ABSTRACT One of the underappreciated effect of obesity is the repression of gut innate immunity, as we recently showed in overweight/obese patients, using Paneth cell phenotype (as a surrogate for Paneth cell function) as a proof- of-concept. Wild type mice fed with western diet (WD) also developed Paneth cell defects. We further showed that WD consumption results in microbiota-mediated increase in deoxycholic acid (a secondary bile acid), which activates the FXR pathway in the ileum. FXR activation in the intestinal epithelium, as well as FXR- mediated type I interferon activation in myeloid cells, collectively trigger Paneth cell defects. The critical questions that need to be addressed before translating these findings to clinic include how individual dietary components that impact Paneth cell function, and if long-term WD consumption renders Paneth cell defects irreversible to dietary switch alone. Our long-term goal is to dissect the cellular and molecular mechanisms of how WD consumption affect the intestinal stem cell (ISC) and Paneth cell biology. These discoveries will facilitate design of trials for obese patients with gut innate immunity dysfunction. The objective of this proposal is to determine how WD consumption induces Paneth cell abnormality. The central hypothesis is that long-term consumption of dietary fructose result in Paneth cell defect due to diminished capacity of Paneth cell replenishment (by Paneth cells themselves and/or intestinal stem cells [ISC]). Our rationale is that identification of the mechanism(s) to restore Paneth cell function will offer new therapeutic opportunities for many patients, such as those with inflammatory bowel disease and graft versus host disease, of which Paneth cells play a critical role in pathogenesis. Our preliminary data suggest that dietary fructose consumption alone is sufficient to trigger Paneth cell defects, and that Paneth cell defects after long-term (≥12 months) WD consumption are not reversible by switching to standard diet. Our specific aims will test the following hypotheses: (Aim1) How dietary fructose directly induces Paneth cell defects; (Aim 2) Long-term WD consumption will impair the capacity of Paneth cells as well as ISCs to repair and replenish defective Paneth cells. Upon conclusion, we will understand the role for dietary fructose in modulating Paneth cell and ISC function. This contribution is significant since it will establish intestinal epithelial fructose catabolism and associated genes as therapeutic targets. The proposed research is innovative because we investigate how long-term exposure to poor diet on gut innate immunity, a heretofore-unexamined process. We also use state-of-the-art ISC culture system and scRNA-seq and scATAC-seq techniques to identify molecular and cellular targets that affect Paneth cell and ISC functions. Identifying the mechanisms of how diets regulate a key disease-relevant cellular phenotype will provide insight into other inflammatory ...