Anti-DEFA5 monoclonal antibody, clone FQS25410(C)

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.

INTRODUCTION: DNA methylation is an epigenetic mechanism that regulates gene expression and represents an important link between genotype, environment, and disease. It is a reversible and inheritable mechanism that could offer treatment targets. We aimed to assess the methylation changes on specific genes previously associated with Crohn's disease (CD) and to study their possible associations with the pathology. METHODS: We included 103 participants and grouped them into 2 cohorts (a first En = 31l and a second validation En = 72l cohort), with active CD (aCD) and inactive CD (iCD) and healthy participants (CTR). DNA was obtained from the peripheral blood and analyzed by the Agena platform. The selected genes were catalase (CA7), alpha-defensin 5 (DEFA5), FasR, FasL, tumor necrosis factor (TNF), TNFRSF1A, TNFRSF1B, PPA2, ABCB1, NOD2, PPAR gamma, and PKC zeta. We used the elastic net algorithm and R software. RESULTS: We studied 240 CpGs. Sixteen CpGs showed differential methylation profiles among aCD, iCD, and CTR. We selected for validation those with the greatest differences: DEFA5 CpG_11; CpG_13; CAT CpG_31.32; TNF CpG_4, CpG_12; and ABCB1 CpG_21. Our results validated the genes DEFA5 (methylation gain) and TNF(methylation loss) with P values < 0.001. In both cases, the methylation level was maintained and did not change with CD activity (aCD vs iCD). The subanalysis comparison between aCD and iCD showed significant differential methylation profiles in other CpGs: TNF, FAS, ABCB1, CAT, and TNFRS1BF genes. DISCUSSION: The methylation status of DEFA5 and TNF genes provides a signature biomarker that characterizes patients with CD and supports the possible implication of the environment and the immune system in CD pathogenesis.