Pro-inflammatory Pathways Contribute to Pathogenesis of Clostridioides difficile Infection in a Murine Model - A Spatial Transcriptomics Study

Clostridioides difficile (C. difficile) is a common cause of antibiotic-induced diarrhea and causes the highest number of nosocomial infections. Only two antibiotics are currently recommended for treating C. difficile infection (CDI), which may contribute to unsatisfactory treatment outcomes and an increased likelihood of recurrence. In this study, we aim to evaluate the difference in gene expression between symptomatic and asymptomatic mice after infection with C. difficile using spatial transcriptomics analysis. We also aim to evaluate the spatial aspect of altered genes between different layers of intestinal mucosa (superficial vs deep) and identify the key pathways. Formalin-fixed paraffin-embedded (FFPE) intestinal sections were utilized for analysis using NanoStringTM platform to evaluate differential gene expressions in the caecum and colon. The IL-17 pathway, including Lcn2, Cxcl2, and S100a8 genes, was significantly upregulated in symptomatic mice. The IL-17 signaling pathway activated downstream signaling through NF-κB and MAPK pathways. Gene expression was significantly altered between the intestinal superficial and deep mucosal layers, highlighting layer-specific differences in gene expression patterns in the intestines of symptomatic and asymptomatic mice. Gene expression patterns in the enteric mucosa explained several clinical signs and lesions in CDI mice. Mice were infected with C. difficile spores and let to develop the disease symptoms. Mice were divided into groups based on the outcome of the infection. Groups included symptomatic and asymptomatic mice. Also, gene expression was measured in different layers of the intestine, superficial, middle and deep.