Lysosomal cholesterol overload in macrophages promotes liver fibrosis in a mouse model of NASH.

Accumulation of lipotoxic lipids, such as free cholesterol, induces hepatocyte death and subsequent inflammation and fibrosis in the pathogenesis of nonalcoholic steatohepatitis (NASH). However, the underlying mechanisms remain unclear. We have previously reported that hepatocyte death locally induces phenotypic changes in the macrophages surrounding the corpse and remnant lipids, thereby promoting liver fibrosis in a murine model of NASH. Here, we demonstrated that lysosomal cholesterol overload triggers lysosomal dysfunction and profibrotic activation of macrophages during the development of NASH. ß-cyclodextrin polyrotaxane (ßCD-PRX), a unique supramolecule, is designed to elicit free cholesterol from lysosomes. Treatment with ßCD-PRX ameliorated cholesterol accumulation and profibrotic activation of macrophages surrounding dead hepatocytes with cholesterol crystals, thereby suppressing liver fibrosis in a NASH model, without affecting the hepatic cholesterol levels. In vitro experiments revealed that cholesterol-induced lysosomal stress triggered profibrotic activation in macrophages predisposed to the steatotic microenvironment. This study provides evidence that dysregulated cholesterol metabolism in macrophages would be a novel mechanism of NASH.

Dietary Supplementation With Eicosapentaenoic Acid Inhibits Plasma Cell Differentiation and Attenuates Lupus Autoimmunity.

Accumulating evidence suggests that cholesterol accumulation in leukocytes is causally associated with the development of autoimmune diseases. However, the mechanism by which fatty acid composition influences autoimmune responses remains unclear. To determine whether the fatty acid composition of diet modulates leukocyte function and the development of systemic lupus erythematosus, we examined the effect of eicosapentaenoic acid (EPA) on the pathology of lupus in drug-induced and spontaneous mouse models. We found that dietary EPA supplementation ameliorated representative lupus manifestations, including autoantibody production and immunocomplex deposition in the kidneys. A combination of lipidomic and membrane dynamics analyses revealed that EPA remodels the lipid composition and fluidity of B cell membranes, thereby preventing B cell differentiation into autoantibody-producing plasma cells. These results highlight a previously unrecognized mechanism by which fatty acid composition affects B cell differentiation into autoantibody-producing plasma cells during autoimmunity, and imply that EPA supplementation may be beneficial for therapy of lupus.

Macrophages rely on extracellular serine to suppress aberrant cytokine production.

A growing body of evidence indicates that cellular metabolism is involved in immune cell functions, including cytokine production. Serine is a nutritionally non-essential amino acid that can be generated by de novo synthesis and conversion from glycine. Serine contributes to various cellular responses, but the role in inflammatory responses remains poorly understood. Here, we show that macrophages rely on extracellular serine to suppress aberrant cytokine production. Depleting serine from the culture media reduced the cellular serine content in macrophages markedly, suggesting that macrophages depend largely on extracellular serine rather than cellular synthesis. Under serine deprivation, macrophages stimulated with lipopolysaccharide showed aberrant cytokine expression patterns, including a marked reduction of anti-inflammatory interleukin-10 expression and sustained expression of interleukine-6. Transcriptomic and metabolomics analyses revealed that serine deprivation causes mitochondrial dysfunction: reduction in the pyruvate content, the NADH/NAD+ ratio, the oxygen consumption rate, and the mitochondrial production of reactive oxygen species (ROS). We also found the role of mitochondrial ROS in appropriate cytokine production. Thus, our results indicate that cytokine production in macrophages is tightly regulated by the nutritional microenvironment.

Dipeptidyl peptidase-4 inhibition prevents nonalcoholic steatohepatitis-associated liver fibrosis and tumor development in mice independently of its anti-diabetic effects.

Nonalcoholic steatohepatitis (NASH) is a hepatic phenotype of the metabolic syndrome, and increases the risk of cirrhosis and hepatocellular carcinoma (HCC). Although increasing evidence points to the therapeutic implications of certain types of anti-diabetic agents in NASH, it remains to be elucidated whether their effects on NASH are independent of their effects on diabetes. Genetically obese melanocortin 4 receptor-deficient (MC4R-KO) mice fed Western diet are a murine model that sequentially develops hepatic steatosis, NASH, and HCC in the presence of obesity and insulin resistance. In this study, we investigated the effect of the dipeptidyl peptidase-4 (DPP-4) inhibitor anagliptin on NASH and HCC development in MC4R-KO mice. Anagliptin treatment effectively prevented inflammation, fibrosis, and carcinogenesis in the liver of MC4R-KO mice. Interestingly, anagliptin only marginally affected body weight, systemic glucose and lipid metabolism, and hepatic steatosis. Histological data and gene expression analysis suggest that anagliptin treatment targets macrophage activation in the liver during the progression from simple steatosis to NASH. As a molecular mechanism underlying anagliptin action, we showed that glucagon-like peptide-1 suppressed proinflammatory and profibrotic phenotypes of macrophages in vitro. This study highlights the glucose metabolism-independent effects of anagliptin on NASH and HCC development.

Upregulation of cancer-associated gene expression in activated fibroblasts in a mouse model of non-alcoholic steatohepatitis.

Non-alcoholic steatohepatitis (NASH), characterized by chronic inflammation and fibrosis, is predicted to be the leading cause of cirrhosis and hepatocellular carcinoma (HCC) in the next decade. Although recent evidence suggests the importance of fibrosis as the strongest determinant of HCC development, the molecular mechanisms underlying NASH-induced carcinogenesis still remain unclear. Here we performed RNA sequencing analysis to compare gene expression profiles of activated fibroblasts prepared from two distinct liver fibrosis models: carbon tetrachloride-induced fibrosis as a model without obesity and HCC and genetically obese melanocortin 4 receptor-deficient (MC4R-KO) mice fed Western diet, which develop steatosis, NASH, and eventually HCC. Our data showed that activated fibroblasts exhibited distinct gene expression patterns in each etiology, and that the 'pathways in cancer' were selectively upregulated in the activated fibroblasts from MC4R-KO mice. The most upregulated gene in these pathways was fibroblast growth factor 9 (FGF9), which was induced by metabolic stress such as palmitate. FGF9 exerted anti-apoptotic and pro-migratory effects in fibroblasts and hepatoma cells in vitro and accelerated tumor growth in a subcutaneous xenograft model. This study reveals upregulation of cancer-associated gene expression in activated fibroblasts in NASH, which would contribute to the progression from NASH to HCC.

Obeticholic acid protects against hepatocyte death and liver fibrosis in a murine model of nonalcoholic steatohepatitis.

Accumulating evidence has suggested that farnesoid X receptor (FXR) agonists, such as obeticholic acid (OCA) are therapeutically useful for non-alcoholic steatohepatitis (NASH). However, it is still unclear how FXR agonists protect against NASH and which cell type is the main target of FXR agonists. In this study, we examined the effects of OCA on the development of NASH using melanocortin 4 receptor-deficient (MC4R-KO) mice that progressively developed hepatic steatosis and NASH on Western diet (WD). Treatment with OCA effectively prevented chronic inflammation and liver fibrosis in WD-fed MC4R-KO mice with only marginal effect on body weight and hepatic steatosis. Hepatic crown-like structure (hCLS) is a unique histological structure characteristic of NASH, which triggers hepatocyte death-induced interstitial fibrosis. Intriguingly, treatment with OCA markedly reduced hCLS formation even after MC4R-KO mice developed NASH, thereby inhibiting the progression of liver fibrosis. As its mechanism of action, OCA suppressed metabolic stress-induced p53 activation and cell death in hepatocytes. Our findings in this study highlight the role of FXR in hepatocytes in the pathogenesis of NASH. Collectively, this study demonstrates the anti-fibrotic effect of OCA in a murine model of NASH with obesity and insulin resistance, which suggests the clinical implication for human NASH.

Synthetic "smart gel" provides glucose-responsive insulin delivery in diabetic mice.

Although previous studies have attempted to create "electronics-free" insulin delivery systems using glucose oxidase and sugar-binding lectins as a glucose-sensing mechanism, no successful clinical translation has hitherto been made. These protein-based materials are intolerant of long-term use and storage because of their denaturing and/or cytotoxic properties. We provide a solution by designing a protein-free and totally synthetic material-based approach. Capitalizing on the sugar-responsive properties of boronic acid, we have established a synthetic polymer gel-based insulin delivery device confined within a single catheter, which exhibits an artificial pancreas-like function in vivo. Subcutaneous implantation of the device in healthy and diabetic mice establishes a closed-loop system composed of "continuous glucose sensing" and "skin layer"-regulated insulin release. As a result, glucose metabolism was controlled in response to interstitial glucose fluctuation under both insulin-deficient and insulin-resistant conditions with at least 3-week durability. Our "smart gel" technology could offer a user-friendly and remarkably economic (disposable) alternative to the current state of the art, thereby facilitating availability of effective insulin treatment not only to diabetic patients in developing countries but also to those patients who otherwise may not be strongly motivated, such as the elderly, infants, and patients in need of nursing care.

CD11c+ resident macrophages drive hepatocyte death-triggered liver fibrosis in a murine model of nonalcoholic steatohepatitis.

Although recent evidence has pointed to the role of organ- and pathogenesis-specific macrophage subsets, it is still unclear which subsets are critically involved in the pathogenesis of nonalcoholic steatohepatitis (NASH). Using melanocortin-4 receptor-deficient (MC4R-KO) mice fed Western diet (WD), which exhibit liver phenotypes similar to those of human NASH, we found a histological structure, termed hepatic crown-like structure (hCLS), in which CD11c+ macrophages surround dead/dying hepatocytes, a prominent feature of NASH. Here, we demonstrate that hCLS-constituting macrophages could be a novel macrophage subset that drives hepatocyte death-triggered liver fibrosis. In an "inducible NASH model," hepatocyte death induces hCLS formation and liver fibrosis sequentially in the short term. In combination with the long-term WD feeding model, we also showed that resident macrophages are a major cellular source of CD11c+ macrophages constituting hCLS, which exhibited gene expression profiles distinct from CD11c- macrophages scattered in the liver. Moreover, depletion of CD11c+ macrophages abolished hCLS formation and fibrogenesis in NASH. Our clinical data suggest the role of CD11c+ macrophages in the disease progression from simple steatosis to NASH. This study sheds light on the role of resident macrophages, in addition to recruited macrophages, in the pathogenesis of NASH.

The H3K9 methyltransferase Setdb1 regulates TLR4-mediated inflammatory responses in macrophages.

Proinflammatory cytokine production in macrophages involves multiple regulatory mechanisms, which are affected by environmental and intrinsic stress. In particular, accumulating evidence has suggested epigenetic control of macrophage differentiation and function mainly in vitro. SET domain, bifurcated 1 (Setdb1, also known as Eset) is a histone 3 lysine 9 (H3K9)-specific methyltransferase and is essential for early development of embryos. Here we demonstrate that Setdb1 in macrophages potently suppresses Toll-like receptor 4 (TLR4)-mediated expression of proinflammatory cytokines including interleukin-6 through its methyltransferase activity. As a molecular mechanism, Setdb1-deficiency decreases the basal H3K9 methylation levels and augments TLR4-mediated NF-κB recruitment on the proximal promoter region of interleukin-6, thereby accelerating interleukin-6 promoter activity. Moreover, macrophage-specific Setdb1-knockout mice exhibit higher serum interleukin-6 concentrations in response to lipopolysaccharide challenge and are more susceptible to endotoxin shock than wildtype mice. This study provides evidence that the H3K9 methyltransferase Setdb1 is a novel epigenetic regulator of proinflammatory cytokine expression in macrophages in vitro and in vivo. Our data will shed insight into the better understanding of how the immune system reacts to a variety of conditions.

Macrophage-inducible C-type lectin underlies obesity-induced adipose tissue fibrosis.

In obesity, a paracrine loop between adipocytes and macrophages augments chronic inflammation of adipose tissue, thereby inducing systemic insulin resistance and ectopic lipid accumulation. Obese adipose tissue contains a unique histological structure termed crown-like structure (CLS), where adipocyte-macrophage crosstalk is known to occur in close proximity. Here we show that Macrophage-inducible C-type lectin (Mincle), a pathogen sensor for Mycobacterium tuberculosis, is localized to macrophages in CLS, the number of which correlates with the extent of interstitial fibrosis. Mincle induces obesity-induced adipose tissue fibrosis, thereby leading to steatosis and insulin resistance in liver. We further show that Mincle in macrophages is crucial for CLS formation, expression of fibrosis-related genes and myofibroblast activation. This study indicates that Mincle, when activated by an endogenous ligand released from dying adipocytes, is involved in adipose tissue remodelling, thereby suggesting that sustained interactions between adipocytes and macrophages within CLS could be a therapeutic target for obesity-induced ectopic lipid accumulation.

Activating transcription factor 4 links metabolic stress to interleukin-6 expression in macrophages.

Chronic inflammation is a molecular element of the metabolic syndrome and type 2 diabetes. Saturated fatty acids (SFAs) are considered to be an important proinflammatory factor. However, it is still incompletely understood how SFAs induce proinflammatory cytokine expression. Hereby we report that activating transcription factor (ATF) 4, a transcription factor that is induced downstream of metabolic stresses including endoplasmic reticulum (ER) stress, plays critical roles in SFA-induced interleukin-6 (Il6) expression. DNA microarray analysis using primary macrophages revealed that the ATF4 pathway is activated by SFAs. Haploinsufficiency and short hairpin RNA-based knockdown of ATF4 in macrophages markedly inhibited SFA- and metabolic stress-induced Il6 expression. Conversely, pharmacological activation of the ATF4 pathway and overexpression of ATF4 resulted in enhanced Il6 expression. Moreover, ATF4 acts in synergy with the Toll-like receptor-4 signaling pathway, which is known to be activated by SFAs. At a molecular level, we found that ATF4 exerts its proinflammatory effects through at least two different mechanisms: ATF4 is involved in SFA-induced nuclear factor-κB activation; and ATF4 directly activates the Il6 promoter. These findings provide evidence suggesting that ATF4 links metabolic stress and Il6 expression in macrophages.

Increased expression of macrophage-inducible C-type lectin in adipose tissue of obese mice and humans.

OBJECTIVE: We have provided evidence that saturated fatty acids, which are released from adipocytes via macrophage-induced adipocyte lipolysis, serve as a naturally occurring ligand for the Toll-like receptor (TLR) 4 complex in macrophages, thereby aggravating obesity-induced adipose tissue inflammation. The aim of this study was to identify the molecule(s) activated in adipose tissue macrophages in obesity. RESEARCH DESIGN AND METHODS: We performed a cDNA microarray analysis of coculture of 3T3-L1 adipocytes and RAW264 macrophages. Cultured adipocytes and macrophages and the adipose tissue of obese mice and humans were used to examine mRNA and protein expression. RESULTS: We found that macrophage-inducible C-type lectin (Mincle; also called Clec4e and Clecsf9), a type II transmembrane C-type lectin, is induced selectively in macrophages during the interaction between adipocytes and macrophages. Treatment with palmitate, a major saturated fatty acid released from 3T3-L1 adipocytes, induced Mincle mRNA expression in macrophages at least partly through the TLR4/nuclear factor (NF)-κB pathway. Mincle mRNA expression was increased in parallel with macrophage markers in the adipose tissue of obese mice and humans. The obesity-induced increase in Mincle mRNA expression was markedly attenuated in C3H/HeJ mice with defective TLR4 signaling relative to control C3H/HeN mice. Notably, Mincle mRNA was expressed in bone-marrow cell (BMC)-derived proinflammatory M1 macrophages rather than in BMC-derived anti-inflammatory M2 macrophages in vitro. CONCLUSIONS: Our data suggest that Mincle is induced in adipose tissue macrophages in obesity at least partly through the saturated fatty acid/TLR4/NF-κB pathway, thereby suggesting its pathophysiologic role in obesity-induced adipose tissue inflammation.

Activating transcription factor 3 constitutes a negative feedback mechanism that attenuates saturated Fatty acid/toll-like receptor 4 signaling and macrophage activation in obese adipose tissue.

Obese adipose tissue is markedly infiltrated by macrophages, suggesting that they may participate in the inflammatory pathways that are activated in obese adipose tissue. Evidence has suggested that saturated fatty acids released via adipocyte lipolysis serve as a naturally occurring ligand that stimulates Toll-like receptor (TLR)4 signaling, thereby inducing the inflammatory responses in macrophages in obese adipose tissue. Through a combination of cDNA microarray analyses of saturated fatty acid-stimulated macrophages in vitro and obese adipose tissue in vivo, here we identified activating transcription factor (ATF)3, a member of the ATF/cAMP response element-binding protein family of basic leucine zipper-type transcription factors, as a target gene of saturated fatty acids/TLR4 signaling in macrophages in obese adipose tissue. Importantly, ATF3, when induced by saturated fatty acids, can transcriptionally repress tumor necrosis factor-alpha production in macrophages in vitro. Chromatin immunoprecipitation assay revealed that ATF3 is recruited to the region containing the activator protein-1 site of the endogenous tumor necrosis factor-alpha promoter. Furthermore, transgenic overexpression of ATF3 specifically in macrophages results in the marked attenuation of proinflammatory M1 macrophage activation in the adipose tissue from genetically obese KKA(y) mice fed high-fat diet. This study provides evidence that ATF3, which is induced in obese adipose tissue, acts as a transcriptional repressor of saturated fatty acids/TLR4 signaling, thereby revealing the negative feedback mechanism that attenuates obesity-induced macrophage activation. Our data also suggest that activation of ATF3 in macrophages offers a novel therapeutic strategy to prevent or treat obesity-induced adipose tissue inflammation.

Fluvastatin accelerates re-endothelialization impaired by local sirolimus treatment.

Sirolimus-eluting stent reduces restenosis after percutaneous coronary intervention. However, accumulating evidence suggests that sirolimus potentially affects re-endothelialization, leading to late thrombosis. Statins have protective effects on endothelium. Recently, statins are reported to increase the number of circulating endothelial progenitor cells (EPCs) and accelerate re-endothelialization after vascular injury. Here, we tested the hypothesis that fluvastatin has beneficial effect on re-endothelialization after local sirolimus treatment. We performed wire-mediated vascular injury to both sides of femoral arteries of wild-type mice and bone marrow chimeric mice. Either sirolimus (100 microg) or DMSO was administered locally to the perivascular area of the injured arteries. All mice received either fluvastatin (5 mg/kg/day) or vehicle by gavage starting at one week before the surgery until sacrifice. At 4 weeks after the surgery, re-endothelialization of the sirolimus-treated artery was significantly less than that of DMSO-treated one in the vehicle-treated mice as determined by the percentage of CD31-positive area (P<0.05). Systemic administration of fluvastatin accelerated the re-endothelialization in the sirolimus-treated artery to the similar degree of that in the DMSO-treated artery (P=NS). Contribution of bone marrow-derived cells to re-endothelialization was seldom observed in bone marrow chimeric mice regardless of fluvastatin administration. Fluvastatin significantly ameliorated proliferation (2.5-folds) and migration activities (2.3-folds) of mature endothelial cells impaired by sirolimus treatment (P<0.05, respectively). Fluvastatin increased endothelial nitric oxide synthase expression and decreased plasminogen activator inhibitor-1 expression in mature endothelial cell in the presence of sirolimus (P<0.05, respectively). Our findings suggest that fluvastatin has protective effects against impaired re-endothelialization after sirolimus treatment.

Atorvastatin attenuates transplant-associated coronary arteriosclerosis in a murine model of cardiac transplantation.

Accelerated coronary arteriosclerosis remains a major problem for the long-term survival of cardiac transplant recipients. However, the pathogenesis of transplant vasculopathy is poorly understood and there is no effective therapy. HMG-CoA reductase inhibitors, or statins, are widely prescribed to lower plasma cholesterol level. Accumulating evidence indicates that statins have various effects on vascular cells which are independent of their lipid-lowering effect. We investigated whether orally administered atorvastatin, one of the most potent statins, inhibits the development of intima hyperplasia in a mouse model of cardiac transplantation. Cardiac allografts from DBA mice were transplanted heterotopically into B10.D2 mice. Mice were administered either vehicle or atorvastatin everyday by gavage. Morphometrical analysis revealed that atorvastatin significantly reduced the development of coronary arteriosclerosis on the cardiac allografts harvested at one month. Immunohistochemical analysis revealed that atorvastatin attenuated infiltration of inflammatory cells with reduced expression of TGF-beta and adhesion molecules. These results suggest that atorvastatin may be effective in preventing transplant-associated arteriosclerosis along with other immunosuppressive agents.

The role of circulating precursors in vascular repair and lesion formation.

The accumulation of smooth muscle cells (SMCs) plays a principal role in atherogenesis, post-angioplasty restenosis and transplantation-associated vasculopathy. Therefore, much effort has been expended in targeting the migration and proliferation of medial smooth muscle cells to prevent occlusive vascular remodeling. Recent evidence suggests that bone marrow-derived circulating precursors can also give rise to endothelial cells and smooth muscle cells that contribute to vascular repair, remodeling, and lesion formation under physiological and pathological conditions. This article overviews recent findings on circulating vascular progenitor cells and describes potential therapeutic strategies that target these cells to treat occlusive vascular diseases.

Effect of deuterium oxide on contraction characteristics and ATPase activity in glycerinated single rabbit skeletal muscle fibers.

We studied the effect of deuterium oxide (D(2)O) on contraction characteristics and ATPase activity of single glycerinated muscle fibers of rabbit psoas. D(2)O increased the maximum isometric force P(0) by about 20%, while the force versus stiffness relation did not change appreciably. The maximum shortening velocity under zero load V(max) did not change appreciably in D(2)O, so that the force-velocity (P-V) curve was scaled depending on the value of P(0). The Mg-ATPase activity of the fibers during generation of steady isometric force P(0) was reduced by about 50% in D(2)O. Based on the Huxley contraction model, these results can be accounted for in terms of D(2)O-induced changes in the rate constants f(1) and g(1) for making and breaking actin-myosin linkages in the isometric condition, in such a way that f(1)/(f(1)+g(1)) increases by about 20%, while (f(1)+g(1)) remains unchanged. The D(2)O effect at the molecular level is discussed in connection with biochemical studies on actomyosin ATPase.

Electrical and mechanical properties and mode of innervation in scorpionfish sound-producing muscle fibres.

To obtain information about the neural mechanism underlying sound production in teleost fish, we studied the electrical and mechanical properties and mode of innervation in the swimbladder muscle (SBM) fibres of scorpionfish Sebastiscus marmoratus. Action potentials of the SBM fibres in response to direct electrical stimulation neither exhibited overshoot nor propagated along the fibre. Stimulation of the motor nerve, however, uniformly evoked action potentials along the fibre. When neuromuscular transmission was blocked by curare, motor nerve stimulation uniformly evoked endplate potentials along the fibre. These results indicate that action potentials propagate along the nerve branches but not along the SBM fibre membrane. In accordance with the above results, histochemical studies showed that motor nerve branches run along the SBM fibres to form many endplates with cholinesterase activity, indicating multiterminal innervation. The SBM consisted of about 600 fibres, while its motor nerve contained about 100 axons, giving an innervation ratio of about 1:6. Like mammalian fast muscle fibres, the SBM fibres exhibited a low succinic dehydrogenase activity and a high ATPase activity. These results are discussed in connection with the function of the SBM fibres in producing sound.

Marked load-bearing ability of Mytilus smooth muscle in both active and catch states as revealed by quick increases in load.

The anterior byssal retractor muscle (ABRM) of the bivalve Mytilus edulis shows a prolonged tonic contraction, called the catch state. To investigate the catch mechanism, details of which still remain obscure, we studied the mechanical responses of ABRM fibres to quick increases in load applied during maximum active isometric force (P(0)) generation and during the catch state. The mechanical response consisted of three components: (1) initial extension of the series elastic component (SEC), (2) early isotonic fibre lengthening with decreasing velocity, and (3) late steady isotonic fibre lengthening. The ABRM fibres could bear extremely large loads up to 10-15P(0) for more than 30-60 s, while being lengthened extremely slowly. If, on the other hand, quick increases in load were applied during the early isometric force development, the ABRM fibres were lengthened rapidly ('give') under loads of 1.5-2P(0). These findings might possibly be explained by two independent systems acting in parallel with each other; one is the actomyosin system producing active shortening and active force generation, while the other is the load-bearing system responsible for the extremely marked load-bearing ability as well as the maintenance of the catch state.