Abstract

Plain Language Summary Background: Although monocyte-to-macrophage differentiation is essential for innate immune defense, its dysregulation can drive excessive inflammation in sepsis. Reticulocalbin 3 (RCN3) is an endoplasmic reticulum chaperone in the secretory pathway implicated in alveolar epithelial maturation and lung injury repair. This study aimed to investigate the effect of RCN3 on monocyte-to-macrophage differentiation in pneumonia-associated sepsis. Methods: Patients with pneumonic sepsis ( n = 10) and age- and sex-matched healthy volunteers ( n = 8) were enrolled from Beijing Chao-Yang Hospital. Peripheral blood was collected within 12 h after diagnosis; peripheral blood mononuclear cells (PBMCs) were isolated and monocytes were purified for transcriptional analyses of RCN3 and macrophage markers. For in vivo validation, myeloid-specific Rcn3 knockout mice received intratracheal lipopolysaccharide (10 mg/kg) or saline, and were assessed 16 h later ( n = 3–6 per group). Mechanistic studies in THP1 cells, human primary circulating monocytes and mouse bone marrow-derived monocytes used RCN3 knockdown and overexpression to evaluate differentiation markers, intracellular oxidative stress, and phagocytosis assays and autophagy flux evaluation. Results: In patients with pneumonic sepsis, RCN3 was significantly increased in circulating monocytes, but not lymphocytes, and its expression correlated with the macrophage marker CD68. RCN3 was also upregulated during monocyte-to-macrophage differentiation in both human and mouse monocytes. Furthermore, RCN3 knockdown attenuated differentiation, phagocytosis, and the lipopolysaccharide (LPS)-induced inflammatory response, whereas RCN3 overexpression had opposite effects. In vivo , myeloid-specific Rcn3 deletion decreased lung interstitial macrophage accumulation after intratracheal LPS instillation and alleviated acute lung injury. Mechanistically, the blunted differentiation caused by RCN3 deficiency was associated