Arginine and NASH – Do macrophages deliver the first hit?
Authors: Ludger Scheja, Johannes Kluwe
Journal: Journal of Hepatology (2014)

Arginase 2 deficiency results in spontaneous steatohepatitis: A novel link between innate immune activation and hepatic de novo lipogenesisJournal of HepatologyVol. 62Issue 2PreviewInnate immune activation has been postulated as a central mechanism for disease progression from hepatic steatosis to steatohepatitis in obesity-related fatty liver disease. Arginase 2 competes with inducible nitric oxide synthase (iNOS) for its substrate and the balance between these two enzymes plays a crucial role in regulating immune responses and macrophage activation. Our aim was to test the hypothesis that arginase 2 deficiency in mice favours progression from isolated hepatic steatosis, induced by high fat feeding, to steatohepatitis. Full-Text PDF The amino acid arginine has multiple biosynthetic functions, serving as a precursor not only in protein synthesis and the urea cycle but also in the generation of polyamines, creatine and nitric oxide (NO). In the context of metabolic syndrome and inflammatory diseases, the conversion of arginine to NO and citrulline by NO synthases (NOS) and also its conversion to ornithine and urea by arginases have been of special interest. Research has focused in particular on two enzyme isoforms, inducible NOS (iNOS) and arginase 1. Induction of iNOS is a hallmark of classically activated (M1) macrophages and type 1 T helper (Th1) cell responses with iNOS producing oxidative stress, thus promoting anti-pathogen responses [1Bogdan C. Nitric oxide and the immune response.Nat Immunol. 2001; 2: 907-916Crossref PubMed Scopus (2736) Google Scholar, 2El Kasmi K.C. Qualls J.E. Pesce J.T. Smith A.M. Thompson R.W. Henao-Tamayo M. et al.Toll-like receptor-induced arginase 1 in macrophages thwarts effective immunity against intracellular pathogens.Nat Immunol. 2008; 9: 1399-1406Crossref PubMed Scopus (501) Google Scholar] but also metabolic disorders, such as insulin resistance [[3]Charbonneau A. Marette A. Inducible nitric oxide synthase induction underlies lipid-induced hepatic insulin resistance in mice: potential role of tyrosine nitration of insulin signaling proteins.Diabetes. 2010; 59: 861-871Crossref PubMed Scopus (93) Google Scholar]. Arginase 1 is a key marker of alternatively activated (M2) macrophages, which confer anti-inflammatory properties by substrate competition with iNOS and through other mechanisms [4Khallou-Laschet J. Varthaman A. Fornasa G. Compain C. Gaston A.T. Clement M. et al.Macrophage plasticity in experimental atherosclerosis.PLoS One. 2010; 5: e8852Crossref PubMed Scopus (429) Google Scholar, 5Ming X.F. Rajapakse A.G. Yepuri G. Xiong Y. Carvas J.M. Ruffieux J. et al.Arginase II promotes macrophage inflammatory responses through mitochondrial reactive oxygen species, contributing to insulin resistance and atherogenesis.J Am Heart Assoc. 2012; 1: e000992Crossref Google Scholar, 6Munder M. Arginase: an emerging key player in the mammalian immune system.Br J Pharmacol. 2009; 158: 638-651Crossref PubMed Scopus (530) Google Scholar]. Interestingly, macrophages and dendritic cells express also the second arginase isoform, arginase 2, a predominantly mitochondrial protein [4Khallou-Laschet J. Varthaman A. Fornasa G. Compain C. Gaston A.T. Clement M. et al.Macrophage plasticity in experimental atherosclerosis.PLoS One. 2010; 5: e8852Crossref PubMed Scopus (429) Google Scholar, 7Dunand-Sauthier I. Irla M. Carnesecchi S. Seguin-Estevez Q. Vejnar C.E. Zdobnov E.M. et al.Repression of arginase-2 expression in dendritic cells by microRNA-155 is critical for promoting T cell proliferation.J Immunol. 2014; 193: 1690-1700Crossref PubMed Scopus (52) Google Scholar, 8Munder M. Eichmann K. Moran J.M. Centeno F. Soler G. Modolell M. Th1/Th2-regulated expression of arginase isoforms in murine macrophages and dendritic cells.J Immunol. 1999; 163: 3771-3777PubMed Google Scholar]. Arg1 and Arg2 knockout mice have been instrumental in understanding the in vivo roles of arginases. As the total deficiency of arginase 1, an essential urea cycle enzyme, is postnatally lethal, most studies have used arginase 2 deficient mice (Arg2−/−) [[9]Shi O. Morris Jr., S.M. Zoghbi H. Porter C.W. O'Brien W.E. Generation of a mouse model for arginase II deficiency by targeted disruption of the arginase II gene.Mol Cell Biol. 2001; 21: 811-813Crossref PubMed Scopus (123) Google Scholar]. The role of arginase 2 in inflammation is, however, less well understood. In this issue, the Journal of Hepatology publishes an interesting study by Navarro et al. [[10]Navarro L.A. Wree A. Povero D. Berk M.P. Eguchi A. Ghosh S. et al.Arginase 2 deficiency results in spontaneous steatohepatitis: a novel link between innate immune activation and hepatic de novo lipogenesis.J Hepatol. 2015; 62: 412-420Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar]. The authors describe a phenotype of spontaneous steatohepatitis in Arg2−/− mice on a chow diet, which is aggravated after feeding a high fat diet. As underlying mechanisms the authors identify hepatic upregulation of genes and proteins associated with de novo lipogenesis, increased hepatic recruitment of F4/80-positive macrophages, and an increased hepatic expression of inflammatory genes, especially iNOS and IL-1β, in Arg2−/− mice. This finding is remarkable, as arginase 2 is not expressed in hepatocytes but rather in extra-hepatic organs, such as intestine and kidney as well as in macrophages and dendritic cells [4Khallou-Laschet J. Varthaman A. Fornasa G. Compain C. Gaston A.T. Clement M. et al.Macrophage plasticity in experimental atherosclerosis.PLoS One. 2010; 5: e8852Crossref PubMed Scopus (429) Google Scholar, 5Ming X.F. Rajapakse A.G. Yepuri G. Xiong Y. Carvas J.M. Ruffieux J. et al.Arginase II promotes macrophage inflammatory responses through mitochondrial reactive oxygen species, contributing to insulin resistance and atherogenesis.J Am Heart Assoc. 2012; 1: e000992Crossref Google Scholar, 6Munder M. Arginase: an emerging key player in the mammalian immune system.Br J Pharmacol. 2009; 158: 638-651Crossref PubMed Scopus (530) Google Scholar, 7Dunand-Sauthier I. Irla M. Carnesecchi S. Seguin-Estevez Q. Vejnar C.E. Zdobnov E.M. et al.Repression of arginase-2 expression in dendritic cells by microRNA-155 is critical for promoting T cell proliferation.J Immunol. 2014; 193: 1690-1700Crossref PubMed Scopus (52) Google Scholar, 8Munder M. Eichmann K. Moran J.M. Centeno F. Soler G. Modolell M. Th1/Th2-regulated expression of arginase isoforms in murine macrophages and dendritic cells.J Immunol. 1999; 163: 3771-3777PubMed Google Scholar]. Depletion of hepatic, but not of adipose tissue macrophages by clodronate at least partially reversed the induction of lipogenic genes and hepatic steatosis that were observed in Arg2−/− mice. This led the authors to the interpretation that hepatic macrophages are pivotal for hepatic steatogenesis in Arg2−/− mice. The classical multiple-hit theory of NASH pathogenesis identifies macrophages as mediators of the transition from steatosis to steatohepatitis, responding to inflammatory second hits, the first hit being hepatocellular lipid accumulation [[11]Lewis J.R. Mohanty S.R. Nonalcoholic fatty liver disease: a review and update.Dig Dis Sci. 2010; 55: 560-578Crossref PubMed Scopus (275) Google Scholar]. This concept is increasingly being challenged. Several recent studies have identified liver macrophages as promoters not only of inflammation in NASH but also of hepatic lipogenesis and lipid accumulation: two studies have demonstrated that depletion of hepatic macrophages prevents obesity-induced hepatic steatosis in rodents. Both studies postulate an influence of macrophage-derived cytokines on hepatocellular lipid metabolism, resulting in hepatocellular lipid accumulation. While Huang et al. identify TNF-α as a mediator of this macrophage-hepatocyte crosstalk, the study by Negrin et al. convincingly demonstrates that hepatocellular lipogenesis is induced by IL-1β, confirming findings of an earlier paper, which has identified IL-1β as a promoter of experimental NASH progression [12Huang W. Metlakunta A. Dedousis N. Zhang P. Sipula I. Dube J.J. et al.Depletion of liver Kupffer cells prevents the development of diet-induced hepatic steatosis and insulin resistance.Diabetes. 2010; 59: 347-357Crossref PubMed Scopus (409) Google Scholar, 13Negrin K.A. RothFlach R.J. DiStefano M.T. Matevossian A. Friedline R.H. Jung D. et al.IL-1 signaling in obesity-induced hepatic lipogenesis and steatosis.PLoS One. 2014; 9: e107265Crossref PubMed Scopus (116) Google Scholar, 14Miura K. Kodama Y. Inokuchi S. Schnabl B. Aoyama T. Ohnishi H. et al.Toll-like receptor 9 promotes steatohepatitis by induction of interleukin-1beta in mice.Gastroenterology. 2010; 139 (e327): 323-334Abstract Full Text Full Text PDF PubMed Scopus (601) Google Scholar]. In line with these studies, the paper by Navarro et al. suggests that macrophages have the potential to deliver the "first hit" in the pathogenesis of NASH by inducing hepatocellular lipid accumulation. One could speculate that Arg2 deficiency creates a macrophage phenotype with a lipogenesis-inducing cytokine signature. More work is warranted to confirm the role of liver macrophages/Kupffer cells in Arg2 deficiency-mediated liver steatosis and inflammation. Bone marrow transplantation experiments using Arg2−/− donors, or tissue specific knockouts, would be a suitable way to address these questions. Moreover, it will be an interesting challenge to identify steatogenic mediators, secreted from Arg2−/− macrophages, a likely candidate being IL-1β, which was strongly upregulated in the livers of Arg2−/− mice. Identifying the downstream signalling events in hepatocytes will be equally important. These could be, for example, increased endoplasmic reticulum stress resulting in SREBP1c activation [[15]Kammoun H.L. Chabanon H. Hainault I. Luquet S. Magnan C. Koike T. et al.GRP78 expression inhibits insulin and ER stress-induced SREBP-1c activation and reduces hepatic steatosis in mice.J Clin Invest. 2009; 119: 1201-1215Crossref PubMed Scopus (592) Google Scholar] or oxidative stress generated by iNOS. iNOS is expressed in hepatocytes as well as liver macrophages and has been shown to be involved in the development of NAFLD in mice, provoked by high fructose feeding [[16]Spruss A. Kanuri G. Uebel K. Bischoff S.C. Bergheim I. Role of the inducible nitric oxide synthase in the onset of fructose-induced steatosis in mice.Antioxid Redox Signal. 2011; 14: 2121-2135Crossref PubMed Scopus (67) Google Scholar]. The findings by Navarro et al. emphasize that lack of arginase 2 promotes NASH progression, implying a therapeutic potential of sustained arginase 2 activity. However, studies of arginase 2 in atherosclerosis models caution against translational enthusiasm. Two papers describe a pro-inflammatory role of arginase 2 in macrophages, connecting arginase 2 with the inflammatory macrophage M1 phenotype [4Khallou-Laschet J. Varthaman A. Fornasa G. Compain C. Gaston A.T. Clement M. et al.Macrophage plasticity in experimental atherosclerosis.PLoS One. 2010; 5: e8852Crossref PubMed Scopus (429) Google Scholar, 5Ming X.F. Rajapakse A.G. Yepuri G. Xiong Y. Carvas J.M. Ruffieux J. et al.Arginase II promotes macrophage inflammatory responses through mitochondrial reactive oxygen species, contributing to insulin resistance and atherogenesis.J Am Heart Assoc. 2012; 1: e000992Crossref Google Scholar]. Strikingly, Arg2 deficiency protected apolipoprotein E-knockout mice from atherosclerotic lesions [[5]Ming X.F. Rajapakse A.G. Yepuri G. Xiong Y. Carvas J.M. Ruffieux J. et al.Arginase II promotes macrophage inflammatory responses through mitochondrial reactive oxygen species, contributing to insulin resistance and atherogenesis.J Am Heart Assoc. 2012; 1: e000992Crossref Google Scholar]. These findings apparently contradict the data by Navarro et al. who claim that Arg2-deficient macrophages display an M1 phenotype. However, it has to be taken into consideration that a detailed characterization of liver macrophages was not the focus of this study, and interpretation was based on the influence of arginase 2 on inflammatory gene expression in whole liver tissue. The divergent data from these studies suggest that effects of arginase 2 on macrophage function could be tissue or organ specific. The study by Navarro et al. provokes the question if an altered arginase 2 activity, or more generally arginine availability, is relevant in human NASH. While in Arg2−/− mice elevated arginine levels and NASH were described [9Shi O. Morris Jr., S.M. Zoghbi H. Porter C.W. O'Brien W.E. Generation of a mouse model for arginase II deficiency by targeted disruption of the arginase II gene.Mol Cell Biol. 2001; 21: 811-813Crossref PubMed Scopus (123) Google Scholar, 10Navarro L.A. Wree A. Povero D. Berk M.P. Eguchi A. Ghosh S. et al.Arginase 2 deficiency results in spontaneous steatohepatitis: a novel link between innate immune activation and hepatic de novo lipogenesis.J Hepatol. 2015; 62: 412-420Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar], several arginine supplementation studies suggest that increasing systemic arginine levels can be beneficial. Insulin resistant subjects and cardiovascular patients, chronically supplemented with arginine, exhibited significantly improved glucose homeostasis and vascular functions [17Bogdanski P. Suliburska J. Grabanska K. Musialik K. Cieslewicz A. Skoluda A. et al.Effect of 3-month L-arginine supplementation on insulin resistance and tumor necrosis factor activity in patients with visceral obesity.Eur Rev Med Pharmacol Sci. 2012; 16: 816-823PubMed Google Scholar, 18Boger R.H. L-Arginine therapy in cardiovascular pathologies: beneficial or dangerous?.Curr Opin Clin Nutr Metab Care. 2008; 11: 55-61Crossref PubMed Scopus (73) Google Scholar, 19Monti L.D. Setola E. Lucotti P.C. Marrocco-Trischitta M.M. Comola M. Galluccio E. et al.Effect of a long-term oral l-arginine supplementation on glucose metabolism: a randomized, double-blind, placebo-controlled trial.Diabetes Obes Metab. 2012; 14: 893-900Crossref PubMed Scopus (61) Google Scholar]. Similarly, studies have demonstrated favourable effects of arginase inhibitors on endothelial function, mostly attributed to increased NO availability for endothelial NO synthase [[20]Pernow J. Jung C. Arginase as a potential target in the treatment of cardiovascular disease: reversal of arginine steal?.Cardiovasc Res. 2013; 98: 334-343Crossref PubMed Scopus (235) Google Scholar]. In light of the study by Navarro et al., it will be important to perform arginine supplementation and arginase inhibitor studies, addressing NAFLD. Taken together, although the current and previous findings suggest that the net biological effects of arginase 2 are context and tissue specific, arginase modulation still seems to offer many therapeutic implications. Future research is warranted to further explore the fascinating, multi-facetted role of arginine metabolism on NAFLD in the context of the metabolic syndrome. Both authors receive grant support from the Collaborative Research Centre 841 funded by the German Research Foundation. The authors declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.