M. Nguyen1, F. Li2, L. Hung2, O. Escobar1, C. Gayer1 1Children’s Hospital Los Angeles,Pediatric Surgery,Los Angeles, CA, USA 2Children’s Hospital Los Angeles,Single Cell, Sequencing, CyTOF Core Laboratory,Los Angeles, CA, USA
Introduction: Farnesoid-X receptor (FXR) is a nuclear receptor involved in bile acid homeostasis. Our lab has shown that FXR activation interferes with the epidermal growth factor receptor (EGFR) pathway, decreasing cell proliferation. Furthermore, we have shown that the intestinal epithelial barrier of FXR knockout (FXRKO) mice is protected from lipopolysaccharide (LPS)-induced injury compared to that of wild-type (WT) mice, as measured by serum fluorescence after oral gavage with FIT-C dextran. We hypothesized that gene expression of molecules in the EGFR pathway and tight junctional proteins would be increased in FXRKO compared to WT mice.
Methods: WT and FXRKO mice were treated with intraperitoneal injections of either normal saline or LPS (n=4-6 in all treatment groups). After sixteen hours, mice were sacrificed, and RNA was extracted from mucosal scrapings of terminal ileal samples. Gene expression profiling was perfomed by RNA-seq, and differentially expressed genes were identified by DESeq2. Genes demonstrating statistically significant (adjusted p<0.05, Benjamini-Hochberg FDR) differential gene expression were cross-referenced with the NIH DAVID Bioinformatics Resources 6.8 database for molecular function and pathway analysis.
Results: Principal component analysis revealed distinct clustering by genotype, indicating a baseline difference between FXRKO and WT mice. We also found distinct clustering by treatment group. A unique LPS response was observed in FXRKO versus WT mice (Figure 1, each row is one of 494 genes with adjusted p=<0.05). Of note, expression of EGFR was upregulated by LPS treatment in FXRKO mice compared to WT mice, as was expression of Pik3ca and Pic3c2a, members of the PI3K family, and Map3k1, a member of the MAPK family. Other genes demonstrating unique LPS response in FXRKO mice included Ddx3x, Yes1, and Arhgap5, which are involved in pathways related to cadherin binding, adherens junctions, and focal adhesions, respectively.
Conclusion: Acute intestinal injury as modelled by LPS injection significantly alters the gene expression profile of FXRKO mice in comparison to WT mice, specifically affecting pathways involved in intestinal barrier integrity and restitution. These data provide avenues for investigation into specific genes and molecular pathways to understand the mechanism through which the intestinal barrier is protected from acute injury in FXRKO mice. Understanding the role of FXR in acute injury will help identify therapeutic targets for the treatment of diseases such as necrotizing enterocolitis and inflammatory bowel disease.
