IMPAIRED PANETH CELL FUNCTION AND MICROBIAL DYSBIOSIS PRECEDE INTESTINAL INFLAMMATION IN P-GLYCOPROTEIN KNOCKOUT MICE

P-glycoprotein (Pgp), encoded by the MDR1 (multidrug resistance 1/ABCB1) gene plays a critical role in maintaining intestinal homeostasis by pumping out xenobiotics/bacterial toxins from the mucosa into the gut lumen. Decrease in Pgp function and expression has been shown in patients with IBD and in animal models of gut inflammation. Also, global Pgp knockout (Pgp KO) mice spontaneously develop severe colitis at 15 wks (similar to the human IBD) but not at 8 wks of age. Previously, we have shown impaired Paneth cell (PC) function with a significant decrease in Lyz1 staining in the ileum of Pgp KO mice at 15 wks associated with a marked reduction in the expression of other antimicrobial peptides (AMPs). Whether impaired Paneth cell function in Pgp KO mice precedes gut inflammation at 8 wks or is a consequence of inflammation (15 wks) is not known. Methods: 8- and 15-weeks old, male Pgp KO and FVB (WT) mice (Taconic, NY) were used to examine changes in epithelial cell subtypes (Paneth cells, goblet cells) in the ileum and colon. Cecal contents were collected for gut microbiome analysis by 16S rRNA sequencing. Results: Pgp KO mice at 8 wks of age showed a significant decrease in Lyz1 staining in the ileum (~50%; p<0.05) associated with a decrease in the mRNA expression of other AMPs, Regs 3a, 3g and Ang4. In addition, microbial analysis showed a significant expansion of Turicibacter sanguinis, Alistipes sp. and Desulfovibrio sp. (shown to be increased in gut inflammation) in Pgp KO mice at 8 wks and was not detected in WT mice. Pgp KO mice (8 wks) showed a marked decrease in SCFA-producing bacteria, Roseburia (12 fold; p<0.05) and Oscillibacter (4 fold; p<0.01) which are known to be decreased in IBD. These microbial changes were also observed in Pgp KO mice at 15 wks of age. Interestingly, Pgp KO mice at 15 wks showed a lower abundance of Bacteroides thetaiotamicron (50 fold lower; p<0.05) which is known to stimulate Paneth cell function of secreting antimicrobial peptides (AMPs). Thus, reduced PC AMPs secretion and SCFA-producing bacteria as well as increased abundance of sulfate-reducing bacteria, Desulfovibrio sp. in Pgp KO mice suggest that these mice may have a defective mucus barrier. We also observed a reduction in the number of MUC2-positive goblet cells in the colon of Pgp KO mice at 8 wks (33%; p<0.05) which was more pronounced at 15 wks (~70%, p<0.05) but remained unaltered in the ileum. This was associated with a decrease in mucus layer thickness by PAS staining in the colon at both 8 and 15 wks (~30-40%; p<0.01) respectively. Conclusion: Our novel findings in Pgp KO mice suggest that impaired Paneth cell function along with gut dysbiosis and a defective colonic mucus barrier that precedes inflammation (at 8 wks) is likely to contribute to the development of inflammation/spontaneous colitis in Pgp KO mice at 15 wks.