Discussion
We showed that CDDO-Me, a potent activator of Nrf2, dramatically improved NASH with lipid droplets and fibrosis in CDAHFD-fed mice and markedly suppressed the recruitment of monocyte-derived macrophages from blood vessels, in addition to activating the Nrf2-dependent pathway. During this study, the CCL3-CCR1 and CCL4-CCR5 axes involved in the chemoattraction of macrophages were identified from the regulated genes in NASH model mice as novel targets of CDDO-Me.
NAFLD occurred in mice fed a choline-deficient, L-amino-acid-defined, high-fat diet and progressed to NASH with inflammation and hepatic injury for 8 weeks. Administration of CDDO-Me to NASH mice improved the NAS calculated from steatosis, inflammation, and hepatic injury. To identify the molecular targets of CDDO-Me, which regulate NASH progression, whole-transcriptome analysis was performed using the livers of NASH mice. The upregulated genes in NASH mice treated with CDDO-Me showed activation of the Nrf2-mediated oxidative stress response originating from NFE2L2 (gene name of Nrf2), indicating that CDDO-Me-regulated genes were mediated by Nrf2 activation. This observation was consistent with our quantitative PCR data and previous studies analysing the downstream factors of Nrf2 activation (Tonelli, Chio, & Tuveson, 2018). Pathway analysis using the expressed genes corresponding to the disease condition in the presence of CDDO-Me during NASH indicated that CDDO-Me regulated the recruitment of immune cells, which was confirmed by immunohistochemical observation. Among the immune cells, numerous macrophages/monocytes were detected in NASH, which were efficiently reduced by CDDO-Me treatment; however, it did not affect the infiltration of lymphoid cells. Hepatic macrophages, which release chemokines and cytokines, considerably affect the progression of NASH from NAFLD, leading to inflammation and fibrosis (Tacke, Puengel, Loomba, & Friedman, 2023). Subtypes with distinct functions are present in monocytes/macrophages of NASH, and the classification of these subtypes is based on the diversity of gene expression in macrophages (Daemen et al., 2021). Using scRNA-seq, we divided hepatic macrophages into seven subtypes in the NASH model: lipid-associated macrophages, monocytes, patrolling monocytes, central vein and capsule macrophages, monocyte-derived Kupffer cells, resident Kupffer cells, and peritoneal macrophages (Guilliams et al., 2022). Subtype-specific genes not expressed in other cells within the liver were selected as marker genes to examine subtype localization. Monocytes and monocyte-derived macrophages, such as lipid-associated macrophages, monocytes, patrolling monocytes, central vein and capsule macrophages, and monocyte-derived Kupffer cells were increased during NASH and were suppressed by CDDO-Me, in contrast to the constant or slight increase in resident Kupffer cells and peritoneal macrophages localized in the tissues. Staining with macrophage subtype-specific antibodies showed that lipid-associated macrophages, monocytes, and patrolling monocytes infiltrated the vessel lumen into the space of Disse, and that the macrophages surrounding the blood vessels disappeared after CDDO-Me treatment. These findings indicate that the major target of CDDO-Me is monocyte/macrophage recruitment from the bone marrow. The marker for resident Kupffer cells showed scattered detection in whole tissues and was not affected by CDDO-Me treatment. Krenkel et al. showed that monocyte-derived macrophages specifically activate various growth factors and cytokines involved in fibrosis progression in NASH (Krenkel et al., 2018). Inhibition of macrophage recruitment by CDDO-Me was mainly determined to be mediated by the CCL3-CCR1 and CCL4-CCR5 axes for the following reasons. Pathway analysis using genes downregulated by CDDO-Me showed decreased phagocyte recruitment, which included CCL3 and CCL4 in the network. Second, CCL3 and CCL4 were released into blood during NASH, and serum CCL3 and CCL4 levels returned to control levels in the presence of CDDO-Me. Third, CCR1, the receptor for CCL3, and CCR5, the receptor for CCL4, were localized in monocytes infiltrated during NASH. Finally, CDDO-Me directly inhibited the expression of Ccl3-Ccr1 andCcl4-Ccr5 in the mouse macrophage cell line RAW264.7, in a dose-dependent manner. In NAFLD model mice, CCL3-deficiency has been reported to attenuate the steatosis and fibrosis associated with M2-macrophages (Xu et al., 2021). CCR5 critically reduces macrophage infiltration into the adipose tissue and subsequent insulin resistance in CCR5-deficient mice (Kitade et al., 2012). Further, CCL2, CCL3, CCL4, and CCL5, have been previously identified as the relevant chemokines in NAFLD/NASH (Marra & Tacke, 2014); we found that CDDO-Me inhibited the expression of these chemokines, except for CCL2. The absence of CCL2 extensively inhibits hepatic steatosis and macrophage accumulation in adipose tissue produced by a high-fat diet (Kanda et al., 2006), whereas CCL2-deficient mice show no effect on macrophage recruitment in obesity (Inouye et al., 2007). Obese mice with deleted CCR2, the receptor of CCL2, show ameliorated steatosis and improved glucose homeostasis (Weisberg et al., 2006), consistent with data obtained using a pharmacological antagonist of CCR2 (Tamura et al., 2010), indicating that the inhibiting CCR2 rather than CCL2 is more effective for treating NASH progression. CCR2 expression was decreased in the presence of CDDO-Me, which may partially inhibit macrophage infiltration during NASH. The upstream pathway indicated that CCL3 and CCL4 was regulated by NF-κB, which is also a target of CDDO-Me. Ahmad et al. reported that CDDO-Me was directly associated with Cys-179 on IKKβ, causing inhibition of the NF-κB pathway in U937 myeloid leukaemia cells (Ahmad, Raina, Meyer, Kharbanda, & Kufe, 2006). The inhibitory effect of CDDO-Me on the NF-κB pathway may predominantly work in myeloid cells because macrophage-specific NF-κB activation is involved in the numerous inflammatory diseases (Mussbacher, Derler, Basílio, & Schmid, 2023).
scRNA-seq data from the NAFLD model indicate that CCR5 is mainly expressed in monocytes/macrophages, while CCR1 is expressed in neutrophils and monocytes (Figure S10). Neutrophil infiltration is often observed in the early phase of NASH from NAFLD (Herrero-Cervera, Soehnlein, & Kenne, 2022), and the CCL3/CCR1 axis may be involved in the progression of the early phase of the disease through neutrophils and monocytes. CDDO-Me may reduce neutrophil infiltration through CCR1 in the early phase and regulate the recruitment of monocytes/macrophages expressing CCR5 in the late phase of NASH, which may lead to efficient inhibition of disease progression. The chemokines CCL3 and CCL4 increase in macrophages during NASH, stimulating the chemokine receptors CCR1 and CCR5 in phagocytes, and enhancing the release of CCL3 and CCL4 from macrophages through autocrine/paracrine mechanisms (Fahey et al., 1992). This activation could sustain inflammation in the tissues for a long time, and inhibition of chemokines and receptors by CDDO-Me may be helpful in arresting chronic inflammatory diseases in various tissue macrophages, for instance, CDDO-Me significantly improved chronic kidney disease with type 2 diabetes in clinical trials. CDDO-Me also ameliorates high-fat diet-induced colon inflammation and radiation-induced lung inflammation in mice. The novel pharmacological activities of CDDO-Me may inhibit macrophage recruitment through autocrine/paracrine mechanisms involving chemokines and their receptors. However, additional whole-transcriptome analysis will be useful for elucidating the pharmacological targets of various medicines.
Overall, this study demonstrates the potent hepatoprotective effect of CDDO-Me in a mouse model of NASH along with the underlying mechanism of reduced macrophage infiltration via CCL3-CCR1 and CCL4-CCR5 inhibition, in addition to Nrf2-mediated hepatoprotective effects.
Acknowledgments: We thank Kenji Yoneda (Yoshindo Inc.) for helpful discussions, Sayuri Kurihara (UBE Corporation) for providing technical assistance. We also thank Makiko Nakagawa and Yuko Nakatani (Yamaguchi University) for support with the NGS analysis, and Noriko Kondo (Yamaguchi University) for assisting with the immunohistochemical analysis. Part of the research was carried out using research equipment shared in the MEXT Project for Promoting Public Utilization of Advanced Research Infrastructure (Program for Supporting the Construction of Core Facilities), grant number JPMXS0440400021-23. The RNA-seq and immunohistochemical analyses were supported by the Institute of Gene Research of the Yamaguchi University Science Research Center.
Data and materials availability: The data and materials described in this report are available upon request.