Regulation of the mouse gene encoding TAFI by TNFα: role of NFκB binding site

Thrombin-activable fibrinolysis inhibitor (TAFI) is a plasma pro-carboxypeptidase, encoded by the gene CPB2, with roles in both inhibition of fibrinolysis and inflammation. In mice, plasma TAFI levels and hepatic CPB2 mRNA expression were found to increase within 24h after intra-peritoneal lipopolysaccharide (LPS) injection. On the other hand, plasma TAFI in humans decrease in experimental endotoxemia and sepsis and we have previously demonstrated that CPB2 mRNA abundance in human hepatoma cells is decreased by inflammatory cytokines. Here, we have evaluated the effects of TNFα on mouse CPB2 expression. Treatment of primary mouse hepatocytes or the mouse hepatic cell line FL83B with TNFα for 12-48h resulted in increases in CPB2 mRNA abundance of up to 2-fold; mouse TAFI protein levels secreted from FL83B cells increased 2.7-fold after 48h treatment with TNFα. When FL83B cells were transfected with reporter plasmids containing the mouse CPB2 5'-flanking region, treatment with TNFα for 24 and 48h resulted in a 1.5-fold increased mouse CPB2 promoter activity. Mutation of a putative NFκB site not conserved in the human gene ablated the increased promoter activity observed following TNFα treatment. This site binds NFκB as assessed by gel mobility shift assays, and TNFα treatment increases the translocation of NFκB from the cytoplasm to the nucleus of mouse hepatocytes. These results demonstrate that the unique NFκB site in the mouse CPB2 promoter is functional and mediates the upregulation of mouse CPB2 expression by TNFα via increase in NFκB translocation to the nucleus.     

The osteopontin level in liver, adipose tissue and serum is correlated with fibrosis in patients with alcoholic liver disease

Background

Osteopontin (OPN) plays an important role in the progression of chronic liver diseases. We aimed to quantify the liver, adipose tissue and serum levels of OPN in heavy alcohol drinkers and to compare them with the histological severity of hepatic inflammation and fibrosis.

Methodology/Principal Findings

OPN was evaluated in the serum of a retrospective and prospective group of 109 and 95 heavy alcohol drinkers, respectively, in the liver of 34 patients from the retrospective group, and in the liver and adipose tissue from an additional group of 38 heavy alcohol drinkers. Serum levels of OPN increased slightly with hepatic inflammation and progressively with the severity of hepatic fibrosis. Hepatic OPN expression correlated with hepatic inflammation, fibrosis, TGFβ expression, neutrophils accumulation and with the serum OPN level. Interestingly, adipose tissue OPN expression also correlated with hepatic fibrosis even after 7 days of alcohol abstinence. The elevated serum OPN level was an independent risk factor in estimating significant (F≥2) fibrosis in a model combining alkaline phosphatase, albumin, hemoglobin, OPN and FibroMeter® levels. OPN had an area under the receiving operator curve that estimated significant fibrosis of 0.89 and 0.88 in the retrospective and prospective groups, respectively. OPN, Hyaluronate (AUROC: 0.88), total Cytokeratin 18 (AUROC: 0.83) and FibroMeter® (AUROC: 0.90) estimated significance to the same extent in the retrospective group. Finally, the serum OPN levels also correlated with hepatic fibrosis and estimated significant (F≥2) fibrosis in 86 patients with chronic hepatitis C, which suggested that its elevated level could be a general response to chronic liver injury.

Conclusion/Significance

OPN increased in the liver, adipose tissue and serum with liver fibrosis in alcoholic patients. Further, OPN is a new relevant biomarker for significant liver fibrosis. OPN could thus be an important actor in the pathogenesis of this chronic liver disease.     

Leptin enhances alpha1(I) collagen gene expression in LX-2 human hepatic stellate cells through JAK-mediated H2O2-dependent MAPK pathways

Leptin, a liver profibrogenic cytokine, induces oxidative stress in hepatic stellate cells (HSCs), with increased formation of the oxidant H2O2, which signals through p38 and extracellular signal-regulated kinase 1/2 (ERK1/2) pathways, stimulating tissue inhibitor of metalloproteinase-1 production. Since oxidative stress is a pathogenic mechanism of liver fibrosis and activation of collagen gene is a marker of fibrogenesis, we evaluated the effects of leptin on collagen I expression. We report here that, in LX-2 human HSCs, leptin enhances the levels of alpha1(I) collagen mRNA, promoter activity and protein. Janus kinase (JAK)1 and JAK2 were activated. H2O2 formation was increased; this was prevented by the JAK inhibitor AG490, suggesting a JAK-mediated process. ERK1/2 and p38 were activated, and the activation was blocked by catalase, consistent with an H2O2-dependent mechanism. AG490 and catalase also prevented leptin-stimulated alpha1(I) collagen mRNA expression. PD098059, an ERK1/2 inhibitor, abrogated ERK1/2 activation and suppressed alpha1(I) collagen promoter activity, resulting in mRNA down-regulation. The p38 inhibitor SB203580 and overexpression of dominant negative p38 mutants abrogated p38 activation and down-regulated the mRNA. While SB203580 had no effect on the promoter activity, it reduced the mRNA half-life from 24 to 4 h, contributing to the decreased mRNA level. We conclude that leptin stimulates collagen production through the H2O2-dependent and ERK1/2 and p38 pathways via activated JAK1 and JAK2. ERK1/2 stimulates alpha1(I) collagen promoter activity, whereas p38 stabilizes its mRNA. Accordingly, interference with leptin-induced oxidative stress by antioxidants provides an opportunity for the prevention of liver fibrosis.     

The rno-miR-34 family is upregulated and targets ACSL1 in dimethylnitrosamine-induced hepatic fibrosis in rats

The mechanisms whereby hepatic fibrosis develops in chronic liver diseases remain incompletely defined. Here, we sought to examine whether microRNA (miRNA) became dysregulated in dimethylnitrosamine-induced hepatic fibrosis in rats. Our microarray analysis revealed that the miR-34 family was upregulated along with other miRNAs in liver fibrotic tissues. Six miRNAs, such as rno-miR-878, were downregulated. The findings were confirmed by RT-PCR assays. Gene ontology analysis further showed that many of these dysregulated miRNAs were involved in lipid/fatty acid metabolism. The acyl-CoA synthetase long-chain family member 1 (ACSL1) gene contained specific binding sites for miR-34a/miR-34c. Additional enhanced green fluorescence protein reporter activity assays indicated that the miR-34 family targeted ACSL1. Our RT-PCR and immunoblotting assays further demonstrated that both the mRNA and protein levels of ACSL1 were markedly reduced in fibrotic liver tissues. Our findings suggest that miRNA becomes dysregulated during hepatic fibrosis, and that the miR-34 family may be involved in the process by targeting ACSL1.     

肝细胞脂肪变性程度对慢性病毒性肝炎弥散度成像的影响

目的：探讨肝细胞脂肪变性对肝脏弥散度成像技术（Realtime Tissue Elastography,RTE）无创评估病毒性肝炎肝纤维化及炎 症程度的影响。方法：91 例确诊慢性乙型或丙型病毒性肝炎患者均于肝脏活检穿刺前行肝脏弥散度成像检查,分析不同肝细胞脂 肪变性程度与肝纤维化程度及肝脏炎症程度之间的弥散度成像LF指数差异。结果：中度脂肪变性组LF 指数最高（P=0.0254）;轻 度纤维化（S1-S2）时,不同的脂肪变性程度组间LF 值的差异无统计学意义（P=0.1105）;中- 重度纤维化时,不同的脂肪变性程度 组间LF值的差异无统计学意义（P=0.0994）。轻度炎症时,不同的脂肪变性程度组间LF值的差异具有统计学意义,中度脂肪变性 组的LF值最高（P=0.0010）;中- 重度炎症时,不同的脂肪变性程度组间LF值的差异具有统计学意义（P<0.05）。结论：应用肝脏弥 散度成像的LF指数判断肝脏纤维化程度不受肝细胞脂肪变性的影响,但对于肝脏炎症程度的判断受肝细胞脂肪变性的影响,尤 其应重视中度肝细胞脂肪变性对于测值的影响。     

TGF-β1和FBRS可指示肝纤维化发展程度

为了探讨转化生长因子(TGF-β1)、外周血纤维化蛋白(FBRS)表达水平变化与肝纤维化发生发展的相关性,本研究选取自2015年5月至2017年6月间在我院诊治的慢性病毒性肝炎患者120例,设为肝炎组。采用免疫组化法检测肝组织TGF-β1的表达水平,酶联免疫分析检测血清中TGF-β1的含量,RT-PCR检测外周血单个核细胞FBRS mRNA的表达水平,分析TGF-β1、FBRS与肝纤维化程度的相关性。研究结果表明:随肝纤维化程度的不同,肝组织TGF-β1、血清TGF-β1表达水平、FBRS mRNA表达水平与肝脏胶原含量同步性升高(p<0.05)。进一步的相关分析表明:肝组织TGF-β1水平、FBRS mRNA与肝纤4项检查,即血清Ⅲ型前胶原(PC-Ⅲ)、Ⅳ型胶原片段(Ⅳ-C)、层粘连蛋白(LN)和透明质酸(HA)水平之间均呈正相关。本研究结果初步得出结论,慢性病毒性肝炎肝组织TGF-β1、血清TGF-β1表达水平、外周血FBRS的表达水平与肝组织纤维化程度呈正相关。     

Th2-associated alternative Kupffer cell activation promotes liver fibrosis without inducing local inflammation

Cirrhosis is the final outcome of liver fibrosis. Kupffer cell-mediated hepatic inflammation is considered to aggravate liver injury and fibrosis. Alternatively-activated macrophages are able to control chronic inflammatory events and trigger wound healing processes. Nevertheless, the role of alternative Kupffer cell activation in liver harm is largely unclear. Thus, we evaluated the participation of alternatively-activated Kupffer cells during liver inflammation and fibrosis in the murine model of carbon tetrachloride-induced hepatic damage. To stimulate alternative activation in Kupffer cells, 20 Taenia crassiceps (Tc) larvae were inoculated into BALBc/AnN female mice. Six weeks post-inoculation, carbon tetrachloride or olive oil were orally administered to Tc-inoculated and non-inoculated mice twice per week during other six weeks. The initial exposure of animals to T. crassiceps resulted in high serum concentrations of IL-4 accompanied by a significant increase in the hepatic mRNA levels of Ym-1, with no alteration in iNOS expression. In response to carbon tetrachloride, recruitment of inflammatory cell populations into the hepatic parenchyma was 5-fold higher in non-inoculated animals than Tc-inoculated mice. In contrast, carbon tetrachloride-induced liver fibrosis was significantly less in non-inoculated animals than in the Tc-inoculated group. The latter showed elevated IL-4 serum levels and low IFN-γ concentrations during the whole experiment, associated with hepatic expression of IL-4, TGF-β, desmin and α-sma, as well as increased mRNA levels of Arg-1, Ym-1, FIZZ-1 and MMR in Kupffer cells. These results suggest that alternative Kupffer cell activation is favored in a Th2 microenvironment, whereby such liver resident macrophages could exhibit a dichotomic role during chronic hepatic damage, being involved in attenuation of the inflammatory response but at the same time exacerbation of liver fibrosis.     

LC3-associated phagocytosis in myeloid cells,a fireman that restrains inflammation and liver fibrosis,via immunoreceptor inhibitory signaling

ABSTRACT

Control of systemic and hepatic inflammation, in particular originating from monocytes/macrophages, is crucial to prevent liver fibrosis and its progression to end-stage cirrhosis. LC3-associated phagocytosis (LAP) is a non-canonical form of autophagy that shifts the monocyte/macrophage phenotype to an anti-inflammatory phenotype. In a recent study, we uncovered LAP as a protective mechanism against inflammation-driven liver fibrosis and systemic inflammation in the context of cirrhosis. We observed that LAP is enhanced in blood and liver monocytes from patients with liver fibrosis or those who progress to cirrhosis. Combining studies in which LAP was pharmacologically or genetically inactivated, we found that LAP limits inflammation in monocytes from cirrhotic patients, and the hepatic inflammatory profile in mice with chronic liver injury, resulting in anti-fibrogenic effects. Mechanistically, LAP-induced anti-inflammatory and antifibrogenic signaling results from enhanced expression of the Fc immunoreceptor FCGR2A/FcγRIIA and activation of an FCGR2A-mediated PTPN6/SHP-1 anti-inflammatory pathway, leading to increased engulfment of IgG into LC3 ⁺ phagosomes. In patients with cirrhosis progressing to multi-organ failure (acute-on chronic liver failure), LAP is lost in monocytes, and can be restored by targeting FCGR2A-mediated PTPN6/SHP-1 signaling. These data suggest that sustaining LAP may open novel therapeutic perspectives for patients with end-stage liver disease.     

Cellular and molecular mechanisms that mediate basal and tumour necrosis factor-alpha-induced regulation of myosin light chain kinase gene activity.

The patients with Crohn's disease (CD) have a 'leaky gut' manifested by an increase in intestinal epithelial tight junction (TJ) permeability. Tumour necrosis factor-alpha (TNF-alpha) is a proto-typical pro-inflammatory cytokine that plays a central role in intestinal inflammation of CD. An important pro-inflammatory action of TNF-alpha is to cause a functional opening of intestinal TJ barrier. Previous studies have shown that TNF-alpha increase in TJ permeability was regulated by an increase in myosin light chain kinase (MLCK) gene activity and protein expression. The major aim of this study was to elucidate the cellular and molecular mechanisms that mediate basal and TNF-alpha-induced increase in MLCK gene activity. By progressive 5' deletion, minimal MLCK promoter was localized between -313 to +118 on MLCK promoter. A p53 binding site located within minimal promoter region was identified as an essential determinant for basal promoter activity. A 4 bp start site and a 5 bp downstream promoter element were required for MLCK gene activity. TNF-alpha-induced increase in MLCK promoter activity was mediated by NF-kappaB activation. There were eight kappaB binding sites on MLCK promoter. The NF-kappaB1 site at +48 to +57 mediated TNF-alpha-induced increase in MLCK promoter activity. The NF-kappaB2 site at -325 to -316 had a repressive role on promoter activity. The opposite effects on promoter activity were due to differences in the NF-kappaB dimer type binding to the kappaB sites. p50/p65 dimer preferentially binds to the NF-kappaB1 site and up-regulates promoter activity; while p50/p50 dimer preferentially binds to the NF-kappaB2 site and down-regulates promoter activity. In conclusion, we have identified the minimal MLCK promoter region, essential molecular determinants and molecular mechanisms that mediate basal and TNF-alpha-induced modulation of MLCK promoter activity in Caco-2 intestinal epithelial cells. These studies provide novel insight into the cellular and molecular mechanisms that regulate basal and TNF-alpha-induced modulation of MLCK gene activity.     

Spontaneous hepatic fibrosis in transgenic mice overexpressing PDGF-A

Platelet derived growth factor (PDGF) plays a central role in repair mechanisms after acute and chronic tissue damage. To further evaluate the role of PDGF-A in liver fibrogenesis in vivo, we generated transgenic mice with hepatocyte-specific overexpression of PDGF-A using the CRP-gene promoter. Transgenic but not wildtype mice showed expression of PDGF-A mRNA in the liver. Hepatic PDGF-A overexpression was accompanied by a significant increase in hepatic procollagen III mRNA expression as well as TGF-beta1 expression. Liver histology showed increased deposition of extracellular matrix in transgenic but not in wildtype mice. PDGF-A-transgenic mice showed positive sinusoidal staining for alpha-SMA indicating an activation of hepatic stellate cells. Since the profibrogenic effect of PDGF-A was accompanied by increased TGF-beta1 protein concentration in the liver of transgenic mice, it can be postulated that PDGF-A upregulates expression of TGF-beta1 which is a strong activator of hepatic stellate cells. Thus, these results point towards a fibrosis induction by PDGF-A via the TGF-beta1 signalling pathway. In conclusion, expression and functional analysis of PDGF-A in the liver of transgenic mice suggest a relevant profibrogenic role of PDGF-A via TGF-beta1 induction. Counteracting PDGF-A may therefore be one of the effects of tyrosine kinase inhibitors which showed protective effects in animal models of liver fibrosis.     

IL-1β Production through the NLRP3 Inflammasome by Hepatic Macrophages Links Hepatitis C Virus Infection with Liver Inflammation and Disease

Chronic hepatitis C virus (HCV) infection is a leading cause of liver disease. Liver inflammation underlies infection-induced fibrosis, cirrhosis and liver cancer but the processes that promote hepatic inflammation by HCV are not defined. We provide a systems biology analysis with multiple lines of evidence to indicate that interleukin-1β (IL-1β) production by intrahepatic macrophages confers liver inflammation through HCV-induced inflammasome signaling. Chronic hepatitis C patients exhibited elevated levels of serum IL-1β compared to healthy controls. Immunohistochemical analysis of healthy control and chronic hepatitis C liver sections revealed that Kupffer cells, resident hepatic macrophages, are the primary cellular source of hepatic IL-1β during HCV infection. Accordingly, we found that both blood monocyte-derived primary human macrophages, and Kupffer cells recovered from normal donor liver, produce IL-1β after HCV exposure. Using the THP-1 macrophage cell-culture model, we found that HCV drives a rapid but transient caspase-1 activation to stimulate IL-1β secretion. HCV can enter macrophages through non-CD81 mediated phagocytic uptake that is independent of productive infection. Viral RNA triggers MyD88-mediated TLR7 signaling to induce IL-1β mRNA expression. HCV uptake concomitantly induces a potassium efflux that activates the NLRP3 inflammasome for IL-1β processing and secretion. RNA sequencing analysis comparing THP1 cells and chronic hepatitis C patient liver demonstrates that viral engagement of the NLRP3 inflammasome stimulates IL-1β production to drive proinflammatory cytokine, chemokine, and immune-regulatory gene expression networks linked with HCV disease severity. These studies identify intrahepatic IL-1β production as a central feature of liver inflammation during HCV infection. Thus, strategies to suppress NLRP3 or IL-1β activity could offer therapeutic actions to reduce hepatic inflammation and mitigate disease.