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 1 and CD8 1 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-a) 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 1 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 upregulation 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)

Recent evidence implicates adaptive immunity as a key player in the mechanisms supporting hepatic inflammation during the progression of nonalcoholic fatty liver disease (NAFLD). In these settings, patients with NAFLD often show an increase in the circulating levels of antibodies against oxidative stress-derived epitopes (OSE). Nonetheless, the actual role of humoral immunity in NAFLD is still unclear. This study investigates the contribution of B-lymphocytes to NAFLD evolution. B-lymphocyte immunostaining of liver biopsies from NAFLD patients showed that B-cells were evident within cell aggregates rich in T-lymphocytes. In these subjects, B/T-lymphocyte infiltration positively correlated with both circulating IgG targeting oxidative stress-derived epitopes (OSE) and interferon-γ (IFN-γ) levels. Furthermore, high prevalence of lymphocyte aggregates identified patients with more severe lobular inflammation and fibrosis. In mouse models of NAFLD, the onset of steatohepatitis was characterized by hepatic B2-lymphocytes maturation to plasma cells and by an elevation in circulating anti-OSE IgG titers. B-cell responses preceded T-cell activation and were accompanied by the up-regulation in the hepatic expression of B-cell Activating Factor (BAFF). Selective B2-cell depletion in mice over-expressing a soluble form of the BAFF/APRIL receptor Transmembrane Activator and Cyclophilin Ligand Interactor (TACI-Ig) prevented plasma cell maturation and Th-1 activation of liver CD4 T-lymphocytes. Furthermore, TACI-Ig mice showed milder steatohepatitis and a decreased progression to fibrosis. Similarly, mice treatment with the BAFF-neutralizing monoclonal antibody Sandy-2 prevented hepatic B2-cell responses and ameliorated steatohepatitis. From these data we conclude that B2-lymphocyte activation is an early event in NAFLD evolution and contributes to the disease progression through the interaction with T-cells. Furthermore, combined clinical and experimental data suggest that elevated circulating anti-OSE IgG can identify a subset of NAFLD patients in whom adaptive immunity has a relevant role in the disease evolution toward fibrosis.

Annexin A1 (AnxA1) is an effector of the resolution of inflammation and is highly effective in terminating acute inflammatory responses. However, its role in chronic settings is less investigated. Because changes in AnxA1 expression within adipose tissue characterize obesity in mice and humans, we queried a possible role for AnxA1 in the pathogenesis of nonalcoholic steatohepatitis (NASH), a disease commonly associated with obesity. NASH was induced in wild-type (WT) and AnxA1 knockout (AnxA1 KO) C57BL/6 mice by feeding a methionine-choline deficient (MCD) diet up to 8 weeks. In MCD-fed WT mice, hepatic AnxA1 increased in parallel with progression of liver injury. This mediator was also detected in liver biopsies from patients with NASH and its degree of expression inversely correlated with the extent of fibrosis. In both humans and rodents, AnxA1 production was selectively localized in liver macrophages. NASH in AnxA1 KO mice was characterized by enhanced lobular inflammation resulting from increased macrophage recruitment and exacerbation of the M1 phenotype. Consistently, in vitro addition of recombinant AnxA1 to macrophages isolated from NASH livers down-modulated M1 polarization through stimulation of interleukin-10 production. Furthermore, the degree of hepatic fibrosis was enhanced in MCD-fed AnxA1 KO mice, an effect associated with augmented liver production of the profibrotic lectin, galectin-3. Accordingly, AnxA1 addition to isolated hepatic macrophages reduced galectin-3 expression. Conclusions: Macrophage-derived AnxA1 plays a functional role in modulating hepatic inflammation and fibrogenesis during NASH progression, suggesting the possible use of AnxA1 analogs for therapeutic control of this disease.

These results indicate that hepatocyte HIF-2α activation is a key feature in both human and experimental NAFLD and significantly contributes to the disease progression through the up-regulation of HRGP production. (Hepatology 2018;67:2196-2214).

In humans, there is large inter-individual variability in the evolution of NAFLD (non-alcoholic fatty liver disease) to NASH (non-alcoholic steatohepatitis). To investigate this issue, NASH was induced with an MCD (methionine-choline-deficient) diet in C57BL/6 and Balb/c mice that are characterized by different biases in Th1/Th2 and macrophage (M1/M2) responses. Following 4 weeks on the MCD diet, steatosis and lobular inflammation were prevalent in C57BL/6 (Th1, M1 oriented) than in Balb/c (Th2, M2 oriented) mice. Consistently, hepatic TNFα (tumour necrosis factor α) mRNA expression and circulating TNFα levels were higher in MCD-fed C57BL/6 than in MCD-fed Balb/c mice. The Th1/Th2 bias did not account for the increased NASH severity, as in both strains MCD feeding did not significantly modify the liver mRNA expression of the Th1 markers IFNγ (interferon γ) and T-bet or that of the Th2 markers IL-4 (interleukin 4) and GATA-3. Conversely, MCD-fed C57BL/6 mice displayed higher liver mRNAs for the macrophage M1 activation markers iNOS (inducible NO synthase), IL-12p40 and CXCL10 (CXC chemokine ligand 10) than similarly treated Balb/c mice, without effects on the M2 polarization markers IL-10 and MGL-1 (macrophage galactose-type C-type lectin-1). Circulating IL-12 was also higher in MCD-fed C57BL/6 than in MCD-fed Balb/c mice. The analysis of macrophages isolated from the livers of MCD-fed animals confirmed an enhanced expression of M1 markers in C57BL/6 mice. Among all of the MCD-treated mice, liver iNOS, IL-12p40 and CXCL10 mRNA levels positively correlated with the frequency of hepatic necro-inflammatory foci. We concluded that the macrophage M1 bias in C57BL/6 mice may account for the increased severity of NASH in this strain, suggesting macrophage responses as important contributors to NAFLD progression.