Vitamin D Receptor in Paneth Cells Modulates Small Intestinal Inflammation

BACKGROUND Paneth cells (PCs) are specialized epithelial cells mainly found in the small intestine. PCs have a key role in shaping microbiome. It is considered as a site of origin for intestinal inflammation. Previous studies indicate that vitamin D3/vitamin D receptor (VDR) are implicated in IBD and infection. However, there is limited study exploring Paneth cells with VDR deficiency in ileitis and dysbiosis. Hypothesis We hypothesize that VDR deficiency in Paneth cells alters the microbiome and makes the host susceptible to small intestinal inflammation (e.g., ileitis). Methods/Models Paneth cells isolated from conditional PC KO (VDRΔPC) mice and the lox controls (VDRloxp) were analyzed by single cell RNA sequencing. Small intestinal microbiome of VDRΔPC mice were detected by Fluorescence in situ hybridization (FISH). To rule out confounding factors that may contribute to the intestinal epithelial VDR regulation, we have established enteroids derived from the VDRΔPC and VDRloxp mice. SAMP1/YitFc mice develop a spontaneous ileitis that is similar in many features to human Crohn’s Disease (CD). We then examined the expression level of VDR in the SAMP1/YitFc mice with ileitis. RESULTS The Paneth cells in the ileal organoids isolated from VDRloxp and VDRΔPC mice were labeled with lysozyme via immunofluorescence staining. The number of lysozyme-positive cell, ie Paneth cell, were counted in each bud of the organoids. There were significantly reduced Paneth cells in the VDRΔPC organoids, compared to the VDRloxp organoids. Further, we observed dislocation of small intestinal microbiome and penetration to epithelial cells in the VDRΔPC mice. The mucus thickness was significantly reduced in the small intestine of the VDRΔPC mice, compared to the VDRloxp mice. Conditional VDR deletion in Paneth cell severely changed antimicrobial peptides, DEFA3, DEFA4 and DEFA21 expression decreased in the ileum of VDRΔPC mice, compared to the VDRloxp mice. Development of ileitis is accelerated by the presence of luminal bacteria and is characterized by discontinuous segmental inflammation in the ileum while sparing the proximal small intestine and colon. We found abnormal Paneth cells in the SAMP1/YitFc mice, compared with the control AKR mice. VDR and VDR target ATG16L1 were significantly reduced in the small intestine of the SAMP1/YitFc mice with ileitis via RT-PCR, WB, and IHC staining. Conclusion And Future Plan Our current observation suggests that PC VDR deletion makes the host susceptible to small intestinal inflammation. Insights gained from understanding how VDR is integrally involved in regulating Paneth cell function may serve as a novel paradigm for understanding the mechanism of host-microbial interactions. This effort can facilitate new therapeutic targets for CD by assessing cellular and molecular responses or resistance to treatment.