Fig 2: Representative immunohistochemistry (IHC) images and quantification of NLRP6 and MUC2 expression in left colon biopsies. All biopsies were paraffin embedded, cut into 3-5 µm sections and incubated with NLRP6 (NBP2-31372, Novus Biologicals, Littleton, CA) and MUC2 (H:300 sc-15334, Santa Cruz Biotechnologies, TX). Images were captured using DP21 microscope camera (Olympus Australia, Melbourne, Australia) attached to an IX71 microscope (Olympus). Images are 400X and scale bar = 50 µm. (A) Quantitative (Quant.) analysis of DAB staining in paraffin embedded left colon mucosal biopsies as analysed by IHC. (B) NLRP6 expression in sections from normal colon (number of images analysed = 18), active UC (n = 43), remission UC (Rem. UC, n = 20), active ileal CD (n = 34), active colonic CD (n = 18) and remission CD (Rem. CD, n = 12). (C) MUC2 expression in sections from normal colon (n = 20), active UC n = 62), remission UC (n = 20), active ileal CD (n = 24), active colonic CD (n = 30), and remission CD (n = 30). The optical intensity of DAB staining was determined using ImageJ software. All data are presented as mean ± standard deviation. Statistical significance was evaluated using Dunn's multiple comparison One-way ANOVA and the significance threshold was P < .05. Here we present contrasting expression patterns for both NLRP6 and MUC2. In active ileal CD, NLRP6 expression is localised to the epithelial cell layer, myofibroblasts, neutrophils and monocytic linage cells residing in the lamina propria. In active UC NLRP6 expression is limited to lamina propria immune cells and absent for the epithelial cell layer. Interestingly, in the normal colon, moderate cytoplasmic NLRP6 expression was present in the epithelial cell layer and scattered within the lamina propria cells. Quantitative analysis of NLRP6 IHC staining confirms the high NLRP6 expression observed in ileal CD (active ileal CD vs active colonic CD, P < .001; active ileal CD vs remission CD, P < .001). MUC2 expression in active IBD and the normal colon is highly variable in distribution and depth. In active CD, both colonic and ileal the expression of MUC2 is increased while in UC it is reduced. The reduced MUC2 expression in active UC is well established and consistent with previous studies in human derived material.[2-4] Interestingly, there was no change in MUC2 expression in ileal and colonic CD despite the increased NLRP6 in ileal CD. Quantitatively, the expression of MUC2 in active ileal CD was greater than in active UC (P = .002).

Fig 3: The effect of disease activity and rosiglitazone treatment on the expression of NLRP6-related genes. (A–D) Paired colon biopsy specimens from quiescent (q) and active disease (a) in ileal CD (n = 6), colonic CD (n = 9), and UC patients (n = 30). Relative gene expression of individual biopsy specimens expressed as log10-fold change. Horizontal lines indicate the median relative expression and error bars represent the interquartile ranges. (E and F) Gene expression of NLRP6 and MUC2 in LS174T cells treated with rosiglitazone (5–50,000 nmol/L). Levels are relative to ethanol-treated control cells. Data are expressed as means ± SEM of 3 independent experiments performed in duplicate. The significance threshold was P < .05. Un, unstimulated.