Commensal-derived acetylcholine enhances mucosal immune education

Article Commensal-derived acetylcholine enhances mucosal immune education https://doi.org/10.1038/s41586-026-10592-7 Received: 28 November 2024 Accepted: 24 April 2026 Deguang Song1, Brianna Duncan-Lowey1, Varnica Khetrapal1, Randy Hamchand2, Tong Deng1, Hailey Brown1, Anchi Wu1, Anjelica L. Martin1, Kaylyn M. Bauer1, Yanyu Zhao2, Mytien T. Nguyen1, Nicole D. Sonnert1, Shana R. Leopold1, Qihao Wu2,3, Jason M. Crawford2 & Noah W. Palm1 ✉ Published online: xx xx xxxx Open access Check for updates The microbiota produces thousands of potentially bioactive small molecules1–3. 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)4–7. 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. The human gut microbiota produces thousands of small-molecule metabolites through de novo synthesis and through the biotransformation of chemicals derived from our diet or produced and secreted by the host8–10. These microbiota metabolites have essential roles in regulating diverse physiological functions, including energy metabolism, inflammation and mucosal barrier integrity11–13. Previous studies from our laboratory and others have used high-throughput GPCR screens of supernatants from commensal strains grown in vitro (that is, in vitro commensal metabolomes) to identify impacts of microbiota metabolites on host physiology4,6. However, the chemical diversity and physiological relevance of these in vitro metabolomes are constrained by the simplicity of the cultivation medium and the unavoidable artificiality of in vitro growth conditions. By contrast, in vivo microbiota metabolomes reflect the dynamic environment of the mammalian gastrointestinal tract, in which commensal microorganisms are exposed to complex environmental cues and precursor chemicals from the host, diet and other microorganisms. However, due to the requirement for large sample volumes, it is not possible to perform unbiased receptorome-wide GPCR screening of in vivo microbiota metabolomes using conventional approaches. To overcome this limitation, we recently developed a highly multiplexed GPCR screening platform (PRESTO-Salsa) that enables receptorome-wide assessment of nearly all non-olfactory GPCRs (>300 receptors) in a single well of a 96-well plate5 and used this technology to directly compare bioactivities of in vitro and in vivo commensal metabolomes. In vitro and in vivo metabolome screens The microbial compositions of commensal communities grown in vitro differ substantially from those grown in vivo, making it infeasible to compare complex microbial communities grown under these two conditions. Thus, to enable direct comparisons between in vitro and in vivo metabolomes, we compared metabolomes from individual strains grown either in standard culture medium or in monocolonized gnotobiotic mice. We selected 100 phylogenetically diverse microbiota strains from public and internal strain collections (Supplementary Table 1). Department of Immunobiology, Yale School of Medicine, New Haven, CT, USA. 2Institute of Biomolecular Design & Discovery and Department of Chemistry, Yale University, West Haven and New Haven, CT, USA. 3Department of Pharmaceutical Sciences, School of Pharmacy, University of Pittsburgh, Pittsburgh, PA, USA. ✉e-mail: 1 Nature | www.nature.com | 1 Article a 99 strains in vitro culture Metabolites enrichment 100 strains monocolonization Multiplexed GPCR screening Commensal metabolome library Acetonitrile:methanol:water T Ethyl acetate A C G C T T T T b A C G C T T Butanol A C G C T A C G C T A C G C T G A G A G A G A G A Number of activating bacteria 40 In vivo 20 0 In vitro 20 c Host-mediated metabolite degradation d e Succinate + M. morganii A. muciniphila Histamine CHRM4 CHRM3 Biotransformation of dietary substrate In vivo microbial metabolic reprogramming PEA+ CHRM2 HRH4 HRH3 DRD4 DRD2 DRD3 ADRA2C ADRA2B ADRA2A GPR120 SUCNR1 FPR2 FPR1 HTR4 HTR6 BDKBR1 HRH2 HCA3 ADRB3 ADRB1 ADRA1D ADRA1A CYSLTR2 MTNR1A MTNR1B 40 + Substrate deficiency 0 6,000 4,000 2,000 0.5343 F G M um e B. diu lo m ng um B. lo ng F ca le n M s or ed is tu iu ca m le ns is 0 tu uc m In vivo 0.0005 M .m 0.0523 CHRM4 (RLU) 2,000 0 A. .m or g M a M or . ed nii ga m iu ni or m i + ga M nii AO i G F 4,000 G 0.5078 0.0204 6,000 In vitro 8,000 In vitro In vivo .p 5,000 Food or hostderived substrate C 10,000 0 M 8,000 0.0002 or 5,000 0.9814 0.0094 0.0090 In vitro In vivo .p 10,000 15,000 Histamine C. portucalensis C DRD3 (RLU) 15,000 In vitro In vivo 0.0021 Succinate MMCM (B12 dependent) Propionate HRH3 (RLU) MAOs MAOi B12 in ip A. M hil m ed a uc iu in m ip hi la G F PEA A. muciniphila SUCNR1 (RLU) M. morganii C. portucalensis Fig. 1 | In vivo and in vitro commensal metabolomes exhibit distinct GPCR activation patterns. a, Schematic of generating commensal metabolomes from bacteria cultured in vitro in medium and in vivo in monoassociated GF mice, followed by GPCRome interaction screening using the multiplexed GPCR screening technology PRESTO-Salsa. b, The number of in vitro or in vivo commensal metabolomes activating each GPCR. c–e, Representative models of in vivo-mediated alterations in GPCR-active commensal metabolites. c, Host MAOs degrade bacterially derived PEA in vivo. MAOi, MAO inhibitor; RLU, relative luciferase units. d, In vivo metabo (...truncated)