Integrative transcriptomic and computational analysis associates Ms4a6c+ macrophages with fibrosis in radiation proctitis and computationally nominates MOTS-c as a candidate modulator

Chronic radiation proctitis (CRP) represents a significant clinical challenge characterized by irreversible fibrosis and a lack of specific pharmacological interventions. This study aimed to elucidate the cellular mechanisms underlying impaired tissue repair to identify novel therapeutic targets.

We employed an integrative framework combining machine learning with bulk and high-resolution single-cell transcriptomic profiling in a murine CRP model. Computational mechanistic analyses included regulon inference, cell-cell communication analysis, and single-cell virtual knockout simulation. The pro-fibrotic association of the identified macrophage subset was further examined using independent single-cell datasets from liver cirrhosis and idiopathic pulmonary fibrosis. Furthermore, structure-based virtual screening and molecular dynamics simulations were conducted to identify potential pharmacological modulators.

We identified Ms4a6c as a key candidate gene exclusively expressed in a distinct, hybrid-activation macrophage subpopulation associated with pro-fibrotic transcriptional programs. These macrophages showed predicted associations with fibrosis via Thbs1-Cd47 paracrine signaling interactions with fibroblasts, and SCENIC regulon analysis identified Cebpb as a candidate upstream transcriptional regulator. Cross-disease analysis in liver cirrhosis and idiopathic pulmonary fibrosis single-cell datasets indicated that Ms4a6c-high macrophage subsets are consistently associated with elevated epithelial-mesenchymal transition and TGF-β signaling activity, supporting a potentially conserved pro-fibrotic role across organ systems. Furthermore, single-cell virtual knockout simulation predicted that the computational depletion of Ms4a6c may reverse pro-fibrotic transcriptomic signatures. Additionally, molecular docking and dynamics simulations identified the mitochondrial-derived peptide MOTS-c as a top-ranking candidate predicted to target Ms4a6c.

This study highlights the potential pathogenic involvement of Ms4a6c+ macrophages and identifies the Cebpb-Ms4a6c-Thbs1-Cd47 axis as a candidate pro-fibrotic signaling cascade that may be conserved across multiple organ fibrosis models. We propose MOTS-c as a potential pharmacological agent for mitigating radiation-induced intestinal fibrosis, pending experimental validation.