Clostridioides difficile colitis promotes neuroinflammatory responses in the prefrontal cortex: a preclinical study in mice

Recurrent and severe Clostridioides difficile infection (CDI) is a significant problem in the elderly. There is growing evidence that intestinal dysbiosis and inflammation, characteristic features of CDI, can influence brain function. We examined whether neuroinflammatory responses occur during CDI and employed a spatial metabolomic and transcriptomic approach, integrated with biomolecular analyses, to characterize these responses in the prefrontal cortex (PFC) of a CDI mouse model. 6-month-old mice were infected with C. difficile and euthanized on days 3, 4, and 7 post-infection (p.i.) for spatial metabolic analysis, bulk RNA-sequencing of the PFC, or systemic and gut inflammation analysis. Infected mice developed diarrhea and weight loss that peaked by day 3 and resolved by day 7. Spatial metabolomics analysis of the infected brain tissues indicated an imbalance of various neuroprotective metabolites in the PFC on day 4. Differential gene expression analysis identified 124 differentially expressed genes on day 7. Gene set enrichment analysis identified upregulation of pathways associated with neurodegenerative diseases and oxidative phosphorylation in infected mice. PFC protein and transcripts levels of GFAP (glial fibrillary acidic protein) were increased in infected mice, indicating a reactive astrocyte response. Increased levels of lipocalin-2 (Lcn2), myeloperoxidase (MPO), calprotectin (S100A8/A9), IL-33, and IL-13 were also found in the PFC of infected mice. Levels of fecal Lcn2, a marker of intestinal epithelial barrier disruption, correlate positively with the levels of Lcn2, MPO, and S100A8/A9 in the PFC. Our findings show that CDI, in a mouse model, induces metabolomic and transcriptomic changes in the PFC, including genes associated with oxidative phosphorylation and neurodegeneration.