Hepatocellular carcinoma in nonalcoholic fatty liver disease: A link between oxidative stress and T‐cell suppression
Authors: Muhammad Asif Qureshi, Yong Ook Kim, Detlef Schuppan
Journal: Hepatology (2016)

The role of the immune system in modulating metabolic diseases, including nonalcoholic fatty liver disease (NAFLD), is increasingly recognized. A recent report by Ma et al. suggests that in NAFLD certain free fatty acids derived from nutrition increase the production of reactive oxygen species (ROS) in hepatic cluster of differentiation 4-positive (CD4+) T cells causing their apoptosis, which in turn compromises immune surveillance and promotes hepatocarcinogenesis.1 Hepatocellular carcinoma (HCC) is the second leading cause of global cancer-associated deaths. It almost exclusively develops in an inflammatory environment, preferably in a cirrhotic liver; and the most common/established risk factor is chronic viral infection. More recently, NAFLD has emerged as an important risk factor for HCC, with an incidence of 0.44/1,000.2 In a prospective cohort study, 5-year HCC incidence in patients with nonalcoholic steatohepatitis (NASH), the highly inflammatory variant of NAFLD, was high but still 3 times lower compared to patients with chronic hepatitis C virus infection.3 However, mechanistic links between NAFLD and the development of HCC, also in noncirrhotic livers, are not clearly understood. Rodent studies showed that obesity promotes carcinogen-induced HCC through inflammatory pathways including enhanced production of interleukin-6 and tumor necrosis factor-α, factors that are prevalent in NASH but also many other chronic liver diseases.4 Ma et al.1 used the chemical carcinogen diethylnitrosamine in mice with an inducible c-Myc oncogene. Moreover, mice were fed a methionine and choline-deficient diet, a choline-deficient and amino acid-defined diet, or a high-fat diet. Diethylnitrosamine-treated c-Myc oncogene mice fed the methionine and choline-deficient, choline-deficient and amino acid-defined, or high-fat diet developed earlier and more tumors compared to their controls fed normal chow. Importantly, the authors demonstrated that hepatic CD4+ (but not CD8+) T cells in these mice were sensitive to increased ROS production upon oxidizing linoleic acid (LA) in their mitochondria, leading to hepatic CD4+ (but not CD8+) T-cell death. This was accompanied by an increased production of interferon-γ and interleukin-17 by expanded CD4+Rorγt+ (T helper 17) T cells. The rodent data were supported by correlative analysis of human biopsies that demonstrated lower relative (but not absolute!) CD4+ T-cell numbers in patients with NASH (and alcoholic hepatitis) when compared to other chronic liver diseases. Based on these findings, the authors suggested that deregulated metabolism of LA decreased immune surveillance and accelerated the development of HCC by ROS-mediated depletion of CD4+ T cells. The authors also studied other immune cells in their models, but this analysis was limited and did not include, e.g., a detailed study of monocytes/macrophages, innate lymphoid cells, and innate natural killer T cells, which are prominent immune cell populations in the liver and appear to play an important role in murine and human NASH.5 While the study proposes an intriguing novel immune mechanism that may underlie inflammation and hepatocarcinogenesis in NASH, there are some shortcomings. The authors largely employed NAFLD/NASH mouse models based on the artificial methionine and choline-deficient and (to a lesser degree) choline-deficient and amino acid-defined and high-fat diets instead of a Western, high-fat (plus high-fructose) diet. Deficiency of methionine depletes the cellular antioxidant glutathione and promotes (hepatocyte) oxidative stress. The combination with depletion of choline and thus the synthesis of phosphatidylcholine, a major constituent of cell membranes, causes severe hepatocyte mitochondrial damage, compromised fatty acid oxidation, lipoapoptosis, and steatohepatitis. Ma et al.1 used a diet highly enriched in LA (12% versus 2% of fatty acids), a fatty acid that is usually consumed in trace amounts. Similarly, it is not clear if the concentration of LA that was used in vitro to induce significant oxidative stress and apoptosis of CD4+ T cells would ever be reached in patients in vivo. The authors also did not rule out the possibility of ROS-related detrimental effects potentially induced by other arachidonic acid-derived LA metabolites such as C18:3 n-3 and docosatetraenoic acid (adrenic acid, C22:4 n-6). In summary, the mouse models used in this study have their limitations, mainly due to metabolic artifacts that incompletely represent the pathogenesis of human NAFLD/NASH toward HCC. Another concern is the use of c-Myc oncogene transgenic mice as the central genetic HCC model. These mice rapidly develop hepatoblastoma, based on overexpression of a single oncogene, which is entirely different from the mechanisms underlying HCC progression in humans. Moreover, selective CD4+ T-cell death induced by fatty acid-mediated ROS production demands further explanation. Thus, the increased sensitivity of CD4+ T cells could be due to a more abundant respiratory (mitochondrial) machinery compared to CD8+ T cells, as shown by measuring extracellular acidification and oxygen consumption rates of CD4+ and CD8+ T cells isolated from C57BL/6 mice, mediated in part by a greater capacity for glutamine oxidation in CD8+ T cells.6 Thus, oxidative capacity of T cells may change in conditions where there is a more pronounced CD8 T-cell response, and this may be model-specific. In summary, the selective CD4+ T-cell depletion in response to LA in vitro and as observed in the mice fed an unusually high amount of LA may be due to both the supraphysiological LA concentrations and the experimental models used. Importantly, it is unlikely that CD4+ T cells are the sole or major immunological determinants in the progression from NAFLD/NASH to HCC (Fig. 1). Thus, T helper 17 cells, which were only marginally addressed by Ma et al.,1 are established promoters of liver inflammation, fibrosis, and HCC.7 Moreover, the closely related type 3 innate lymphoid cells, which were not addressed in the article, may also play a potential role in liver inflammation and progression to fibrosis and HCC. It is also not clear if CD4+ T-cell depletion promotes NAFLD progression to HCC by undermining immunosurveillance, as postulated, or rather interacts or interferes with other immune cells and mediators in the NASH/HCC microenvironment. Thus, apart from the (relative) decrease of intrahepatic CD4+ T cells and, e.g., unchanged numbers of not only CD8+ T cells but other immune cells, as seen in the studied model(s), functional adaptation or plasticity may have affected disease outcome. For example, in CD4+ T cell-depleted tissues, other adaptive immune cells, including CD8+ T cells and especially natural killer T cells, or innate immune cells such as monocytes, macrophages, dendritic cells, innate natural killer T cells, natural killer cells, and innate lymphoid cells very likely affect NAFLD-associated inflammation, liver damage, and possibly progression to HCC.8 Three reports that appeared in tandem in Nature Medicine and that were summarized in a commentary even highlighted a central role of CD8+, and not of CD4+, T cells in adipose tissue inflammation and likely NAFLD.9 Moreover, Ma et al.1 did not study the numbers and function of the other mentioned adaptive and innate immune cells with or without CD4+ T-cell depletion or the role of depletion of these cells on the function of CD4+ T cells. The lower (in fact comparable to normal) CD4/CD8 T-cell ratio of intrahepatic T cells that was found in biopsies from patients with NASH and alcoholic steatohepatitis is interesting but correlative, without functional assessment. Moreover, total CD4+ T-cell counts were still increased. Another relevant tumor promoting factor could be the lack of CD4+ T cell-mediated clearance of premalignant senescent hepatocytes, which in the absence of "senescence surveillance" contributes to the pathogenesis of HCC.10 Finally, in the context of diethylnitrosamine-induced HCC, different arms of the adaptive immune system (B cells versus T cells) demand further investigation as they may differentially modulate the antitumor response in HCC development.11 It is therefore important to further investigate quantitative and functional changes in this and other arms of the adaptive and innate immune system as well as to study other players in the NAFLD/HCC microenvironment, such as (myo-)fibroblasts, lack of senescence surveillance, endothelial cells/angiogenesis, activated progenitor cells/cholangiocytes as potential cancer stem cells, and the recently identified genetic predispositions for severe NAFLD/NASH (Fig. 1). In summary, the findings by Ma et al.1 represent an interesting contribution to the field, which, however, needs further validation in other model systems with relevance for NAFLD/NASH in the rodent and especially human systems. There is no doubt that a single study can never comprehensively address these issues in a complex disease like NASH (and HCC), nor provide a shortcut to the magic bullet. In summary, the discussed study has added an interesting facet to the underlying pathogenesis of HCC in NASH, the overall importance of which needs to be confirmed in further studies. Muhammad Asif Qureshi, M.D., Ph.D. Yong Ook Kim, Ph.D. Detlef Schuppan, M.D., Ph.D. Institute of Translational Immunology, Research Center for Immune Therapy, University Medical Center, Johannes Gutenberg University, Langenbeckstraße 1, 55131 Mainz, Germany