Noah W. Palm

The microbiota produces thousands of potentially bioactive small molecules^1,2,3. High-throughput bioactivity screens of in vitro commensal cultures have exposed microbiota metabolites that shape host physiology by activating diverse G-protein-coupled receptors (GPCRs)^4,5,6,7. However, owing to technical limitations, the GPCRome-wide bioactivities of in vivo metabolomes, which result from complex diet–microorganism–host interactions, remain unclear. Here we used a multiplexed GPCR screening technology to assess GPCRome-wide bioactivities of 100 commensal strains grown in vivo in monoassociated germ-free mice or in vitro in bacterial culture medium. In vivo and in vitro commensal metabolomes exhibited distinct GPCR activation patterns due to (1) host-mediated metabolite degradation; (2) in vivo microbial metabolic reprogramming; and (3) biotransformation of dietary substrates. Notably, we found that multiple commensal strains produced acetylcholine (ACh) in vivo through the conversion of dietary choline, including select Bifidobacterium strains that dominate the microbiome in early life and a probiotic Pediococcus strain. Mechanistically, we identified and characterized the bacterial enzymes that mediate this biotransformation in Bifidobacterium breve and Pediococcus pentosaceus, and generated an isogenic mutant B. breve strain lacking ACh production. Mice colonized with ACh-producing B. breve exhibited enhanced intestinal immunoglobulin A (IgA) production, altered microbiota composition and increased resistance to enteric infection. These findings underscore the profound impacts of the in vivo environment on microbiota metabolism and reveal a diet–microbiome–host axis that strengthens mucosal immune defences and reinforces host–microbiota mutualism.

Most people in the USA manage their health by taking at least one prescription drug, and drugs classified as non-antibiotics can adversely affect the gut microbiome and disrupt intestinal homeostasis^1,2. Here we identify medications that are associated with an increased risk of gastrointestinal infections across a population cohort of more than one million individuals monitored over 15 years. Notably, the cardiac glycoside digoxin and other drugs identified in this epidemiological study are sufficient to alter the composition of the microbiome and the risk of infection with Salmonella enterica subsp. enterica serovar Typhimurium (S. Tm) in mice. The effect of digoxin treatment on S. Tm infection is transmissible through the microbiome, and characterization of this interaction highlights a digoxin-responsive β-defensin that alters the microbiome composition and consequent immune surveillance of the invading pathogen. Combining epidemiological and experimental approaches thus provides an opportunity to uncover drug–host–microbiome–pathogen interactions that increase the risk of infections in humans.

Gut microbes secrete specialized small molecules that broadly influence human physiology. Despite their potential significance, the variety of functional small molecules known in the gut is relatively limited. Here, we screened the supernatants from human fecal-derived bacterial cultures to explore their agonist effects on the human G protein-coupled receptors (GPCRs), melatonin receptor types 1A and 1B (MTNR1A, MTNR1B). Chemical analysis of the supernatant-soluble molecules of Clostridium sporogenes, a prominent gut commensal identified in the screen, led to the characterization of agonists for these two melatonin receptors. Specifically, through bioactivity-assisted isolation and characterization, we identified three small molecules, 1-3, including two previously uncharacterized metabolites, which were synthesized to confirm their structures. While the structure of 1 features a urea core symmetrically disubstituted with tryptamine moieties, 2 and 3 harbor a monomeric tryptamine functionalized with methyl carbamate and N-acetyl groups, respectively. These structural characterization efforts illuminated downstream functional consequences of tryptamine metabolism in C. sporogenes. Additional GPCR screening analyses revealed that 2 activates melatonin receptors and the purinergic P2RY11 receptor, whereas 1 serves as an agonist for the semiorphan receptor GPR55. Interestingly, 1 also exhibits significant inhibitory activity against inflammatory soluble epoxide hydrolase with a half-maximal inhibitory concentration of 420 nM. Single-cell RNA sequencing analysis of the gut tissue from mice orally treated with 1 relative to the solvent vehicle control revealed that 1 specifically decreased the frequency of GPR55- and granzyme K-expressing effector-like CD8 T cells in the intraepithelial lymphocyte population. Overall, this study broadens our understanding of tryptamine-derived signaling at the human-microbe interface.

Specialized immune cells that reside in tissues orchestrate diverse biological functions by communicating with parenchymal cells¹. The contribution of the innate immune compartment in the meninges and the central nervous system (CNS) is well-characterized; however, whether cells of the adaptive immune system reside in the brain and are involved in maintaining homeostasis is unclear^2,3,4. Here we show that the subfornical organ (SFO) of the brain is a nucleus for parenchymal αβ T cells in the steady-state brain in both mice and humans. Using unbiased transcriptomics, we show that these extravascular T cells in the brain are distinct from meningeal T cells: they secrete IFNγ robustly and express tissue-residence proteins such as CXCR6, which are required for their retention in the brain and for normal adaptive behaviour. These T cells are primed in the periphery by the microbiome, and traffic from the white adipose and gastrointestinal tissues to the brain. Once established, their numbers can be modulated by alterations to either the gut microbiota or the composition of adipose tissue. In summary, we find that CD4 T cells reside in the brain at steady state and are anatomically concentrated in the SFO in mice and humans; that they are transcriptionally and functionally distinct from meningeal T cells; and that they secrete IFNγ to maintain CNS homeostasis through homeostatic fat–brain and gut–brain axes.

Our current understanding of immunity to pathogens suggests that anatomic coupling of antigens with danger signals is a required feature for the formation of immune memory. However, in the context of pathogen-independent inflammation, the stringency of this anatomical coupling is unclear. Here, we demonstrate that multiple modes of skin injury were sufficient to induce a humoral response to antigens introduced in the gut. Skin damage induced a narrow subset of endocrine cytokines that were necessary and sufficient for the priming of antigens introduced at various distal tissues. Thus, in addition to “local priming” of antigen entering through damaged skin, there also exists another paradigm of “remote priming” where anatomical coupling is not essential because of the dissemination of damage-associated intermediaries. Our findings have implications for understanding the fundamental mechanisms of the formation of humoral memory with wide implications for diseases such as food allergy and in vaccinology.

Microbiota-mediated drug metabolism can affect pharmacological efficacy. Here we conducted a systematic comparative metabolomics investigation of drug metabolism modes by evaluating the impacts of human gut commensal bacteria on 127 G-protein-coupled receptor (GPCR)-targeted drugs. For the most extensively metabolized drugs in our screen, we elucidated both conventional and unconventional drug transformations and the corresponding activities of generated metabolites. Comparisons of drug metabolism by a gut microbial community versus individual species revealed both taxon intrinsic and collaborative processes that influenced the activity of the metabolized drugs against target GPCRs. We also observed iloperidone inactivation by generating unconventional metabolites. The human gut commensal bacteria mixture incorporated sulfur in the form of a thiophene motif, whereas Morganella morganii used a cascade reaction to incorporate amino-acid-derived tricyclic systems into the drug metabolites. Our results reveal a broad impact of human gut commensal bacteria on GPCR-targeted drug structures and activities through diverse microbiota-mediated biotransformations.

Chronic autoimmune diseases often lead to long-term sequelae and require lifelong immunosuppression because of an incomplete understanding of the triggers and drivers in genetically predisposed patients. Gut bacteria that escape the gut barrier, known as translocating gut pathobionts, have been implicated as instigators and perpetuators of extraintestinal autoimmune diseases in mice. The gut microbial contributions to autoimmunity in humans remain largely unclear, including whether specific pathological human adaptive immune responses are triggered by such pathobionts. Here, we show that the translocating pathobiont Enterococcus gallinarum can induce both human and mouse interferon-γ ⁺ T helper 17 (T _H 17) differentiation and immunoglobulin G3 (IgG3) subclass switch of anti– E. gallinarum RNA antibodies, which correlated with anti-human RNA autoantibody responses in patients with systemic lupus erythematosus (SLE) and autoimmune hepatitis, two extraintestinal autoimmune diseases. E. gallinarum RNA, but not human RNA, triggered Toll-like receptor 8 (TLR8), and TLR8-mediated human monocyte activation promoted human T _H 17 induction by E. gallinarum . Translocation of the pathobiont triggered increased anti-RNA autoantibody titers that correlated with renal autoimmune pathophysiology in murine gnotobiotic lupus models and with disease activity in patients with SLE. These studies elucidate cellular mechanisms of how a translocating gut pathobiont induces systemic human T cell– and B cell–dependent autoimmune responses and provide a framework for developing host- and microbiota-derived biomarkers and targeted therapies in autoimmune diseases.

The spirochete Borrelia burgdorferi causes Lyme disease. In some patients, an excessive, dysregulated proinflammatory immune response can develop in joints leading to persistent arthritis even after antibiotic therapy. In such patients, persistence of antigenic B. burgdorferi peptidoglycan (PG ^Bb ) fragments within joint tissues may contribute to immunopathogenesis pre- and post-antibiotic treatment. In live B. burgdorferi cells, the outer membrane shields the polymeric PG ^Bb sacculus from exposure to the immune system. However, unlike most diderm bacteria, B. burgdorferi releases PG ^Bb turnover products into its environment due to the absence of recycling activity. In this study, we identified the released PG ^Bb fragments using a mass spectrometry-based approach. By characterizing the L,D-carboxypeptidase activity of B. burgdorferi protein BB0605 (renamed DacA), we found that PG ^Bb turnover largely occurs at sites of PG ^Bb synthesis. In parallel, we demonstrated that the lytic transglycosylase activity associated with BB0259 (renamed MltS) releases PG ^Bb fragments with 1,6-anhydro bond on their N -acetylmuramyl residues. Stimulation of human cell lines with various synthetic PG ^Bb fragments revealed that 1,6-anhydromuramyl-containing PG ^Bb fragments are poor inducers of a NOD2-dependent immune response relative to their hydrated counterparts found in the polymeric PG ^Bb isolated from dead bacteria. We also showed that the activity of the human N -acetylmuramyl-L-alanine amidase PGLYRP2, which reduces the immunogenicity of PG ^Bb material, is low in joint (synovial) fluids relative to serum. Altogether, our findings suggest that MltS activity helps B. burgdorferi evade PG-based immune detection by NOD2 during growth despite shedding PG ^Bb fragments and that PG ^Bb -induced immunopathology likely results from host sensing of PG ^Bb material from dead (lysed) spirochetes. Additionally, our results suggest the possibility that natural variation in PGLYRP2 activity may contribute to differences in susceptibility to PG-induced inflammation across tissues and individuals. Author summary: During bacterial infection, the presence of peptidoglycan– a polymeric element of bacterial cell walls–triggers a host inflammatory response. Although generally protective during acute phases, inflammation, when chronic, can contribute to disease development. Recent work has suggested that the persistence of pro-inflammatory peptidoglycan derived from the Lyme disease spirochete Borrelia burgdorferi in joints may contribute to persistent arthritis in some patients despite appropriate antibiotic therapy. Interestingly, B. burgdorferi sheds peptidoglycan turnover products into the environment during growth. Here, we show that these shed products from live spirochetes are poor effectors of an immune response by the human NOD2 immune receptor due to the formation of an anhydro bond on the N -acetyl-muramic residue during peptidoglycan hydrolysis by a B. burgdorferi lytic transglycosylase. We also show that human N -acetylmuramyl-L-alanine amidase activity, which abrogates the NOD2-dependent response to the immunogenic peptidoglycan isolated from lysed B. burgdorferi cells, is low in human joint fluids relative to serum. Based on our findings, we propose that immunopathogenesis by peptidoglycan material more likely derives from lysed spirochetes (killed by an immune attack or antibiotics) than live ones and that the level of human peptidoglycan hydrolytic enzymes across tissues and individuals influences susceptibility to chronic inflammation.