<i>Lactobacillus rhamnosus</i> confers protection against enteropathogenic bacteria by enhancing mucosal immunity and epithelial barrier function.

Abstract

<h4>Introduction</h4>Probiotics such as <i>Lactobacillus rhamnosus</i> represent promising alternatives to antibiotics for combating enteric infections, yet their mechanisms of action remain incompletely understood. This study aimed to elucidate the protective mechanisms of <i>L. rhamnosus</i> CIQ249 against enteropathogenic bacterial infection, focusing on the intestinal physical barrier and mucosal immune responses.<h4>Methods</h4>The intestinal colonization ability of CIQ249 was assessed using cFDA-SE labeling and flow cytometry. Growth performance and intestinal morphology were evaluated in mice. Antimicrobial activity of CIQ249 cell-free supernatant was tested against various pathogens, and pathogen damage was visualized by scanning electron microscopy. Protective effects against <i>Salmonella typhimurium</i> and <i>Escherichia coli</i> K99 were examined in a mouse model. Tight junction protein expression was analyzed <i>in vitro</i> and <i>in vivo</i> using immunofluorescence, qRT-PCR, and Western blot. Immune responses- including cytokine production, dendritic cell (DC) activation, T follicular helper (Tfh) cell differentiation, and IgA secretion-were assessed by ELISA, flow cytometry, and immunohistochemistry. Transcriptomic changes in porcine intestinal epithelial cells (PIEC) were analyzed by RNA-seq.<h4>Results</h4>CIQ249 demonstrated strong intestinal colonization and increased villus height and the villus-to-crypt ratio, contributing to improved growth performance. Its cell-free supernatant selectively inhibited enteropathogens and induced structural damage in <i>S. typhimurium</i> and <i>E. coli</i> K99. CIQ249 protected mice from lethal pathogen challenge, preserved intestinal architecture by upregulating tight junction proteins ZO-1 and Claudin-1. It also enhanced mucosal and systemic cytokine levels (IFN-γ, IL-2, IL-4, IL-17, and IL-27), activated DCs, promoted differentiation of CXCR5+CD4+ Tfh and IgA-secreting plasma cells in Peyer's patches, leading to sIgA production. Transcriptome analysis revealed broad modulation of immune- and barrier-related pathways, with validation of key genes (e.g., IL-10, Masp2, Igf2).<h4>Conclusion</h4>CIQ249 enhances mucosal defense against enteropathogenic bacteria through a dual mechanism-strengthening the epithelial barrier and activating a coordinated DC-Tfh-IgA immune axis. These findings provide a multi-level mechanistic basis for its application as a microecological agent against intestinal infections.