Fecal Dysbiosis and Inflammation in Intestinal-Specific Cftr Knockout Mice
Authors: Woode Rowena , Young Sarah , Williams Estela , Ericsson Aaron , Clarke Lane 
Published: May 2023

The genetic disease cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The CFTR anion channel is expressed in epithelial cells to promote fluid secretion, and in immune cells to regulate the immune response. People with CF experience intestinal inflammation and dysbiosis, characterized by decreased microbial diversity, loss of commensal bacteria, and an increase in pathogenic species. Currently, the impact of intestinal epithelial-specific CFTR loss versus global CFTR loss on intestinal inflammation and the fecal microbiome is unknown. We hypothesized that epithelial-specific Cftr knockout will induce intestinal dysbiosis and inflammation. Fecal pellets were collected using sterile technique from single-housed B6.Cg-Tg(Vil1-cre)-Cftrf10/f10 intestinal-specific Cftr knockout (iCftr KO) mice and wildtype (WT) littermates. Fecal calprotectin, a marker of inflammation, was measured via ELISA kit, and a DNA extraction kit was used to obtain fecal microbial DNA for subsequent 16S rRNA sequencing. These experimental endpoints were measured in iCftr KO and WT mice maintained on either a commercially available chow diet with an osmotic polyethylene glycol (PEG) laxative in their drinking water, or a nutritionally complete liquid diet (Peptamen) and tap water to prevent intestinal obstruction. Fecal calprotectin was significantly increased in iCftr KO mice compared to WT in both PEG (2.75±1.39μg/g WT, 19.86±3.97μg/g iCftr KO, n = 9 pairs, p = 0.001) and Peptamen-fed mice (12.04±2.83μg/g WT, 36.05±6.63μg/g iCftr KO, n = 9 pairs, p = 0.007). Measurement of fecal microbial α-diversity of WT and iCftr KO PEG mice revealed no significant difference in either Shannon H diversity (3.79±0.19 WT, 3.56±0.12 iCftr KO, n = 10 pairs, p = 0.273) or Chao-1 richness indices (202.9±12.8 WT, 184.5±8.68 iCftr KO, n = 10 pairs, p = 0.250). PERMANOVA results of β-diversity calculated using the Bray-Curtis dissimilarity demonstrated that the fecal microbiome of iCftr KO PEG mice were significantly different from WT PEG mice (n = 10 pairs, p = 0.0001, F = 6.511). iCftr KO PEG mice showed enrichment of several potential pathobionts, including several species from the genus Clostridium. Peptamen-fed iCftr KO mice had a significant decrease in α-diversity using both Shannon H diversity (3.58±0.08 WT, 2.797±0.17 iCftr KO, n = 10 pairs, p < 0.001) and Chao-1 richness indices (207.8±15.29 WT, 100.0±10.93 iCftr KO, n = 10 pairs, p < 0.001). PERMANOVA results for Peptamen-fed mice revealed a significant difference between the microbial composition of iCftr KO and WT mice (n = 10 pairs, p = 0.0001, F = 5.671). Peptamen-fed iCftr KO mice also had significant enrichment of several Clostridium species, including C. difficile. Based on the above data, we conclude that intestinal epithelial-specific loss of Cftr function is sufficient to induce intestinal inflammation and dysbiosis. Funding: Cystic Fibrosis Foundation grant CLARKE20G0, Crohn's and Colitis Foundation grant #693938 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.