The intestinal epithelial cell (IEC) circadian clock maintains 24-hour rhythms in the gut microbiome and mucosal immunity, and colonic inflammation both damps epithelial clock function and generates de novo circadian oscillations in colonic regulatory T cell (Treg) abundance and activity (Butler et al., iScience 2025). The mechanistic basis of these observations remains unresolved: the feeding-independent mediators by which the IEC clock sustains microbiome rhythmicity are unidentified; the clock status of Tregs within the inflamed colon has never been measured; the extrinsic cues that drive the emergent Treg rhythmicity in colitis are unknown (candidate cues include glucocorticoid availability, microbial metabolites, and dietary components); the functional impact of diurnal Treg accumulation on local inflammation is undemonstrated; and published reports of worsened, unchanged, and improved colitis in IEC-Bmal1-deficient mice await reconciliation. The problem is to supply this missing causal account: identify the mediators and cues, establish the functional consequences for colonic inflammation, and resolve the conflicting colitis phenotypes reported across genetic models and protocols.

Background

Circadian (~24 h) clocks are transcription-translation feedback circuits built from core genes such as Bmal1 (which encodes the BMAL1 transcription factor, the obligate positive arm of the clock); nearly every mammalian cell runs one, coordinated by a hypothalamic master clock synchronized to the light-dark cycle. In the intestine, circadian clocks in intestinal epithelial cells (IECs — the absorptive and secretory cells lining the gut) impose daily rhythms on antigen presentation, antimicrobial peptide production, mucus and tight-junction barrier components, and nutrient uptake, and the gut microbiome itself oscillates in composition and metabolite output over the day. Food timing is generally regarded as the dominant entrainer of microbiome rhythmicity, but work from the Kiessling group showed that the IEC clock itself drives microbial rhythmicity to maintain gastrointestinal homeostasis (Heddes et al., Nature Communications 2022) and that restricting feeding to the active phase ameliorates intestinal inflammation (Niu et al., Cellular & Molecular Immunology 2024), while others showed that epithelial Bmal1 deficiency alters the microbiome and protects from high-fat-diet obesity (Yu et al., Nature Communications 2021). Regulatory T cells (Tregs — Foxp3+ CD4+ T cells that suppress other immune cells) are essential for intestinal tolerance; glucocorticoid receptor-deficient Tregs fail to control transfer colitis (Rocamora-Reverte et al., Frontiers in Immunology 2019), and endogenous glucocorticoid oscillations drive diurnal T cell redistribution and responses (Shimba et al., Immunity 2018), while diurnal microbial metabolites such as glycitein time inflammatory rhythms in arthritis (Ma et al., Cell Metabolism 2024). Clock–colitis coupling is well documented — whole-animal or epithelial circadian disruption alters dextran sulfate sodium (DSS, a chemical administered in drinking water to induce colitis in mice) colitis outcomes (Taleb et al., Frontiers in Cellular and Infection Microbiology 2022; Jochum et al., Inflammatory Bowel Diseases 2023) — but reports disagree in sign, from worsened to unchanged to (most recently, Hua et al., EMBO Reports 2025) improved colitis after IEC-specific Bmal1 deletion. Butler et al. (iScience 2025; bioRxiv 2025.02.20.639289) added two key observations: (i) IEC-specific Bmal1 deletion (Villin-Cre — a genetic system in which the villin promoter drives Cre recombinase to delete a floxed target gene only in intestinal epithelial cells) damps colonic microbiome rhythmicity despite persisting feeding and rest-activity rhythms, implying a feeding-independent epithelial-clock pathway acting through candidate mediators (antimicrobial peptides, major histocompatibility complex class II antigen-presentation molecules, toll-like receptors — a family of innate receptors that sense microbial products — and barrier components such as the rhythmically expressed tight-junction gene Cldn8); and (ii) acute DSS colitis damps the IEC clock yet induces de novo 24 h oscillations in lamina-propria (LP — the intestinal connective-tissue layer beneath the epithelium) Treg numbers, proliferation, and suppressive-marker expression. Clock gene oscillations were demonstrable in LP Tregs of naive but not colitic mice simply because the colitis experiment was not run; the cues and consequences of this emergent Treg rhythmicity were untested, colonic tissue cytokines showed no clear day-night difference, and IEC-Bmal1 deletion in the Butler et al. constitutive Villin-Cre line left DSS severity unchanged. A follow-up from the same group (Hunter et al., npj Biological Timing and Sleep 2026) showed the converse dependence — flattening feeding rhythms produces a striking loss of rhythmic secretory IgA (the antibody secreted across the epithelium into the gut lumen) and significantly diminishes microbiome and short-chain fatty acid (bacterial fermentation products) rhythmicity while the IEC clock keeps ticking — so both feeding timing and the epithelial clock contribute to microbiome rhythmicity, but the molecular mediators of the clock arm and everything on the Treg side remain open.

Problem Statement

Establish the causal mechanistic account, left open by Butler et al. (iScience 2025), of how the intestinal epithelial circadian clock and colonic inflammation jointly govern microbiome and mucosal immune rhythmicity. Concretely: (1) identify the feeding-independent mediators by which the IEC BMAL1-dependent clock maintains rhythmic microbiome composition and metabolite output (candidate mediators include antimicrobial peptides, MHC class II and other immune sensors such as toll-like receptors, and epithelial barrier components), and quantify the relative contributions of the epithelial clock versus feeding-timing cues (e.g., rhythmic IgA secretion) to microbial rhythmicity; (2) determine whether the Treg clock itself is damped or persists in the inflamed colonic environment, that is, whether core clock-gene oscillations can be detected in lamina-propria Tregs isolated from mice with colitis; (3) identify the extrinsic cues that drive the de novo 24 h rhythmicity of colonic Treg abundance, proliferation, and suppressive activity during colitis (candidate cues include daily glucocorticoid availability, microbial metabolites, and dietary components), including whether daily variation in glucocorticoid availability modulates Treg suppressive function; (4) determine the functional consequence — if any — of this diurnal Treg accumulation and activity for the local inflammatory response in the colon across the 24 h day, distinguishing the possibility that nocturnal Treg expansion suppresses local inflammation from the possibilities that no clear day-night difference exists or that night-time Treg accumulation coincides with reduced suppressive capacity; and (5) account for the divergent colitis phenotypes reported after Bmal1 loss in the intestinal epithelium (and, for comparison, in the whole-animal Bmal1(-/-) model) — no effect on acute DSS severity in Butler et al. (constitutive Villin-Cre × Bmal1-floxed; 2–2.5% DSS for 6–7 days), worsened acute DSS colitis in Niu et al. 2024 (the same group's constitutive Villin-Cre × Bmal1-floxed Bmal1(IEC-/-) line on a C57BL/6J background; 2% DSS for 5 days in constant darkness, the dramatic survival loss being confined to the compound Bmal1(IEC-/-) × IL-10(-/-) chronic model), augmented DSS responses in Jochum et al. 2023 (TS4-Cre), more severe DSS colitis in global Bmal1(-/-) mice (Taleb et al. 2022), and protection in the tamoxifen-inducible Villin-Cre model (Hua et al. 2025), which activates deletion only in adult animals — assessing whether model features such as Cre driver and transgene, developmental versus adult timing of deletion, institute-specific commensal microbiota, genetic background, and DSS dose, treatment length, washout and severity scoring contribute to the divergent outcomes. Butler et al. suggest that a different Cre driver could explain the inconsistency, but this remains an untested hypothesis: two constitutive Villin-Cre × Bmal1-floxed lines held in different facilities gave opposite acute-DSS outcomes. These components address one objective: a mechanistic, causally tested explanation of the gut circadian-immune-microbiome axis in health and inflammation, of the kind required before circadian logic (e.g., timing of glucocorticoid dosing or Treg-directed therapy) can be rationally applied to IBD treatment. All five components are jointly required.

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