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.