Tim Hendrikx

Ldl receptor knock-out mice are a physiological model particularly vulnerable to study the onset of inflammation in non-alcoholic fatty liver disease

Background & Aims Non-alcoholic steatohepatitis (NASH) involves steatosis combined with inflammation, which can progress into fibrosis and cirrhosis. Exploring the molecular mechanisms of NASH is highly dependent on the availability of animal models. Currently, the most commonly used animal models for NASH imitate particularly late stages of human disease. Thus, there is a need for an animal model that can be used for investigating the factors that potentiate the inflammatory response within NASH. We have previously shown that 7-day high-fat-high-cholesterol (HFC) feeding induces steatosis and inflammation in both APOE2ki and Ldlr−/− mice. However, it is not known whether the early inflammatory response observed in these mice will sustain over time and lead to liver damage. We hypothesized that the inflammatory response in both models is sufficient to induce liver damage over time. Methods APOE2ki and Ldlr−/− mice were fed a chow or HFC diet for 3 months. C57Bl6/J mice were used as control. Results Surprisingly, hepatic inflammation was abolished in APOE2ki mice, while it was sustained in Ldlr−/− mice. In addition, increased apoptosis and hepatic fibrosis was only demonstrated in Ldlr−/− mice. Finally, bone-marrow-derived-macrophages of Ldlr−/− mice showed an increased inflammatory response after oxidized LDL (oxLDL) loading compared to APOE2ki mice. Conclusion Ldlr−/− mice, but not APOE2ki mice, developed sustained hepatic inflammation and liver damage upon long term HFC feeding due to increased sensitivity for oxLDL uptake. Therefore, the Ldlr−/− mice are a promising physiological model particularly vulnerable for investigating the onset of hepatic inflammation in non-alcoholic steatohepatitis.

Internalization of Modified Lipids by CD36 and SR-A Leads to Hepatic Inflammation and Lysosomal Cholesterol Storage in Kupffer Cells

Background & Aims Non-alcoholic steatohepatitis (NASH) is characterized by steatosis and inflammation, which can further progress into fibrosis and cirrhosis. Recently, we demonstrated that combined deletion of the two main scavenger receptors, CD36 and macrophage scavenger receptor 1 (MSR1), which are important for modified cholesterol-rich lipoprotein uptake, reduced NASH. The individual contributions of these receptors to NASH and the intracellular mechanisms by which they contribute to inflammation have not been established. We hypothesize that CD36 and MSR1 contribute independently to the onset of inflammation in NASH, by affecting intracellular cholesterol distribution inside Kupffer cells (KCs). Methods & Results Ldlr−/− mice were transplanted with wild-type (Wt), Cd36−/− or Msr1−/− bone marrow and fed a Western diet for 3months. Cd36−/−- and Msr1−/−- transplanted (tp) mice showed a similar reduction in hepatic inflammation compared to Wt-tp mice. While the total amount of cholesterol inside KCs was similar in all groups, KCs of Cd36−/−- and Msr1−/−-tp mice showed increased cytoplasmic cholesterol accumulation, while Wt-tp mice showed increased lysosomal cholesterol accumulation. Conclusion CD36 and MSR1 contribute similarly and independently to the progression of inflammation in NASH. One possible explanation for the inflammatory response related to expression of these receptors could be abnormal cholesterol trafficking in KCs. These data provide a new basis for prevention and treatment of NASH.

The Cholesterol Derivative 27-Hydroxycholesterol Reduces Steatohepatitis in Mice

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.

Trapping of oxidized LDL in lysosomes of Kupffer cells is a trigger for hepatic inflammation

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.

Lysosomal cholesterol accumulation: driver on the road to inflammation during atherosclerosis and non-alcoholic steatohepatitis

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.

Plasma cholesteryl ester transfer protein is predominantly derived from Kupffer cells

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‐Guérin 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)

Bone marrow-specific caspase-1/11 deficiency inhibits atherosclerosis development in Ldlr(-/-) mice.

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.

Protective Role of Plant Sterol and Stanol Esters in Liver Inflammation: Insights from Mice and Humans

The inflammatory component of non–alcoholic steatohepatitis (NASH) can lead to irreversible liver damage. Therefore there is an urgent need to identify novel interventions to combat hepatic inflammation. In mice, omitting cholesterol from the diet reduced hepatic inflammation. Considering the effects of plant sterol/stanol esters on cholesterol metabolism, we hypothesized that plant sterol/stanol esters reduces hepatic inflammation. Indeed, adding plant sterol/stanol esters to a high-fat-diet reduced hepatic inflammation as indicated by immunohistochemical stainings and gene expression for inflammatory markers. Finally, adding sterol/stanol esters lowered hepatic concentrations of cholesterol precursors lathosterol and desmosterol in mice, which were highly elevated in the HFD group similarly as observed in severely obese patients with NASH. In vitro, in isolated LPS stimulated bone marrow derived macrophages desmosterol activated cholesterol efflux whereas sitostanol reduced inflammation. This highly interesting observation that plant sterol/stanol ester consumption leads to complete inhibition of HFD-induced liver inflammation opens new venues in the treatment and prevention of hepatic inflammation.

Plasma Cathepsin D Levels: A Novel Tool to Predict Pediatric Hepatic Inflammation

OBJECTIVES:Nonalcoholic steatohepatitis (NASH) is the most severe form of a hepatic condition known as nonalcoholic fatty liver disease (NAFLD). NASH is histologically characterized by hepatic fat accumulation, inflammation, and ballooning, and eventually coupled with fibrosis that, in turn, may progress to end-stage liver disease even in young individuals. Hence, there is a critical need for specific noninvasive markers to predict hepatic inflammation at an early age. We investigated whether plasma levels of cathepsin D (CatD), a lysosomal protease, correlated with the severity of liver inflammation in pediatric NAFLD.METHODS:Liver biopsies from children (n=96) with NAFLD were histologically evaluated according to the criteria of Kleiner (NAFLD activity score) and the Brunt’s criteria. At the time of liver biopsy, blood was taken and levels of CatD, alanine aminotransferase (ALT), and cytokeratin-18 (CK-18) were measured in plasma.RESULTS:Plasma CatD levels were significantly lower in subjects with liver inflammation compared with steatotic subjects. Furthermore, we found that CatD levels were gradually reduced and corresponded with increasing severity of liver inflammation, steatosis, hepatocellular ballooning, and NAFLD activity score. CatD levels correlated with pediatric NAFLD disease progression better than ALT and CK-18. In particular, CatD showed a high diagnostic accuracy (area under receiver operating characteristic curve (ROC-AUC): 0.94) for the differentiation between steatosis and hepatic inflammation, and reached almost the maximum accuracy (ROC-AUC: 0.998) upon the addition of CK-18.CONCLUSIONS:Plasma CatD holds a high diagnostic value to distinguish pediatric patients with hepatic inflammation from children with steatosis.

Sialic Acid-Binding Immunoglobulin-like Lectin G Promotes Atherosclerosis and Liver Inflammation by Suppressing the Protective Functions of B-1 Cells

Summary Atherosclerosis is initiated and sustained by hypercholesterolemia, which results in the generation of oxidized LDL (OxLDL) and other metabolic byproducts that trigger inflammation. Specific immune responses have been shown to modulate the inflammatory response during atherogenesis. The sialic acid-binding immunoglobulin-like lectin G (Siglec-G) is a negative regulator of the functions of several immune cells, including myeloid cells and B-1 cells. Here, we show that deficiency of Siglec-G in atherosclerosis-prone mice inhibits plaque formation and diet-induced hepatic inflammation. We further demonstrate that selective deficiency of Siglec-G in B cells alone is sufficient to mediate these effects. Levels of B-1 cell-derived natural IgM with specificity for OxLDL were significantly increased in the plasma and peritoneal cavity of Siglec-G-deficient mice. Consistent with the neutralizing functions of OxLDL-specific IgM, Siglec-G-deficient mice were protected from OxLDL-induced sterile inflammation. Thus, Siglec-G promotes atherosclerosis and hepatic inflammation by suppressing protective anti-inflammatory effector functions of B cells.