Alcoholic and nonalcoholic fatty liver disease (ALD and NAFLD) are the predominant causes of liver-related mortality in Western countries. We have shown that limiting classical (M1) Kupffer cell (KC) polarization reduces alcohol-induced liver injury. Herein, we investigated whether favoring alternatively activated M2 KCs may protect against ALD and NAFLD. Ongoing alcohol drinkers and morbidly obese patients, with minimal hepatic injury and steatosis, displayed higher hepatic expression of M2 genes, as compared to patients with more severe liver lesions; individuals with limited liver lesions showed negligible hepatocyte apoptosis but significant macrophage apoptosis. Experiments in mouse models of ALD or NAFLD further showed that BALB/c or resveratrol-treated mice fed alcohol or a high-fat diet displayed preponderant M2 KC polarization, M1 KC apoptosis, and resistance to hepatocyte steatosis and apoptosis, as compared to control C57BL6/J mice. In vitro experiments in isolated KC, peritoneal, and Raw264.7 macrophages demonstrated that M1 macrophage apoptosis was promoted by conditioned medium from macrophages polarized into an M2 phenotype by either interleukin (IL)4, resveratrol, or adiponectin. Mechanistically, IL10 released from M2 cells promoted M1 death, and anti-IL10 antibodies blunted the proapoptic effects of M2-conditioned media. IL10 secreted by M2 KCs promoted selective M1 death by a mechanism involving activation of arginase in high inducible nitric oxide synthase-expressing M1 KCs. In alcohol-exposed mice, neutralization of IL10 impaired M1 apoptosis. Conclusion: These data uncover a novel mechanism regulating the M1/M2 balance that relies on apoptotic effects of M2 KCs towards their M1 counterparts. They suggest that promoting M2-induced M1 KC apoptosis might prove a relevant strategy to limit alcohol-and high fat-induced inflammation and hepatocyte injury. (HEPATOLOGY 2014;59:130-142)

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T-cell regulation in adipose tissue provides a link between inflammation and insulin resistance. Because of alterations in adipose tissue T-cell composition in obesity, we aimed to identify the antigen-presenting cells in adipose tissue of obese mice and patients with insulin resistance. Dendritic cells (DCs) and T cells were studied in mice and in two cohorts of obese patients. In lean mice, only CD11c+ DCs were detected in adipose tissue. Adoptive transfer of naive CD4+ T cells in Rag1−/− mice led to a predominant Th1 response in adipose tissue. In contrast, during obesity DCs (human CD11c+CD1c+ and mouse CD11chighF4/80low) accumulated in adipose tissue. CD11chighF4/80low DCs from obese mice induced Th17 differentiation. In patients, the presence of CD11c+CD1c+ DCs correlated with the BMI and with an elevation in Th17 cells. In addition, these DCs led to ex vivo Th17 differentiation. CD1c gene expression further correlated with homeostatic model assessment-insulin resistance in the subcutaneous adipose tissue of obese patients. We show for the first time the presence and accumulation of specific DCs in adipose tissue in mouse and human obesity. These DCs were functional and could be important regulators of adipose tissue inflammation by regulating the switch toward Th17 cell responses in obesity-associated insulin resistance.

The incidence of chronic liver disease is constantly increasing, owing to the obesity epidemic. However, the causes and mechanisms of inflammation-mediated liver damage remain poorly understood. Endoplasmic reticulum (ER) stress is an initiator of cell death and inflammatory mechanisms. Although obesity induces ER stress, the interplay between hepatic ER stress, NLRP3 inflammasome activation and hepatocyte death signaling has not yet been explored during the etiology of chronic liver diseases. Steatosis is a common disorder affecting obese patients; moreover, 25% of these patients develop steatohepatitis with an inherent risk for progression to hepatocarcinoma. Increased plasma LPS levels have been detected in the serum of patients with steatohepatitis. We hypothesized that, as a consequence of increased plasma LPS, ER stress could be induced and lead to NLRP3 inflammasome activation and hepatocyte death associated with steatohepatitis progression. In livers from obese mice, administration of LPS or tunicamycin results in IRE1α and PERK activation, leading to the overexpression of CHOP. This, in turn, activates the NLRP3 inflammasome, subsequently initiating hepatocyte pyroptosis (caspase-1, -11, interleukin-1β secretion) and apoptosis (caspase-3, BH3-only proteins). In contrast, the LPS challenge is blocked by the ER stress inhibitor TUDCA, resulting in: CHOP downregulation, reduced caspase-1, caspase-11, caspase-3 activities, lowered interleukin-1β secretion and rescue from cell death. The central role of CHOP in mediating the activation of proinflammatory caspases and cell death was characterized by performing knockdown experiments in primary mouse hepatocytes. Finally, the analysis of human steatohepatitis liver biopsies showed a correlation between the upregulation of inflammasome and ER stress markers, as well as liver injury. We demonstrate here that ER stress leads to hepatic NLRP3 inflammasome pyroptotic death, thus contributing as a novel mechanism of inflammation-mediated liver injury in chronic liver diseases. Inhibition of ER-dependent inflammasome activation and cell death pathways may represent a potential therapeutic approach in chronic liver diseases.

Data concerning the fine structure of the 12q13-15 amplicon which contains MDM2 and CDK4 in well-differentiated and dedifferentiated liposarcomas (WDLPS/DDLPS) are scarce. We investigated a series of 38 WDLPS/DDLPS using fluorescence in situ hybridization analysis with 17 probes encompassing the 12q13-15 region. In addition, using quantitative RT-PCR we studied the expression of MDM2, CDK4, DDIT3 (CHOP/GADD153), DYRK2, HMGA2, TSPAN31 and YEATS4 (GAS41) in 11 cases. We showed that CDK4 (12q14.1) belonged to a distinct amplicon than MDM2 (12q15). There was no continuity in the amplified sequences between MDM2 and CDK4. Moreover, while MDM2 was amplified and overexpressed in all cases, CDK4 was not amplified or overexpressed in 13% of cases. The centromeric border of the CDK4 amplicon was located immediately downstream the 5 0 end of DDIT3, a gene known for being involved in myxoid liposarcoma translocations. DDIT3 was amplified in 3 cases and overexpressed in 9 cases. The overexpression of DDIT3 was correlated to the CDK4 amplification and not to its own amplification status. This suggested that the CDK4 amplicon, as well as the overexpression of DDIT3, might be generated by the disruption of a fragile region in 5 0 DDIT3. HMGA2 was always amplified and rearranged indicating that it plays a central role in WDLPS/DDLPS. HMGA2 rearrangement frequently resulted in a loss of the 3 0 end region that is a binding site for let-7. We also found a frequent amplification and overexpression of YEATS4, an oncogene that inactivates P53, suggesting that YEATS4 might play an important role together with MDM2 in WDLPS/DDLPS oncogenesis.

BackgroundObesity modulates inflammation and activation of immune pathways which can lead to liver complications. We aimed at identifying expression patterns of inflammatory and immune response genes specifically associated with obesity and NASH in the liver of morbidly obese patients.Methodology/Principal FindingsExpression of 222 genes was evaluated by quantitative RT-PCR in the liver of morbidly obese patients with histologically normal liver (n = 6), or with severe steatosis without (n = 6) or with NASH (n = 6), and in lean controls (n = 5). Hepatic expression of 58 out of 222 inflammatory and immune response genes was upregulated in NASH patients. The most notable changes occurred in genes encoding chemokines and chemokine receptors involved in leukocyte recruitment, CD and cytokines involved in the T cell activation towards a Th1 phenotype, and immune semaphorins. This regulation seems to be specific for the liver since visceral adipose tissue expression and serum levels of MCP1, IP10, TNFα and IL6 were not modified. Importantly, 47 other genes were already upregulated in histologically normal liver (e.g. CRP, Toll-like receptor (TLR) pathway). Interestingly, serum palmitate, known to activate the TLR pathway, was increased with steatosis.Conclusion/SignificanceThe liver of obese patients without histological abnormalities already displayed a low-grade inflammation and could be more responsive to activators of the TLR pathway. NASH was then characterized by a specific gene signature. These findings help to identify new potential actors of the pathogenesis of NAFLD.

OBJECTIVE-Osteopontin (OPN) plays an important role in the development of insulin resistance and liver complications in dietary murine models. We aimed to determine the expression pattern of OPN and its receptor CD44 in obese patients and mice according to insulin resistance and liver steatosis.RESEARCH DESIGN AND METHODS-OPN and CD44 expressions were studied in 52 morbidly obese patients and in mice. Cellular studies were performed in HepG2 cells. RESULTS-HepaticOPN and CD44 expressions were strongly correlated with liver steatosis and insulin resistance in obese patients and mice. This increased OPN expression could be due to the accumulation of triglycerides, since fat loading in HepG2 promotes OPN expression. In contrast, OPN expression in adipose tissue (AT) was enhanced independently of insulin resistance and hepatic steatosis in obese patients. The elevated OPN expression in AT was paralleled with the AT macrophage infiltration, and both phenomena were reversed after weight loss. The circulating OPN level was slightly elevated in obese patients and was not related to liver steatosis. Further, AT did not appear to secrete OPN. In contrast, bariatric surgery-induced weight loss induced a strong increase in circulating OPN. CONCLUSIONS-The modestly elevated circulating OPN levels in morbidly obese patients were not related to liver steatosis and did not appear to result from adipose tissue secretion. In subcutaneous AT, expression of OPN was directly related to macrophage accumulation independently from liver complications. In contrast, hepatic OPN and CD44 expressions were related to insulin resistance and steatosis, suggesting their local implication in the progression of liver injury. Diabetes 58: 125-133, 2009

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