Proteomic reprogramming of ileal epithelial cells during homologous superimposed intestinal trematode infection reveals coordinated restoration of intestinal homeostasis

Background Intestinal helminth infections trigger complex host responses, determining parasite survival and tissue homeostasis. Primary Echinostoma caproni infection disrupts epithelial metabolism, differentiation, and repair in an IL-25-deficient environment, as shown in a previous study by our research group; however, the adaptive mechanisms during homologous superimposed infections remain unclear. Methodology/Principal findings Male ICR mice were assigned to control, primary infection, and homologous superimposed infection groups, and ileal epithelial cells were isolated for proteomic profiling using liquid chromatography–tandem mass spectrometry (LC–MS/MS) with data-dependent acquisition (DDA) and sequential window acquisition of all theoretical mass spectra (SWATH). Differential protein expression was analyzed with Elastic Net regression, partial least squares discriminant analysis, and fold-change ranking, while functional enrichment and protein–protein interaction networks were explored using gene set enrichment analysis (GSEA) and STRING. Notably, homologous superimposed infection revealed proteomic signatures associated with lysosomal and peroxisomal lipid metabolism, PPAR pathway activation, cytoskeletal reorganization, epithelial barrier reinforcement, a specialized antimicrobial peptide repertoire, and interactions between IgE receptor-associated proteins, consistent with a restoration of intestinal homeostasis influenced by IL-25. Conclusions Host adaptation to repeated E. caproni exposure involves coordinated metabolic, signaling, and tissue repair responses that partially restore intestinal homeostasis, with IL-25 emerging as a central regulator linking metabolic reprogramming, epithelial integrity, and anti-helminth immunity, thereby providing a proteomic framework for understanding how repeated helminth exposure drives partial resistance through integrated epithelial and immunometabolic adaptations. Author summary We investigated how repeated intestinal worm infections affect the cells lining the small intestine in mice. Infections with intestinal trematodes can disrupt the normal balance of the gut, leading to tissue damage and altered metabolism. Using a proteomics approach, we measured changes in thousands of proteins in intestinal epithelial cells during a first infection and after a second, repeated infection. We found that the first infection caused stress in the cells, impaired oxygen use, and reduced the activity of pathways that normally help repair tissue. In contrast, the repeated infection triggered a coordinated response that restored many cellular functions. Cells increased protein activity related to fat metabolism, tissue structure, barrier integrity, and antimicrobial defense. We also observed evidence that the immune signaling molecule interleukin-25 plays a central role in coordinating these protective and repair processes. These results suggest that the gut epithelium can adapt to repeated infections by reorganizing its metabolic and structural functions, which may help limit tissue damage and promote partial resistance to parasites. Our study provides a detailed map of the molecular changes that underlie this adaptation, improving our understanding of how the intestinal lining responds to repeated worm infections.