Johannes Kluwe

TLR4 enhances TGF-beta signaling and hepatic fibrosis.

Hepatic injury is associated with a defective intestinal barrier and increased hepatic exposure to bacterial products. Here we report that the intestinal bacterial microflora and a functional Toll-like receptor 4 (TLR4), but not TLR2, are required for hepatic fibrogenesis. Using Tlr4-chimeric mice and in vivo lipopolysaccharide (LPS) challenge, we demonstrate that quiescent hepatic stellate cells (HSCs), the main precursors for myofibroblasts in the liver, are the predominant target through which TLR4 ligands promote fibrogenesis. In quiescent HSCs, TLR4 activation not only upregulates chemokine secretion and induces chemotaxis of Kupffer cells, but also downregulates the transforming growth factor (TGF)-β pseudoreceptor Bambi to sensitize HSCs to TGF-β–induced signals and allow for unrestricted activation by Kupffer cells. LPS-induced Bambi downregulation and sensitization to TGF-β is mediated by a MyD88–NF-κB–dependent pathway. Accordingly, Myd88-deficient mice have decreased hepatic fibrosis. Thus, modulation of TGF-β signaling by a TLR4-MyD88–NF-κB axis provides a novel link between proinflammatory and profibrogenic signals.

CCR1 and CCR5 promote hepatic fibrosis in mice

Hepatic fibrosis develops as a response to chronic liver injury and almost exclusively occurs in a proinflammatory environment. However, the role of inflammatory mediators in fibrogenic responses of the liver is only poorly understood. We therefore investigated the role of CC chemokines and their receptors in hepatic fibrogenesis. The CC chemokines MIP-1alpha, MIP-1beta, and RANTES and their receptors CCR1 and CCR5 were strongly upregulated in 2 experimental mouse models of fibrogenesis. Neutralization of CC chemokines by the broad-spectrum CC chemokine inhibitor 35k efficiently reduced hepatic fibrosis, and CCR1- and CCR5-deficient mice displayed substantially reduced hepatic fibrosis and macrophage infiltration. Analysis of fibrogenesis in CCR1- and CCR5-chimeric mice revealed that CCR1 mediates its profibrogenic effects in BM-derived cells, whereas CCR5 mediates its profibrogenic effects in resident liver cells. CCR5 promoted hepatic stellate cell (HSC) migration through a redox-sensitive, PI3K-dependent pathway. Both CCR5-deficient HSCs and CCR1- and CCR5-deficient Kupffer cells displayed strong suppression of CC chemokine-induced migration. Finally, we detected marked upregulation of RANTES, CCR1, and CCR5 in patients with hepatic cirrhosis, confirming activation of the CC chemokine system in human fibrogenesis. Our data therefore support a role for the CC chemokine system in hepatic fibrogenesis and suggest distinct roles for CCR1 and CCR5 in Kupffer cells and HSCs.

Toll-like receptors as targets in chronic liver diseases

Toll-like receptors (TLRs) recognise pathogen-associated molecular patterns (PAMPs) to detect the presence of pathogens. In addition to their role in innate immunity, TLRs also play a major role in the regulation of inflammation, even under sterile conditions such as injury and wound healing. This involvement has been suggested to depend, at least in part, on the ability of TLRs to recognise several endogenous TLR ligands termed damage-associated molecular patterns (DAMPs). The liver not only represents a major target of bacterial PAMPs in many disease states but also upregulates several DAMPs following injury. Accordingly, TLR-mediated signals have been implicated in a number of chronic liver diseases. Here, we will summarise recent findings on the role TLRs and TLR ligands in the pathophysiology of liver fibrosis and cirrhosis, viral hepatitis, alcoholic liver disease, non-alcoholic fatty liver disease and hepatocellular carcinoma, and highlight the potential role of TLR agonists, antagonists and probiotics for the treatment of chronic liver disease.

Hepatic stellate cell lipid droplets: A specialized lipid droplet for retinoid storage

The majority of retinoid (vitamin A and its metabolites) present in the body of a healthy vertebrate is contained within lipid droplets present in the cytoplasm of hepatic stellate cells (HSCs). Two types of lipid droplets have been identified through histological analysis of HSCs within the liver: smaller droplets bounded by a unit membrane and larger membrane-free droplets. Dietary retinoid intake but not triglyceride intake markedly influences the number and size of HSC lipid droplets. The lipids present in rat HSC lipid droplets include retinyl ester, triglyceride, cholesteryl ester, cholesterol, phospholipids and free fatty acids. Retinyl ester and triglyceride are present at similar concentrations, and together these two classes of lipid account for approximately three-quarters of the total lipid in HSC lipid droplets. Both adipocyte-differentiation related protein and TIP47 have been identified by immunohistochemical analysis to be present in HSC lipid droplets. Lecithin:retinol acyltransferase (LRAT), an enzyme responsible for all retinyl ester synthesis within the liver, is required for HSC lipid droplet formation, since Lrat-deficient mice completely lack HSC lipid droplets. When HSCs become activated in response to hepatic injury, the lipid droplets and their retinoid contents are rapidly lost. Although loss of HSC lipid droplets is a hallmark of developing liver disease, it is not known whether this contributes to disease development or occurs simply as a consequence of disease progression. Collectively, the available information suggests that HSC lipid droplets are specialized organelles for hepatic retinoid storage and that loss of HSC lipid droplets may contribute to the development of hepatic disease.

Hepatic macrophages but not dendritic cells contribute to liver fibrosis by promoting the survival of activated hepatic stellate cells in mice.

Although it is well established that hepatic macrophages play a crucial role in the development of liver fibrosis, the underlying mechanisms remain largely elusive. Moreover, it is not known whether other mononuclear phagocytes such as dendritic cells (DCs) contribute to hepatic stellate cell (HSC) activation and liver fibrosis. We show for the first time that hepatic macrophages enhance myofibroblast survival in a nuclear factor kappa B (NF‐κB)–dependent manner and thereby promote liver fibrosis. Microarray and pathway analysis revealed no induction of HSC activation pathways by hepatic macrophages but a profound activation of the NF‐κB pathway in HSCs. Conversely, depletion of mononuclear phagocytes during fibrogenesis in vivo resulted in suppressed NF‐κB activation in HSCs. Macrophage‐induced activation of NF‐κB in HSCs in vitro and in vivo was mediated by interleukin (IL)−1 and tumor necrosis factor (TNF). Notably, IL‐1 and TNF did not promote HSC activation but promoted survival of activated HSCs in vitro and in vivo and thereby increased liver fibrosis, as demonstrated by neutralization in coculture experiments and genetic ablation of IL‐1 and TNF receptor in vivo. Coculture and in vivo ablation experiments revealed only a minor contribution to NF‐κB activation in HSCs by DCs, and no contribution of DCs to liver fibrosis development, respectively. Conclusion: Promotion of NF‐κB–dependent myofibroblast survival by macrophages but not DCs provides a novel link between inflammation and fibrosis. (Hepatology 2013;58:1461–1473)

Modulation of hepatic fibrosis by c-Jun-N-terminal kinase inhibition

BACKGROUND & AIMS c-Jun N-terminal kinase (JNK) is activated by multiple profibrogenic mediators; JNK activation occurs during toxic, metabolic, and autoimmune liver injury. However, its role in hepatic fibrogenesis is unknown. METHODS JNK phosphorylation was detected by immunoblot analysis and confocal immunofluorescent microscopy in fibrotic livers from mice after bile duct ligation (BDL) or CCl(4) administration and in liver samples from patients with chronic hepatitis C and non-alcoholic steatohepatitis. Fibrogenesis was investigated in mice given the JNK inhibitor SP600125 and in JNK1- and JNK2-deficient mice following BDL or CCl(4) administration. Hepatic stellate cell (HSC) activation was determined in primary mouse HSCs incubated with pan-JNK inhibitors SP600125 and VIII. RESULTS JNK phosphorylation was strongly increased in livers of mice following BDL or CCl(4) administration as well as in human fibrotic livers, occurring predominantly in myofibroblasts. In vitro, pan-JNK inhibitors prevented transforming growth factor (TGF) beta-, platelet-derived growth factor-, and angiotensin II-induced murine HSC activation and decreased platelet-derived growth factor and TGF-beta signaling in human HSCs. In vivo, pan-JNK inhibition did not affect liver injury but significantly reduced fibrosis after BDL or CCl(4). JNK1-deficient mice had decreased fibrosis after BDL or CCl(4), whereas JNK2-deficient mice displayed increased fibrosis after BDL but fibrosis was not changed after CCl(4). Moreover, patients with chronic hepatitis C who displayed decreased fibrosis in response to the angiotensin receptor type 1 blocker losartan showed decreased JNK phosphorylation. CONCLUSIONS JNK is involved in HSC activation and fibrogenesis and represents a potential target for antifibrotic treatment approaches.

Toll-like receptors, wound healing, and carcinogenesis.

Following acute injury, the concerted action of resident and nonresident cell populations evokes wound healing responses that entail a temporary increase in inflammation, extracellular matrix production, and proliferation to ultimately restore normal organ architecture. However, chronic injury evokes a perpetuating wound healing response promoting the development of fibrosis, organ failure, and cancer. Recent evidence points toward toll-like receptors (TLRs) as important regulators of inflammatory signals in wound healing. Here, we will review the activation of TLRs by different endogenous and bacterial TLR ligands during wound healing, and the contribution of TLR-induced signals to injury, fibrogenesis, regeneration, and carcinogenesis. We will discuss the hypothesis that TLRs act as sensors of danger signals in injured tissue to switch the wound healing response toward fibrogenesis and regeneration as a protective response to imminent danger at the cost of an increased long-term risk of developing scars and cancer.

Absence of hepatic stellate cell retinoid lipid droplets does not enhance hepatic fibrosis but decreases hepatic carcinogenesis

Objective Hepatic stellate cells (HSCs) contain a number of bioactive metabolites or their precursors including retinoids in their characteristic lipid droplets. The loss of lipid droplets and retinoids is a hallmark of HSC activation, but it remains unclear whether this loss promotes HSC activation, liver fibrogenesis or carcinogenesis. Design Spontaneous and experimental fibrogenesis as well as a diethylnitrosamine-induced hepatocarcinogenesis were investigated in lecithin-retinol acyltransferase (LRAT)-deficient mice which lack retinoid-containing lipids droplets in their HSCs. Results Following HSC activation, LRAT expression was rapidly lost, emphasising its importance in lipid droplet biology in HSCs. Surprisingly, there was no difference in fibrosis induced by bile duct ligation (BDL) or by eight injections of carbon tetrachloride (CCl4) between wild-type and LRAT-deficient mice. To exclude the possibility that the effects on fibrogenesis were missed due to the rapid downregulation of LRAT following HSC activation, acute as well as spontaneous liver fibrosis was investigated. However, there was no increased fibrosis in 3-, 8- and 12-month-old LRAT-deficient mice and in LRAT-deficient mice after a single injection of CCl4 compared with wild-type mice. To determine whether the absence of retinoids in HSCs affects hepatocarcinogenesis, wild-type and LRAT-deficient mice were injected with diethylnitrosamine. LRAT deficiency decreased diethylnitrosamine-induced injury and tumour load and increased the expression of the retinoic acid responsive genes Cyp26a1, RARb and p21, suggesting that the lower tumour load of LRAT-deficient mice was a result of increased retinoid signalling and subsequent p21-mediated inhibition of proliferation. Conclusions The absence of retinoid-containing HSC lipid droplets does not promote HSC activation but reduces hepatocarcinogenesis.

Role and Cellular Source of Nicotinamide Adenine Dinucleotide Phosphate Oxidase in Hepatic Fibrosis

Reactive oxygen species (ROS) generated by nicotinamide adenine dinucleotide phosphate oxidase (NOX) is required for liver fibrosis. This study investigates the role of NOX in ROS production and the differential contribution of NOX from bone marrow (BM)‐derived and non–BM‐derived liver cells. Hepatic fibrosis was induced by bile duct ligation (BDL) for 21 days or by methionine‐choline‐deficient (MCD) diet for 10 weeks in wild‐type (WT) mice and mice deficient in p47phox (p47phox knockout [KO]), a component of NOX. The p47phox KO chimeric mice were generated by the combination of liposomal clodronate injection, irradiation, and BM transplantation of p47phox KO BM into WT recipients and vice versa. Upon BDL, chimeric mice with p47phox KO BM‐derived cells, including Kupffer cells, and WT endogenous liver cells showed a ∼25% reduction of fibrosis, whereas chimeric mice with WT BM‐derived cells and p47phox KO endogenous liver cells, including hepatic stellate cells, showed a ∼60% reduction of fibrosis. In addition, p47phox KO compared to WT mice treated with an MCD diet showed no significant changes in steatosis and hepatocellular injury, but a ∼50% reduction in fibrosis. Cultured WT and p47phox KO hepatocytes treated with free fatty acids had a similar increase in lipid accumulation. Free fatty acids promoted a 1.5‐fold increase in ROS production both in p47phox KO and in WT hepatocytes. Conclusion: NOX in both BM‐derived and non–BM‐derived cells contributes to liver fibrosis. NOX does not play a role in experimental steatosis and the generation of ROS in hepatocytes, but exerts a key role in fibrosis. (HEPATOLOGY 2010;)

Angiotensin‐converting‐enzyme 2 inhibits liver fibrosis in mice

The renin‐angiotensin system (RAS) plays a major role in liver fibrosis. Recently, a homolog of angiotensin‐converting‐enzyme 1 (ACE1), termed ACE2, has been identified that appears to be a negative regulator of the RAS by degrading Ang II to Ang1‐7. The aim of this study was to characterize the long‐term effects of gene deletion of ACE2 in the liver, to define the role of ACE2 in acute and chronic liver disease, and to characterize the role of Ang1‐7 in hepatic stellate cell (HSC) activation. Ace2 knockout (KO) mice and wild‐type (wt) littermates underwent different models of acute and chronic liver injury. Liver pathology was analyzed by histology, immunohistochemistry, alpha smooth muscle actin (α‐SMA) immunoblotting, and quantitative polymerase chain reaction (qPCR). Murine HSCs were isolated by collagenase‐pronase‐perfusion, and density gradient centrifugation. One‐year‐old ace2 KO mice spontaneously developed an inflammatory cell infiltration and mild hepatic fibrosis that was prevented by treatment with irbesartan. Ace2 KO mice showed increased liver fibrosis following bile duct ligation for 21 days or chronic carbon tetrachloride (CCl4) treatment. In contrast, ace2 KO mice subjected to acute liver injury models did not differ from wt littermates. Treatment with recombinant ACE2 attenuated experimental fibrosis in the course of cholestatic and toxic liver injury. HSCs express the Ang1‐7 receptor Mas and Ang1‐7 inhibited Ang II‐induced phosphorylation of extracellular signal‐regulated kinase (ERK)‐1/2 in cultured HSCs. Conclusion: ACE2 is a key negative regulator of the RAS and functions to limit fibrosis through the degradation of Ang II and the formation of Ang1‐7. Whereas loss of ACE2 activity worsens liver fibrosis in chronic liver injury models, administration of recombinant ACE2 shows therapeutic potential. (HEPATOLOGY 2009.)