Suppression of macrophage infiltration in the livers of NASH mice treated with CDDO-Me
To identify the inflammatory cells regulated by CDDO-Me, the expression of marker genes in leukocyte subtypes was visualized using a heatmap. The representative macrophage markers, Adgre1 and Cd86 , were remarkably elevated in NASH mice and their increased expression was significantly inhibited by CDDO-Me treatment (Figure 6a). The neutrophil markers Ptprc , Cxcr4 , and Itgb2 were upregulated, similar to those of macrophages, but their inhibition by CDDO-Me treatment was weak. The markers for T and B cells remained almost unchanged (Figure 6a and Figure S7). To confirm the leukocyte subtypes in the liver, immunohistochemical staining with the macrophage marker F4/80 and the neutrophil marker LY6G, was performed in liver sections from NASH mice treated with CDDO-Me. CDDO-Me treatment decreased the number of macrophages in the livers of NASH mice (Figure 6b,c). The number of neutrophils infiltrating the livers of NASH mice was comparatively low, and their infiltration was decreased by half by CDDO-Me treatment (Figure 6b,d). The hepatic macrophage subtypes targeted by CDDO-Me have been analysed based on marker genes using scRNA-seq of NASH in previous reports (Guilliams M et al., 2022). Most marker genes in lipid-associated macrophages were highly expressed in NASH, and CDDO-Me significantly inhibited their increased expression along with a decrease in lipid accumulation (Figure 7a,b). The expression of marker genes for monocytes and monocyte-derived macrophages, such as monocyte-derived Kupffer cells and patrolling monocytes, was significantly increased in the liver during NASH, and this increase was suppressed by CDDO-Me treatment (Figure 7a,b). Marker genes of tissue-localized macrophages, such as resident Kupffer cells and peritoneal macrophages were almost constant in the NASH group, independent of CDDO-Me administration (Figure 7a,b). The localization of representative macrophage subtypes was examined by immunohistochemistry using specific antibodies against marker genes. GPNNB, a marker of lipid-associated macrophages, was not detected in the control liver sections, whereas GPNNB-positive cells were present in the area surrounding the lipid droplets in NASH (Figure 7c,d). CDDO-Me strongly suppressed lipid-associated macrophages staining in NASH. The number of cells stained for markers of monocytes and patrolling monocytes, CCR2 and SPN, respectively, was significantly increased in NASH and was decreased by CDDO-Me administration (Figure 7c,e,f). These cells were frequently observed in the surrounding blood vessels. CLEC4F-positive cells indicated that resident Kupffer cells widely existed throughout the normal liver, and that staining was slightly decreased by NASH, independent of CDDO-Me treatment (Figure 7c,g). These observations indicated that monocyte-derived macrophages infiltrated the liver through blood vessels, while CDDO-Me effectively inhibited recruitment.