Optimization of prophylactic vaccine regimens to elicit strong, long-lasting immunity is an urgent need highlighted by the COVID-19 pandemic. Stronger vaccine immunogenicity is frequently reported in individuals living in high-income countries compared to individuals living in low- and middle-income countries. While numerous host genetic and immune factors may influence vaccine responses, geographic restrictions to vaccine effectiveness may also be influenced by the intestinal microbiota, which modulates host immune systems. However, the potential role of the gut microbiota on responses to HIV-1 vaccines has not yet been explored. We analyzed the bacteriome by targeted 16S sequencing and the virome by virus-like particle sequencing of 154 fecal samples collected from healthy individuals in Uganda, Rwanda, and the United States early (week 2) and late (week 26) after vaccination with multivalent adenovirus serotype 26 (Ad26)-vectored mosaic HIV-1 vaccines. Vaccination did not affect the enteric bacteriome or virome regardless of geographic location. However, geography was the major driver of microbiota differences within this cohort. Differences in overall bacterial and viral diversity and in specific microbial taxa, including Bacteroidota and Bacillota, between participants from the United States and East African countries correlated with differential immune responses, including specific antibody titers, antibody functionality, and cellular immune responses to vaccination regimens. These findings support the microbiota as a putative modifier of vaccine immunogenicity.IMPORTANCEOur research examined how gut bacteria might influence vaccine effectiveness in different parts of the world. We studied adults from the United States, Rwanda, and Uganda who received an experimental HIV vaccine. We found that participants from East Africa had more diverse gut bacteria than those from the United States, but their immune responses to the vaccine were weaker. This is the first study to directly show this relationship between higher gut bacterial diversity and reduced vaccine effectiveness in the same group of people. We also identified specific types of bacteria that were linked to either stronger or weaker immune responses. These findings are particularly relevant now as we use vaccines globally to fight diseases like COVID-19, as they suggest that regional differences in gut bacteria Bacteroidota and Bacillota might help explain why vaccines work better in some places than others. This could inform how we design and test future vaccines.

Oral antibiotics can predispose to joint inflammation, but this phenomenon remains poorly understood. Here, we leverage mouse models of alphavirus-induced arthritis to investigate the roles of gut commensals, metabolites, and host immune mechanisms in promoting musculoskeletal inflammation. Mice treated with a short course of oral antibiotics exhibited worsened arthritis after chikungunya (CHIKV) or Mayaro virus infections. This phenotype was associated with loss of short-chain fatty acids (SCFAs), greater intestinal permeability, and activation of gut-associated immune cells and required TLR4 signaling, MyD88 expression, monocytes, antigen-specific and bystander CD4 + T cells, and proinflammatory cytokines. Administration of exogenous SCFAs or colonization of mice with bacterial species that generate SCFAs mitigated CHIKV-induced joint inflammation. scRNA-seq revealed that gut-derived SCFAs ameliorate the inflammatory phenotype of synovial CD4 + T cells, infiltrating monocytes, and resident osteoclast-like cells. Thus, antibiotic-triggered gut dysbiosis exacerbates alphavirus arthritis by shaping the inflammatory profile of both infiltrating and resident immune cells in joint tissues.

Background & Aims:Rare cases of non-hepatotropic virus (NHV) infection in humans can cause severe hepatitis and even acute liver failure. Clinically relevant animal models of NHV-induced hepatitis are limited, contributing to the incomplete understanding of pathological mechanisms. Murine norovirus (MNV) elicits hepatosplenomegaly in mice lacking the antiviral immune effector Signal Transducer and Activator of Transcription-1 (STAT1), providing a model to investigate mechanisms of NHV-induced hepatic pathology. Methods:STAT1-sufficient and -deficient (Stat1 Het, Stat1 KO) littermates infected intravenously (i.v.) with MNV strain CR6 were assessed for hepatic inflammation and viral burden. Cell types and molecular pathways associated with hepatic pathology in CR6-infected Stat1 KO mice were identified by flow cytometry and RNAseq of liver tissue. The relative importance of hematopoietic vs non-hematopoietic expression of STAT1 in restricting CR6 replication and maintaining tissue homeostasis was assessed in bone marrow chimeras. Results:MNV CR6 Stat1 KO mice developed severe hepatitis with patchy hepatocellular necrosis and localized enrichment of CR6-infected myeloid cells, particularly macrophages. Gene set enrichment analysis (GSEA) of hepatic biopsies isolated from CR6-infected Stat1 KO mice suggested dysregulated myeloid cell activation and indicated similarities between murine and human hepatic pathologies. STAT1 expression in hematopoietic cells was protective against hepatic viral dissemination, but hematopoietic STAT1-deficiency permitted persistent hepatic MNV infection, facilitating dysregulated myeloid cell activation and hepatic fibrosis. Conclusions:These results demonstrate that the role of STAT1 extends beyond restricting MNV dissemination and suggest that STAT1-dependent regulation of myeloid cell activation prevents acute hepatic necroinflammation and secondary fibrosis. This model of MNV-induced hepatitis may prove valuable in elucidating mechanisms of rare clinical complications.

Human norovirus is the leading cause of viral gastroenteritis, yet effective vaccines and therapeutics remain elusive. Using murine norovirus as a model, we found that mucosal immunoglobulin A (IgA) is both necessary and sufficient for protection against infection, whereas CD8 + T cells are dispensable. Robust intestinal IgA production requires at least 4 weeks of enteric infection, consistent with kinetics of human norovirus RNA clearance. Systemic vaccination elicits high titers of neutralizing serum IgG but fails to prevent enteric norovirus infection, phenocopying a recent human norovirus vaccine failure. In contrast, prophylactic delivery of dimeric anti-norovirus IgA via mRNA lipid nanoparticles confers sterilizing immunity. Together, these findings define a critical role for mucosal IgA in norovirus protection and identify IgA-based treatments as a therapeutic approach for human norovirus.

Double-stranded RNA (dsRNA) is recognized by cellular receptors as a sign of viral infection, triggering the innate immune response. Increasing evidence shows that cellular dysregulation, for example in immune disorders and neurodegenerative diseases, can also lead to accumulation of endogenously produced dsRNA that stimulates a viral-like immune response. Additionally, dsRNA contamination in RNA therapeutics can lead to harmful side effects via a similar pathway. Despite the clinical relevance of dsRNA, reliable tools for its detection remain limited. At present, dsRNA detection relies almost exclusively on the monoclonal antibodies J2 and K1, which suffer from sequence bias and low sensitivity, limiting their reliability. To address this challenge, we aimed to repurpose naturally occurring dsRNA-binding domains (dsRBDs) to produce reliable, pan-specific affinity reagents for dsRNA. We first systematically screened the dsRBDs of the three human adenosine deaminases acting on RNA (ADARs). This analysis identified ADAR3 dsRBDs as promising candidates due to their reduced sequence dependence compared to the dsRBDs of ADAR1 and ADAR2. We then engineered ADAR3-derived dsRBD constructs having varying linker lengths and domain combinations, allowing us to specifically vary the length cutoff of dsRNA detected, thus creating dsRNA accumulation detected by ADAR3 RBDs (dsRADAR) affinity reagents. Finally, we demonstrate the superior performance of dsRADAR over currently available dsRNA antibodies in a cell model of viral infection and a tissue model of gastric inflammation. Together, dsRADAR provides a sensitive and reliable approach for imaging and quantifying diverse dsRNA structures in a variety of biological contexts. Graphic Abstract:

The gut virome is a complex ecosystem characterized by the interplay of diverse viral entities, predominantly bacteriophages and eukaryotic viruses. The gut virome has a critical role in human health by shaping microbial community profiles, modulating host immunity and influencing metabolic processes. Different viral metagenomics approaches have revealed the remarkable diversity of the gut virome, showing individual-specific patterns that evolve over time and adapt dynamically to environmental factors. Perturbations in this community are increasingly associated with chronic immune and inflammatory conditions, metabolic disorders and neurological conditions, highlighting its potential as a diagnostic biomarker and therapeutic target. The early-life gut virome is particularly influential in establishing lifelong health trajectories through its interactions with diet, immune pathways and others, thereby contributing to inflammatory and metabolic regulation. This Review synthesizes current knowledge of gut virome composition, dynamics and functional relevance, critically evaluating evidence distinguishing causal from correlative roles in disease pathogenesis. The interactions of the virome with other microbiome components and host immunity are examined, and emerging translational applications, including phage therapy and biomarker development, are discussed. Integrating these insights while acknowledging methodological challenges provides a comprehensive framework for understanding the complex roles of the gut virome in health and disease. The gut virome is a complex ecosystem and has a critical role in human health. This Review outlines gut virome composition and functional relevance, and its role in human health and disease. Methodological challenges in advancing our knowledge of the gut virome are also discussed.

Paramyxoviruses are significant human and animal pathogens that include mumps virus (MuV), Newcastle disease virus (NDV) and the murine parainfluenza virus Sendai (SeV). Despite their importance, few host factors implicated in paramyxovirus infection are known. Using a recombinant SeV expressing destabilized eGFP (rSeVCdseGFP) in a loss-of-function CRISPR screen, we identified the CMP-sialic acid transporter (CST) gene SLC35A1 and the UDP-galactose transporter (UGT) gene SLC35A2 as essential for paramyxovirus infection. As expected, SLC35A1 knockout (KO) cells showed drastic reduction in infections with SeV, NDV and MuV due to the lack of cell surface sialic acids receptors. However, SLC35A2 KO cells revealed unknown critical roles for this factor in virus-cell and cell-to-cell fusion events for the different paramyxoviruses. While UGT was essential for virus-cell fusion during SeV entry to the cell, it was not required for NDV or MuV entry. Importantly, UGT promoted the formation of syncytia during MuV infection, suggesting a role in cell-to-cell virus spread. Our findings demonstrate that paramyxoviruses can bind to or enter A549 cells in the absence of canonical galactose-bound sialic-acid decorations and show that UGT facilitates paramyxovirus fusion processes involved in entry and spread.

Background and Aims:In inflammatory bowel disease, protein misfolding in the endoplasmic reticulum (ER) potentiates epithelial barrier dysfunction and impairs mucosal healing. Tauroursodeoxycholic acid (TUDCA), a naturally occurring bile acid, acts as a chemical chaperone to reduce protein aggregation and colitis severity in preclinical models. We conducted an open label trial evaluating oral TUDCA as therapy in patients with active ulcerative colitis (UC). Methods:Patients with moderate-to-severely active UC (Mayo score ≥6, endoscopic subscore ≥1) received oral TUDCA at 1.75 or 2 g/day for 6 weeks. Exclusion criteria included known hepatic disorders or change in UC therapy within 60 days. Clinical disease activity questionnaires, endoscopy with biopsy, blood, and stool were collected at enrollment and after 6 weeks. The primary outcome measure was change in ER stress markers while safety, tolerability and change in UC disease activity were secondary outcomes. Results:Thirteen participants completed the study with eleven evaluable for clinical response. TUDCA was well-tolerated with transient dyspepsia being the most common side effect. Mucosal biopsies revealed significant reductions in ER stress and inflammation as well as an increase in markers of epithelial restitution. Clinical, endoscopic, and histologic disease activity were significantly improved at week 6 (mean total Mayo Score: 9 to 4.5, p<0.001). Conclusions:Six weeks of oral TUDCA treatment was well-tolerated in patients with active ulcerative colitis and promoted mucosal healing, lessened ER stress, and reduced clinical disease activity. A randomized controlled trial of adjunctive TUDCA therapy in patients with UC is warranted. Trial registration:ClinicalTrials.gov (NCT04114292).

Neonatal mice are frequently used to model diseases that affect human infants. Microbial community composition has been shown to impact disease progression in these models. Despite this, the maturation of the early-life murine microbiome has not been well-characterized. We address this gap by characterizing the assembly of the bacterial microbiota of C57BL/6 and BALB/c litters from birth to adulthood across multiple independent litters. The fecal microbiome of young pups is dominated by only a few pioneering bacterial taxa. These taxa are present at low levels in the microbiota of multiple maternal body sites, precluding a clear identification of maternal source. The pup microbiota begins diversifying after 14 days, coinciding with the beginning of coprophagy and the consumption of solid foods. Pup stool bacterial community composition and diversity are not significantly different from dams from day 21 onwards. Short-read shotgun sequencing-based metagenomic profiling of young pups enabled the assembly of metagenome-assembled genomes for strain-level analysis of these pioneer Ligilactobacillus, Streptococcus, and Proteus species. Assembly of the murine microbiome occurs over the first weeks of postnatal life and is largely complete by day 21. This detailed view of bacterial community development across multiple commonly employed mouse strains informs experimental design, allowing researchers to better target interventions before, during, or after the maturation of the bacterial microbiota. The source of pioneer bacterial strains appears heterogeneous, as the most abundant taxa identified in young pup stool were found at low levels across multiple maternal body sites, suggesting diverse routes for seeding of the murine microbiome.

Mouse models are vital tools for discerning the relative contributions of host and microbial genetics to disease, often requiring the transplantation of microbiota between different mouse strains. Transfer methods include antibiotic treatment of recipients and colonization using either co-housing with donors or the transplantation of fecal or cecal donor material. However, the efficiency and dynamics of these methods in reconstituting recipients with donor microbes is not well understood. We thus directly compared co-housing, fecal transplantation, and cecal transplantation methods. Donor mice from Taconic Biosciences, possessing distinct microbial communities, served as the microbial source for recipient mice from Jackson Laboratories, which were treated with antibiotics to disrupt their native microbiota. We monitored bacterial and viral populations longitudinally over the course of antibiotics treatment and reconstitution using 16S rRNA gene sequencing, quantitative PCR, and shotgun sequencing of viral-like particles. As expected, antibiotic treatment rapidly depleted microbial biomass and diversity, with slow and incomplete natural recovery of the microbiota in non-transplanted control mice. While all transfer methods reconstituted recipient mice with donor microbiota, co-housing achieved this more rapidly for both bacterial and viral communities. This study provides valuable insights into microbial transfer methods, enhancing reproducibility and informing best practices for microbiota transplantation in mouse models.

Although oral antibiotics can predispose to joint inflammation, this phenomenon remains poorly understood. Here, we leverage mouse models of alphavirus-induced arthritis to investigate the roles of gut commensals, metabolites, and host immune mechanisms in promoting musculoskeletal inflammation. Mice treated with a short course of oral antibiotics exhibited worsened arthritis after chikungunya (CHIKV) or Mayaro virus infections. This phenotype was associated with loss of short chain fatty acids (SCFA), greater intestinal permeability, and activation of gut-associated immune cells, and required TLR4 signaling, MyD88 expression, monocytes, antigen-specific and bystander CD4 + T cells, and pro-inflammatory cytokines. Administration of exogenous SCFA or colonization of mice with bacterial species that generate SCFA mitigated CHIKV-induced joint inflammation. Single-cell RNA sequencing revealed that gut-derived SCFA ameliorate the inflammatory phenotype of synovial CD4 + T cells, infiltrating monocytes, and resident osteoclast-like cells. Thus, antibiotic-triggered gut dysbiosis exacerbates alphavirus arthritis by shaping the inflammatory profile of both infiltrating and resident immune cells in joint tissues.

Autophagy is an essential cellular process which functions to maintain homeostasis in response to stressors such as starvation or infection. Here, we report that a subset of autophagy factors including ATG-3 play an antiviral role in Orsay virus infection of Caenorhabditis elegans. Orsay virus infection does not modulate autophagic flux, and re-feeding after starvation limits Orsay virus infection and blocks autophagic flux, suggesting that the role of ATG-3 in Orsay virus susceptibility is independent of its role in maintaining autophagic flux. atg-3 mutants phenocopy rde-1 mutants, which have a defect in RNA interference (RNAi), in susceptibility to Orsay virus infection and transcriptional response to infection. However, atg-3 mutants do not exhibit defects in RNAi. Additionally, atg-3 limits viral infection at a post-entry step, similar to rde-1 mutants. Differential expression analysis using RNA sequencing revealed that antiviral sqt-2, which encodes a collagen trimer protein, is depleted in naïve and infected atg-3 mutants, as well as in infected WT animals, as are numerous other collagen genes. These data suggest that ATG-3 has a role in collagen organization pathways that function in antiviral defense in C. elegans.

SummaryTo examine the effect of early-life infection withCryptosporidium parvumon the development of oral tolerance, we developed a low-dose infection model in neonatal mice.C. parvuminfection in neonatal mice results in immunopathology in the colon. IL-1β released duringC. parvuminfection blocked the formation of colonic goblet cell associated antigen passages, which normally serve as a conduit for antigen uptake and development of peripheral regulatory T cells (pTregs), responsible for long-term oral tolerance. Following infection withC. parvum, adoptively transferred OT-II cells, which respond to ovalbumin (ova), developed reduced frequency of Foxp3+Rorγt+cells in mesenteric lymph nodes with an expansion of TH1-like Tregs in the colon. The altered pTreg profile was accompanied by a strong TH1 immune response and robust IgG2c antibody responses to orally administered ova. Our findings suggest that intestinal inflammation and altered pTreg development leads to loss of oral tolerance during early life infection withC. parvum.

The gut microbiota has emerged as a pivotal regulator of host inflammatory processes after traumatic brain injury (TBI). However, the mechanisms by which the gut microbiota communicates to the brain in TBI are still under investigation. We previously reported that gut microbiota depletion (GMD) using antibiotics after TBI resulted in increased microglial activation, reduced neurogenesis, and reduced T cell infiltration. In the present study, we have demonstrated that intestinal T cells contribute to the pool of cells infiltrating the brain after TBI. Depletion or genetic deletion of T cells before injury reversed GMD induced reductions in post-TBI neurogenesis. Short-chain fatty acid supplementation increased T regulatory and T helper1 cell infiltration to the brain along with restoring neurogenesis and microglia activation after TBI with GMD. These data suggest that T cell subsets are essential cellular mediators by which the gut microbiota modulates TBI pathogenesis, a finding with important therapeutic implications.

The associations of the gut microbiome and virome with human health and disease are increasingly numerous and clear. The mechanistic roles of bacteriophages (phages) in the microbiome, however, are especially unclear, as their cultivation is exceedingly difficult and their diversity so immense. We use viral tagging (VT), a technique wherein fluorescently stained uncultivated viruses are allowed to adsorb to host cells and then host cells are singly sorted. This method identifies interacting phage-bacteria pairs to better sample and characterize the phages in human stool samples from healthy and inflammatory bowel disease (IBD)-affected patients. First, we apply VT to uncultivated bacteria from a healthy human sample, demonstrating far-reaching ability to observe diverse bacteria and phages alike. We also use VT with a cultured Faecalibacterium prausnitzii isolate, a bacterial host of interest due to its anti-inflammatory effects and strong negative correlation with IBD. Comparing VT with virome sequencing and phage identification from single amplified genomes shows that it is a practical technique for phage discovery, especially when it is used to focus on individual bacterial cultivars for which genomes have been sequenced. VT can detect phages so rare as to be undetectable in standard virome sequencing, which is biased toward the most abundant phage species even at high sequencing depth. Remarkably, VT also identified novel prophage integration events in F. prausnitzii, demonstrating that VT interactions can extend beyond the level of surface attachment and constitute active infection events. In total, VT identified at least 328 unique and highly diverse phage-host pairs, almost all of which are entirely uncharacterized, and several phages that are differentially abundant in IBD patients compared to healthy controls. Taken together, we show that VT is an extremely powerful tool to move beyond the cultivation and abundance biases inherent to current techniques and suggest that the phage-host pairs identified by VT here are crucial first step to enable future mechanistic studies of phage-bacteria-human interactions.

BACKGROUND:The intestinal microbiota regulates normal brain physiology and the pathogenesis of several neurological disorders. While prior studies suggested that this operates through immune cells, the underlying mechanisms remain unclear. Leveraging 2 well-characterized murine models of low-grade glioma occurring in the setting of the neurofibromatosis type 1 (NF1) cancer predisposition syndrome, we sought to determine the impact of the gut microbiome on optic glioma progression. METHODS:Neurofibromatosis type 1 (Nf1)-mutant mice genetically engineered to develop optic pathway gliomas (Nf1OPG mice) by 3 months of age were reared under germ-free (GF) conditions, treated with specific cocktails of antibiotics, or given fecal matter transplants (FMTs). Intestinal microbial species were identified by 16S genotyping. Neutralizing transforming growth factor-beta (TGFβ) antibodies were delivered systemically, while in vitro experiments used isolated murine microglia and T cells. Single-cell RNA sequencing analysis was performed using established methods. RESULTS:Nf1 OPG mice raised in a GF environment or postnatally treated with vancomycin did not harbor optic gliomas or exhibit OPG-induced retinal nerve fiber layer thinning, which was reversed following conventionally raised mouse FMT or colonization with Bacteroides species. Moreover, this intestinal microbiota-regulated gliomagenesis was mediated by circulating TGFβ, such that systemic TGFβ neutralization reduced Nf1-OPG growth. TGFβ was shown to act on tumor-associated monocytes to induce Ccl3 expression and recruit CD8+ T cells necessary for glioma growth. CONCLUSIONS:Taken together, these findings establish, for the first time, a mechanistic relationship between Bacteroides in the intestinal microbiome and NF1-LGG pathobiology, suggesting both future predictive risk assessment strategies and therapeutic opportunities.

Human astroviruses (HAstVs) are a global cause of pediatric gastroenteritis and can cause disseminated infection in immunocompromised hosts. Murine astrovirus (muAstV) causes acute asymptomatic infections in immunocompetent mice and chronic infection in immunodeficient models and has provided important insights into AstV pathogenesis in vivo. MuAstV can protect immunodeficient mice from other enteric viruses via robust induction of antiviral cytokine interferon-lambda (IFN-λ), with recent findings implicating goblet cells and enterocytes as both cell types infected by muAstV as well as potential sources of IFN-λ in vivo. However, the viral sensing pathways that regulate induction of IFN-λ, as well as the specific activity of IFN-λ in viral control and regulation of cellular tropism, remain to be defined for muAstV. Here, we leveraged single-cell RNA sequencing (scRNA-seq) to provide additional evidence of muAstV tropism for multiple intestinal epithelial cells (IECs) including goblet cells and diverse enterocyte types. Significantly, enterocytes appear to serve as the dominant source of IFN-λ in response to muAstV infection. Moreover, we report that this induction of IFN-λ in response to muAstV is regulated by the MDA5-MAVS pathway, with enhanced infection and expansion of infected cell numbers observed when either Mda5 or Mavs is disrupted. Leveraging mice conditionally deficient for Ifnlr1 or Mavs, we characterized the specific cellular requirements for IFN-λ signaling and MAVS to control muAstV infection. While IFN-λ signaling acts predominantly on secretory cells, including goblet cells, to limit muAstV infection, we found that IECs broadly require MAVS to control muAstV but with no specific IEC type implicated, suggesting a potential synergistic requirement across IECs. Our study highlights the MDA5-MAVS-IFN-λ signaling axis as critical for regulation of muAstV infection, providing further insights into the innate immune regulation of this enteric pathogen

Paramyxoviruses are significant human and animal pathogens that include mumps virus (MuV), Newcastle disease virus (NDV) and the murine parainfluenza virus Sendai (SeV). Despite their importance, few host factors implicated in paramyxovirus infection are known. Using a recombinant SeV expressing destabilized eGFP (rSeVCdseGFP) in a loss-of-function CRISPR screen, we identified the CMP-sialic acid transporter (CST) gene SLC35A1 and the UDP-galactose transporter (UGT) gene SLC35A2 as essential for paramyxovirus infection. As expected, SLC35A1 knockout (KO) cells showed drastic reduction in infections with SeV, NDV and MuV due to the lack of cell surface sialic acids receptors. However, SLC35A2 KO cells revealed unknown critical roles for this factor in virus-cell and cell-to-cell fusion events for the different paramyxoviruses. While UGT was essential for virus-cell fusion during SeV entry to the cell, it was not required for NDV or MuV entry. Importantly, UGT promoted the formation of syncytia during MuV infection, suggesting a role in cell-to-cell virus spread. Our findings demonstrate that paramyxoviruses can bind to or enter A549 cells in the absence of canonical galactose-bound sialic-acid decorations and show that UGT facilitates paramyxovirus fusion processes involved in entry and spread.

Rotavirus (RV) is the leading cause of life-threatening gastroenteritis in children under five despite effective RV vaccines availability, necessitating novel protective strategies for high-risk populations. Gut microbiota can modulate RV susceptibility and vaccine immunogenicity, yet underlying mechanisms are poorly characterized. Here, we demonstrate that microbiota-derived indole metabolites confer protection against RV infection. In healthy adults, higher fecal indole-3-acetic acid (IAA) and indole-3-propionic acid (IPA) levels associated with reduced fecal RV shedding following challenge. In human intestinal enteroids (HIEs), IAA pre-treatment inhibited human RV Wa G1P[8] replication via the aryl hydrocarbon receptor (AhR) pathway. In mice, treatment with AhR agonist indole-3-carbinol (I3C) significantly reduced fecal shedding of murine RV strain EDIM-Cambridge. Finally, Zambian infants with active RV infection exhibited lower fecal IAA and IPA levels than age-matched healthy controls. These findings demonstrate that microbiota-derived AhR ligands consistently inhibit RV infection across three experimental models and hold promise for protecting at-risk pediatric populations.

To examine the effect of early-life infection with Cryptosporidium parvum on the development of oral tolerance, we developed a low-dose infection model in neonatal mice. C. parvum infection in neonatal mice results in immunopathology in the colon. IL-1β released during C. parvum infection blocked the formation of colonic goblet cell associated antigen passages, which normally serve as a conduit for antigen uptake and development of peripheral regulatory T cells (pTregs), responsible for long-term oral tolerance. Following infection with C. parvum, adoptively transferred OT-II cells, which respond to ovalbumin (ova, a model food antigen), underwent expansion of TH1-like Tregs in the colon while the frequency of Foxp3+Rorγt+ cells in mesenteric lymph nodes decreased. The altered pTreg profile was accompanied by a strong TH1 immune response and robust IgG2c antibody responses to orally administered ova. Our findings suggest that intestinal inflammation and altered pTreg cells lead to loss of oral tolerance during early life infection with C. parvum.