Exercise-associated 5-HIAA restrains macrophage NLRP3 inflammasome-mediated pyroptosis through AHR signaling in experimental colitis.

Abstract

BACKGROUND AND AIMS: Exercise is associated with reduced intestinal inflammation, but the immunometabolic mediators linking physical activity to mucosal immune regulation remain incompletely defined. Exercise-associated metabolomic profiling identified the serotonin-derived metabolite 5-hydroxyindoleacetic acid (5-HIAA) as a potential bioactive mediator. We therefore investigated whether 5-HIAA could inhibit macrophage NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome-dependent pyroptosis and reproduce selected protective effects of exercise against experimental colitis. METHODS: Prior to dextran sulfate sodium (DSS) administration, mice were subjected to a treadmill exercise preconditioning regimen. Untargeted metabolomics was used to identify exercise-associated metabolic changes in colonic tissue. The protective effects of exogenously administered 5-HIAA were further investigated in a DSS-induced colitis model and LPS/nigericin-induced pyroptosis models in THP-1-derived macrophages and primary bone marrow-derived macrophages. Aryl hydrocarbon receptor (AHR) silencing in vitro and systemic administration of AAV-F4/80-shAhr in vivo were used to investigate the involvement of AHR in the effects of 5-HIAA. RESULTS: Exercise preconditioning alleviated DSS-induced colitis and was associated with a distinct colonic metabolic profile in which 5-HIAA showed higher relative abundance. Exogenous 5-HIAA reproduced several protective phenotypes of exercise, including reduced body-weight loss, preserved colon length, improved mucosal histology, lower pro-inflammatory cytokine concentrations, and reduced macrophage-associated interleukin-1β (IL-1β) staining. In macrophages, 5-HIAA attenuated NLRP3 inflammasome activation and pyroptotic membrane damage, as indicated by reduced caspase-1 cleavage, gasdermin D (GSDMD) processing, lactate dehydrogenase (LDH) release, and propidium iodide (PI) uptake. AHR silencing weakened the anti-pyroptotic effects of 5-HIAA in vitro. Systemic administration of AAV-F4/80-shAhr attenuated its protective effects against colitis, although the efficiency and cellular specificity of silencing were not directly validated. CONCLUSIONS: These findings identify 5-HIAA as an exercise-associated candidate immunometabolite and support an AHR-dependent mechanism through which 5-HIAA restrains NLRP3 inflammasome-associated pyroptotic responses in macrophages.

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