Genes regulated by CDDO-Me treatment in macrophages
To elucidate the molecules participating in macrophage recruitment
regulated by CDDO-Me during NASH, network analysis was performed using
CDDO-Me-downregulated genes. The network indicated that CDDO-Me
inhibited the NF-κB pathway (Figure S8), leading to inhibition of cell
infiltration through chemokine ligands CCL4, and human CCL3L3
corresponding to mouse CCL3 (Figure 8a). The expression levels of
chemokines and receptors associated with macrophage recruitment were
visualized on a heatmap (Figure 8b).
CDDO-Me markedly inhibited the
expression of chemokine ligands, Ccl3 and Ccl4 , and
chemokine receptors, Ccr1 and Ccr5, which were increased
in NASH mice; this pattern was consistent with that of NAS, an indicator
of NASH. The expression levels of representative chemokines and their
receptors were confirmed using quantitative PCR (Figure S9). Serum
protein levels of CCL3 and CCL4, which were upregulated in NASH mice
were inhibited in a dose-dependent manner by CDDO-Me treatment (Figure
8c,d), indicating that the chemokine ligands released into blood may
function as migration factors for monocyte-derived macrophages. The
number of cells expressing CCR1 and CCR5 increased in the surrounding
blood vessels during NASH and almost disappeared in the presence of
CDDO-Me (Figure 8e-g). Analysis of mouse NAFLD using scRNA-seq data from
public datasets (https://www.livercellatlas.org/) suggested thatCcl3 and the receptor Ccr1 were expressed in
monocytes/macrophages and neutrophils, and that Ccl4 and the
receptor Ccr5 were detected in macrophages/monocytes and Kupffer
cells (Figure S10). CCR1- and CCR5-expressing cells corresponded to
cells expressing CCR2, a monocyte marker (Figure 8h), indicating that
CCR1 and CCR5 expressed in monocyte-derived macrophages were regulated
by CDDO-Me. To examine whether CDDO-Me directly participates in the
expression of chemokine receptors and ligands, we measured their
expression levels in RAW264.7 murine macrophages after CDDO-Me
treatment. CDDO-Me inhibited the expression levels of Ccr1 andCcr5 , and simultaneously blocked Ccl3 and Ccl4 , the
ligands of these receptors, respectively in RAW264.7 cells (Figure
8i-8l). Taken together with these observations, CDDO-Me directly
inhibits the CCL3-CCR1 and CCL4-CCR5 axes in the macrophages of NASH
mice, contributing to the improvement of non-alcoholic steatohepatitis
and fibrosis through the prevention of monocyte-derived macrophage
migration.