Anti-DEFA5 monoclonal antibody, clone 9D9

Metabolic syndrome (MetS), characterized as obesity, insulin resistance, and non-alcoholic fatty liver diseases (NAFLD), is associated with vitamin D insufficiency/deficiency in epidemiological studies, while the underlying mechanism is poorly addressed. On the other hand, disorder of gut microbiota, namely dysbiosis, is known to cause MetS and NAFLD. It is also known that systemic inflammation blocks insulin signaling pathways, leading to insulin resistance and glucose intolerance, which are the driving force for hepatic steatosis. Vitamin D receptor (VDR) is highly expressed in the ileum of the small intestine, which prompted us to test a hypothesis that vitamin D signaling may determine the enterotype of gut microbiota through regulating the intestinal interface. Here, we demonstrate that high-fat-diet feeding (HFD) is necessary but not sufficient, while additional vitamin D deficiency (VDD) as a second hit is needed, to induce robust insulin resistance and fatty liver. Under the two hits (HFD+VDD), the Paneth cell-specific alpha-defensins including u-defensin 5 (DEFA5), MMP7 which activates the pro-defensins, as well as tight junction genes, and MUC2 are all suppressed in the ileum, resulting in mucosal collapse, increased gut permeability, dysbiosis, endotoxemia, systemic inflammation which underlie insulin resistance and hepatic steatosis. Moreover, under the vitamin D deficient high fat feeding (HFD+VDD), Helicobacter hepaticus, a known murine hepatic-pathogen, is substantially amplified in the ileum, while Akkermansia muciniphila, a beneficial symbiotic, is diminished. Likewise, the VD receptor (VDR) knockout mice exhibit similar phenotypes, showing down regulation of alpha-defensins and MMP7 in the ileum, increased Helicobacter hepaticus and suppressed Akkermansia muciniphila. Remarkably, oral administration of DEFA5 restored eubiosys, showing suppression of Helicobacter hepaticus and increase of Akkermansia muciniphila in association with resolving metabolic disorders and fatty liver in the HFD+VDD mice. An in vitro analysis showed that DEFA5 peptide could directly suppress Helicobacter hepaticus, Thus, the results of this study reveal critical roles of a vitamin D/VDR axis in optimal expression of defensins and tight junction genes in support of intestinal integrity and eubiosis to suppress NAFLD and metabolic disorders.

Background and aims: Reduced ileal Paneth cell alpha-defensin expression has been reported to be associated with Crohn's disease, especially in patients carrying NOD2 mutations. The aim of this study was to independently assess whether NOD2, alpha-defensins and Crohn's disease are linked. Methods: Using quantitative real-time polymerase chain reaction (RT-PCR), we measured the mRNA expression levels of key Paneth cell antimicrobial peptides (DEFA5, DEFA6, LYZ, PLA2G2A), inflammatory cytokines [interkelukin 6 (IL6) and IL8], and a marker of epithelial cell content, villin (VIL1) in 106 samples from both affected ileum (inflamed Crohn's disease cases, n = 44) and unaffected ileum (non-inflamed; Crohn's disease cases, n = 51 and controls, n = 11). Anti-human defensin 5 (HD-5) and haematoxylin/eosin immunohistochemical staining was performed on parallel sections from NOD2 wild-type and NOD2 mutant ileal Crohn's disease tissue. Results: In Crohn's disease patients, DEFA5 and DEFA6 mRNA expression levels were 1.9- and 2.2-fold lower, respectively, in histologically confirmed inflamed ileal mucosa after adjustment for confounders (DEFA5, p < 0.001; DEFA6, p = 0.001). In contrast to previous studies, we found no significant association between alpha-defensin expression and NOD2 genotype. HD-5 protein data supports these RNA findings. The reduction in HD-5 protein expression appears due to surface epithelial cell loss and reduced Paneth cell numbers as a consequence of tissue damage. Conclusions: Reduction in alpha-defensin expression is independent of NOD2 status and is due to loss of surface epithelium as a consequence of inflammatory changes rather than being the inciting event prior to inflammation in ileal Crohn's disease.