In a recent Immunity paper, Lobel and colleagues integrate cross-cohort human microbiome meta-analyses with mechanistic studies in mice to uncover a dietary sulfur amino acid-microbiota-immune axis that enhances anti-tumor immunity. Sulfur amino acids expand the mucus-associated bacterium Mucispirillum schaedleri and trigger an NKT-cDC1 immune circuit.

Understanding the effect of gut microbiota function on immune checkpoint inhibitor (ICI) responses is urgently needed. Here, we integrated 821 fecal metagenomes from 12 datasets to identify differentially abundant genes and construct random forest models to predict ICI response. Gene markers demonstrated excellent predictive performance, with an average area under the curve (AUC) of 0.810. Pathway analyses revealed that quorum sensing (QS), ABC transporters, flagellar assembly, and amino acid biosynthesis pathways were enriched between responders (R) and non-responders (NRs) across 12 datasets. Furthermore, luxS, manA, fliC, and trpB exhibited consistent changes between R and NR across 12 datasets. Follow-up microbiota transplant experiments showed that inter-species signaling by different QS autoinducer-2 (AI-2) molecules (synthesized by luxS) can act on overall community function to promote the colonization of Akkermansia muciniphila, which is associated with superior ICI responses. Together, our data highlight the role of gut microbiota function in modulating the microbiome and antitumor immunity.

Gut symbiont Bacteroides fragilis can produce α-galactosylceramides (BfaGCs), sphingolipids with immunomodulatory functions that regulate colonic natural killer T (NKT) cells. However, their synthesis pathway and whether other human gut bacteria can produce them are unclear. Here, using genetic and metabolomic approaches, we mapped the sphingolipid biosynthesis pathway of B. fragilis and determined that α-galactosyltransferase (agcT) is essential and sufficient for colonic NKT cell regulation in mice. The distribution of agcT is restricted to only a few species among Bacteroidales. However, structural homologues of AgcT, such as BgsB, are widely distributed in gut microbiota and produce α-glycosyldiacylglycerols (aGDGs), particularly in Enterococcus. Analysis of infant gut metagenomes revealed that B. fragilis predominantly accounts for agcT abundance regardless of the cohort, but bgsB-encoding bacteria were taxonomically diverse and showed dynamic changes with host age. In addition, aGDGs from bgsB-encoding species act as antagonistic ligands for BfaGC-mediated NKT cell activation in vitro and in vivo. Our findings highlight the distinct natures of immunoactive glycolipid-producing symbionts and their relevance in the human gut microbiome, particularly in early life.

The gut microbiota plays a crucial role in maintaining intestinal homeostasis and influencing various aspects of host physiology, including immune function. Recent advances have highlighted the emerging importance of the complement system, particularly the C3 protein, as a key player in microbiota-host interactions. Traditionally known for its role in innate immunity, the complement system is now recognized for its interactions with microbial communities within the gut, where it promotes immune tolerance and protects against enteric infections. This Review explores the gut complement system as a possibly novel frontier in microbiota-host communication and examines its role in shaping microbial diversity, modulating inflammatory responses, and contributing to intestinal health. We discuss the dynamic interplay between microbiota-derived signals and complement activation, with a focus on the C3 protein and its effect on both the gut microbiome and host immune responses. Furthermore, we highlight the therapeutic potential of targeting complement pathways to restore microbial balance and treat diseases such as inflammatory bowel disease and colorectal cancer. By elucidating the functions of the gut complement system, we offer insights into its potential as a target for microbiota-based interventions aimed at restoring intestinal homeostasis and preventing disease.

The gut microbiota is a crucial regulator of anti-tumor immunity during immune checkpoint inhibitor therapy. Fecal transplantation can improve the efficacy of anti-PD-1 therapy in patients with melanoma. However, the increased efficacy from fecal transplants is variable, and the mechanisms by which gut bacteria promote anti-tumor immunity remain unclear. Here, we show that the gut microbiome and specific gut bacteria downregulate PD-L2 expression on dendritic cells and its binding partner, repulsive guidance molecule B (RGMb), to promote anti-tumor immunity. Moreover, antibody-mediated blockade of the PD-L2–RGMb pathway or conditional deletion of RGMb in T cells, combined with anti-PD-1 or anti-PD-L1 antibodies, promotes anti-tumor responses in multiple mouse tumor models that do not respond to anti-PD-1 or anti-PD-L1 alone in germ-free, antibiotic-treated mice and in mice with non-responder patient microbiota. We further isolated the bacterial molecules that downregulate PD-L2 and promote anti-tumor immunity. Mechanistically, the bacterial molecules suppress PD-L2 expression in a TLR4/MyD88-dependent manner. These studies identify the downregulation of the PD-L2–RGMb pathway as a specific mechanism by which the gut microbiota can promote responses to PD-1 checkpoint blockade. The results also define a potentially effective immunological strategy for treating patients who do not respond to PD-1 cancer immunotherapy. Quark Ventures (A31696), National Institutes of Health/National Cancer Institute (F32-CA247072) Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)

Commensal bacteria of the Bacteroidetes phylum are the primary producers of sphingolipids in the gut lumen. These lipids serve dual roles as bacterial virulence factors and regulators of the host mucosal immune system, including regulatory T cells and invariant natural killer T cells (iNKT). Sphingolipid composition is significantly altered in fecal samples of patients with inflammatory bowel disease (IBD). However, the specific mechanisms by which bacterial sphingolipids modulate mucosal homeostasis and regulate intestinal inflammation remain unclear. In this study, we investigated the impact of bacterial sphingolipids on intestinal inflammation by mono-colonizing mice with Bacteroides fragilis strains that either express or lack sphingolipids during DSS-induced colitis. We discovered that B. fragilis sphingolipids exacerbate intestinal inflammation. Mice mono-colonized with B. fragilis lacking sphingolipids exhibited less severe DSS-induced colitis. This amelioration of colitis was associated with increased production of interleukin-22 (IL-22) by innate lymphoid cell type 3 (ILC3). Consistent with the inhibitory effect of sphingolipids on IL-22 production, mice colonized with B. fragilis lacking sphingolipids showed enhanced epithelial STAT3 activity, intestinal cell proliferation, and antimicrobial peptide production following DSS treatment compared to those colonized with B. fragilis producing sphingolipids. Additionally, colitis severity in mice colonized with B. fragilis lacking sphingolipids was exacerbated upon IL-22 blockade. Furthermore, our study reveals that bacterial sphingolipids restrict epithelial IL-18 production following DSS treatment and interfere with IL-22 production by a subset of ILC3 cells expressing both the interleukin-18 receptor (IL-18R) and major histocompatibility complex class II (MHC II). These findings indicate that B. fragilis -derived sphingolipids exacerbate mucosal inflammation by impeding epithelial IL-18 expression, resulting in compromised production of IL-22 by ILC3 cells. Highlights B. fragilis -derived sphingolipids exacerbate DSS-induced colitis in mono-colonized C57BL/6 mice. B. fragilis -derived sphingolipids constrain ILC3-derived IL-22, leading to reduced colonic epithelial cell proliferation and compromised barrier function. B. fragilis -derived sphingolipids restrict epithelial NLRC4 inflammasome activation and IL-18 secretion. B. fragilis -derived sphingolipids modulate IL-22 production by IL18R + MHC II + ILC3s.

Canonically, the complement system is known for its rapid response to remove microbes in the bloodstream. However, relatively little is known about a functioning complement system on intestinal mucosal surfaces. Herein, we report the local synthesis of complement component 3 (C3) in the gut, primarily by stromal cells. C3 is expressed upon commensal colonization and is regulated by the composition of the microbiota in healthy humans and mice, leading to an individual host's specific luminal C3 levels. The absence of membrane attack complex (MAC) components in the gut ensures that C3 deposition does not result in the lysis of commensals. Pathogen infection triggers the immune system to recruit neutrophils to the infection site for pathogen clearance. Basal C3 levels directly correlate with protection against enteric infection. Our study reveals the gut complement system as an innate immune mechanism acting as a vigilant sentinel that combats pathogens and spares commensals.

The human gut microbiome constantly converts natural products derived from the host and diet into numerous bioactive metabolites 1 – 3 . Dietary fats are essential micronutrients that undergo lipolysis to release free fatty acids (FAs) for absorption in the small intestine 4 . Gut commensal bacteria modify some unsaturated FAs—for example, linoleic acid (LA)—into various intestinal FA isomers that regulate host metabolism and have anticarcinogenic properties 5 . However, little is known about how this diet–microorganism FA isomerization network affects the mucosal immune system of the host. Here we report that both dietary factors and microbial factors influence the level of gut LA isomers (conjugated LAs (CLAs)) and that CLAs in turn modulate a distinct population of CD4 + intraepithelial lymphocytes (IELs) that express CD8αα in the small intestine. Genetic abolition of FA isomerization pathways in individual gut symbionts significantly decreases the number of CD4 + CD8αα + IELs in gnotobiotic mice. Restoration of CLAs increases CD4 + CD8αα + IEL levels in the presence of the transcription factor hepatocyte nuclear factor 4γ (HNF4γ). Mechanistically, HNF4γ facilitates CD4 + CD8αα + IEL development by modulating interleukin-18 signalling. In mice, specific deletion of HNF4γ in T cells leads to early mortality from infection by intestinal pathogens. Our data reveal a new role for bacterial FA metabolic pathways in the control of host intraepithelial immunological homeostasis by modulating the relative number of CD4 + T cells that were CD4 + CD8αα + .

Supplementary Table 1 from The CHRNA5-CHRNA3-CHRNB4 Nicotinic Receptor Subunit Gene Cluster Affects Risk for Nicotine Dependence in African-Americans and in European-Americans

Bioactive metabolites produced by symbiotic microbiota causally impact host health and disease, nonetheless, incomplete functional annotation of genes as well as complexities and dynamic nature of microbiota make understanding species-level contribution in production and actions difficult. Alpha-galactosylceramides produced by Bacteroides fragilis (BfaGC) are one of the first modulators of colonic immune development, but biosynthetic pathways and the significance of the single species in the symbiont community still remained elusive. To address these questions at the microbiota level, we have investigated the lipidomic profiles of prominent gut symbionts and the metagenome-level landscape of responsible gene signatures in the human gut. We first elucidated the chemical diversity of sphingolipid biosynthesis pathways of major bacterial species. In addition to commonly shared ceramide backbone synthases showing two distinct intermediates, alpha-galactosyltransferase (agcT), the necessary and sufficient component for BfaGC production and host colonic type I natural killer T (NKT) cell regulation by B. fragilis, was characterized by forward-genetics based targeted metabolomic screenings. Phylogenetic analysis of agcT in human gut symbionts revealed that only a few ceramide producers have agcT and hence can produce aGCs, on the other hand, structurally conserved homologues of agcT are widely distributed among species lacking ceramides. Among them, alpha-glucosyl-diacylglycerol(aGlcDAG)-producing glycosyltransferases with conserved GT4-GT1 domains are one of the most prominent homologs in gut microbiota, represented by Enterococcus bgsB . Of interest, aGlcDAGs produced by bgsB can antagonize BfaGC-mediated activation of NKT cells, showing the opposite, lipid structure-specific actions to regulate host immune responses. Further metagenomic analysis of multiple human cohorts uncovered that the agcT gene signature is almost exclusively contributed by B. fragilis , regardless of age, geographical and health status, where the bgsB signature is contributed by >100 species, of which abundance of individual microbes is highly variable. Our results collectively showcase the diversities of gut microbiota producing biologically relevant metabolites in multiple layers-biosynthetic pathways, host immunomodulatory functions and microbiome-level landscapes in the host.

Canonically, complement is a serum-based host defense system that protects against systemic microbial invasion. Little is known about the production and function of complement components on mucosal surfaces. Here we show gut complement component 3 (C3), central to complement function, is regulated by the composition of the microbiota in healthy humans and mice, leading to host-specific gut C3 levels. Stromal cells in intestinal lymphoid follicles (LFs) are the predominant source of intestinal C3. During enteric infection with Citrobacter rodentium or enterohemorrhagic Escherichia coli, luminal C3 levels increase significantly and are required for protection. C. rodentium is remarkably more invasive to the gut epithelium of C3-deficient mice than of wild-type mice. In the gut, C3-mediated phagocytosis of C. rodentium functions to clear pathogens. Our study reveals that variations in gut microbiota determine individuals’ intestinal mucosal C3 levels, dominantly produced by LF stromal cells, which directly correlate with protection against enteric infection. Highlights Gut complement component 3 (C3) is induced by the microbiome in healthy humans and mice at a microbiota-specific level. Gut stromal cells located in intestinal lymphoid follicles are a major source of luminal C3 During enteric infections with Citrobacter rodentium or enterohemorrhagic Escherichia coli, gut luminal C3 levels increase and are required for protection. C. rodentium is significantly more invasive of the gut epithelium in C3-deficient mice when compared to WT mice. In the gut, C3-mediated opsonophagocytosis of C. rodentium functions to clear pathogens.

The gut microbiota is a crucial regulator of anti-tumour immunity during immune checkpoint inhibitor therapy. Several bacteria that promote an anti-tumour response to immune checkpoint inhibitors have been identified in mice(1-6). Moreover, transplantation of faecal specimens from responders can improve the efficacy of anti-PD-1 therapy in patients with melanoma(7,8). However, the increased efficacy from faecal transplants is variable and how gut bacteria promote anti-tumour immunity remains unclear. Here we show that the gut microbiome downregulates PD-L2 expression and its binding partner repulsive guidance molecule b (RGMb) to promote anti-tumour immunity and identify bacterial species that mediate this effect. PD-L1 and PD-L2 share PD-1 as a binding partner, but PD-L2 can also bind RGMb. We demonstrate that blockade of PD-L2-RGMb interactions can overcome microbiome-dependent resistance to PD-1 pathway inhibitors. Antibody-mediated blockade of the PD-L2-RGMb pathway or conditional deletion of RGMb in T cells combined with an anti-PD-1 or anti-PD-L1 antibody promotes anti-tumour responses in multiple mouse tumour models that do not respond to anti-PD-1 or anti-PD-L1 alone (germ-free mice, antibiotic-treated mice and even mice colonized with stool samples from a patient who did not respond to treatment). These studies identify downregulation of the PD-L2-RGMb pathway as a specific mechanism by which the gut microbiota can promote responses to PD-1 checkpoint blockade. The results also define a potentially effective immunological strategy for treating patients who do not respond to PD-1 cancer immunotherapy.

Supplementary Table 2 from The CHRNA5-CHRNA3-CHRNB4 Nicotinic Receptor Subunit Gene Cluster Affects Risk for Nicotine Dependence in African-Americans and in European-Americans

Neuroepithelial crosstalk is critical for gut physiology. However, the mechanisms by which sensory neurons communicate with epithelial cells to mediate gut barrier protection at homeostasis and during inflammation are not well understood. Here, we find that Nav1.8+CGRP+ nociceptor neurons are juxtaposed with and signal to intestinal goblet cells to drive mucus secretion and gut protection. Nociceptor ablation led to decreased mucus thickness and dysbiosis, while chemogenetic nociceptor activation or capsaicin treatment induced mucus growth. Mouse and human goblet cells expressed Ramp1, receptor for the neuropeptide CGRP. Nociceptors signal via the CGRP-Ramp1 pathway to induce rapid goblet cell emptying and mucus secretion. Notably, commensal microbes activated nociceptors to control homeostatic CGRP release. In the absence of nociceptors or epithelial Ramp1, mice showed increased epithelial stress and susceptibility to colitis. Conversely, CGRP administration protected nociceptor-ablated mice against colitis. Our findings demonstrate a neuron-goblet cell axis that orchestrates gut mucosal barrier protection.

Commensal bacteria within the gut microbiome contribute to development of host tolerance to infection, however, identifying specific microbes responsible for this response is difficult. Here we describe methods for developing microfluidic organ-on-a-chip models of small and large intestine lined with epithelial cells isolated from duodenal, jejunal, ileal, or colon organoids derived from wild type or transgenic mice. To focus on host-microbiome interactions, we carried out studies with the mouse Colon Chip and demonstrated that it can support co-culture with living gut microbiome and enable assessment of effects on epithelial adhesion, tight junctions, barrier function, mucus production, and cytokine release. Moreover, infection of the Colon Chips with the pathogenic bacterium, Salmonella typhimurium , resulted in epithelial detachment, decreased tight junction staining, and increased release of chemokines (CXCL1, CXCL2, and CCL20) that closely mimicked changes previously seen in mice. Symbiosis between microbiome bacteria and the intestinal epithelium was also recapitulated by populating Colon Chips with complex living mouse or human microbiome. By taking advantage of differences in the composition between complex microbiome samples cultured on each chip using 16s sequencing, we were able to identify Enterococcus faecium as a positive contributor to host tolerance, confirming past findings obtained in mouse experiments. Thus, mouse Intestine Chips may represent new experimental in vitro platforms for identifying particular bacterial strains that modulate host response to pathogens, as well as for investigating the cellular and molecular basis of host-microbe interactions.

In this issue of Cell Host & Microbe, Yeung et al. (2020) and Lin et al. (2020) expose laboratory mice to a natural environment and use immune and microbiota characterization to show that fungi promote more human-like immunity. These studies will help develop animal models to more accurately resemble human immune responses.

Coronavirus disease 2019 (COVID-19), the global pandemic caused by SARS-CoV-2, has resulted thus far in greater than 933,000 deaths worldwide; yet disease pathogenesis remains unclear. Clinical and immunological features of patients with COVID-19 have highlighted a potential role for changes in immune activity in regulating disease severity. However, little is known about the responses in human lung tissue, the primary site of infection. Here we show that pathways related to neutrophil activation and pulmonary fibrosis are among the major up-regulated transcriptional signatures in lung tissue obtained from patients who died of COVID-19 in Wuhan, China. Strikingly, the viral burden was low in all samples, which suggests that the patient deaths may be related to the host response rather than an active fulminant infection. Examination of the colonic transcriptome of these patients suggested that SARS-CoV-2 impacted host responses even at a site with no obvious pathogenesis. Further proteomics analysis validated our transcriptome findings and identified several key proteins, such as the SARS-CoV-2 entry-associated protease cathepsins B and L and the inflammatory response modulator S100A8/A9, that are highly expressed in fatal cases, revealing potential drug targets for COVID-19.

Although maternal antibodies protect newborn babies from infection(1,2), little is known about how protective antibodies are induced without prior pathogen exposure. Here we show that neonatal mice that lack the capacity to produce IgG are protected from infection with the enteric pathogen enterotoxigenic Escherichia coli by maternal natural IgG antibodies against the maternal microbiota when antibodies are delivered either across the placenta or through breast milk. By challenging pups that were fostered by either maternal antibody-sufficient or antibody-deficient dams, we found that IgG derived from breast milk was crucial for protection against mucosal disease induced by enterotoxigenic E. coli. IgG also provides protection against systemic infection by E. coli. Pups used the neonatal Fc receptor to transfer IgG from milk into serum. The maternal commensal microbiota can induce antibodies that recognize antigens expressed by enterotoxigenic E. coli and other Enterobacteriaceae species. Induction of maternal antibodies against a commensal Pantoea species confers protection against enterotoxigenic E. coli in pups. This role of the microbiota in eliciting protective antibodies to a specific neonatal pathogen represents an important host defence mechanism against infection in neonates. Neonatal mice are protected against infection with the enteric pathogen enterotoxigenic Escherichia coli by maternally derived natural antibodies as well as by maternal commensal microbiota that induce antibodies that recognize antigens expressed by Enterobacteriaceae.