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BACKGROUND & AIMS: Previous single-cell RNA-sequencing analyses have shown that Trem2-expressing macrophages are present in the liver during obesity, non-alcoholic steatohepatitis (NASH) and cirrhosis. Herein, we aimed to functionally characterize the role of bone marrow-derived TREM2-expressing macrophage populations in NASH.METHODS: We used bulk RNA sequencing to assess the hepatic molecular response to lipid-dependent dietary intervention in mice. Spatial mapping, bone marrow transplantation in two complementary murine models and single-cell sequencing were applied to functionally characterize the role of TREM2+ macrophage populations in NASH.RESULTS: We found that the hepatic transcriptomic profile during steatohepatitis mirrors the dynamics of recruited bone marrow-derived monocytes that already acquire increased expression of Trem2 in the circulation. Increased Trem2 expression was reflected by elevated levels of systemic soluble TREM2 in mice and humans with NASH. In addition, soluble TREM2 levels were superior to traditionally used laboratory parameters for distinguishing between different fatty liver disease stages in two separate clinical cohorts. Spatial transcriptomics revealed that TREM2+ macrophages localize to sites of hepatocellular damage, inflammation and fibrosis in the steatotic liver. Finally, using multiple murine models and in vitro experiments, we demonstrate that hematopoietic Trem2 deficiency causes defective lipid handling and extracellular matrix remodeling, resulting in exacerbated steatohepatitis, cell death and fibrosis.CONCLUSIONS: Our study highlights the functional properties of bone marrow-derived TREM2+ macrophages and implies the clinical relevance of systemic soluble TREM2 levels in the context of NASH.LAY SUMMARY: Our study defines the origin and function of macrophages (a type of immune cell) that are present in the liver and express a specific protein called TREM2. We find that these cells have an important role in protecting against non-alcoholic steatohepatitis (a progressive form of fatty liver disease). We also show that the levels of soluble TREM2 in the blood could serve as a circulating marker of non-alcoholic fatty liver disease.

Purpose Neuromuscular training (NMT) is effective at reducing football injuries. The purpose of this study was to document the use of NMT to prevent anterior cruciate ligament injuries and lateral ankle sprains in adult amateur football and to identify barriers for using NMT. Methods A preseason and in-season online survey was completed by players and coaches of 164 football teams. The survey contained questions concerning injury history, type and frequency of NMT, and barriers when NMT was not used. Results A total of 2013 players (40% female) and 180 coaches (10% female) completed the preseason survey, whereas 1253 players and 140 coaches completed the in-season survey. Thirty-four percent (preseason) to 21% (in-season) of players used NMT, but only 8% (preseason) to 5% (in-season) performed adequate NMT (i.e. both balance and plyometric exercises, at least twice per week). In the subpopulation of players with an injury history, 12% (preseason) and 7% (in-season) performed adequate NMT. With respect to the coaches, only 5% (preseason) and 2% (in-season) implemented adequate NMT. Most important barriers for using NMT for both players and coaches were a lack of belief in its effectiveness, a lack of knowledge, the belief that stretching is sufficient, and not feeling the need for it. Conclusion Most amateur football teams do not implement essential components of NMT. The results highlight the urgent need for developing strategies to enhance the adequate use of NMT in amateur football. Level of evidence II.

ObjectiveAlcohol-associated liver disease is accompanied by microbial dysbiosis, increased intestinal permeability and hepatic exposure to translocated microbial products that contribute to disease progression. A key strategy to generate immune protection against invading pathogens is the secretion of IgA in the gut. Intestinal IgA levels depend on the polymeric immunoglobulin receptor (pIgR), which transports IgA across the epithelial barrier into the intestinal lumen and hepatic canaliculi. Here, we aimed to address the function of pIgR during ethanol-induced liver disease.DesignpIgR and IgA were assessed in livers from patients with alcohol-associated hepatitis and controls. Wild-type andpIgR-deficient (pIgR-/-) littermates were subjected to the chronic-binge (NIAAA model) and Lieber-DeCarli feeding model for 8 weeks. HepaticpIgRre-expression was established inpIgR-/-mice using adeno-associated virus serotype 8 (AAV8)-mediatedpIgRexpression in hepatocytes.ResultsLivers of patients with alcohol-associated hepatitis demonstrated an increased colocalisation of pIgR and IgA within canaliculi and apical poles of hepatocytes.pIgR-deficient mice developed increased liver injury, steatosis and inflammation after ethanol feeding compared with wild-type littermates. Furthermore, mice lackingpIgRdemonstrated increased plasma lipopolysaccharide levels and more hepatic bacteria, indicating elevated bacterial translocation. Treatment with non-absorbable antibiotics prevented ethanol-induced liver disease inpIgR-/-mice. Injection of AAV8 expressingpIgRintopIgR-/-mice prior to ethanol feeding increased intestinal IgA levels and ameliorated ethanol-induced steatohepatitis compared withpIgR-/-mice injected with control-AAV8 by reducing bacterial translocation.ConclusionOur results highlight that dysfunctional hepatic pIgR enhances alcohol-associated liver disease due to impaired antimicrobial defence by IgA in the gut.

The prevalence of non-alcoholic fatty liver disease (NAFLD), ranging from simple steatosis to inflammatory steatohepatitis (NASH) and cirrhosis, continues to rise, making it one of the major chronic liver diseases and indications for liver transplantation worldwide. The pathological processes underlying NAFLD not only affect the liver but are also likely to have systemic effects. In fact, growing evidence indicates that patients with NAFLD are at increased risk for developing atherosclerosis. Indeed, cardiovascular complications are the leading cause of mortality in NAFLD patients. Here, we aim to address common pathophysiological molecular pathways involved in chronic fatty liver disease and atherosclerosis. In particular, we focus on the role of oxidized lipids and the formation of oxidation-specific epitopes, which are important targets of host immunity. Acting as metabolic danger signals, they drive pro-inflammatory processes and thus contribute to disease progression. Finally, we summarize encouraging studies indicating that oxidized lipids are promising immunological targets to improve intervention strategies for NAFLD and potentially limit the risk of developing atherosclerosis.

Previous single-cell RNA-sequencing analyses have shown that Trem2-expressing macrophages are present in the liver during obesity, non-alcoholic steatohepatitis (NASH) and cirrhosis. Herein, we aimed to functionally characterize the role of bone marrow-derived TREM2-expressing macrophage populations in NASH.We used bulk RNA sequencing to assess the hepatic molecular response to lipid-dependent dietary intervention in mice. Spatial mapping, bone marrow transplantation in two complementary murine models and single-cell sequencing were applied to functionally characterize the role of TREM2We found that the hepatic transcriptomic profile during steatohepatitis mirrors the dynamics of recruited bone marrow-derived monocytes that already acquire increased expression of Trem2 in the circulation. Increased Trem2 expression was reflected by elevated levels of systemic soluble TREM2 in mice and humans with NASH. In addition, soluble TREM2 levels were superior to traditionally used laboratory parameters for distinguishing between different fatty liver disease stages in two separate clinical cohorts. Spatial transcriptomics revealed that TREM2Our study highlights the functional properties of bone marrow-derived TREM2Our study defines the origin and function of macrophages (a type of immune cell) that are present in the liver and express a specific protein called TREM2. We find that these cells have an important role in protecting against non-alcoholic steatohepatitis (a progressive form of fatty liver disease). We also show that the levels of soluble TREM2 in the blood could serve as a circulating marker of non-alcoholic fatty liver disease.

As a mediator between lipid metabolism dysfunction, oxidative stress and inflammation, oxidized low-density lipoprotein (oxLDL) is a promising therapeutical target in a wide range of metabolic diseases. In mice, pneumococcal immunization increases anti-phosphorylcholine and oxLDL antibody levels, and reduces atherosclerosis, non-alcoholic steatohepatitis and Niemann&ndash, Pick disease burden. These findings suggest that pneumococcal vaccination may be a useful preventive and therapeutical strategy in metabolic disease patients. In this pilot clinical trial, our aim was to determine whether the administration of a pneumococcal vaccine increases anti-phosphorylcholine and anti-oxLDL antibody levels in metabolic disease patients. The following patients were enrolled: four patients with familial partial lipodystrophy (all women, mean age 32 years old), three familial hypercholesterolemia patients (one girl, two boys, mean age 13 years), and two Niemann&ndash, Pick type B (NP-B) patients (two men, mean age 37.5 years old). Participants received one active dose of a 13-valent conjugated pneumococcal vaccine (Prevenar 13) and were followed-up for four weeks. Four weeks after Prevenar 13 vaccination, no differences were observed in patients&rsquo, levels of anti-oxLDL IgM or IgG antibodies. In addition, we observed a reduction in anti-phosphorylcholine (anti-PC) IgM antibody levels, whereas no differences were observed in anti-PC IgG antibody titers. These findings indicate that Prevenar 13 vaccination does not induce an immune response against oxLDL in patients with metabolic diseases. Therefore, Prevenar 13 is not suited to target the metabolic disruptor and pro-inflammatory mediator oxLDL in patients.

Alterations of gut microbes play a role in the pathogenesis and progression of many disorders including liver and gastrointestinal diseases. Both qualitative and quantitative changes in gut microbiota have been associated with liver disease. Intestinal dysbiosis can disrupt the integrity of the intestinal barrier leading to pathological bacterial translocation and the initiation of an inflammatory response in the liver. In order to sustain symbiosis and protect from pathological bacterial translocation, antimicrobial proteins (AMPs) such as a-defensins and C-type lectins are expressed in the gastrointestinal tract. In this review, we provide an overview of the role of AMPs in different chronic liver disease such as alcoholic steatohepatitis, non-alcoholic fatty liver disease, and cirrhosis. In addition, potential approaches to modulate the function of AMPs and prevent bacterial translocation are discussed.

Objectives To investigate the utility of ultrasonic (US) perfluorohexane (PFH)-loaded monocyte imaging for detection of liver inflammation in fatty liver disease. Methods C57Bl6 mice were injected intraperitoneally with tumor necrosis factor α and assessed by US PFH-loaded monocyte imaging 3 hours later. Echogenic monocytes were injected intravenously, leading to a transient increase in liver tissue intensity on a US perfusion scan. The contrast wash-out time constant was hypothesized to reflect the degree of inflammation. Next, we evaluated US PFH-loaded monocyte imaging in Ldlr−/− mice fed a 1-week high-fat/high-cholesterol diet as model for early developing nonalcoholic steatohepatitis. Adjunct analyses included tissue markers of liver inflammation. Results Tumor necrosis factor α–injected mice showed a reduced wash-out time constant (mean ± SEM, 0.013 ± 0.003; n = 8) compared to controls (0.054 ± 0.009; n = 7; P = .0006), indicative of increased inflammatory adhesion molecule expression on the endothelium. The Ldlr−/− mice fed the high-fat/high-cholesterol diet showed liver inflammation, as reflected by increased (3- to 4-fold) infiltration of inflammatory cells and increased (3- to 4-fold) gene expression of tumor necrosis factor α, integrin αM, intracellular adhesion molecule, and vascular cell adhesion molecule. However, in these mice, no difference was detected in the wash-out time constant as assessed by US PFH-loaded monocyte imaging (high-fat/high-cholesterol, 0.050 ± 0.017; n = 5; chow, 0.048 ± 0.006; n = 6; P = .91). Conclusions Our results indicate that US PFH-loaded monocyte imaging is able to detect vascularly expressed inflammatory adhesion molecules in the mouse liver on direct endothelial stimulation. However, in our mouse model of early developing nonalcoholic steatohepatitis, we did not detect inflammation by this method, which may suggest that the time-dependent relationship between parenchymal and endothelial inflammation remains a fundamental issue to be addressed.

ObjectiveAntimicrobial C-type lectin regenerating islet-derived 3 gamma (REG3G) is suppressed in the small intestine during chronic ethanol feeding. Our aim was to determine the mechanism that underlies REG3G suppression during experimental alcoholic liver disease.DesignInterleukin 22 (IL-22) regulates expression of REG3G. Therefore, we investigated the role of IL-22 in mice subjected to chronic-binge ethanol feeding (NIAAA model).ResultsIn a mouse model of alcoholic liver disease, we found that type 3 innate lymphoid cells produce lower levels of IL-22. Reduced IL-22 production was the result of ethanol-induced dysbiosis and lower intestinal levels of indole-3-acetic acid (IAA), a microbiota-derived ligand of the aryl hydrocarbon receptor (AHR), which regulates expression of IL-22. Importantly, faecal levels of IAA were also found to be lower in patients with alcoholic hepatitis compared with healthy controls. Supplementation to restore intestinal levels of IAA protected mice from ethanol-induced steatohepatitis by inducing intestinal expression of IL-22 and REG3G, which prevented translocation of bacteria to liver. We engineered Lactobacillus reuteri to produce IL-22 (L. reuteri/IL-22) and fed them to mice along with the ethanol diet; these mice had reduced liver damage, inflammation and bacterial translocation to the liver compared with mice fed an isogenic control strain and upregulated expression of REG3G in intestine. However, L. reuteri/IL-22 did not reduce ethanol-induced liver disease in Reg3g–/– mice.ConclusionEthanol-associated dysbiosis reduces levels of IAA and activation of the AHR to decrease expression of IL-22 in the intestine, leading to reduced expression of REG3G; this results in bacterial translocation to the liver and steatohepatitis. Bacteria engineered to produce IL-22 induce expression of REG3G to reduce ethanol-induced steatohepatitis.

Atherosclerotic cardiovascular disease causes heart attacks and strokes, which are the leading causes of mortality worldwide1. The formation of atherosclerotic plaques is initiated when low-density lipoproteins bind to heparan-sulfate proteoglycans (HSPGs)2 and become trapped in the subendothelial space of large and medium size arteries, which leads to chronic inflammation and remodelling of the artery wall2. A proliferation-inducing ligand (APRIL) is a cytokine that binds to HSPGs3, but the physiology of this interaction is largely unknown. Here we show that genetic ablation or antibody-mediated depletion of APRIL aggravates atherosclerosis in mice. Mechanistically, we demonstrate that APRIL confers atheroprotection by binding to heparan sulfate chains of heparan-sulfate proteoglycan 2 (HSPG2), which limits the retention of low-density lipoproteins, accumulation of macrophages and formation of necrotic cores. Indeed, antibody-mediated depletion of APRIL in mice expressing heparan sulfate-deficient HSPG2 had no effect on the development of atherosclerosis. Treatment with a specific anti-APRIL antibody that promotes the binding of APRIL to HSPGs reduced experimental atherosclerosis. Furthermore, the serum levels of a form of human APRIL protein that binds to HSPGs, which we termed non-canonical APRIL (nc-APRIL), are associated independently of traditional risk factors with long-term cardiovascular mortality in patients with atherosclerosis. Our data reveal properties of APRIL that have broad pathophysiological implications for vascular homeostasis. The heparan sulfate proteoglycan-binding cytokine APRIL has a protective role against atherosclerotic disease.

Alterations in the bacteria that reside in our gastrointestinal tract play a role in the pathogenesis and progression of many disorders including liver and gastrointestinal diseases. Both qualitative (composition) and quantitative (amount) changes in gut microbes are associated with increased susceptibility to liver disease. Importantly, the intestinal microbiota is involved in the regulation of many host signalling pathways via the generation of different metabolites. Hence, dysbiosis influences disease development and progression by directly affecting the host-bacteria metabolic interaction. Microbe-derived harmful metabolites can translocate to distant organs due to increased intestinal permeability as observed during dysbiosis. Contrary, certain bacterial metabolites such as tryptophan metabolites contribute to intestinal and systemic homeostasis. Here, we provide an overview of current evidence describing to what extent microbial metabolites modulate the development of chronic liver diseases such as alcoholic steatohepatitis and nonalcoholic fatty liver disease with a special emphasis on indoles.

Recently, the importance of lysosomes in the context of the metabolic syndrome has received increased attention. Increased lysosomal cholesterol storage and cholesterol crystallization inside macrophages have been linked to several metabolic diseases, such as atherosclerosis and non-alcoholic fatty liver disease (NAFLD). Two-hydroxypropyl-beta-cyclodextrin (HP-B-CD) is able to redirect lysosomal cholesterol to the cytoplasm in Niemann-Pick type C1 disease, a lysosomal storage disorder. We hypothesize that HP-B-CD ameliorates liver cholesterol and intracellular cholesterol levels inside Kupffer cells (KCs). Hyperlipidemic low-density lipoprotein receptor knockout (Ldlr(-/-)) mice were given weekly, subcutaneous injections with HP-B-CD or control PBS. In contrast to control injections, hyperlipidemic mice treated with HP-B-CD demonstrated a shift in intracellular cholesterol distribution towards cytoplasmic cholesteryl ester (CE) storage and a decrease in cholesterol crystallization inside KCs. Compared to untreated hyperlipidemic mice, the foamy KC appearance and liver cholesterol remained similar upon HP-B-CD administration, while hepatic campesterol and 7alpha-hydroxycholesterol levels were back increased. Thus, HP-B-CD could be a useful tool to improve intracellular cholesterol levels in the context of the metabolic syndrome, possibly through modulation of phyto- and oxysterols, and should be tested in the future. Additionally, these data underline the existence of a shared etiology between lysosomal storage diseases and NAFLD.

Background: Liver surgery requires mobilization of the liver resulting in injury. Mobilization is the predominant cause of hepatocyte damage during liver surgery and jeopardizes post-operative liver function. Previously, mobilization-induced liver injury was found to be associated with inflammation. So far, anti-inflammatory drugs to potentially prevent liver inflammation following liver mobilization were not tested. In this study, we aimed to establish an in vivo mouse model of mobilization-induced liver injury and to evaluate the effect of anti-inflammatory pre-treatment before liver mobilization on liver inflammation. Methods: To develop a mouse model for mobilization-induced liver injury, C57BL/6 mice (n = 8) underwent surgery during which the liver was mobilized by gentle manipulation of the lobes with cotton-wool applicators for 15 minutes. In two control groups, the liver was left alone or was subjected to laparotomy only. An additional group was added that received anti-TNF treatment (Infliximab) 2 days prior to surgery. Liver samples were obtained 2 hours after mobilization and liver inflammation was analyzed by histology and inflammatory gene expression. Results: Gentle liver mobilization resulted in acute liver inflammation as indicated by increased recruitment of inflammatory cells and elevated inflammatory gene expression compared to controls. Infliximab pre-treatment had no effect on the inflammatory response in the liver. Conclusion: Our current model provides an excellent opportunity to study the effects of pre-treatment with anti-inflammatory drugs on mobilization-induced liver inflammation. Further studies are needed to investigate whether anti-inflammatory mediators can be used to prevent liver inflammation.

Aim: Nonalcoholic steatohepatitis (NASH) is a liver disease characterized by lipid accumulation and inflammation. Here, we aimed to evaluate plasma IL-1Ra as a marker for NASH and to determine whether diagnosis of NASH can be further improved by adding IL-1Ra measurements. Materials & methods: Therefore, plasma concentrations of IL-1Ra were measured from 146 subjects of a biopsy-proven NASH cohort with matched controls. Results: NASH patients had higher levels of plasma IL-1Ra compared with patients with steatosis or healthy controls. Conclusion: Our data confirm that IL-1Ra can be a useful tool in the diagnosis of hepatic inflammation and suggest that measuring plasma IL-1Ra levels in addition to ALT will improve the diagnosis for NASH at all stages of the disease.

Despite the consistent rise of non-alcoholic steatohepatitis (NASH) worldwide, the mechanisms that govern the inflammatory aspect of this disease remain unknown. Previous research showed an association between hepatic inflammation and lysosomal lipid accumulation in blood-derived hepatic macrophages. Additionally, in vitro findings indicated that lipids, specifically derived from the oxidized low-density lipoprotein (oxLDL) particle, are resistant to removal from lysosomes. On this basis, we investigated whether lysosomal lipid accumulation in blood-derived hepatic macrophages is causally linked to hepatic inflammation and assessed to what extent increasing anti-oxLDL IgM autoantibodies can affect this mechanism. By creating a proof-of-concept mouse model, we demonstrate a causal role for lysosomal lipids in blood-derived hepatic macrophages in mediating hepatic inflammation and initiation of fibrosis. Furthermore, our findings show that increasing anti-oxLDL IgM autoantibody levels reduces inflammation. Hence, therapies aimed at improving lipid-induced lysosomal dysfunction and blocking oxLDL-formation deserve further investigation in the context of NASH.

Due to the obesity epidemic, non-alcoholic steatohepatitis (NASH) is a prevalent liver disease, characterized by fat accumulation and inflammation of the liver. However, due to a lack of mechanistic insight, diagnostic and therapeutic options for NASH are poor. Recent evidence has indicated cathepsin D (CTSD), a lysosomal enzyme, as a marker for NASH. Here, we investigated the function of CTSD in NASH by using an in vivo and in vitro model. In addition to diminished hepatic inflammation, inhibition of CTSD activity dramatically improved lipid metabolism, as demonstrated by decreased plasma and liver levels of both cholesterol and triglycerides. Mechanistically, CTSD inhibition resulted in an increased conversion of cholesterol into bile acids and an elevated excretion of bile acids via the feces, indicating that CTSD influences lipid metabolism. Consistent with these findings, treating Wt BMDMs with PepA in vitro showed a similar decrease in inflammation and an analogous effect on cholesterol metabolism. Conclusion: CTSD is a key player in the development of hepatic inflammation and dyslipidemia. Therefore, aiming at the inhibition of the activity of CTSD may lead to novel treatments to combat NASH.

Diet-related health issues such as nonalcoholic fatty liver disease and cardiovascular disorders are known to have a major inflammatory component. However, the exact pathways linking diet-induced changes (e.g., hyperlipidemia) and the ensuing inflammation have remained elusive so far. We identified biological processes related to innate immunity and oxidative stress as prime response pathways in livers of low-density lipoprotein receptor-deficient mice on a Western-type diet using RNA sequencing and in silico functional analyses of transcriptome data. The observed changes were independent of the presence of microbiota and thus indicative of a role for sterile triggers. We further show that malondialdehyde (MDA) epitopes, products of lipid peroxidation and markers for enhanced oxidative stress, are detectable in hepatic inflammation predominantly on dying cells and stimulate cytokine secretion as well as leukocyte recruitment in vitro and in vivo. MDA-induced cytokine secretion in vitro was dependent on the presence of the scavenger receptors CD36 and MSR1. Moreover, in vivo neutralization of endogenously generated MDA epitopes by intravenous injection of a specific MDA antibody results in decreased hepatic inflammation in low-density lipoprotein receptor-deficient mice on a Western-type diet. Conclusion Accumulation of MDA epitopes plays a major role during diet-induced hepatic inflammation and can be ameliorated by administration of an anti-MDA antibody. (Hepatology 2017;65:1181-1195).

Many studies show an association between the accumulation of cholesterol inside lysosomes and the progression towards inflammatory disease states that are closely related to obesity. While in the past, the knowledge regarding lysosomal cholesterol accumulation was limited to its association with plaque severity during atherosclerosis, recently, a growing body of evidence indicates a causal link between lysosomal cholesterol accumulation and inflammation. These findings make lysosomal cholesterol accumulation an important target for intervention in metabolic diseases that are characterized by the presence of an inflammatory response. In this review, we aim to show the importance of cholesterol trapping inside lysosomes to the development of inflammation by focusing upon cardiovascular disease and non-alcoholic steatohepatitis (NASH) in particular. We summarize current data supporting the hypothesis that lysosomal cholesterol accumulation plays a key role in the development of inflammation during atherosclerosis and NASH. In addition, potential mechanisms by which disturbed lysosomal function can trigger the inflammatory response, the challenges in improving cholesterol trafficking in macrophages and recent successful research directions will be discussed.

Background & Aims: Non-alcoholic steatohepatitis (NASH) is characterized by steatosis and inflammation. The transition from steatosis towards NASH represents a key step in pathogenesis, as it will set the stage for further severe liver damage. Under normal conditions, lipoproteins that are endocytosed by Kupffer cells (KCs) are easily transferred from the lysosomes into the cytoplasm. Oxidized LDL (oxLDL) that is taken up by the macrophages in vitro is trapped within the lysosomes, while acetylated LDL (acLDL) is leading to normal lysosomal hydrolysis, resulting in cytoplasmic storage. We have recently demonstrated that hepatic inflammation is correlated with lysosomal trapping of lipids. So far, a link between lysosomal trapping of oxLDL and inflammation was not established. We hypothesized that lysosomal trapping of oxLDL in KCs will lead to hepatic inflammation. Methods: Ldlr(-/-) mice were injected with LDL, acLDL and oxLDL and sacrificed after 2, 6 and 24h. Results: Electron microscopy of KCs demonstrated that after oxLDL injection, small lipid inclusions were present inside the lysosomes after all time points and were mostly pronounced after 6 and 24h. In contrast, no lipid inclusions were present inside KCs after LDL or acLDL injection. Hepatic expression of several inflammatory genes and scavenger receptors was higher after oxLDL injections compared with LDL or acLDL. Conclusions: These data suggest that trapping of oxLDL inside lysosomes of KCs in vivo is causally linked to increased hepatic inflammatory gene expression. Our novel observations provide new bases for prevention and treatment of NASH.

Background & Aims Non-alcoholic steatohepatitis is characterized by hepatic steatosis with inflammation. Although steatosis is benign and reversible, inflammation can increase liver damage. Hepatic inflammation has been associated with accumulation of cholesterol in lysosomes of Kupffer cells. 27-Hydroxycholesterol (27HC), a derivative of cholesterol formed by CYP27A1, can mobilize cholesterol from the lysosomes to the cytoplasm. We investigated whether 27HC can change the intracellular distribution cholesterol and reduce hepatic inflammation in mice. Methods We transplanted bone marrow from irradiated wild-type or Cyp27a1 −/− mice to mice that do not express the low density lipoprotein receptor ( Ldlr −/− ), which are hyperlipidemic; 9 weeks later, mice were fed either regular chow or a high-fat, high-cholesterol (HFC) diet for 3 months. In a separate experiment, Ldlr −/− mice were given subcutaneous injections of 27HC and placed on regular chow or HFC diets for 3 weeks. Blood and liver tissues samples were collected and analyzed for intracellular cholesterol distribution and inflammation. Results In Ldlr −/− mice that received bone marrow transplants from Cyp27a1 −/− mice, lysosomes of Kupfer cells had a greater accumulation of cholesterol than those of mice that received bone marrow from wild-type mice, after the HFC diet. Liver histology and gene expression analyses showed increased inflammation and liver damage in mice given bone marrow transplants from Cyp27a1 −/− mice and placed on the HFC diet. Administration of 27HC to Ldlr −/− mice, following the HFC diet, reduced the accumulation of lysosomal cholesterol and hepatic inflammation, compared with mice that were not given 27HC. Conclusions Accumulation of cholesterol in lysosomes of Kupfer cells promotes hepatic inflammation in mice. The cholesterol derivative 27HC reduces accumulation of cholesterol in lysosomes and might be used to treat non-alcoholic steatohepatitis.

Non-alcoholic steatohepatitis (NASH) is characterized by liver lipid accumulation and inflammation. The mechanisms that trigger hepatic inflammation are poorly understood and subsequently, no specific non-invasive markers exist. We previously demonstrated a reduction in the plasma lysosomal enzyme, cathepsin D (CatD), in children with NASH compared to children without NASH. Recent studies have raised the concept that non-alcoholic fatty liver disease (NAFLD) in adults is distinct from children due to a different histological pattern in the liver. Yet, the link between plasma CatD to adult NASH was not examined. In the current manuscript, we investigated whether plasma CatD in adults correlates with NASH development and regression. Biopsies were histologically evaluated for inflammation and NAFLD in three complementary cohorts of adults (total n = 248). CatD and alanine aminotransferase (ALT) were measured in plasma. Opposite to our previous observations with childhood NASH, we observed increased levels of plasma CatD in patients with NASH compared to adults without hepatic inflammation. Furthermore, after surgical intervention, we found a reduction of plasma CatD compared to baseline. Our observations highlight a distinct pathophysiology between NASH in children and adults. The observation that plasma CatD correlated with NASH development and regression is promising for NASH diagnosis.

Non-alcoholic steatohepatitis (NASH) is characterized by liver steatosis and inflammation. Currently, the underlying mechanisms leading to hepatic inflammation are not fully understood and consequently, therapeutic options are poor. Non-alcoholic steatohepatitis (NASH) and atherosclerosis share the same etiology whereby macrophages play a key role in disease progression. Macrophage function can be modulated via activation of receptor-ligand binding of Notch signaling. Relevantly, global inhibition of Notch ligand Delta-Like Ligand-4 (DLL4) attenuates atherosclerosis by altering the macrophage-mediated inflammatory response. However, the specific contribution of macrophage DLL4 to hepatic inflammation is currently unknown. We hypothesized that myeloid DLL4 deficiency in low-density lipoprotein receptor knock-out (LdIr(-/-) mice reduces hepatic inflammation. Irradiated LdIr(-/-) mice were transplanted (tp) with bone marrow from wild type (Wt) or DLL4(f/f)LysMCre(+0) (DLL4(del)) mice and fed either chow or high fat, high cholesterol (HFC) diet for 11 weeks. Additionally, gene expression was assessed in bone marrow-derived macrophages (BMDM) of DLL4(f/f)LysM-Cre(WT) and DLL(f/f)LysMCre(+/0) mice. In contrast to our hypothesis, inflammation was not decreased in HFC-fed DLL4(del)-transplanted mice. In line, in vitro, there was no difference in the expression of inflammatory genes between DLL4-deficient and wildtype bone marrow derived macrophages. These results suggest that myeloid DLL4 deficiency does not contribute to hepatic inflammation in vivo. Since, macrophage-DLL4 expression in our model was not completely suppressed, it can't be totally excluded that complete DLL4 deletion in macrophages might lead to different results. Nevertheless, the contribution of non-myeloid Kupffer cells to notch signaling with regard to the pathogenesis of steatohepatitis is unknown and as such it is possible that, DLL4 on Kupffer cells promote the pathogenesis of steatohepatitis.

The symbiotic gut microbiota play pivotal roles in host physiology and the development of cardiovascular diseases, but the microbiota-triggered pattern recognition signaling mechanisms that impact thrombosis are poorly defined. In this article, we show that germ-free (GF) and Toll-like receptor-2 (Tlr2)-deficient mice have reduced thrombus growth after carotid artery injury relative to conventionally raised controls. GF Tlr2-/- and wild-type (WT) mice were indistinguishable, but colonization with microbiota restored a significant difference in thrombus growth between the genotypes. We identify reduced plasma levels of von Willebrand factor (VWF) and reduced VWF synthesis, specifically in hepatic endothelial cells, as a critical factor that is regulated by gut microbiota and determines thrombus growth in Tlr2-/- mice. Static platelet aggregate formation on extracellular matrix was similarly reduced in GF WT, Tlr2-/-, and heterozygous Vwf-/- mice that are all characterized by a modest reduction in plasma VWF levels. Defective platelet matrix interaction can be restored by exposure to WT plasma or to purified VWF depending on the VWF integrin binding site. Moreover, administration of VWF rescues defective thrombus growth in Tlr2-/- mice in vivo. These experiments delineate an unexpected pathway in which microbiota-triggered TLR2 signaling alters the synthesis of proadhesive VWF by the liver endothelium and favors platelet integrin–dependent thrombus growth. © 2017 by The American Society of Hematology.

To investigate the utility of ultrasonic (US) perfluorohexane (PFH)-loaded monocyte imaging for detection of liver inflammation in fatty liver disease.C57Bl6 mice were injected intraperitoneally with tumor necrosis factor α and assessed by US PFH-loaded monocyte imaging 3 hours later. Echogenic monocytes were injected intravenously, leading to a transient increase in liver tissue intensity on a US perfusion scan. The contrast wash-out time constant was hypothesized to reflect the degree of inflammation. Next, we evaluated US PFH-loaded monocyte imaging in LdlrTumor necrosis factor α-injected mice showed a reduced wash-out time constant (mean ± SEM, 0.013 ± 0.003; n = 8) compared to controls (0.054 ± 0.009; n = 7; P = .0006), indicative of increased inflammatory adhesion molecule expression on the endothelium. The LdlrOur results indicate that US PFH-loaded monocyte imaging is able to detect vascularly expressed inflammatory adhesion molecules in the mouse liver on direct endothelial stimulation. However, in our mouse model of early developing nonalcoholic steatohepatitis, we did not detect inflammation by this method, which may suggest that the time-dependent relationship between parenchymal and endothelial inflammation remains a fundamental issue to be addressed.

Background Lipid oxidation of membrane phospholipids is accompanied by the formation of oxidation-specific epitopes (OSE). These epitopes are recognized by specific antibodies and represent danger-associated molecular patterns that are generated during chronic inflammatory processes. In a murine model for hepatic inflammation during non-alcoholic fatty liver disease (NAFLD), increased antibody levels targeting OSE were found to be protective. Here, our aim was to determine an association between OSE-specific antibody titers and NAFLD in humans. Methods IgM and IgG levels with specificity for various OSE were assessed in the plasma of patients with NAFLD (n = 71) and healthy controls (n = 68). Antibody titers were comprehensively analyzed in patients with NAFLD after classification by histological evaluation of liver biopsies. Statistical analysis was performed to determine significant correlations and odds ratios. To study the specificity for NAFLD, plasma antibody titers were measured in patients with hepatitis C (n = 40) and inflammatory bowel disease (n = 62). Results IgM titers against OSE were lower in patients with NAFLD compared to controls. Further biopsy-based classification of patients with NAFLD did not show any difference in IgM levels. Plasma IgM titers towards the P1 mimotope demonstrated an inverse correlation with markers for obesity, systemic inflammation, and liver damage. In contrast, hepatitis C and increased disease activity during inflammatory bowel disease was not associated with reduced IgM titers. Conclusions Our data highlight the importance of immune recognition of OSE by IgM antibodies in the pathophysiology of NAFLD. Electronic supplementary material The online version of this article (doi:10.1186/s12916-016-0652-0) contains supplementary material, which is available to authorized users.

Background and aims Atherosclerosis is a chronic inflammatory disease of medium and large vessels and is typically characterized by the predominant accumulation of low-density lipoprotein (LDL)-cholesterol inside macrophages that reside in the vessel walls. Previous studies clearly demonstrated an association specifically between the oxidized type of LDL (oxLDL) and atherosclerotic lesion formation. Further observations revealed that these atherosclerotic lesions displayed enlarged, lipid-loaded lysosomes. By increasing natural antibodies against oxLDL, pneumococcal vaccination has been shown to reduce atherosclerosis in LDL receptor knockout ( Ldlr −/− ) mice. Relevantly, loss of the lysosomal membrane protein Niemann-Pick Type C1 (NPC1) led to lysosomal accumulation of various lipids and promoted atherosclerosis. Yet, the importance of lysosomal oxLDL accumulation inside macrophages, compared to non-modified LDL, in atherosclerosis has never been established. Methods By transplanting NPC1 bone marrow into lethally irradiated Ldlr −/− mice, a hematopoietic mouse model for lysosomal cholesterol accumulation was created. Through injections with heat-inactivated pneumococci, we aimed to demonstrate the specific contribution of lysosomal oxLDL accumulation inside macrophages in atherosclerosis development. Results While there were no differences in plaque morphology, a reduction in plaque size and plaque inflammation was found in immunized NPC1 mut -transplanted mice, compared to non-immunized NPC1 mut -transplanted mice. Conclusions Lysosomal oxLDL accumulation within macrophages contributes to murine atherosclerosis. Future intervention strategies should focus specifically on preventing oxLDL, unlike non-modified LDL, from being internalized into lysosomes. Such an intervention can have an additive effect to current existing treatments against atherosclerosis.

The combustion engine has a history of development for far more than 100 years. The last few decades has resulted in an increasing interest in electro mobility and can be described as a new technology era regarding road transport. Due to climate changes and the growing scarcity of energy resources, countries and cities are constantly trying to reduce CO2 emissions. Road transport is considered to be a large part of these emissions. The majority of vehicle manufacturers (such as BMW, Volkswagen and Renault) as well as new entrants such as Tesla are developing and realizing EVs with high speed. The following study recaptures the acceptance of private electro mobility in Germany. Potential users associate EVs with driving pleasure and fun. On the other hand, top speed is not considered to be a critical argument for purchasing an EV. Nowadays users place increasing importance on environmentally friendliness (which can be described as a “lifestyle" nowadays) and cost efficiency. The typical EV customer is considered to be technique affine with a modern lifestyle. These users are ready to pay a higher purchasing price for an EV, however are not yet willing to pay the current market prices which are demanded. Furthermore, the research summarizes the disadvantages of EVs which affect the consumer demand, such as the current charging infrastructure which is perceived as not fully developed yet. Additionally, individuals criticized security standards of EVs, as long term tests have not been conducted sufficiently, especially regarding the liability of both the electric engine and lithium-ion batteries. It is of great importance for EV manufacturers to work on improving the range and the purchasing cost. Concluding, the overall willingness of purchasing an EV in the future as well as to date is considered to be high. However, in the foreseeable future, the internal combustion engine will keep its importance.

Recent investigations have suggested that inflammasome activation plays an important role during atherosclerosis. Upon activation, the inflammasome induces processing and release of pro-inflammatory cytokines interleukin 1β (IL-1β) and interleukin 18 (IL-18) via activation of caspase-1/11. Previously, it was shown that complete caspase-1 deficiency is protective against atherosclerosis development. However, while macrophages are the main inflammatory cells involved in atherosclerosis, the exact role of macrophage-specific caspase-1/11 activation during development of cardiovascular disease has never been investigated. We hypothesized that hematopoietic caspase-1/11 deficiency leads to reduced atherosclerosis development. To investigate the specific contribution of hematopoietic caspase-1/11 activation to atherosclerosis development, Ldlr-/- mice received a transplant (tp) of wild-type (WT) or caspase-1/11-/- bone marrow, to create WT-tp mice and caspase-1/11-/--tp mice, and fed a high-fat, high-cholesterol diet for 12 weeks. Our results showed an increase in anti-inflammatory blood leukocytes in caspase-1/11-/--tp mice compared with WT-tp mice, as indicated by a decreased level of Ly6Chigh monocytes and an increased level of Ly6Clow monocytes. In line with our hypothesis, hematopoietic deletion of caspase-1/11 resulted in a strong reduction in atherosclerotic plaque size. Furthermore, necrotic core content was dramatically decreased in caspase-1/11-/--tp mice. Our data indicate that hematopoietic caspase-1/11 activation is involved in vascular inflammation and atherosclerosis, and plays an important role in cardiovascular disease progression. In this study, we investigated the contribution of hematopoietic caspase-1/11 to atherosclerosis development by transferring wild-type or caspase-1/11 deficient bone marrow cells into hyperlipidemic Ldlr-/- recipient mice. Hematopoietic deletion of caspase-1/11 resulted in smaller plaque size and reduced cell death in the plaque area compared to controls. These data indicate that hematopoietic caspase-1/11 activation plays an important role in vascular inflammation and atherosclerosis.

Contains fulltext : 152584.pdf (Publisher’s version ) (Closed access) The role of Kupffer cells (KCs) in the pathophysiology of the liver has been firmly established. Nevertheless, KCs have been underexplored as a target for diagnosis and treatment of liver diseases owing to the lack of noninvasive diagnostic tests. We addressed the hypothesis that cholesteryl ester transfer protein (CETP) is mainly derived from KCs and may predict KC content. Microarray analysis of liver and adipose tissue biopsies, obtained from 93 obese subjects who underwent elective bariatric surgery, showed that expression of CETP is markedly higher in liver than adipose tissue. Hepatic expression of CETP correlated strongly with that of KC markers, and CETP messenger RNA and protein colocalized specifically with KCs in human liver sections. Hepatic KC content as well as hepatic CETP expression correlated strongly with plasma CETP concentration. Mechanistic and intervention studies on the role of KCs in determining the plasma CETP concentration were performed in a transgenic (Tg) mouse model expressing human CETP. Selective elimination of KCs from the liver in CETP Tg mice virtually abolished hepatic CETP expression and largely reduced plasma CETP concentration, consequently improving the lipoprotein profile. Conversely, augmentation of KCs after Bacille-Calemette-Guerin vaccination largely increased hepatic CETP expression and plasma CETP. Also, lipid-lowering drugs fenofibrate and niacin reduced liver KC content, accompanied by reduced plasma CETP concentration. CONCLUSIONS: Plasma CETP is predominantly derived from KCs, and plasma CETP level predicts hepatic KC content in humans.(Hepatology 2015;62:1710-1722).