Resolving Resident Colonic Muscularis Macrophage Diversity and Plasticity During Colitis

Background Immune cell populations in the intestinal muscularis propria during colitis are poorly resolved. Maintaining homeostasis in this niche is critical, highlighted by the poorer prognosis of inflammatory bowel disease associated with muscularis propria inflammation.Methods This study utilizes single-cell RNA sequencing to survey the immune cell populations within the muscularis propria of normal colon and dextran sodium sulfate-induced colitis. Findings are validated by immunohistochemistry, flow cytometry and cell-lineage tracing in vivo, and in vitro assays with muscularis macrophages (MM phi).Results In na & iuml;ve conditions, transcriptional duality is observed in MM phi s with 2 major subpopulations: conventional resident Cx3cr1+ MM phi s and Lyve1+ MM phi s. The Lyve1+ population is phagocytic and expresses several known MM phi markers in mouse and human, confirming their identity as a bona fide MM phi subset. Single-cell transcriptomics indicate that resident MM phi s are retained during colitis and exhibit plasticity toward an inflammatory profile. Lyve1+ MM phi s, which express anti-inflammatory marker CD163, are absent during colitis, as confirmed by flow cytometry. In contrast, lineage tracing finds that resident Cx3cr1+ MM phi s remain during colitis and are not completely replaced by the inflammatory infiltrating monocytes. In vitro studies provide biological evidence of the plasticity of resident Cx3cr1+ MM phi s in response to lipopolysaccharide (LPS), mirroring transcriptional observations in vivo of their inflammatory plasticity. Potential markers for colitic MM phi s, validated in animal models and in individuals with ulcerative colitis, are identified.Conclusions Our findings contribute to the understanding of the immune system in the muscularis propria niche during colitis by resolving the heterogeneity and origins of colitic MM phi s. Involvement of the muscularis propria accompanies a poorer prognosis in IBD. This study characterizes muscularis macrophage subpopulations during colitis, highlighting the loss of anti-inflammatory LYVE-1+ macrophages and inflammatory plasticity in resident CX3CR1+ macrophages, providing insights into prognostic and therapeutic targets.