Previous studies have shown that human nonalcoholic steatohepatitis (NASH) is often associated with the presence of circulating antibodies against protein adducted by lipid peroxidation products. Here we used the methionine-choline deficient (MCD) model of NASH to characterize the possible involvement of adaptive immunity in NASH. In mice fed up to 8 weeks with the MCD diet the extension of liver injury and lobular inflammation paralleled the development of immunoglobulin G (IgG) against malonyldialdehyde (MDA) and 4-hydroxynonenal (4-HNE)-derived antigens as well as with the hepatic recruitment of CD4+ and CD8+ T-lymphocytes responsive to the same antigens. Moreover, in these animals the individual IgG reactivity against MDA-adducts positively correlated with transaminase release and hepatic tumor necrosis factor alpha (TNF-α) expression. To substantiate the role of immune responses triggered by oxidative stress in the progression of NASH, mice were immunized with MDA-adducted bovine serum albumin (MDA-BSA) before feeding the MCD diet. MDA-BSA immunization did not affect control mice livers, but further stimulated transaminase release, lobular inflammation, and the hepatic expression of proinflammatory cytokine in MCD-fed mice. The increased severity of NASH in immunized MCD-fed mice involved liver recruitment and the T helper (Th)-1 activation of CD4+ T cells that, in turn, further stimulated macrophage M1 responses. Moreover, hepatic fibrosis was also evident in these animals in relation with an IL-15-mediated increase of natural killer T-cells (NKT) and the up-regulation in liver production of osteopontin by NKT cells and hepatic macrophages. Conclusion: These results indicate that oxidative stress can contribute to the progression of NASH by stimulating both humoral and cellular immune responses, pointing to the possible role of adaptive immunity in the pathogenesis of the disease. (Hepatology 2014;59:886–897)
A key issue in understanding the pathogenesis of nonalcoholic fatty liver disease (NAFLD) concerns the identification of the mechanisms responsible for switching from simple steatosis to steatohepatitis (NASH). This aspect is clinically relevant because steatosis does not appear to adversely affect the long-term outcome of NAFLD, whereas parenchymal injury and inflammation are the driving forces for the disease evolution to fibrosis/cirrhosis.[2, 3] Oxidative stress is one of the features of NAFLD/NASH and hepatic oxidative stress markers, such as 4-hydroxynonenal (4-HNE) and 8-hydroxydeoxyguanosine, correlate with the severity of necroinflammation and fibrosis,[5, 6] suggesting that oxidative injury might be involved in triggering steatohepatitis. In this scenario, recent evidence indicates that lipid peroxidation products originating from the oxidation of phospholipids can act as damage-associated molecular patterns (DAMPs) and promote inflammation through the interaction with soluble and cell-associated pattern recognition receptors.[7, 8] A further mechanism by which oxidative stress can stimulate inflammation involves adaptive immunity. Indeed, in atherosclerosis as well as in several autoimmune diseases the interaction of lipid peroxidation products with cellular proteins leads to the formation of immunogenic adducts that induce both humoral and cellular immune responses.[9, 10]
Previous studies from our laboratory have shown that high titers of immunoglobulin G (IgG) against some of the antigens originating from oxidative stress, namely malondialdehyde (MDA)-derived adducts, are detectable in about 40% of adult NAFLD/NASH patients and in 60% of children with NASH.[11, 12] In these latter, high antibody titers associated with more severe lobular inflammation and 13-fold increased risk of an NAFLD activity score ≥5, while in adults anti-MDA IgG is an independent predictor of fibrosis. From this background, we sought to investigate the possible contribution of immune reactions triggered by oxidative stress in modulating hepatic inflammation in NASH. For the experiments, we relied on a rodent model of NASH based on mice feeding with a methionine-choline deficient (MCD).
Recent studies have implicated the contribution of adaptive immunity in promoting fat inflammation in obesity, since CD4+/CD8+ T cells are recruited into the adipose tissue and provide stimulation for the macrophage production of proinflammatory mediators.[25, 26] Lymphocytes are often detected in the lobular infiltrates of NASH, but the actual role of adaptive immunity in the pathogenesis of the disease is still poorly understood. We previously reported that subsets of adult and pediatric NAFLD/NASH patients show antibody responses against oxidative stress-related antigens, such as MDA-derived adducts, that associate with an increased severity of lobular inflammation or fibrosis.[10, 11] Similar antibodies are also detectable in rats with NASH induced by enteral nutrition with a high-fat diet, while preventing oxidative stress with N-acetylcysteine attenuates both the IgG formation and the severity of steatohepatitis. In the present study, we observed that the progression of MCD-induced NASH parallels with the development of IgG against lipid peroxidation-derived adducts and the liver recruitment of CD4+ and CD8+ T-lymphocytes recognizing the same antigens. The possible contribution of adaptive immunity to the progression of experimental NASH is further substantiated by the observation that stimulating immune responses against MDA-protein adducts, one of the antigens recognized by the antibodies detected in both human and rodent NASH, promotes parenchymal injury and inflammation in mice fed the MCD diet. We are well aware that NASH induced by the MCD diet does not reproduce some of the key features of the human disease such as obesity and insulin resistance; however, in this study we exploited the capacity of this model to cause oxidative stress and extensive steatohepatitis rapidly progressing to fibrosis. Our data are not in contrast with a recent report Bieghs et al. showing that IgM targeting oxidized LDLs reduce NASH in LDL receptor-deficient mice receiving a high-fat/cholesterol diet. These discrepancies, in fact, can be explained considering that the two experimental settings are quite different for the immune responses involved and the mechanisms leading to NASH. In Bieghs et al.'s work mice were immunized with heat-inactivated pneumococci leading to the production of natural IgM against bacterial antigens that crossreact with oxidized phosphatidylcholine in LDLs. Feeding a high-fat/cholesterol diet to LDL receptor-deficient mice causes Kupffer cell engulfment by oxidized LDLs that, in turn, promotes Kupffer cell activation and hepatic inflammation. In this scenario, the IgM interaction with oxidized LDLs reduces their uptake by Kupffer cells, lowering the proinflammatory stimuli. These conditions are quite different from those occurring in MCD-induced NASH, where parenchymal injury, oxidative stress, and inflammation result from the impairment of hepatocyte lipid secretion. Furthermore, our data indicate that the immunization with MDA adducts mainly stimulates IgG production and T-cell responses and that these latter are mainly responsible for promoting inflammation.
Concerning the mechanisms by which adaptive immunity contributes the evolution of NASH, we observed that hepatic CD4+ T cells are increased in MCD-fed immunized mice in parallel with a stimulation in the liver expression of IFN-γ and CD40L (CD154). CD40L is a costimulatory molecule predominantly expressed by CD4+ T cells and activated platelets that, by the interaction with its receptor CD40 on macrophages and lymphocytes, has a key role in orchestrating inflammation and immunity in several diseases, including atherosclerosis and obesity.[30, 31] In line with this, CD4+ T-cell depletion prevents the up-regulation of IFN-γ and CD40L and ameliorates lobular inflammation, indicating that Th-1 activation of CD4+ T-lymphocytes plays a major role in promoting NASH. It is noteworthy that Th-1 activation characterizes CD4+ T-cell responses to LDL-derived oxidation antigens in atherosclerosis. In this setting, CD4+ T cell or IFN-γ deficiency have been shown to ameliorate plaque inflammation and the disease progression. Interestingly, an increase in circulating IFN-γ-producing CD4+ T-cells has been observed in either pediatric and adult NASH patients in conjunction with an enhanced liver IFN-γ production,[32, 33] suggesting the possible relevance of these mechanisms to the human disease. A recent report indicates that an increase in hepatic CD8+ T cells also characterizes pediatric NASH. In our hands, CD8+ T-cell recruitment is evident in NASH livers and is further promoted by preimmunization. However, immunization does not affect the expression CD8+ T-cell activation markers, suggesting that in our experimental setting effector T cells do not significantly contribute to hepatic inflammation. Nonetheless, the involvement of CD8+ T cells in NASH requires further investigations. In a similar manner, more studies are needed to clarify the role of B-cell responses. Indeed, the presence of circulating anti-MDA IgG might not be just a hallmark of immune activation against oxidative stress-derived epitopes, but might influence the disease evolution by causing antibody-mediated injury. Furthermore, B cells have been shown to drive CD4+ T-cell activation and cytokine production in the adipose tissue during obesity and modulate the progression of liver injury to fibrosis.[35, 36]
Accumulating data indicate that NKT cells can orchestrate inflammation in autoimmune liver diseases and modulate hepatic fibrogenesis.[37, 38] In line with these observations, recent reports point to an involvement of NKT cells in NASH. Indeed, while steatosis is characterized by the lowering of the liver NKT pool as a consequence of IL-12 production and Tim-3/galectin-9 signaling,[20-22] NKT expansion is a feature of advanced NASH in either rodents and humans.[23, 39, 40] In the former, NKT depletion prevents hepatic inflammation and fibrosis.[23, 39] We observed that an increase in NKT cells characterizes the enhanced severity of NASH in immunized MCD-fed mice, as opposed to NKT cell depletion present in similarly treated naïve animals, further supporting the contribution of NKT cells to NASH progression. Changes in the hepatic levels of IL-15 have been proposed to modulate the NKT pool in NASH.[20, 22] IL-15 is a pleiotropic cytokine responsible for macrophage, T, NK, and NKT cell survival and maturation. In healthy livers, hepatocyte constitutively produce IL-15 to create a T-cell favorable environment, while an increased hepatocyte and macrophage IL-15 expression in response to injury is critical for driving both innate and adaptive immunity.[22, 43] We observed that IL-15 is selectively up-regulated in immunized, but not in naïve MCD-fed mice concomitantly with NKT cell recruitment, suggesting that parenchymal damage and inflammation resulting from Th-1 responses promote an IL-15-mediated expansion of NKT cells, which, in turn, might participate in the evolution of NASH.
According to Syn et al., OPN generated by NKT cells contributes to fibrosis in NASH. OPN is a cytokine produced by either immune and parenchymal cells that modulates both inflammation and tissue healing. In the liver, OPN production by NKT cells drives concanavalin A-induced hepatitis, but OPN can also stimulate collagen synthesis by HSCs through a transforming growth factor beta (TGF-β1)-independent pathway. An up-regulation in liver OPN is evident in either humans and rodents with advanced NASH,[16, 23, 46] while OPN-deficient A/J mice are protected against steatohepatitis and fibrosis induced by feeding the MCD diet.[46, 47] In our hands, hepatic OPN is specifically up-regulated in MCD-fed immunized mice concomitant with the recruitment of OPN-expressing NKT cells. Moreover, we observed that hepatic macrophages also contribute to OPN production. This is consistent with the capacity of Th-1 cytokines to stimulate OPN synthesis in macrophages. However, we cannot exclude that other liver cells, such as cholangiocytes, might also generate OPN. Different from that reported by Sahai et al. using A/J mice, we did not observe changes in hepatic OPN expression in naïve C57BL/6 mice receiving the MCD diet for 4 weeks. This discrepancy might reflect strain differences in the mice susceptibility to steatohepatitis and further support the importance of OPN in NASH evolution. Furthermore, the capacity of OPN to stimulate HSC might account for the increase in collagen deposition observed in MCD-fed immunized mice in spite of the fact that these animals have a high hepatic IFN-γ production that should antagonize fibrogenesis.
In conclusion, the results presented indicate that immune responses triggered by oxidative stress-derived antigens contribute to hepatic inflammation in experimental NASH by promoting the Th-1 activation of CD4+ T-lymphocytes. NASH in immunized animals is also associated with an increase in liver NKT cells that likely participate in the disease evolution by generating osteopontin. Altogether, these data support recent observations in humans about the possible involvement of adaptive immunity in the mechanisms leading to NAFLD evolution.