Sofie M. A. Walenbergh

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.

Specific immunization strategies against oxidized low-density lipoprotein: a novel way to reduce nonalcoholic steatohepatitis in mice.

Nonalcoholic steatohepatitis (NASH) is characterized by hepatic lipid accumulation combined with inflammation, which can ultimately progress into cirrhosis. Recently, we demonstrated that deletion of scavenger receptors (SRs) CD36 and SR‐A in hematopoietic cells reduced hepatic inflammation. In addition to uptake of modified lipoproteins, CD36 and SR‐A are also involved in other functions that can activate the inflammatory response. Therefore, the actual trigger for SR activation during NASH is unclear. Here, we hypothesized that hepatic inflammation is triggered by recognition of oxidized LDL (oxLDL) by Kupffer cells (KCs). To inhibit recognition of oxLDL by KCs, low‐density lipoprotein receptor (Ldlr−/−) mice were immunized with heat‐inactivated pneumococci, which were shown to induce the production of anti‐oxLDL immunoglobulin M (IgM) antibodies, due to molecular mimicry with oxLDL. The mice received a high‐fat, high‐cholesterol diet during the last 3 weeks to induce NASH. Immunization with pneumococci increased anti‐oxLDL IgM levels and led to a reduction in hepatic inflammation, as shown by reduced macrophage, neutrophil, and T cell infiltration, and reduced gene expression of tumor necrosis factor (Tnf), interleukin‐6 (Il‐6), interleukin‐1β (Il‐1b), monocyte chemoattractant protein 1 (Mcp1), and fibrosis‐related genes. In immunized mice, KCs were smaller and showed fewer cholesterol crystals compared with nonimmunized mice. Conclusion: Antibodies to oxLDL play an important role in the pathogenesis of NASH. Therefore, the potential of phosphorylcholine‐based vaccination strategies as a novel tool for the prevention and therapy of NASH should be tested in the future. (HEPATOLOGY 2012;56:894–903)

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.

Non-alcoholic steatohepatitis: the role of oxidized low-density lipoproteins.

Non-alcoholic steatohepatitis (NASH) is hallmarked by lipid accumulation in the liver (steatosis) along with inflammation (hepatitis). The transition from simple steatosis towards NASH represents a key step in pathogenesis, as it will set the stage for further severe liver damage. Yet, the pathogenesis behind hepatic inflammation is still poorly understood. It is of relevance to better understand the underlying mechanisms involved in NASH in order to apply new knowledge to potential novel therapeutic approaches. In the current review, we propose oxidized cholesterol as a novel risk factor for NASH. Here, we summarize mouse and human studies that provide possible mechanisms for the involvement of oxidized low-density lipoproteins in NASH and consequent potential novel diagnostic tools and treatment strategies for 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.

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.

Hematopoietic overexpression of Cyp27a1 reduces hepatic inflammation independently of 27-hydroxycholesterol levels in Ldlr−/− mice

BACKGROUND & AIMS Non-alcoholic steatohepatitis (NASH) is characterized by hepatic lipid accumulation and inflammation. Currently, the underlying mechanisms, leading to hepatic inflammation, are still unknown. The breakdown of free cholesterol inside Kupffer cells (KCs) by the mitochondrial enzyme CYP27A1 produces 27-hydroxycholesterol (27HC). We recently demonstrated that administration of 27HC to hyperlipidemic mice reduced hepatic inflammation. In line, hematopoietic deletion of Cyp27a1 resulted in increased hepatic inflammation. In the current manuscript, the effect of hematopoietic overexpression of Cyp27a1 on the development of NASH and cholesterol trafficking was investigated. We hypothesized that Cyp27a1 overexpression in KCs will lead to reduced hepatic inflammation. METHODS Irradiated Ldlr(-/-) mice were transplanted (tp) with bone marrow from mice overexpressing Cyp27a1 (Cyp27a1(over)) and wild type (Wt) mice and fed either chow or a high-fat, high-cholesterol (HFC) diet for 3 months. Additionally, gene expression was assessed in bone marrow-derived macrophages (BMDM) from Cyp27a1(over) and Wt mice. RESULTS In line with our hypothesis, hepatic inflammation in HFC-fed Cyp27a1(over)-tp mice was reduced and KCs were less foamy compared to Wt-tp mice. Remarkably, these changes occurred even though plasma and liver levels of 27HC did not differ between both groups. BMDM from Cyp27a1(over) mice revealed reduced inflammatory gene expression and increased expression of cholesterol transporters compared to Wt BMDM after lipopolysaccharide (LPS) stimulation. CONCLUSIONS Our data suggest that overexpression of Cyp27a1 in KCs reduces hepatic inflammation independently of 27HC levels in plasma and liver, further pointing towards KCs as specific target for improving the therapy of NASH.

Weekly Treatment of 2-Hydroxypropyl-β-cyclodextrin Improves Intracellular Cholesterol Levels in LDL Receptor Knockout Mice

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-β-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 7α-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.