<b>Gut microbiota butyrate mediated RUNX3 promotes Nr4a1</b> ^<b>high</b> <b>ZFP36</b> ^<b>high</b> <b>resident macrophages via NR4A1/ERK1/2 MAPK to maintain gut homeostasis</b>

Gut homeostasis is critical for systemic health, and its disruption is implicated in various diseases, including metabolic disorders. Gut-resident macrophages play a pivotal role in maintaining intestinal homeostasis, yet the mechanisms underlying their differentiation and function remain incompletely understood. Using single-cell RNA sequencing (scRNA-seq), we found a key regulatory axis in which gut microbiota-derived butyrate induces the differentiation of Nr4a1^highZFP36^high resident macrophages via RUNX3 to sustain gut homeostasis. Butyrate could upregulate RUNX3 expression in gut-resident macrophages through lncRNA lncLy6c-mediated H3K4me3 modification. Genetic ablation of RUNX3 in myeloid cells of <i>RUNX3</i>^{<i>fl/fl-Lyz2-Cre</i>} mice led to a marked reduction in resident macrophages and increased susceptibility to DSS-induced colitis. RUNX3-mediated resident macrophages exhibited elevated ZFP36 expression alongside suppressed pro-inflammatory cytokines and chemokines. Further mechanistic studies revealed that RUNX3 drives the differentiation of Nr4a1^highZFP36^high resident macrophages via the Nr4a1-dependent activation of the ERK1/2 MAPK pathway. Consistently, high expression levels of RUNX3, Nr4a1, and ZFP36 were observed in colon-resident macrophages from healthy human donors. Collectively, our findings demonstrate that butyrate-RUNX3 signaling orchestrates the differentiation of Nr4a1^highZFP36^high resident macrophages through the Nr4a1/ERK1/2 MAPK pathway, thereby safeguarding gut homeostasis.