Thrombospondin-1-mediated macrophage efferocytosis dysfunction exacerbates intestinal pathology in Hirschsprung's disease.

Hirschsprung's disease (HSCR) is a prevalent pediatric congenital enteropathy characterized by disrupted intestinal homeostasis, persistent inflammation, and progressive intestinal fibrosis. However, the cellular and molecular mechanisms underpinning these pathological hallmarks remain incompletely elucidated. Given the pivotal role of macrophages in inflammation resolution and tissue repair, and the established function of thrombospondin-1 (THBS1) in regulating macrophage polarization and efferocytosis, we focused on THBS1 to dissect the mechanisms of HSCR-associated pathology. Single-cell RNA sequencing (scRNA-seq) was performed on 3 paired colon biopsies of aganglionic HSCR lesions and proximal ganglionic controls. Inflammation and fibrosis markers were assessed in additional HSCR samples (n = 5) by histopathology, qRT-PCR, and immunofluorescence. Macrophage efferocytosis was quantified by flow cytometry and immunofluorescence. Mechanistic studies employed recombinant THBS1 (rTHBS1), siRNA/lentiviral transduction, CD36 inhibition, and Rac1 FRET biosensors in bone marrow-derived macrophages (BMDMs), with in vivo confirmation in DSS-induced colitis. scRNA-seq revealed stromal-specific THBS1 downregulation in HSCR as a top candidate regulator of dysregulated macrophage function. HSCR macrophages exhibited proinflammatory hyperactivation and impaired pro-resolving polarization with reduction of CD206+ proportion and downregulation of TGFB1. Mechanistically, THBS1 sustains intestinal homeostasis by mediating macrophage pro-resolving functions and enhances efferocytosis via activation of the CD36/Rac1 signaling axis. Inhibition of THBS1 abrogates macrophage efferocytosis by appropriate 50%, leading to unresolved inflammation and intestinal fibrosis. Critically, rTHBS1 treatment rescued efferocytosis in THBS1-deficient BMDMs. Notably, impaired macrophage THBS1 signaling was consistently observed in human HSCR specimens. This study identifies stromal THBS1-CD36/Rac1 signaling as a critical regulator of macrophage efferocytosis and resolution in HSCR. THBS1 deficiency drives persistent post-surgical inflammation and fibrosis, establishing it as a novel therapeutic target and potential biomarker for precision interventions to improve long-term outcomes in this pediatric disorder.