Bacterial Sphingolipids Exacerbate Colitis by Inhibiting ILC3-derived IL-22 Production.

BACKGROUND & AIMS: Gut bacterial sphingolipids, primarily produced by Bacteroidetes, have dual roles as bacterial virulence factors and regulators of the host mucosal immune system, including regulatory T cells and invariant natural killer T cells. Patients with inflammatory bowel disease display altered sphingolipids profiles in fecal samples. However, how bacterial sphingolipids modulate mucosal homeostasis and regulate intestinal inflammation remains unclear. METHODS: We used dextran sodium sulfate (DSS)-induced colitis in mice monocolonized with Bacteroides fragilis strains expressing or lacking sphingolipids to assess the influence of bacterial sphingolipids on intestinal inflammation using transcriptional, protein, and cellular analyses. Colonic explant and organoid were used to study the function of bacterial sphingolipids. Host mucosal immune cells and cytokines were profiled and characterized using flow cytometry, enzyme-linked immunosorbent assay, and Western blot, and cytokine function in vivo was investigated by monoclonal antibody injection. RESULTS: B fragilis sphingolipids exacerbated intestinal inflammation. Mice monocolonized with B fragilis lacking sphingolipids exhibited less severe DSS-induced colitis. This amelioration of colitis was associated with increased production of interleukin (IL)-22 by ILC3. Mice colonized with B fragilis lacking sphingolipids following DSS treatment showed enhanced epithelial STAT3 activity, intestinal cell proliferation, and antimicrobial peptide production. Protection against DSS colitis associated with B fragilis lacking sphingolipids was reversed on IL22 blockade. Furthermore, bacterial sphingolipids restricted epithelial IL18 production following DSS treatment and interfered with IL22 production by a subset of ILC3 cells expressing both IL18R and major histocompatibility complex class II. CONCLUSIONS: B fragilis-derived sphingolipids exacerbate mucosal inflammation by impeding epithelial IL18 expression and concomitantly suppressing the production of IL22 by ILC3 cells.

Bile acids modified by the intestinal microbiota promote colorectal cancer growth by suppressing CD8+ T cell effector functions.

Concentrations of the secondary bile acid, deoxycholic acid (DCA), are aberrantly elevated in colorectal cancer (CRC) patients, but the consequences remain poorly understood. Here, we screened a library of gut microbiota-derived metabolites and identified DCA as a negative regulator for CD8+ T cell effector function. Mechanistically, DCA suppressed CD8+ T cell responses by targeting plasma membrane Ca2+ ATPase (PMCA) to inhibit Ca2+-nuclear factor of activated T cells (NFAT)2 signaling. In CRC patients, CD8+ T cell effector function negatively correlated with both DCA concentration and expression of a bacterial DCA biosynthetic gene. Bacteria harboring DCA biosynthetic genes suppressed CD8+ T cells effector function and promoted tumor growth in mice. This effect was abolished by disrupting bile acid metabolism via bile acid chelation, genetic ablation of bacterial DCA biosynthetic pathway, or specific bacteriophage. Our study demonstrated causation between microbial DCA metabolism and anti-tumor CD8+ T cell response in CRC, suggesting potential directions for anti-tumor therapy.

Gut complement induced by the microbiota combats pathogens and spares commensals.

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.

Microbiota-dependent regulation of costimulatory and coinhibitory pathways via innate immune sensors and implications for immunotherapy.

Our bodies are inhabited by trillions of microorganisms. The host immune system constantly interacts with the microbiota in barrier organs, including the intestines. Over decades, numerous studies have shown that our mucosal immune system is dynamically shaped by a variety of microbiota-derived signals. Elucidating the mediators of these interactions is an important step for understanding how the microbiota is linked to mucosal immune homeostasis and gut-associated diseases. Interestingly, the efficacy of cancer immunotherapies that manipulate costimulatory and coinhibitory pathways has been correlated with the gut microbiota. Moreover, adverse effects of these therapies in the gut are linked to dysregulation of the intestinal immune system. These findings suggest that costimulatory pathways in the immune system might serve as a bridge between the host immune system and the gut microbiota. Here, we review mechanisms by which commensal microorganisms signal immune cells and their potential impact on costimulation. We highlight how costimulatory pathways modulate the mucosal immune system through not only classical antigen-presenting cells but also innate lymphocytes, which are highly enriched in barrier organs. Finally, we discuss the adverse effects of immune checkpoint inhibitors in the gut and the possible relationship with the gut microbiota.

Functional and metagenomic level diversities of human gut symbiont-derived glycolipids.

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.

Gut microbial fatty acid isomerization modulates intraepithelial T cells.

The human gut microbiome constantly converts natural products derived from the host and diet into numerous bioactive metabolites1-3. Dietary fats are essential micronutrients that undergo lipolysis to release free fatty acids (FAs) for absorption in the small intestine4. Gut commensal bacteria modify some unsaturated FAs-for example, linoleic acid (LA)-into various intestinal FA isomers that regulate host metabolism and have anticarcinogenic properties5. 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+.

Targeting PD-L2-RGMb overcomes microbiome-related immunotherapy resistance.

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 mice1-6. Moreover, transplantation of faecal specimens from responders can improve the efficacy of anti-PD-1 therapy in patients with melanoma7,8. 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.

Kinetic and mechanistic diversity of intestinal immune homeostasis characterized by rapid removal of gut bacteria.

Symbiotic microbiota critically contribute to host immune homeostasis in effector cell-specific manner. For exclusion of microbial component, germ-free animals have been the gold standard method. However, total removal of the entire gut microbiota of an animal from birth significantly skews physiological development. On the other hand, removal of gut microbiota from conventional mice using oral antibiotics has its own limitations, especially lack of consistency and the requirement for long-term treatment period. Here, we introduce an improved regimen to quickly remove gut microbiota and to maintain sterility, that is well received by animals without refusal. Rapid and consistent exclusion of resident bacteria in the gut lumen revealed kinetic differences among colonic lymphocyte subsets, which cannot be observed with typical germ-free animal models. Furthermore, the proposed method distinguished the mechanism of microbiota contribution as a direct stimulus to capable effector cells and a homeostatic cue to maintain such cell types.

Microbiome induced complement synthesized in the gut protects against enteric infections.

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.

Nociceptor neurons direct goblet cells via a CGRP-RAMP1 axis to drive mucus production and gut barrier protection.

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.

Impact of the Host Microbiome on Vaccine Responsiveness: Lessons Learned and Future Perspective.

Vaccination shows high variability in the elicited immune responses among individuals and populations for reasons still poorly understood. An increasing number of studies is supporting the evidence that gut microbiota, along with other interplaying variables, is able to modulate both humoral and cellular responses to infection and vaccination. Importantly, vaccine immunogenicity is often suboptimal at the extremes of age and also in low- and middle-income countries (LMICs), where the microbiota is believed to have an important role on immune responses. Still, contrasting findings and lack of causal evidence are calling for sophisticated methodologies to be able to overcome scientific and technical challenges to better decipher the immunomodulatory role of microbiota. In this perspective, we briefly review the status of the vaccine field in relation to the microbiome and offer possible scientific approaches to better understand the impact of the host microbiome on vaccine responsiveness.

Host immunomodulatory lipids created by symbionts from dietary amino acids.

Small molecules derived from symbiotic microbiota critically contribute to intestinal immune maturation and regulation1. However, little is known about the molecular mechanisms that control immune development in the host-microbiota environment. Here, using a targeted lipidomic analysis and synthetic approach, we carried out a multifaceted investigation of immunomodulatory α-galactosylceramides from the human symbiont Bacteroides fragilis (BfaGCs). The characteristic terminal branching of BfaGCs is the result of incorporation of branched-chain amino acids taken up in the host gut by B. fragilis. A B. fragilis knockout strain that cannot metabolize branched-chain amino acids showed reduced branching in BfaGCs, and mice monocolonized with this mutant strain had impaired colonic natural killer T (NKT) cell regulation, implying structure-specific immunomodulatory activity. The sphinganine chain branching of BfaGCs is a critical determinant of NKT cell activation, which induces specific immunomodulatory gene expression signatures and effector functions. Co-crystal structure and affinity analyses of CD1d-BfaGC-NKT cell receptor complexes confirmed the interaction of BfaGCs as CD1d-restricted ligands. We present a structural and molecular-level paradigm of immunomodulatory control by interactions of endobiotic metabolites with diet, microbiota and the immune system.

Exploring the Gut-Brain Axis for the Control of CNS Inflammatory Demyelination: Immunomodulation by Bacteroides fragilis' Polysaccharide A.

The symbiotic relationship between animals and their resident microorganisms has profound effects on host immunity. The human microbiota comprises bacteria that reside in the gastrointestinal tract and are involved in a range of inflammatory and autoimmune diseases. The gut microbiota's immunomodulatory effects extend to extraintestinal tissues, including the central nervous system (CNS). Specific symbiotic antigens responsible for inducing immunoregulation have been isolated from different bacterial species. Polysaccharide A (PSA) of Bacteroides fragilis is an archetypical molecule for host-microbiota interactions. Studies have shown that PSA has beneficial effects in experimental disease models, including experimental autoimmune encephalomyelitis (EAE), the most widely used animal model for multiple sclerosis (MS). Furthermore, in vitro stimulation with PSA promotes an immunomodulatory phenotype in human T cells isolated from healthy and MS donors. In this review, we discuss the current understanding of the interactions between gut microbiota and the host in the context of CNS inflammatory demyelination, the immunomodulatory roles of gut symbionts. More specifically, we also discuss the immunomodulatory effects of B. fragilis PSA in the gut-brain axis and its therapeutic potential in MS. Elucidation of the molecular mechanisms responsible for the microbiota's impact on host physiology offers tremendous promise for discovering new therapies.

Harnessing Colon Chip Technology to Identify Commensal Bacteria That Promote Host Tolerance to Infection.

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.

Fiber Sets up the Battleground for Intestinal Prevotella.

In this issue of Cell Host & Microbe, Gálvez et al. employed metagenomic and phylogenetic analysis to systemically characterize murine Prevotella spp. Isolation of representative strains identified key fitness determinants, including arabinoxylan utilization loci, in a dominant murine colonizing strain and conserved in human gut microbiota, collectively facilitating the understanding of niches in the gut microbiota.

Commensal Microbiota Modulation of Natural Resistance to Virus Infection.

Interferon (IFN)-Is are crucial mediators of antiviral immunity and homeostatic immune system regulation. However, the source of IFN-I signaling under homeostatic conditions is unclear. We discovered that commensal microbes regulate the IFN-I response through induction of IFN-ß by colonic DCs. Moreover, the mechanism by which a specific commensal microbe induces IFN-ß was identified. Outer membrane (OM)-associated glycolipids of gut commensal microbes belonging to the Bacteroidetes phylum induce expression of IFN-ß. Using Bacteroides fragilis and its OM-associated polysaccharide A, we determined that IFN-ß expression was induced via TLR4-TRIF signaling. Antiviral activity of this purified microbial molecule against infection with either vesicular stomatitis virus (VSV) or influenza was demonstrated to be dependent on the induction of IFN-ß. In a murine VSV infection model, commensal-induced IFN-ß regulated natural resistance to virus infection. Due to the physiological importance of IFN-Is, discovery of an IFN-ß-inducing microbial molecule represents a potential approach for the treatment of some human diseases.

Transcriptional and proteomic insights into the host response in fatal COVID-19 cases.

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.

An Immunologic Mode of Multigenerational Transmission Governs a Gut Treg Setpoint.

At the species level, immunity depends on the selection and transmission of protective components of the immune system. A microbe-induced population of RORγ-expressing regulatory T cells (Tregs) is essential in controlling gut inflammation. We uncovered a non-genetic, non-epigenetic, non-microbial mode of transmission of their homeostatic setpoint. RORγ+ Treg proportions varied between inbred mouse strains, a trait transmitted by the mother during a tight age window after birth but stable for life, resistant to many microbial or cellular perturbations, then further transferred by females for multiple generations. RORγ+ Treg proportions negatively correlated with IgA production and coating of gut commensals, traits also subject to maternal transmission, in an immunoglobulin- and RORγ+ Treg-dependent manner. We propose a model based on a double-negative feedback loop, vertically transmitted via the entero-mammary axis. This immunologic mode of multi-generational transmission may provide adaptability and modulate the genetic tuning of gut immune responses and inflammatory disease susceptibility.