Gut microbiota depletion accelerates hematoma resolution and neurological recovery after intracerebral hemorrhage via p-coumaric acid-promoted Treg differentiation.

Abstract

Hematoma volume significantly influences the prognosis of patients with intracerebral hemorrhage (ICH). Effective resolution of hematoma through enhanced clearance mechanisms and reduced hematoma lysis is essential for neurological recovery following ICH. Regulatory T cells (Tregs), known for their anti-inflammatory properties, exert neuroprotective effects in various central nervous system disorders. Additionally, gut microbiota profoundly impacts Treg development through multiple regulatory pathways. Nonetheless, the precise roles of Tregs and gut microbiota in facilitating hematoma resolution after ICH remain unclear. This study, therefore, aimed to investigate the contributions of Tregs and gut microbiota to hematoma resolution post-ICH, as well as the underlying mechanisms. Methods: The impact of gut microbiota depletion on neurological deficits and hematoma resolution, including erythrophagocytosis and erythrocyte lysis, was assessed using antibiotic cocktail (ABX) gavage administered prior to ICH induction in mice. Flow cytometry analysis and targeted cell depletion techniques were employed to identify peripheral immune cell populations mediating the beneficial effects of gut microbiota depletion on neurological recovery and hematoma resolution. The functional roles of Tregs in erythrophagocytosis, erythrocyte lysis, and associated downstream molecular signaling pathways were investigated through adoptive Treg transfer experiments. The mechanisms underlying Treg population expansion post-microbiota depletion in ICH mice were explored using multi-omics analysis of serum and fecal metabolites via mass spectrometry and fecal microbial composition using 16S rRNA sequencing. Additionally, the effects of p-coumaric acid (PCA) gavage and clindamycin-mediated depletion of PCA-metabolizing gut microbiota on Treg abundance, hematoma resolution, and neurological recovery post-ICH were assessed. Results: Gut microbiota depletion by ABX gavage increased brain Treg populations, thereby enhancing erythrophagocytosis, suppressing erythrocyte lysis, and ultimately promoting hematoma resolution and neurological recovery. Adoptive Treg transfer experiments further established that Tregs facilitate scavenger pathway-mediated erythrophagocytosis and suppress complement-mediated erythrocyte lysis. These effects occurred via upregulation of efferocytosis receptors (MERTK and AXL), ligands (Gas6 and C1q), and the hemoglobin scavenger receptor CD163, alongside downregulation of complement C3 expression and reduced formation of membrane attack complexes (MACs). Multi-omics analysis demonstrated that ABX gavage eliminated PCA-metabolizing microbiota, thereby increasing PCA concentrations in serum and feces. Elevated PCA levels promoted peripheral Treg differentiation by inhibiting the PKCθ-AKT-FoxO1/3a signaling pathway, leading to higher brain Treg numbers. PCA gavage and clindamycin treatment similarly enhanced brain Treg populations, accelerated hematoma resolution, and improved neurological recovery following ICH. Conclusion: Gut microbiota depletion facilitates hematoma resolution and neurological recovery through PCA-mediated induction of Treg differentiation.