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• W3118454925 abstract "•Kupffer cells are magnetically divided into Fe-hi and Fe-lo fractions in a NASH model•Fe-hi Kupffer cells exert proinflammatory and profibrotic properties in NASH•MiT/TFE transcription factors mediate iron-induced Kupffer cells' phenotypic changes•MiT/TFE transcription factors are activated in Kupffer cells in murine and human NASH Although recent evidence suggests the involvement of iron accumulation in the pathogenesis of nonalcoholic steatohepatitis (NASH), the underlying mechanisms remain poorly understood. Previously, we reported a unique histological structure termed “crown-like structure (CLS),” where liver-resident macrophages (Kupffer cells) surround dead hepatocytes, scavenge their debris, and induce inflammation and fibrosis in NASH. In this study, using magnetic column separation, we show that iron-rich Kupffer cells exhibit proinflammatory and profibrotic phenotypic changes during the development of NASH, at least partly, through activation of MiT/TFE transcription factors. Activation of MiT/TFE transcription factors is observed in Kupffer cells forming CLSs in murine and human NASH. Iron chelation effectively attenuates liver fibrosis in a murine NASH model. This study provides insight into the pathophysiologic role of iron in NASH. Our data also shed light on a unique macrophage subset rich in iron that contributes to CLS formation and serves as a driver of liver fibrosis. Although recent evidence suggests the involvement of iron accumulation in the pathogenesis of nonalcoholic steatohepatitis (NASH), the underlying mechanisms remain poorly understood. Previously, we reported a unique histological structure termed “crown-like structure (CLS),” where liver-resident macrophages (Kupffer cells) surround dead hepatocytes, scavenge their debris, and induce inflammation and fibrosis in NASH. In this study, using magnetic column separation, we show that iron-rich Kupffer cells exhibit proinflammatory and profibrotic phenotypic changes during the development of NASH, at least partly, through activation of MiT/TFE transcription factors. Activation of MiT/TFE transcription factors is observed in Kupffer cells forming CLSs in murine and human NASH. Iron chelation effectively attenuates liver fibrosis in a murine NASH model. This study provides insight into the pathophysiologic role of iron in NASH. Our data also shed light on a unique macrophage subset rich in iron that contributes to CLS formation and serves as a driver of liver fibrosis. Nonalcoholic fatty liver disease (NAFLD) is commonly observed in patients with the metabolic syndrome and is one of the most prevalent chronic liver diseases in the world. The term NAFLD refers to a spectrum of chronic liver disease ranging from simple steatosis to nonalcoholic steatohepatitis (NASH), of which the latter predisposes patients to cirrhosis and hepatocellular carcinoma (Tilg and Moschen, 2010Tilg H. Moschen A.R. Evolution of inflammation in nonalcoholic fatty liver disease: the multiple parallel hits hypothesis.Hepatology. 2010; 52: 1836-1846Crossref PubMed Scopus (1449) Google Scholar). As proposed by the multiple parallel hits hypothesis, the pathogenesis of NASH involves a multistep process; in this context, metabolic stresses such as lipid accumulation and insulin resistance and inflammatory stimuli such as proinflammatory cytokines and oxidative stress have been intensively investigated (Tilg and Moschen, 2010Tilg H. Moschen A.R. Evolution of inflammation in nonalcoholic fatty liver disease: the multiple parallel hits hypothesis.Hepatology. 2010; 52: 1836-1846Crossref PubMed Scopus (1449) Google Scholar). Among various etiologies, aberrant iron metabolism is also considered to be a “hit” in the pathogenesis of NASH (Nelson et al., 2012Nelson J.E. Klintworth H. Kowdley K.V. Iron metabolism in nonalcoholic fatty liver disease.Curr. Gastroenterol. Rep. 2012; 14: 8-16Crossref PubMed Scopus (77) Google Scholar). For instance, 10%–50% patients with NAFLD exhibit hepatic iron accumulation and/or hyperferritinemia (Valenti et al., 2006Valenti L. Dongiovanni P. Piperno A. Fracanzani A.L. Maggioni M. Rametta R. Loria P. Casiraghi M.A. Suigo E. Ceriani R. et al.α1-antitrypsin mutations in NAFLD: high prevalence and association with altered iron metabolism but not with liver damage.Hepatology. 2006; 44: 857-864Crossref PubMed Scopus (71) Google Scholar; Manousou et al., 2011Manousou P. Kalambokis G. Grillo F. Watkins J. Xirouchakis E. Pleguezuelo M. Leandro G. Arvaniti V. Germani G. Patch D. et al.Serum ferritin is a discriminant marker for both fibrosis and inflammation in histologically proven non-alcoholic fatty liver disease patients.Liver Int. 2011; 31: 730-739Crossref PubMed Scopus (91) Google Scholar; Nelson et al., 2012Nelson J.E. Klintworth H. Kowdley K.V. Iron metabolism in nonalcoholic fatty liver disease.Curr. Gastroenterol. Rep. 2012; 14: 8-16Crossref PubMed Scopus (77) Google Scholar; Ryan et al., 2018Ryan J.D. Armitage A.E. Cobbold J.F. Banerjee R. Borsani O. Dongiovanni P. Neubauer S. Morovat R. Wang L.M. Pasricha S.R. et al.Hepatic iron is the major determinant of serum ferritin in NAFLD patients.Liver Int. 2018; 38: 164-173Crossref PubMed Scopus (36) Google Scholar). High-fat-diet feeding decreases duodenal expression of oxidoreductases in mice, resulting in suppression of iron absorption (Sonnweber et al., 2012Sonnweber T. Ress C. Nairz M. Theurl I. Schroll A. Murphy A.T. Wroblewski V. Witcher D.R. Moser P. Ebenbichler C.F. et al.High-fat diet causes iron deficiency via hepcidin-independent reduction of duodenal iron absorption.J. Nutr. Biochem. 2012; 23: 1600-1608Crossref PubMed Scopus (82) Google Scholar). Moreover, phlebotomy effectively ameliorates liver damage (Sumida et al., 2006Sumida Y. Kanemasa K. Fukumoto K. Yoshida N. Sakai K. Nakashima T. Okanoue T. Effect of iron reduction by phlebotomy in Japanese patients with nonalcoholic steatohepatitis: a pilot study.Hepatol. Res. 2006; 36: 315-321Crossref PubMed Scopus (52) Google Scholar; Valenti et al., 2011Valenti L. Moscatiello S. Vanni E. Fracanzani A.L. Bugianesi E. Fargion S. Marchesini G. Venesection for non-alcoholic fatty liver disease unresponsive to lifestyle counselling-a propensity score-adjusted observational study.QJM. 2011; 104: 141-149Crossref PubMed Scopus (50) Google Scholar). Because iron increases oxidative stress through the Fenton reaction, iron overload may be involved in the pathogenesis of various chronic diseases including NAFLD (Galaris and Pantopoulos, 2008Galaris D. Pantopoulos K. Oxidative stress and iron homeostasis: mechanistic and health aspects.Crit. Rev. Clin. Lab. Sci. 2008; 45: 1-23Crossref PubMed Scopus (232) Google Scholar). However, the pathophysiologic role of iron in NASH remains controversial (O’Brien and Powell, 2012O’Brien J. Powell L.W. Non-alcoholic fatty liver disease: is iron relevant?.Hepatol. Int. 2012; 6: 332-341Crossref PubMed Scopus (24) Google Scholar). Liver comprises parenchymal hepatocytes and a variety of stromal cells including macrophages and hepatic stellate cells, all of which interact with each other to remodel tissue structure during the development of NASH. We previously demonstrated that hepatocyte death, a prominent feature of NASH, triggers a phenotypic change of resident macrophages in the liver (Kupffer cells), leading to liver fibrosis (Itoh et al., 2013Itoh M. Kato H. Suganami T. Konuma K. Marumoto Y. Terai S. Sakugawa H. Kanai S. Hamaguchi M. Fukaishi T. et al.Hepatic crown-like structure: a unique histological feature in non-alcoholic steatohepatitis in mice and humans.PLoS One. 2013; 8: e82163Crossref PubMed Scopus (82) Google Scholar, Itoh et al., 2017Itoh M. Suganami T. Kato H. Kanai S. Shirakawa I. Sakai T. Goto T. Asakawa M. Hidaka I. Sakugawa H. et al.CD11c+ resident macrophages drive hepatocyte death-triggered liver fibrosis in a murine model of nonalcoholic steatohepatitis.JCI Insight. 2017; 2: e92902Crossref PubMed Scopus (39) Google Scholar). Indeed, in murine and human NASH, there is a unique histological structure termed “crown-like structure (CLS),” in which dead hepatocytes with large lipid droplets are surrounded by CD11c-positive Kupffer cells; in contrast, CD11c-negative Kupffer cells are scattered throughout the liver (Itoh et al., 2013Itoh M. Kato H. Suganami T. Konuma K. Marumoto Y. Terai S. Sakugawa H. Kanai S. Hamaguchi M. Fukaishi T. et al.Hepatic crown-like structure: a unique histological feature in non-alcoholic steatohepatitis in mice and humans.PLoS One. 2013; 8: e82163Crossref PubMed Scopus (82) Google Scholar, Itoh et al., 2017Itoh M. Suganami T. Kato H. Kanai S. Shirakawa I. Sakai T. Goto T. Asakawa M. Hidaka I. Sakugawa H. et al.CD11c+ resident macrophages drive hepatocyte death-triggered liver fibrosis in a murine model of nonalcoholic steatohepatitis.JCI Insight. 2017; 2: e92902Crossref PubMed Scopus (39) Google Scholar). During the maturation of CLSs, Kupffer cells become positive for CD11c through cross talk with dead hepatocytes and acquire proinflammatory and profibrotic properties (Itoh et al., 2017Itoh M. Suganami T. Kato H. Kanai S. Shirakawa I. Sakai T. Goto T. Asakawa M. Hidaka I. Sakugawa H. et al.CD11c+ resident macrophages drive hepatocyte death-triggered liver fibrosis in a murine model of nonalcoholic steatohepatitis.JCI Insight. 2017; 2: e92902Crossref PubMed Scopus (39) Google Scholar). Thus, CD11c-positive Kupffer cells may be a novel macrophage subset crucial for liver fibrosis in NASH (Itoh et al., 2017Itoh M. Suganami T. Kato H. Kanai S. Shirakawa I. Sakai T. Goto T. Asakawa M. Hidaka I. Sakugawa H. et al.CD11c+ resident macrophages drive hepatocyte death-triggered liver fibrosis in a murine model of nonalcoholic steatohepatitis.JCI Insight. 2017; 2: e92902Crossref PubMed Scopus (39) Google Scholar). However, the molecular mechanism underlying the phenotypic change of Kupffer cells remains to be elucidated. There is considerable evidence that iron metabolism plays a critical role in macrophages at steady state and in response to infections (Soares and Hamza, 2016Soares M.P. Hamza I. Macrophages and iron metabolism.Immunity. 2016; 44: 492-504Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). Because iron is an essential micronutrient for both microbial pathogens and their mammalian hosts, iron deficiency in macrophages is thought to predispose the host to bacterial infections (Schaible and Kaufmann, 2004Schaible U.E. Kaufmann S.H.E. Iron and microbial infection.Nat. Rev. Microbiol. 2004; 2: 946-953Crossref PubMed Scopus (691) Google Scholar). With regard to the potential underlying mechanism, low intracellular iron selectively impairs the Toll-like receptor 4 signaling in macrophages (Wang et al., 2009Wang L. Harrington L. Trebicka E. Hai N.S. Kagan J.C. Hong C.C. Lin H.Y. Babitt J.L. Cherayil B.J. Selective modulation of TLR4-activated inflammatory responses by altered iron homeostasis in mice.J. Clin. Invest. 2009; 119: 3322-3328PubMed Google Scholar). Moreover, increasing evidence suggests a role for iron metabolism in non-infectious diseases such as atherosclerosis, cancer, and neurological disorders (da Silva et al., 2017da Silva M.C. Breckwoldt M.O. Vinchi F. Correia M.P. Stojanovic A. Thielmann C.M. Meister M. Muley T. Warth A. Platten M. et al.Iron induces anti-tumor activity in tumor-associated macrophages.Front. Immunol. 2017; 8: 1479Crossref PubMed Scopus (74) Google Scholar; Gillen et al., 2018Gillen K.M. Mubarak M. Nguyen T.D. Pitt D. Significance and in vivo detection of iron-laden microglia in white matter multiple sclerosis lesions.Front. Immunol. 2018; 9: 255Crossref PubMed Scopus (27) Google Scholar; Guo et al., 2018Guo L. Akahori H. Harari E. Smith S.L. Polavarapu R. Karmali V. Otsuka F. Gannon R.L. Braumann R.E. Dickinson M.H. et al.CD163+ macrophages promote angiogenesis and vascular permeability accompanied by inflammation in atherosclerosis.J. Clin. Invest. 2018; 128: 1106-1124Crossref PubMed Scopus (106) Google Scholar). Iron overloading skews macrophages toward a proinflammatory phenotype in a mouse model of skin wound healing (Sindrilaru et al., 2011Sindrilaru A. Peters T. Wieschalka S. Baican C. Baican A. Peter H. Hainzl A. Schatz S. Qi Y. Schlecht A. et al.An unrestrained proinflammatory M1 macrophage population induced by iron impairs wound healing in humans and mice.J. Clin. Invest. 2011; 121: 985-997Crossref PubMed Scopus (658) Google Scholar), suggesting a role for iron metabolism in macrophage function and polarization in sterile inflammation in vivo. In addition, a recent study revealed a positive association between hepatic iron deposition in reticuloendothelial system cells (presumably Kupffer cells) and the severity of NASH (Maliken et al., 2013Maliken B.D. Nelson J.E. Klintworth H.M. Beauchamp M. Yeh M.M. Kowdley K.V. Hepatic reticuloendothelial system cell iron deposition is associated with increased apoptosis in nonalcoholic fatty liver disease.Hepatology. 2013; 57: 1806-1813Crossref PubMed Scopus (56) Google Scholar). These observations led us to hypothesize that aberrant iron metabolism in Kupffer cells is crucial for chronic inflammation in NASH. Here, we show that iron-rich Kupffer cells exhibit proinflammatory and profibrotic phenotypic changes during the development of NASH. Using magnetic column separation, we divided Kupffer cells prepared from a murine NASH model into two fractions: ferromagnetic Fe-hi and remaining Fe-lo. Fe-hi Kupffer cells were responsible for upregulation of proinflammatory and profibrotic genes. With regard to the molecular mechanism, MiT/TFE transcription factors were involved in iron accumulation-mediated phenotypic changes of Kupffer cells. Furthermore, we confirmed activation of MiT/TFE transcription factors in Kupffer cells in murine and human NASH. This study provides insight into the pathophysiologic role of iron in NASH and sheds light on a unique macrophage subset rich in iron. In this study, we employed genetically obese MC4R-KO (knockout) mice fed Western diet (WD) as a murine NASH model. The mice developed simple steatosis after 4 weeks of WD feeding, NASH-like liver phenotypes (micro- and macrovesicular steatosis, ballooning degeneration, massive infiltration of inflammatory cells, and pericellular fibrosis) at 16 weeks and multiple liver tumors at 1 year (Figure S1) (Itoh et al., 2011Itoh M. Suganami T. Nakagawa N. Tanaka M. Yamamoto Y. Kamei Y. Terai S. Sakaida I. Ogawa Y. Melanocortin 4 receptor-deficient mice as a novel mouse model of nonalcoholic steatohepatitis.Am. J. Pathol. 2011; 179: 2454-2463Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar; Wang et al., 2016Wang X. Zheng Z. Caviglia J.M. Corey K.E. Herfel T.M. Cai B. Masia R. Chung R.T. Lefkowitch J.H. Schwabe R.F. et al.Hepatocyte TAZ/WWTR1 promotes inflammation and fibrosis in nonalcoholic steatohepatitis.Cell Metab. 2016; 24: 848-862Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar; Hasenour et al., 2020Hasenour C.M. Kennedy A.J. Bednarski T. Trenary I.A. Eudy B.J. da Silva R.P. Boyd K.L. Young J.D. Vitamin E does not prevent Western diet-induced NASH progression and increases metabolic flux dysregulation in mice.J. Lipid Res. 2020; 61: 707-721Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar). To investigate the effect of iron loading on NASH, MC4R-KO mice fed standard diet (SD) (normal liver) or WD for 8 weeks (simple steatosis) received a single intraperitoneal injection of iron-dextran and were analyzed on day 1 (Figures 1A–1C ). Iron accumulation detected by Perls' staining was mostly observed in F4/80+ macrophages (or Kupffer cells) (Figure 1B), suggesting the uptake of iron-dextran through phagocytotic activity. Efficient iron loading was confirmed by elevated mRNA levels of iron metabolism-related genes such as Hepcidin and Bmp6 (Figure 1C). In this experimental setting, iron loading resulted in a significant increase in mRNA levels of proinflammatory and profibrotic genes such as Itgax (CD11c, a marker for Kupffer cells exhibiting phenotypic changes in NASH), Tnfa (tumor necrosis factor alpha), Ccl3 (chemokine (C–C motif) ligand 3), and Timp1 (tissue inhibitor of metalloproteinase 1) in the livers of MC4R-KO mice fed WD, versus only a modest increase in MC4R-KO mice fed SD. Notably, Kupffer cells isolated from iron-treated MC4R-KO mice fed WD showed higher mRNA levels of proinflammatory and profibrotic genes than those from vehicle-treated mice (Figure 1D). Moreover, macrophage depletion using clodronate liposomes abolished the iron loading-induced increase in these mRNA levels in the livers of MC4R-KO mice fed WD (Figure S2). Collectively, these results indicate that Kupffer cells are responsible for iron-mediated proinflammatory and profibrotic changes in iron-treated steatotic livers. Moreover, we examined the long-term effect of iron loading on the development of NASH (Figures 1E–1H). MC4R-KO mice were fed WD for 16 weeks and received once-weekly injection of iron-dextran during the last 11 week (Figure 1E). Repeated injection of iron-dextran did not change body weight, liver weight, or blood parameters (serum ALT and AST, and blood glucose), but significantly decreased hepatic triglyceride contents and expression of the lipogenic gene Fasn (Figure S3). Notably, iron loading significantly increased mRNA levels of proinflammatory and profibrotic genes, together with type III collagen-positive areas, whereas it did not affect CLS formation (Figures 1F–1H). These findings indicate that iron loading accelerates proinflammatory and profibrotic changes in the liver during the development of NASH in MC4R-KO mice fed WD. To further elucidate the role of iron metabolism in the pathogenesis of NASH, we examined the effects of the iron chelator 2,2′-dipyridyl (2-DP) on liver fibrosis in an inducible NASH model (Figure 2A), in which a single injection of carbon tetrachloride (CCl4, a potent hepatotoxic chemical) into MC4R-KO mice fed WD for 5 weeks (simple steatosis) recapitulates NASH-like liver phenotypes in the short term (Itoh et al., 2017Itoh M. Suganami T. Kato H. Kanai S. Shirakawa I. Sakai T. Goto T. Asakawa M. Hidaka I. Sakugawa H. et al.CD11c+ resident macrophages drive hepatocyte death-triggered liver fibrosis in a murine model of nonalcoholic steatohepatitis.JCI Insight. 2017; 2: e92902Crossref PubMed Scopus (39) Google Scholar). Using this model, we demonstrated that hepatocyte death triggers a phenotypic change in Kupffer cells, inducing CLS formation and fibrogenesis in the liver (Itoh et al., 2017Itoh M. Suganami T. Kato H. Kanai S. Shirakawa I. Sakai T. Goto T. Asakawa M. Hidaka I. Sakugawa H. et al.CD11c+ resident macrophages drive hepatocyte death-triggered liver fibrosis in a murine model of nonalcoholic steatohepatitis.JCI Insight. 2017; 2: e92902Crossref PubMed Scopus (39) Google Scholar). In this study, daily injection of 2-DP effectively decreased mRNA levels of proinflammatory and profibrotic genes and liver fibrosis, whereas it did not affect the number of CLSs, body weight, liver weight, hepatic triglyceride contents, or blood parameters (Figures 2B–2D and S4). These results confirm the role of iron metabolism in liver fibrosis in the NASH model. As liver is composed of hepatocytes and non-parenchymal cells (NPCs) such as Kupffer cells, hepatic stellate cells, and sinusoidal cells, we next sought to determine which cell types are responsible for iron accumulation-mediated proinflammatory and profibrotic responses. First, primary NPCs prepared from the livers of MC4R-KO mice fed WD for 5 weeks (simple steatosis) were treated with FeCl3 for 48 h in culture (Figure 3A). Iron loading significantly increased mRNA levels of proinflammatory and profibrotic genes (Figure 3B). Conversely, primary NPCs prepared from the livers of MC4R-KO mice fed WD for 14 weeks (NASH) were treated with 2-DP for 12 h in culture (Figure 3C). Iron chelation significantly suppressed mRNA expression of these genes (Figure 3D). These observations suggest that iron metabolism in NPCs is involved in proinflammatory and profibrotic responses in the NASH model. We have already demonstrated that there is a small subset of Kupffer cells that become positive for CD11c through interactions with dead hepatocytes during the development of NASH, thereby inducing sustained inflammation and fibrosis in the liver (Itoh et al., 2013Itoh M. Kato H. Suganami T. Konuma K. Marumoto Y. Terai S. Sakugawa H. Kanai S. Hamaguchi M. Fukaishi T. et al.Hepatic crown-like structure: a unique histological feature in non-alcoholic steatohepatitis in mice and humans.PLoS One. 2013; 8: e82163Crossref PubMed Scopus (82) Google Scholar, Itoh et al., 2017Itoh M. Suganami T. Kato H. Kanai S. Shirakawa I. Sakai T. Goto T. Asakawa M. Hidaka I. Sakugawa H. et al.CD11c+ resident macrophages drive hepatocyte death-triggered liver fibrosis in a murine model of nonalcoholic steatohepatitis.JCI Insight. 2017; 2: e92902Crossref PubMed Scopus (39) Google Scholar). Because Kupffer cells are a major cellular source of iron accumulation in the liver (Theurl et al., 2016Theurl I. Hilgendorf I. Nairz M. Tymoszuk P. Haschka D. Asshoff M. He S. Gerhardt L.M.S. Holderried T.A.W. Seifert M. et al.On-demand erythrocyte disposal and iron recycling requires transient macrophages in the liver.Nat. Med. 2016; 22: 945-951Crossref PubMed Scopus (193) Google Scholar), we narrowed down the target cell type to Kupffer cells. Iron is the most predominant cellular metal with ferromagnetic features. Thus, using magnetic column separation, we divided NPCs into two fractions: ferromagnetic Fe-hi and remaining Fe-lo (Figure 4A). Depending on the cellular iron contents, Fe-hi cells were enriched on the magnetic column, whereas Fe-lo cells were in the flow through (Orr et al., 2014Orr J.S. Kennedy A. Anderson-Baucum E.K. Webb C.D. Fordahl S.C. Erikson K.M. Zhang Y. Etzerodt A. Moestrup S.K. Hasty A.H. Obesity alters adipose tissue macrophage iron content and tissue iron distribution.Diabetes. 2014; 63: 421-432Crossref PubMed Scopus (91) Google Scholar; da Silva et al., 2017da Silva M.C. Breckwoldt M.O. Vinchi F. Correia M.P. Stojanovic A. Thielmann C.M. Meister M. Muley T. Warth A. Platten M. et al.Iron induces anti-tumor activity in tumor-associated macrophages.Front. Immunol. 2017; 8: 1479Crossref PubMed Scopus (74) Google Scholar; Hubler et al., 2018Hubler M.J. Erikson K.M. Kennedy A.J. Hasty A.H. MFehi adipose tissue macrophages compensate for tissue iron perturbations in mice.Am. J. Physiol. Cell Physiol. 2018; 315: C319-C329Crossref PubMed Scopus (10) Google Scholar). Each fraction was subjected to fluorescence-activated cell sorting (FACS) analysis, and Fe-hi and Fe-lo Kupffer cells (CD11b+ F4/80hi) were purified (Figure 4B). Fe-hi Kupffer cells displayed higher cellular iron content than Fe-lo ones (Figure 4C). The number of Fe-hi and Fe-lo Kupffer cells was higher in MC4R-KO mice fed WD during the course of NASH development than in wild-type mice fed SD (Figure 4D). The ratio of Fe-hi to Fe-lo Kupffer cell number was significantly elevated in MC4R-KO mice fed WD for 16 weeks (NASH) (Figure 4E). In this experimental setting, mRNA expression of Itgax was markedly increased in Fe-hi Kupffer cells from MC4R-KO mice fed WD during the course of NASH development, whereas there was only a modest increase in Fe-lo cells (Figure 4F). Fe-hi Kupffer cells also expressed higher levels of mRNAs encoding proinflammatory and profibrotic genes than Fe-lo Kupffer cells. These findings strongly suggest that Fe-hi Kupffer cells expressing CD11c are responsible for inflammation and fibrosis in the livers of MC4R-KO mice fed WD. Next, we compared gene expression profiles between NASH liver-derived CD11c-positive Kupffer cells and iron-treated cultured macrophages to clarify how iron metabolism is involved in the phenotypic change of CD11c-positive Kupffer cells with proinflammatory and profibrotic properties (Figure 5A). We found that 305 genes were commonly upregulated in CD11c-positive Kupffer cells from NASH livers (versus CD11c-negative Kupffer cells from normal livers) and iron-treated RAW264 macrophages (versus untreated RAW264 macrophages). As expected, genes related to “cytokine-cytokine receptor interaction” and “chemokine signaling pathway” were enriched in these 305 genes, indicative of the proinflammatory and profibrotic phenotypes. Moreover, pathway analysis revealed that the two most enriched pathways were “phagosome” and “lysosome” (Figure 5B). These results are consistent with our previous findings that CD11c-positive Kupffer cells aggregate around dead hepatocytes to scavenge accumulated lipids and cell debris within CLSs (Itoh et al., 2013Itoh M. Kato H. Suganami T. Konuma K. Marumoto Y. Terai S. Sakugawa H. Kanai S. Hamaguchi M. Fukaishi T. et al.Hepatic crown-like structure: a unique histological feature in non-alcoholic steatohepatitis in mice and humans.PLoS One. 2013; 8: e82163Crossref PubMed Scopus (82) Google Scholar, Itoh et al., 2017Itoh M. Suganami T. Kato H. Kanai S. Shirakawa I. Sakai T. Goto T. Asakawa M. Hidaka I. Sakugawa H. et al.CD11c+ resident macrophages drive hepatocyte death-triggered liver fibrosis in a murine model of nonalcoholic steatohepatitis.JCI Insight. 2017; 2: e92902Crossref PubMed Scopus (39) Google Scholar). It is also known that cellular iron accumulation triggers lysosomal dysfunction through increased production of reactive oxygen species (Boya and Kroemer, 2008Boya P. Kroemer G. Lysosomal membrane permeabilization in cell death.Oncogene. 2008; 27: 6434-6451Crossref PubMed Scopus (957) Google Scholar). Moreover, ChIP Atlas enrichment analysis identified TFE3 as a potential transcription factor enriched in the enhancer regions of these 305 genes (Table S1). TFE3 is a member of the MiT/TFE transcription factors, which regulate lysosomal biogenesis and functions in response to lysosomal dysfunction (Puertollano et al., 2018Puertollano R. Ferguson S.M. Brugarolas J. Ballabio A. The complex relationship between TFEB transcription factor phosphorylation and subcellular localization.EMBO J. 2018; 37: e98804Crossref PubMed Scopus (167) Google Scholar). In this study, knockdown of two MiT/TFE transcription factors, Tfe3 (Transcription factor E3) and Tfeb (Transcription factor EB), effectively suppressed iron treatment-induced upregulation of phagosome- and lysosome-related genes such as Atp6v0d2 (ATPase, H+ transporting, lysosomal 38kDa, V0 subunit d2), Cd63, and Ctsk (Cathepsin K) (Figures 5C and 5D). We validated these small interfering RNAs (siRNAs) by examining the effect of each of them on Tfe3 and Tfeb mRNA expression (Figure 5E). Moreover, using acridine orange staining, we observed that iron treatment induced lysosomal dysfunction in RAW264 macrophages (Figure 5F). In addition, iron treatment increased nuclear localization of TFE3 in primary hepatic macrophages isolated from steatotic livers, whereas it did not increase mRNA levels of Tfe3 and Tfeb (Figures 5G and S5). Furthermore, Atp6v0d2 expression was significantly elevated in the livers of MC4R-KO mice receiving a single injection (Figure 5H) or repeated injection (Figure 5I) of iron-dextran (related data are shown in Figures 1A–1C and 1E–1H, respectively). 2-DP treatment significantly suppressed Atp6v0d2 expression in the inducible NASH model (Figure 5J; related data were shown in Figure 2). Finally, Atp6v0d2 expression was markedly upregulated in Fe-hi Kupffer cells prepared from MC4R-KO mice fed WD for 8 weeks (Figure 5K; related data are shown in Figure 4). These findings, taken together, indicate activation of MiT/TFE transcription factors in CD11c-positive Kupffer cells, probably through lysosomal dysfunction induced by iron accumulation. Next, we investigated how MiT/TFE transcription factors influence proinflammatory and profibrotic properties in macrophages. Chloroquine and bafilomycin A1, which potently induce lysosomal dysfunction, markedly upregulated expression of Itgax, Tnfa, Ccl3, and Timp1 in RAW264 macrophages (Figure 6A). Similarly, FeCl3 treatment dose-dependently increased the expression of these genes, except for Timp1 (Figure 6B). Similar results were observed in primary hepatic macrophages prepared from steatotic livers (Figure 6C). Moreover, iron-induced upregulation of these genes was significantly inhibited by knockdown of Tfe3 and Tfeb in RAW264 macrophages (Figure 6D). As addressed above, Itgax (CD11c) is a marker for Kupffer cells constituting CLSs in NASH. Therefore, our observations indicate that iron accumulation in Kupffer cells is involved in lysosomal dysfunction and subsequent activation of MiT/TFE transcription factors, which contributes to phenotypic changes and sustained inflammation during the development of NASH. To prove this notion in vivo, we performed immunohistochemical analysis of TFE and TFEB. Nuclear immunostaining of TFE3, indicating activation of TFE3, was observed in macrophages forming CLSs in the livers of MC4R-KO mice fed WD for 16 weeks (NASH), whereas there was only faint staining of TFE3, probably in hepatocytes, in wild-type mice fed SD (normal liver) (Figures 7A and 7B ). We confirmed that CD11c-positive Kupffer cells in CLSs were the main cell type expressing TFE3 in the NASH model (Figure 7C). Notably, most CLSs were positive for TFE3 immunostaining (Figures 7B and 7C). Similarly, more than half of CLSs were immunostained with TFEB (Figure 7D). A single injection of iron-dextran to lean mice (normal liver) increased immunostaining of TFE3 in Kupffer cells (Figure 7E). Moreover, TFE3 immunostaining was observed in macrophages forming CLSs in a different experimental model of NASH induced by deficiency of methionine and choline (Figures 7F and 7G). As shown in Figure 7H, deficiency of methionine and choline in wild-type mice exhibited histological feat" @default.
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• W3118454925 title "Iron-rich Kupffer cells exhibit phenotypic changes during the development of liver fibrosis in NASH" @default.
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