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

INTRODUCTION

Probiotics such as Lactobacillus rhamnosus 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 L. rhamnosus CIQ249 against enteropathogenic bacterial infection, focusing on the intestinal physical barrier and mucosal immune responses.

METHODS

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 Salmonella typhimurium and Escherichia coli K99 were examined in a mouse model. Tight junction protein expression was analyzed in vitro and in vivo 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.

RESULTS

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 S. typhimurium and E. coli 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).

CONCLUSION

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.