Pneumococcal Endopeptidase O Attenuates Colitis by Inhibiting the Macrophage–CCL2 Axis and Reshaping Gut Microbiota

Background: Ulcerative colitis (UC) is a chronic inflammatory disease characterized by persistent immune activation and gut microbiota dysbiosis, for which current therapies remain limited by incomplete efficacy and substantial adverse effects. This study aimed to investigate the protective effects of pneumococcal endopeptidase O (PepO) on UC and its potential mechanisms of action. Methods: The efficacy of PepO was evaluated in a dextran sulfate sodium (DSS)-induced acute colitis mouse model. Clinical phenotypes were assessed using the disease activity index (DAI), colon length, histopathological analysis, and inflammatory cytokine levels. Macrophage infiltration and key inflammatory mediators were analyzed via immunofluorescence, flow cytometry, ELISA, and qPCR. Mechanisms were further validated by macrophage depletion using clodronate liposomes and recombinant CCL2 supplementation. In addition, gut microbiota composition was analyzed via 16S rRNA sequencing, and causality was confirmed through antibiotic-induced microbiota depletion and fecal microbiota transplantation (FMT) experiments. Results: PepO administration significantly improved disease activity, colon length, and ameliorated histopathological damage in the mice. Mechanistically, PepO directly inhibited macrophage CCL2 production, reducing macrophage infiltration and the release of pro-inflammatory cytokines in colitis. Additionally, PepO remodelled the gut microbiota and markedly increased the abundance of beneficial bacteria, such as Lactobacillus and Akkermansia . Antibiotic-induced microbiota depletion completely abolished PepO’s protective effect, while FMT successfully replicated its protective actions, including suppression of CCL2 expression and macrophage infiltration. Conclusion: This study shows that PepO exerts potent anti-colitic effects through a dual mechanism involving direct inhibition of the macrophage-CCL2 axis and microbiota-dependent enhancement of gut immune homeostasis. The illustration compares DSS-induced colitis and PepO administration effects. On the left, DSS-induced colitis shows a decrease in MUC2, ZO-1, occludin and Ki67 in epithelial cells, with increased apoptosis. The lamina propria has elevated cytokines IL-6, IL-1 beta, TNF-alpha and increased CCL2 and F4/80 positive macrophages. On the right, PepO administration shows increased MUC2, ZO-1, occludin and Ki67, with reduced apoptosis. Akkermansia and Lactobacillus are increased in the lumen. The lamina propria shows reduced cytokines IL-6, IL-1 beta, TNF-alpha and reduced inflammation, with inhibited macrophage activation and increased CCL2 and macrophage recruitment. Arrows indicate promotion or inhibition of processes.Comparison of DSS-induced colitis and PepO administration effects on gut cells and inflammation. Keywords: CCL2, gut microbiota, intestinal barrier, macrophages, pneumococcal endopeptidase O (PepO), ulcerative colitis