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.