Interferons and tuft cell numbers are bottlenecks for persistent murine norovirus infection.

Noroviruses (NoVs) are a leading cause of viral gastroenteritis. Despite global clinical relevance, our understanding of how host factors, such as antiviral cytokines interferons (IFNs), modulate NoV population dynamics is limited. Murine NoV (MNoV) is a tractable in vivo model for the study of host regulation of NoV. A persistent strain of MNoV, CR6, establishes a reservoir in intestinal tuft cells for chronic viral shedding in stool. However, the influence of host innate immunity and permissive cell numbers on viral population dynamics is an open question. We generated a pool of 20 different barcoded viruses (CR6BC) by inserting 6-nucleotide barcodes at the 3' position of the NS4 gene and used this pool as our viral inoculum for in vivo infections of different mouse lines. We found that over the course of persistent CR6 infection, shed virus was predominantly colon-derived, and viral barcode richness decreased over time irrespective of host immune status, suggesting that persistent infection involves a series of reinfection events. In mice lacking the IFN-λ receptor, intestinal barcode richness was enhanced, correlating with increased viral intestinal replication. IL-4 treatment, which increases tuft cell numbers, also increased barcode richness, indicating the abundance of permissive tuft cells to be a bottleneck during CR6 infection. In mice lacking type I IFN signaling (Ifnar1-/-) or all IFN signaling (Stat1-/-), barcode diversity at extraintestinal sites was dramatically increased, implicating different IFNs as critical bottlenecks at specific tissue sites. Of interest, extraintestinal barcodes were overlapping but distinct from intestinal barcodes, indicating that disseminated virus represents a distinct viral population than that replicating in the intestine. Barcoded viruses are a valuable tool to explore the influence of host factors on viral diversity in the context of establishment and maintenance of infection as well as dissemination and have provided important insights into how NoV infection proceeds in immunocompetent and immunocompromised hosts.

Regulation of host/pathogen interactions in the gastrointestinal tract by type I and III interferons.

Interferons (IFNs) are an integral component of the host innate immune response during viral infection. Recent advances in the study of type I and III IFNs suggest that though both types counteract viral infection, type III IFNs act predominantly at epithelial barrier sites, while type I IFNs drive systemic responses. The dynamics and specific roles of type I versus III IFNs have been studied in the context of infection by a variety of enteric pathogens, including reovirus, rotavirus, norovirus, astrovirus, and intestinal severe acute respiratory syndrome coronavirus 2, revealing shared patterns of regulatory influence. An important role for the gut microbiota, including the virome, in regulating homeostasis and priming of intestinal IFN responses has also recently emerged.

Antibiotic-associated neutropenia is marked by depletion of intestinal Lachnospiraceae in pediatric patients.

Hematologic side effects are associated with prolonged antibiotic exposure in up to 34% of patients. Neutropenia, reported in 10-15% of patients, increases the risk of sepsis and death. Murine studies have established a link between the intestinal microbiota and normal hematopoiesis. We sought to identify predisposing factors, presence of microbiota-derived metabolites, and changes in intestinal microbiota composition in otherwise healthy pediatric patients who developed neutropenia after prolonged courses of antibiotics. In this multi-center study, patients with infections requiring anticipated antibiotic treatment of two or more weeks were enrolled. Stool samples were obtained at the start and completion of antibiotics and at the time of neutropenia. We identified 10 patients who developed neutropenia on antibiotics and 29 controls matched for age, sex, race, and ethnicity. Clinical data demonstrated no association between neutropenia and type of infection or type of antibiotic used; however intensive care unit admission and length of therapy were associated with neutropenia. Reduced intestinal microbiome richness and decreased abundance of Lachnospiraceae family members correlated with neutropenia. Untargeted stool metabolomic profiling revealed several metabolites that were depleted exclusively in patients with neutropenia, including members of the urea cycle pathway, pyrimidine metabolism and fatty acid metabolism that are known to be produced by Lachnospiraceae . Our study confirms a relationship between intestinal microbiota disruption and abnormal hematopoiesis and identifies taxa and metabolites likely to contribute to microbiota-sustained hematopoiesis. As the microbiome is a key determinant of stem cell transplant and immunotherapy outcomes, these findings are likely to be of broad significance. Key Points: Neutropenia occurred in 17% of patients receiving prolonged antibiotic therapy.We found no association between neutropenia and type of infection or class of antibiotic used. Development of neutropenia after prolonged antibiotic treatment was associated with decreased prevalence of Lachnospiraceae and Lachnospiraceae metabolites such as citrulline.

A new piece of the microbiota pie: Mining 'omics for DNA inversion states.

In this issue of Cell Host & Microbe, Carasso et al. survey invertible DNA sites in Bacteroidales from patients with inflammatory bowel disease (IBD) and healthy control individuals. They identify complex functional interactions between Bacteroides fragilis, an invertible promoter, a capsular polysaccharide, a bacteriophage, and the human host. The establishment of 'omics approaches to characterizing genomic targets and functional roles is still required.

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.

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.

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.

Microbiota produced indole metabolites disrupt host cell mitochondrial energy production and inhibit Cryptosporidium parvum growth.

Cryptosporidiosis is a leading cause of life-threatening diarrhea in young children in resource-poor settings. Susceptibility rapidly declines with age, associated with changes in the microbiota. To explore microbial influences on susceptibility, we screened 85 microbiota- associated metabolites enriched in the adult gut for their effects on C. parvum growth in vitro. We identified eight inhibitory metabolites in three main classes: secondary bile salts/acids, a vitamin B 6 precursor, and indoles. Growth restriction of C. parvum by indoles did not depend on the host aryl hydrocarbon receptor (AhR) pathway. Instead, treatment impaired host mitochondrial function and reduced total cellular ATP, as well as directly reduced the membrane potential in the parasite mitosome, a degenerate mitochondria. Oral administration of indoles, or reconstitution of the gut microbiota with indole producing bacteria, delayed life cycle progression of the parasite in vitro and reduced severity of C. parvum infection in mice. Collectively, these findings indicate that microbiota metabolites contribute to colonization resistance to Cryptosporidium infection.

Microbiota-produced indole metabolites disrupt mitochondrial function and inhibit Cryptosporidium parvum growth.

Cryptosporidiosis is a leading cause of life-threatening diarrhea in young children in resource-poor settings. To explore microbial influences on susceptibility, we screened 85 microbiota-associated metabolites for their effects on Cryptosporidium parvum growth in vitro. We identify eight inhibitory metabolites in three main classes: secondary bile salts/acids, a vitamin B6 precursor, and indoles. Growth restriction of C. parvum by indoles does not depend on the host aryl hydrocarbon receptor (AhR) pathway. Instead, treatment impairs host mitochondrial function and reduces total cellular ATP, as well as directly reducing the membrane potential in the parasite mitosome, a degenerate mitochondria. Oral administration of indoles, or reconstitution of the gut microbiota with indole-producing bacteria, delays life cycle progression of the parasite in vitro and reduces the severity of C. parvum infection in mice. Collectively, these findings indicate that microbiota metabolites impair mitochondrial function and contribute to colonization resistance to Cryptosporidium infection.

Age-associated features of norovirus infection analysed in mice.

Norovirus (NoV) is the leading global cause of viral gastroenteritis. Young children bear the highest burden of disease and play a key role in viral transmission throughout the population. However, which host factors contribute to age-associated variability in NoV severity and shedding are not well-defined. The murine NoV (MNoV) strain CR6 causes persistent infection in adult mice and targets intestinal tuft cells. Here we find that natural transmission of CR6 from infected dams occurred only in juvenile mice. Direct oral CR6 inoculation of wild-type neonatal mice led to accumulation of viral RNA in the ileum and prolonged shedding in the stool that was replication-independent. This viral exposure induced both innate and adaptive immune responses including interferon-stimulated gene expression and MNoV-specific antibody responses. Interestingly, viral uptake depended on passive ileal absorption of luminal virus, a process blocked by cortisone acetate administration, which prevented ileal viral RNA accumulation. Neonates lacking interferon signalling in haematopoietic cells were susceptible to productive infection, viral dissemination and lethality, which depended on the canonical MNoV receptor CD300LF. Together, our findings reveal developmentally associated aspects of persistent MNoV infection, including distinct tissue and cellular tropism, mechanisms of interferon regulation and severity of infection in the absence of interferon signalling. These emphasize the importance of defining viral pathogenesis phenotypes across the developmental spectrum and highlight passive viral uptake as an important contributor to enteric infections in early life.

Genetic mapping reveals Pou2af2/OCA-T1-dependent tuning of tuft cell differentiation and intestinal type 2 immunity.

Chemosensory epithelial tuft cells contribute to innate immunity at barrier surfaces, but their differentiation from epithelial progenitors is not well understood. Here, we exploited differences between inbred mouse strains to identify an epithelium-intrinsic mechanism that regulates tuft cell differentiation and tunes innate type 2 immunity in the small intestine. Balb/cJ (Balb) mice had fewer intestinal tuft cells than C57BL/6J (B6) mice and failed to respond to the tuft cell ligand succinate. Most of this differential succinate response was determined by the 50- to 67-Mb interval of chromosome 9 (Chr9), such that congenic Balb mice carrying the B6 Chr9 interval had elevated baseline numbers of tuft cells and responded to succinate. The Chr9 locus includes Pou2af2, which encodes the protein OCA-T1, a transcriptional cofactor essential for tuft cell development. Epithelial crypts expressed a previously unannotated short isoform of Pou2af2 predicted to use a distinct transcriptional start site and encode a nonfunctional protein. Low tuft cell numbers and the resulting lack of succinate response in Balb mice were explained by a preferential expression of the short isoform and could be rescued by expression of full-length Pou2af2. Physiologically, Pou2af2 isoform usage tuned innate type 2 immunity in the small intestine. Balb mice maintained responsiveness to helminth pathogens while ignoring commensal Tritrichomonas protists and reducing norovirus burdens.

Infection of neonatal mice with the murine norovirus strain WU23 is a robust model to study norovirus pathogenesis.

Noroviruses are the leading cause of severe childhood diarrhea and foodborne disease worldwide. While they are a major cause of disease in all age groups, infections in the very young can be quite severe, with annual estimates of 50,000-200,000 fatalities in children under 5 years old. In spite of the remarkable disease burden associated with norovirus infections, very little is known about the pathogenic mechanisms underlying norovirus diarrhea, principally because of the lack of tractable small animal models. The development of the murine norovirus (MNV) model nearly two decades ago has facilitated progress in understanding host-norovirus interactions and norovirus strain variability. However, MNV strains tested thus far either do not cause intestinal disease or were isolated from extraintestinal tissue, raising concerns about translatability of research findings to human norovirus disease. Consequently, the field lacks a strong model of norovirus gastroenteritis. Here we provide a comprehensive characterization of a new small animal model system for the norovirus field that overcomes prior weaknesses. Specifically, we demonstrate that the WU23 MNV strain isolated from a mouse naturally presenting with diarrhea causes a transient reduction in weight gain and acute self-resolving diarrhea in neonatal mice of several inbred mouse lines. Moreover, our findings reveal that norovirus-induced diarrhea is associated with infection of subepithelial cells in the small intestine and systemic spread. Finally, type I interferons (IFNs) are critical to protect hosts from norovirus-induced intestinal disease whereas type III IFNs exacerbate diarrhea. This latter finding is consistent with other emerging data implicating type III IFNs in the exacerbation of some viral diseases. This new model system should enable a detailed investigation of norovirus disease mechanisms.

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.

Innate and Peripheral Immune Alterations after Traumatic Brain Injury Are Regulated in a Gut Microbiota-Dependent Manner in Mice.

Traumatic brain injury (TBI) patients are at high risk for disruption of the gut microbiome. Previously, we have demonstrated that broad-spectrum antibiotic exposure after TBI drastically alters the gut microbiota and modulates neuroinflammation, neurogenesis, and long-term fear memory. However, these data did not determine if the impact of antibiotic exposure on the brain's response to injury was mediated directly by antibiotics or indirectly via modulation of the gut microbiota. We designed two different approaches to address this knowledge gap. One was utilizing fecal microbiota transplantation (FMT) from control and antibiotic-treated mice (treated with vancomycin, neomycin, ampicillin, and metronidazole [VNAM]) into germ-free (GF) mice prior to injury, and the other was exposing specific pathogen-free (SPF) mice to a 2-week period of antibiotics prior to injury but discontinuing antibiotics 72 h prior to injury. GF mice receiving FMT from VNAM-treated mice (GF-VNAM) demonstrated reduced gut bacterial alpha diversity and richness compared with GF mice receiving control FMT. At 7 days post-injury, GF-VNAM had increased microglial activation, reduced infiltration of T cells, and decreased neurogenesis. Similarly, SPF mice exposed to antibiotics prior to but not after injury demonstrated similar alterations in neuroinflammation and neurogenesis compared with control mice. These data support our hypothesis implicating the gut microbiota as an important modulator of the neuroinflammatory process and neurogenesis after TBI and provide an exciting new approach for neuroprotective therapeutics for TBI.

Tuft-cell-intrinsic and -extrinsic mediators of norovirus tropism regulate viral immunity.

Murine norovirus (MNoV) is a model for human norovirus and for interrogating mechanisms of viral tropism and persistence. We previously demonstrated that the persistent strain MNoVCR6 infects tuft cells, which are dispensable for the non-persistent strain MNoVCW3. We now show that diverse MNoV strains require tuft cells for chronic enteric infection. We also demonstrate that interferon-λ (IFN-λ) acts directly on tuft cells to cure chronic MNoVCR6 infection and that type I and III IFNs signal together via STAT1 in tuft cells to restrict MNoVCW3 tropism. We then develop an enteroid model and find that MNoVCR6 and MNoVCW3 similarly infect tuft cells with equal IFN susceptibility, suggesting that IFN derived from non-epithelial cells signals on tuft cells in trans to restrict MNoVCW3 tropism. Thus, tuft cell tropism enables MNoV persistence and is determined by tuft cell-intrinsic factors (viral receptor expression) and -extrinsic factors (immunomodulatory signaling by non-epithelial cells).

Environmentally-triggered contraction of the norovirus virion determines diarrheagenic potential.

Noroviruses are the leading cause of severe childhood diarrhea and foodborne disease worldwide. While they are a major cause of disease in all age groups, infections in the very young can be quite severe with annual estimates of 50,000-200,000 fatalities in children under 5 years old. In spite of the remarkable disease burden associated with norovirus infections in people, very little is known about the pathogenic mechanisms underlying norovirus diarrhea, principally because of the lack of tractable small animal models. We recently demonstrated that wild-type neonatal mice are susceptible to murine norovirus (MNV)-induced acute self-resolving diarrhea in a time course mirroring human norovirus disease. Using this robust pathogenesis model system, we demonstrate that virulence is regulated by the responsiveness of the viral capsid to environmental cues that trigger contraction of the VP1 protruding (P) domain onto the particle shell, thus enhancing receptor binding and infectivity. The capacity of a given MNV strain to undergo this contraction positively correlates with infection of cells expressing low abundance of the virus receptor CD300lf, supporting a model whereby virion contraction triggers infection of CD300lflo cell types that are responsible for diarrhea induction. These findings directly link environmentally-influenced biophysical features with norovirus disease severity.

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.

Advances in understanding interferon-mediated immune responses to enteric viruses in intestinal organoids.

Interferons (IFN) are antiviral cytokines with critical roles in regulating pathogens at epithelial barriers, but their capacity to restrict human enteric viruses has been incompletely characterized in part due to challenges in cultivating some viruses in vitro, particularly human norovirus. Accordingly, advancements in the development of antiviral therapies and vaccine strategies for enteric viral infections have been similarly constrained. Currently emerging is the use of human intestinal enteroids (HIEs) to investigate mechanisms of human enteric viral pathogenesis. HIEs provide a unique opportunity to investigate host-virus interactions using an in vitro system that recapitulates the cellular complexity of the in vivo gastrointestinal epithelium. This approach permits the exploration of intestinal epithelial cell interactions with enteric viruses as well as the innate immune responses mediated by IFNs and IFN-stimulated genes. Here, we describe recent findings related to the production, signaling, and function of IFNs in the response to enteric viral infections, which will ultimately help to reveal important aspects of pathogenesis and facilitate the future development of therapeutics and vaccines.