AB4 inhibited the activation of NLRP3 inflammasome through targeting CD1d
CD1d molecule has the role of antigen presentation, and several studies have confirmed that CD1d-related immune pathways have important effects on UC (Fuss et al., 2004; Huang et al., 2016; Lee et al., 2019; Mizoguchi, Mizoguchi, Takedatsu, Blumberg, & Bhan, 2002; Olszak et al., 2014). One of our recent works showed that macrophage CD1d could inhibit NLRP3 inflammasome expression during inflammation (Cui et al., 2020). To further investigate the mechanism by which AB4 alleviated the progression of colitis, we next investigated whether AB4 inhibited NLRP3 inflammasome expression in macrophages through CD1d signaling. In 3.0% DSS-induced WT mice colitis, compared with the control group, the expression level of CD1d protein in the colon tissue of the DSS group was significantly decreased. Compared with the DSS group, AB4 (5, 10, and 15mg/kg) significantly enhanced CD1d protein (Supporting Information Fig. S5) expression in colonic homogenates of mice. Strikingly, AB4 enhanced CD1d protein expression in colonic macrophages (Fig. 7A). To investigate whether the protective effect of AB4 on colitis depended on the CD1d signaling pathway, we constructed 3% DSS-induced colitis in macrophage-specific CD1d-knockout (CD1d-/-) mice and WT mice. Inflammation index values (i.e., body weight loss, DAI score, and colon length) demonstrated that CD1d-/- mice were more susceptible to DSS-induced colitis compared with WT mice (Fig. 7B-D), supporting a critical role of macrophage CD1d in the disease development. However, macrophage-specific CD1d depletion reversed the protective effects of AB4 (15mg/kg) on body weight loss, DAI score, and colon shortening in DSS-induced colitis (Fig. 7B-D). H&E staining and F4/80+ immunofluorescent revealed macrophage-specific CD1d depletion abolished AB4’s protective effects both in the colonic morphometry (Fig. 7E) and macrophagic observation (Fig. 7F). Western Blot and ELISA results confirmed that macrophage-specific CD1d depletion reversed the inhibition of the protein expression of p-AKT/AKT, p-STAT1/STAT1, PRDX1, p-P65/P65, p-IκBα/IκBα, NLRP3, ASC, Caspase-1 p20, IL-1β, and IL-18 by AB4 (Fig. 7G and H). Collectively, these data suggested that AB4 might target CD1d thus reducing the AKT-STAT1-PRDX1-NF-κB signaling pathway, eventually inhibiting the activation of NLRP3 inflammasome and ameliorating DSS-induced colitis in mice.