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.