Gastroenterology

Gastroenterology

Volume 163, Issue 5, November 2022, Pages 1377-1390.e11
Gastroenterology

Original Research
Full Report: Gut Microbiota
Histone Deacetylase Inhibition by Gut Microbe-Generated Short-Chain Fatty Acids Entrains Intestinal Epithelial Circadian Rhythms

https://doi.org/10.1053/j.gastro.2022.07.051Get rights and content

Background & Aims

The circadian clock orchestrates ∼24-hour oscillations of gastrointestinal epithelial structure and function that drive diurnal rhythms in gut microbiota. Here, we use experimental and computational approaches in intestinal organoids to reveal reciprocal effects of gut microbial metabolites on epithelial timekeeping by an epigenetic mechanism.

Methods

We cultured enteroids in media supplemented with sterile supernatants from the altered Schaedler Flora (ASF), a defined murine microbiota. Circadian oscillations of bioluminescent PER2 and Bmal1 were measured in the presence or absence of individual ASF supernatants. Separately, we applied machine learning to ASF metabolomics to identify phase-shifting metabolites.

Results

Sterile filtrates from 3 of 7 ASF species (ASF360 Lactobacillus intestinalis, ASF361 Ligilactobacillus murinus, and ASF502 Clostridium species) induced minimal alterations in circadian rhythms, whereas filtrates from 4 ASF species (ASF356 Clostridium species, ASF492 Eubacterium plexicaudatum, ASF500 Pseudoflavonifactor species, and ASF519 Parabacteroides goldsteinii) induced profound, concentration-dependent phase shifts. Random forest classification identified short-chain fatty acid (SCFA) (butyrate, propionate, acetate, and isovalerate) production as a discriminating feature of ASF “shifters.” Experiments with SCFAs confirmed machine learning predictions, with a median phase shift of 6.2 hours in murine enteroids. Pharmacologic or botanical histone deacetylase (HDAC) inhibitors yielded similar findings. Further, mithramycin A, an inhibitor of HDAC inhibition, reduced SCFA-induced phase shifts by 20% (P < .05) and conditional knockout of HDAC3 in enteroids abrogated butyrate effects on Per2 expression. Key findings were reproducible in human Bmal1-luciferase enteroids, colonoids, and Per2-luciferase Caco-2 cells.

Conclusions

Gut microbe-generated SCFAs entrain intestinal epithelial circadian rhythms by an HDACi-dependent mechanism, with critical implications for understanding microbial and circadian network regulation of intestinal epithelial homeostasis.

Section snippets

ASF Strain Information

All strains used in this study originate from the ASF, a defined mouse gut microbiota. ASF strains were a gift to the Papin laboratory from Drs Michael Wannemuehler and Gregory Phillips. The members used in this study are: Clostridium species ASF356, Lactobacillus intestinalis ASF360, Ligilactobacillus murinus ASF361, Eubacterium plexicaudatum ASF492, Pseudoflavonifractor species ASF500, Clostridium species ASF502, and Parabacteroides goldsteinii ASF519. Mucispirillum schaedleri ASF457 was

Metabolites From ASF Species Differentially Modulate the Intestinal Epithelial Clock

Incubating PER2::LUC enteroids with bacterial supernatants from ASF356, ASF492, ASF500, and ASF519 significantly altered the circadian phase (Figure 1B), whereas supernatants from ASF360, ASF361, and ASF502 did not (Figure 1A). Mann-Whitney U test indicated significant phase delays in hours with ASF356 (10% median, 7.09 hours; P < .05; 25% median, 10.89 hours; P < .05), ASF492 (25% median, 3.37 hours; P < .05), ASF500 (10% median, 2.2 hours; P < .05; 25% median, 7.67 hours; P < .05), and ASF519

Discussion

In these ex vivo intestinal organoid studies of microbial metabolites and intestinal epithelial circadian timekeeping, we used a combination of tissue culture experiments and systems metabolomics to elucidate gut microbial influences on host circadian rhythms. Recently, Kuang et al21 demonstrated cross-talk between intestinal microbiota and the intestinal circadian clock and role of SCFA and HDAC epigenetic pathways in mice. Our present results complement their findings by revealing: (1) SCFAs

CRediT Authorship Contributions

Jibraan A. Fawad, MBBS (Formal analysis: Lead; Investigation: Lead; Methodology: Lead; Project administration: Lead; Validation: Lead; Visualization: Lead; Writing – original draft: Lead).

Deborah H. Luzader, PhD (Conceptualization: Lead; Formal analysis: Lead; Investigation: Lead; Methodology: Lead; Project administration: Lead; Software: Lead; Validation: Lead; Writing – original draft: Lead).

Gabriel F. Hanson, BS (Data curation: Supporting; Formal analysis: Supporting; Investigation:

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    Conflicts of interest S.R.M. is a consultant for Takeda (short bowel syndrome). D.T.B. is now a full-time employee of Lumen Biosciences. G.L.M. is now a full-time employee of Vedanta Biosciences. A.E.R. is now a full-time employee at Procter & Gamble. The contributions of D.T.B., G.L.M., and A.E.R. to the paper predated their transition to industry and they declare no conflict of interest with its contents. The remaining authors disclose no conflicts.

    Funding This work was supported by National Institutes of Health grants R01 DK117005 (C.I.H., S.R.M.), U19 AI116491 (S.R.M., C.I.H.), R01AT010253 (J.A.P.), Pendleton Laboratory Endowment (S.R.M.), and R01DK114123 (T.A), the University of Virginia Trans-University Microbiome Initiative, and the National Research Foundation of Korea 2020R1A6A3A03038405 (M.P.).

    Authors share co-first authorship.

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