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Despite the increased awareness of differences in the inflammatory response between men and women, only limited research has focused on the biological factors underlying these sex differences. The cholesterol derivative 27-hydroxycholesterol (27HC) has been shown to have opposite inflammatory effects in independent experiments using mouse models of atherosclerosis and non-alcoholic steatohepatitis (NASH), pathologies characterized by cholesterol-induced inflammation. As the sex of mice in thesein vivomodels differed, we hypothesized that 27HC exerts opposite inflammatory effects in males compared to females. To explore whether the sex-opposed inflammatory effects of 27HC translated to humans, plasma 27HC levels were measured and correlated with hepatic inflammatory parameters in obese individuals. To investigate whether 27HC exerts sex-opposed effects on inflammation, we injected 27HC into female and male Niemann-Pick disease type C1 mice (Npc1(nih)) that were used as an extreme model of cholesterol-induced inflammation. Finally, the involvement of estrogen signaling in this mechanism was studied in bone marrow-derived macrophages (BMDMs) that were treated with 27HC and 17 beta-estradiol (E2). Plasma 27HC levels showed opposite correlations with hepatic inflammatory indicators between female and male obese individuals. Likewise, hepatic 27HC levels oppositely correlated between female and maleNpc1(nih)mice. Twenty-seven hydroxycholesterol injections reduced hepatic inflammation in femaleNpc1(nih)mice in contrast to maleNpc1(nih)mice, which showed increased hepatic inflammation after 27HC injections. Furthermore, 27HC administration also oppositely affected inflammation in female and male BMDMs cultured in E2-enriched medium. Remarkably, female BMDMs showed higher ER alpha expression compared to male BMDMs. Our findings identify that the sex-opposed inflammatory effects of 27HC are E2-dependent and are potentially related to differences in ER alpha expression between females and males. Hence, the individual's sex needs to be taken into account when 27HC is employed as a therapeutic tool as well as in macrophage estrogen research in general. (c) 2020 The Authors.The Journal of Pathologypublished by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Although phytosterols, plant-derived sterol-like components, are well known for their cholesterol-lowering properties, their atherogenic potential is still under debate. Although they are known to share structural similarities with cholesterol, it is unclear whether their oxidized forms (oxyphytosterols) have the capacity to mediate proinflammatory responses in macrophages. In the present study, bone marrow-derived macrophages were treated with oxidized low-density lipoproteins, oxyphytosterols (7keto-sito/campesterol [7keto-sit/camp] or 7-beta-hydroxy-sito/campesterol [7OH-sit/camp]), nonoxidized phytosterol (-sitosterol), or carrier-control (cyclodextrin) in a dose- and time-dependent manner. Inflammatory cytokine release, activity, and the corresponding mRNA expression levels were analyzed. 7OH-sit/camp, rather than 7keto-sit/camp, induced a modest proinflammatory response in wild-type cells derived from C57Bl/6 mice. The observed mild inflammatory effects are independent of the low-density lipoprotein receptor and Cluster of differentiation 36/Scavenger receptor-a. These data suggest that exogenously added oxyphytosterols do not affect macrophage-mediated inflammatory responses, at least in vitro.

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.

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.

Scientific reports 7(1), 11670 (2017). doi:10.1038/s41598-017-10549-x, Published by Nature Publishing Group, London

Oxysterols (oxidised cholesterol) may play a role in the pathogenesis of CVD. Similar to cholesterol, plant sterols are susceptible to oxidation. However, less is known about the potential atherogenicity of oxidised plant sterols (oxyphytosterols). In the present study, the atherogenicity of a mixture of oxyphytosterols was examined by feeding female LDL receptor-deficient (LDLR+/ −) mice for 35 weeks a control diet (atherogenic high-fat diet;n9), an oxysterol diet (control diet+0·025 % (w/w) oxysterols;n12) or an oxyphytosterol diet (control diet+0·025 % (w/w) oxyphytosterols;n12). In the LDLR+/ −mice, serum levels of cholesterol, lipoprotein profiles, cholesterol exposure and inflammatory markers at the end of the experiment were comparable between the three diet groups. Nevertheless, the proportion of severe atherosclerotic lesions was significantly higher after oxysterol (41 %;P= 0·004) and oxyphytosterol (34 %;P= 0·011) diet consumption than after control diet consumption (26 %). Oxyphytosterol levels in the lesions were the highest in the oxyphytosterol group. Here, we show that not only dietary oxysterols but also dietary oxyphytosterols increase the proportion of severe atherosclerotic lesions. This suggests that plant sterols when oxidised may increase atherosclerotic lesion severity instead of lowering the size and severity of lesions when fed in their non-oxidised form. Therefore, this finding might give an indication as to where to find the answer in the current hot debate about the potential atherogenicity of plant sterols. However, to what extent these results can be extrapolated to the human situation warrants further investigation.

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.

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.

Non-alcoholic steatohepatitis (NASH) is a common liver disease characterized by hepatic lipid accumulation (steatosis) and inflammation. Currently, therapeutic options are poor and the long-term burden to society is constantly increasing. Previously, macrophage stimulating protein (MSP)-a serum protein mainly secreted by liver-was shown to inhibit oxidized low-density lipoprotein (OxLDL)/lipopolysaccharides (LPS)-induced inflammation in mouse macrophages. Additionally, MSP could reduce palmitic acid (PA)-induced lipid accumulation and lipogenesis in the HepG2 cell line. Altogether, these data suggest MSP as a suppressor for metabolic inflammation. However, so far the potential of MSP to be used as a treatment for NASH was not investigated. We hypothesized that MSP reduces lipid accumulation and hepatic inflammation. To investigate the effects of MSP in the early stage of NASH, low-density lipoprotein receptor (Ldlr(-/-)) mice were fed either a regular chow or a high fat, high cholesterol (HFC) diet for 7 days. Recombinant MSP or saline (control) was administrated to the mice by utilizing subcutaneously-implanted osmotic mini-pumps for the last 4 days. As expected, mice fed an HFC diet showed increased plasma and hepatic lipid accumulation, as well as enhanced hepatic inflammation, compared with chow-fed controls. Upon MSP administration, the rise in cholesterol and triglyceride levels after an HFC diet remained unaltered. Surprisingly, while hepatic macrophage and neutrophil infiltration was similar between the groups, MSP-treated mice showed increased gene expression of pro-inflammatory and pro-apoptotic mediators in the liver, compared with saline-treated controls. Contrary to our expectations, MSP did not ameliorate NASH. Observed changes in inflammatory gene expression suggest that further research is needed to clarify the long-term effects of MSP.

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.

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.

PLoS one 9(10), e110758 (2014). doi:10.1371/journal.pone.0110758, Published by PLoS, Lawrence, Kan.

Contains fulltext : 125609.pdf (Publisher’s version ) (Open Access) BACKGROUND & AIMS: While non-alcoholic steatohepatitis (NASH) is characterized by hepatic steatosis combined with inflammation, the mechanisms triggering hepatic inflammation are unknown. In Ldlr(-/-) mice, we have previously shown that lysosomal cholesterol accumulation in Kupffer cells (KCs) correlates with hepatic inflammation and cholesterol crystallization. Previously, cholesterol crystals have been shown to induce the activation of inflammasomes. Inflammasomes are protein complexes that induce the processing and release of pro-inflammatory cytokines IL-1b and IL-18 via caspase-1 activation. Whereas caspase-1 activation is independent of caspase-11 in the canonical pathway of inflammasome activation, caspase-11 was found to trigger caspase-1-dependent IL-1b and IL-18 in response to non-canonical inflammasome activators. So far, it has not been investigated whether inflammasome activation stimulates the formation of cholesterol crystals. We hypothesized that inflammasome activation in KCs stimulates cholesterol crystallization, thereby leading to hepatic inflammation. METHODS: Ldlr (-/-) mice were transplanted (tp) with wild-type (Wt) or caspase-1/11(-/-) (dKO) bone marrow and fed either regular chow or a high-fat, high-cholesterol (HFC) diet for 12 weeks. In vitro, bone marrow derived macrophages (BMDM) from wt or caspase-1/11(-/-) mice were incubated with oxLDL for 24h and autophagy was assessed. RESULTS: In line with our hypothesis, caspase-1/11(-/-)-tp mice had less severe hepatic inflammation than Wt-tp animals, as evident from liver histology and gene expression analysis in isolated KCs. Mechanistically, KCs from caspase-1/11(-/-)-tp mice showed less cholesterol crystals, enhanced cholesterol efflux and increased autophagy. In wt BMDM, oxLDL incubation led to disturbed autophagy activity whereas BMDM from caspase-1/11(-/-) mice had normal autophagy activity. CONCLUSION: Altogether, these data suggest a vicious cycle whereby disturbed autophagy and decreased cholesterol efflux leads to newly formed cholesterol crystals and thereby maintain hepatic inflammation during NASH by further activating the inflammasome.

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. (C) 2014 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.