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.

Chronic intestinal inflammation is a poorly understood manifestation of Cystic Fibrosis (CF), which may be refractory to ion channel CFTR modulator therapy. People with CF exhibit intestinal dysbiosis which has potential for stimulating intestinal and systemic inflammation. CFTR is expressed in organ epithelia and in the leukocyte population. Here, we investigate the contribution of intestinal epithelial-specific loss of Cftr (iCftr KO) to dysbiosis and inflammation in mice treated with either of two anti-obstructive dietary regimens necessary to maintain CF mouse models (PEG laxative or a liquid diet, LiqD). Feces collected from iCftr KO mice and their wildtype (WT) sex-matched littermates were used to measure fecal calprotectin and to perform 16S rRNA sequencing to characterize the gut microbiome. Fecal calprotectin was elevated in iCftr KO relative to WT samples of mice consuming either PEG or LiqD. PEG iCftr KO mice did not show a change in α-diversity versus WT but demonstrated a significant difference in microbial composition (β-diversity) with increases in phylumProteobacteria, familyPeptostreptococcaceae, four genera ofClostridiaincludingC. innocuum, and mucolytic genusAkkermansia. Fecal microbiome analysis of LiqD iCftr KO mice showed both decreased α-diversity and differences in microbial composition with increases inProteobacteriafamilyEnterobacteriaceae,FirmicutesfamiliesClostridiaceaeandPeptostreptococcaceae, and enrichment ofClostridium perfringens,C. innocuum,C. difficile, mucolyticRuminococcus gnavus, and reduction ofAkkermansia. It was concluded that epithelial-specific loss of Cftr is a major driver of CF intestinal dysbiosis and inflammation with significant similarities to previous studies of global Cftr KO mice.