in vitro, demonstrating Mendelian segregation of chromosomes during meiosis. ALI culture provides an accessible model that will enable innovative studies into Cryptosporidium biology and host interactions.

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.

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.

Hematopoiesis and the bacterial microbiome.

Recent studies have revealed that the intestinal bacterial microbiome plays an important role in the regulation of hematopoiesis. A correlation between adverse hematologic effects and imbalance of the intestinal microbiome, or dysbiosis, is evident in several human conditions, such as inflammatory bowel disease, obesity, and, critically, in the setting of antibiotic exposure. Here we review the effects of gut dysbiosis on the hematological compartment and our current understanding of the mechanisms through which changes in the bacterial microbiome affect hematopoiesis.

Expression of Ifnlr1 on Intestinal Epithelial Cells Is Critical to the Antiviral Effects of Interferon Lambda against Norovirus and Reovirus.

Lambda interferon (IFN-λ) has potent antiviral effects against multiple enteric viral pathogens, including norovirus and rotavirus, in both preventing and curing infection. Because the intestine includes a diverse array of cell types, however, the cell(s) upon which IFN-λ acts to exert its antiviral effects is unclear. Here, we sought to identify IFN-λ-responsive cells by generation of mice with lineage-specific deletion of the receptor for IFN-λ, Ifnlr1 We found that expression of IFNLR1 on intestinal epithelial cells (IECs) in the small intestine and colon is required for enteric IFN-λ antiviral activity. IEC Ifnlr1 expression also determines the efficacy of IFN-λ in resolving persistent murine norovirus (MNoV) infection and regulates fecal shedding and viral titers in tissue. Thus, the expression of Ifnlr1 by IECs is necessary for the response to both endogenous and exogenous IFN-λ. We further demonstrate that IEC Ifnlr1 expression is required for the sterilizing innate immune effects of IFN-λ by extending these findings in Rag1-deficient mice. Finally, we assessed whether our findings pertained to multiple viral pathogens by infecting mice specifically lacking IEC Ifnlr1 expression with reovirus. These mice phenocopied Ifnlr1-null animals, exhibiting increased intestinal tissue titers and enhanced reovirus fecal shedding. Thus, IECs are the critical cell type responding to IFN-λ to control multiple enteric viruses. This is the first genetic evidence that supports an essential role for IECs in IFN-λ-mediated control of enteric viral infection, and these findings provide insight into the mechanism of IFN-λ-mediated antiviral activity.IMPORTANCE Human noroviruses (HNoVs) are the leading cause of epidemic gastroenteritis worldwide. Type III interferons (IFN-λ) control enteric viral infections in the gut and have been shown to cure mouse norovirus, a small-animal model for HNoVs. Using a genetic approach with conditional knockout mice, we identified IECs as the dominant IFN-λ-responsive cells in control of enteric virus infection in vivo Upon murine norovirus or reovirus infection, Ifnlr1 depletion in IECs largely recapitulated the phenotype seen in Ifnlr1-/- mice of higher intestinal tissue viral titers and increased viral shedding in the stool. Moreover, IFN-λ-mediated sterilizing immunity against murine norovirus requires the capacity of IECs to respond to IFN-λ. These findings clarify the mechanism of action of this cytokine and emphasize the therapeutic potential of IFN-λ for treating mucosal viral infections.

Antibiotics impair murine hematopoiesis by depleting the intestinal microbiota.

Bone marrow suppression is an adverse effect associated with many antibiotics, especially when administered for prolonged treatment courses. Recent advances in our understanding of steady-state hematopoiesis have allowed us to explore the effects of antibiotics on hematopoietic progenitors in detail using a murine model. Antibiotic-treated mice exhibited anemia, thrombocytosis, and leukopenia, with pronounced pan-lymphopenia as demonstrated by flow cytometric analysis of peripheral blood. Bone marrow progenitor analysis revealed depletion of hematopoietic stem cells and multipotent progenitors across all subtypes. Granulocytes and B cells were also diminished in the bone marrow, whereas the number of CD8+ T cells increased. Reductions in progenitor activity were not observed when cells were directly incubated with antibiotics, suggesting that these effects are indirect. Hematopoietic changes were associated with a significant contraction of the fecal microbiome and were partially rescued by fecal microbiota transfer. Further, mice raised in germ-free conditions had hematopoietic abnormalities similar to those seen in antibiotic-treated mice, and antibiotic therapy of germ-free mice caused no additional abnormalities. The effects of antibiotics were phenocopied in Stat1-deficient mice, with no additional suppression by antibiotics in these mice. We conclude that microbiome depletion as a result of broad-spectrum antibiotic treatment disrupts basal Stat1 signaling and alters T-cell homeostasis, leading to impaired progenitor maintenance and granulocyte maturation. Methods to preserve the microbiome may reduce the incidence of antibiotic-associated bone marrow suppression.

Paneth cell defects in Crohn's disease patients promote dysbiosis.

BACKGROUND: Paneth cell dysfunction has been implicated in a subset of Crohn's disease (CD) patients. We previously stratified clinical outcomes of CD patients by using Paneth cell phenotypes, which we defined by the intracellular distribution of antimicrobial proteins. Animal studies suggest that Paneth cells shape the intestinal microbiome. However, it is unclear whether Paneth cell phenotypes alter the microbiome complexity in CD subjects. Therefore, we analyzed the correlation of Paneth cell phenotypes with mucosal microbiome composition and ileal RNA expression in pediatric CD and noninflammatory bowel disease (non-IBD) patients. METHODS: Pediatric CD (n = 44) and non-IBD (n = 62) patients aged 4 to 18 were recruited prior to routine endoscopic biopsy. Ileal mucosal samples were analyzed for Paneth cell phenotypes, mucosal microbiome composition, and RNA transcriptome. RESULTS: The prevalence of abnormal Paneth cells was higher in pediatric versus adult CD cohorts. For pediatric CD patients, those with abnormal Paneth cells showed significant changes in their ileal mucosal microbiome, highlighted by reduced protective microbes and enriched proinflammatory microbes. Ileal transcriptome profiles showed reduced transcripts for genes that control oxidative phosphorylation in CD patients with abnormal Paneth cells. These transcriptional changes in turn were correlated with specific microbiome alterations. In non-IBD patients, a subset contained abnormal Paneth cells. However, this subset was not associated with alterations in the microbiome or host transcriptome. CONCLUSION: Paneth cell abnormalities in human subjects are associated with mucosal dysbiosis in the context of CD, and these changes are associated with alterations in oxidative phosphorylation, potentially in a feedback loop. FUNDING: The research was funded by Helmsley Charitable Trust (to T.S. Stappenbeck, R.J. Xavier, and D.P.B. McGovern), Crohn's and Colitis Foundation of America (to N.H. Salzman, T.S. Stappenbeck, R.J. Xavier, and C. Huttenhower), and Doris Duke Charitable Foundation grant 2014103 (to T.C. Liu).

Altered Virome and Bacterial Microbiome in Human Immunodeficiency Virus-Associated Acquired Immunodeficiency Syndrome.

Human immunodeficiency virus (HIV) infection is associated with increased intestinal translocation of microbial products and enteropathy as well as alterations in gut bacterial communities. However, whether the enteric virome contributes to this infection and resulting immunodeficiency remains unknown. We characterized the enteric virome and bacterial microbiome in a cohort of Ugandan patients, including HIV-uninfected or HIV-infected subjects and those either treated with anti-retroviral therapy (ART) or untreated. Low peripheral CD4 T cell counts were associated with an expansion of enteric adenovirus sequences and this increase was independent of ART treatment. Additionally, the enteric bacterial microbiome of patients with lower CD4 T counts exhibited reduced phylogenetic diversity and richness with specific bacteria showing differential abundance, including increases in Enterobacteriaceae, which have been associated with inflammation. Thus, immunodeficiency in progressive HIV infection is associated with alterations in the enteric virome and bacterial microbiome, which may contribute to AIDS-associated enteropathy and disease progression.

Homeostatic Control of Innate Lung Inflammation by Vici Syndrome Gene Epg5 and Additional Autophagy Genes Promotes Influenza Pathogenesis.

Mutations in the autophagy gene EPG5 are linked to the multisystem human disease Vici syndrome, which is characterized in part by pulmonary abnormalities, including recurrent infections. We found that Epg5-deficient mice exhibited elevated baseline innate immune cellular and cytokine-based lung inflammation and were resistant to lethal influenza virus infection. Lung transcriptomics, bone marrow transplantation experiments, and analysis of cellular cytokine expression indicated that Epg5 plays a role in lung physiology through its function in macrophages. Deletion of other autophagy genes including Atg14, Fip200, Atg5, and Atg7 in myeloid cells also led to elevated basal lung inflammation and influenza resistance. This suggests that Epg5 and other Atg genes function in macrophages to limit innate immune inflammation in the lung. Disruption of this normal homeostatic dampening of lung inflammation results in increased resistance to influenza, suggesting that normal homeostatic mechanisms that limit basal tissue inflammation support some infectious diseases.

Disease-specific alterations in the enteric virome in inflammatory bowel disease.

Decreases in the diversity of enteric bacterial populations are observed in patients with Crohn's disease (CD) and ulcerative colitis (UC). Less is known about the virome in these diseases. We show that the enteric virome is abnormal in CD and UC patients. In-depth analysis of preparations enriched for free virions in the intestine revealed that CD and UC were associated with a significant expansion of Caudovirales bacteriophages. The viromes of CD and UC patients were disease and cohort specific. Importantly, it did not appear that expansion and diversification of the enteric virome was secondary to changes in bacterial populations. These data support a model in which changes in the virome may contribute to intestinal inflammation and bacterial dysbiosis. We conclude that the virome is a candidate for contributing to, or being a biomarker for, human inflammatory bowel disease and speculate that the enteric virome may play a role in other diseases.

Irgm1 protects hematopoietic stem cells by negative regulation of IFN signaling.

The IFN-inducible immunity-related p47 GTPase Irgm1 has been linked to Crohn disease as well as susceptibility to tuberculosis. Previously we demonstrated that HSC quiescence and function are aberrant in mice lacking Irgm1. To investigate the molecular basis for these defects, we conducted microarray expression profiling of Irgm1-deficient HSCs. Cell-cycle and IFN-response genes are up-regulated in Irgm1(-/-) HSCs, consistent with dysregulated IFN signaling. To test the hypothesis that Irgm1 normally down-regulates IFN signaling in HSCs, we generated Irgm1(-/-)Ifngr1(-/-) and Irgm1(-/-)Stat1(-/-) double-knockout animals. Strikingly, hyperproliferation, self-renewal, and autophagy defects in Irgm1(-/-) HSCs were normalized in double-knockout animals. These defects were also abolished in Irgm1(-/-)Irgm3(-/-) double-knockout animals, indicating that Irgm1 may regulate Irgm3 activity. Furthermore, the number of HSCs was reduced in aged Irgm1(-/-) animals, suggesting that negative feedback inhibition of IFN signaling by Irgm1 is necessary to prevent hyperproliferation and depletion of the stem cell compartment. Collectively, our results indicate that Irgm1 is a powerful negative regulator of IFN-dependent stimulation in HSCs, with an essential role in preserving HSC number and function. The deleterious effects of excessive IFN signaling may explain how hematologic abnormalities arise in patients with inflammatory conditions.