Cholesterol and Liver Inflammation: from Mice to Human

Transcription

1 Cholesterol and Liver Inflammation: from Mice to Human Sofie M.A. Walenbergh 1, Tim Hendrikx 1 and Ronit Shiri-Sverdlov 1 1 Introduction Nowadays, obesity is seen as one of the leading health concerns worldwide. The dramatically increasing occurrence of obesity in Western countries is accompanied with the development of the metabolic syndrome. This syndrome refers to a cluster of multiple metabolic abnormalities, the so-called deadly quartet, which increases the susceptibility for the development of atherosclerotic cardiovascular disease and has rapidly evolved as the leading cause of death in the developed world (Roger et al., 2011). The metabolic syndrome is a significant predictor of atherosclerosis; a chronic inflammatory cardiovascular disease that starts developing from a young age and slowly leads to formation of lesions (made of excess fat deposits) and narrowing of the arteries over the years. Rupture of these atherosclerotic lesions is the cause of severe cardiovascular manifestations that appear later in life, such as stroke and myocardial infarction (Weber & Noels, 2011). Apart from atherosclerosis, the metabolic syndrome is also a strong predictor for disease development in the liver, termed non-alcoholic fatty liver disease (NAFLD). NAFLD involves a cluster of liver disease pathologies starting with the simplest form, termed hepatic steatosis, whereby excessive fat is stored and accumulates inside the liver. While steatosis is a reversible liver condition, it is the presence of hepatic inflammation, or non-alcoholic steatohepatitis (NASH) that precedes and sets the stage for more advanced stages of the disease, including fibrosis, cirrhosis and hepatocellular carcinoma. By then, patients await liver transplantation as the only curative treatment option left (Kopec & Burns, 2011). 1 Department of Molecular Genetics, Faculty of Health, Medicine and Life Sciences, Maastricht University, The Netherlands

2 Altogether, the presence of inflammation in different tissues, such as the arteries during atherosclerosis and the liver during NASH, are both common features of adult and pediatric obesity. As these patients have a clearly increased mortality risk, there is a critical need for treatment options against these inflammatory obesity-related diseases. In order to develop such treatment options, the etiology of these diseases needs to be clarified first. 2 Cholesterol as a Consistent Risk Factor The disease spectrum of the metabolic syndrome is strongly associated with a disturbed lipid profile. Individuals with the metabolic syndrome often demonstrate decreased levels of high-density lipoproteins (HDL) in plasma, while levels of triglycerides (TGs) and low-density lipoprotein (LDL)-cholesterol are increased (Grundy et al., 2004). Therefore, cholesterol may play an important role in inflammatory diseases related to the metabolic syndrome. Due to mutations in the LDL receptor (LDLR) gene, the main characteristic of patients with familial hypercholesterolemia is the presence of extremely high LDLcholesterol levels in plasma. As expected, these patients demonstrate high morbidity and mortality rates due to the development of coronary heart disease early in life. Application of a lipid-lowering therapy reduced the amount of plasma LDL-cholesterol and contributed to a prolonged lifespan of these patients, suggesting that LDLcholesterol is the main lipid involved in cardiovascular disease (Raal et al., 2011). Additional human research demonstrated increased cholesterol deposition in livers of NASH patients compared to control subjects, and in line, a clear elevation of total cholesterol levels in plasma (Caballero et al., 2009; Puri et al., 2007). Despite the common association between obesity and NAFLD, non-obese patients with NAFLD had a greater intake of dietary cholesterol compared to their control group (Musso et al., 2003; Yasutake et al., 2009), strengthening the role of cholesterol during the pathogenesis of NAFLD even outside the scope of the metabolic syndrome. Data from numerous mouse experiments confirm the role of cholesterol as found in human cardiovascular disease and NAFLD and can be summarized as follows. First, similar to the genetic cause of cardiovascular disease during familial hypercholesterolemia, transgenic mouse models have been developed that lack the LDLR gene. When fed a high-fat, high-cholesterol (HFC) diet, these mice fully mimic a human-like lipoprotein profile with typical hypercholesterolemia. As a result, the LDLR knockout (LDLR / ) mouse model has been established as an excellent animal model to study the development of atherosclerosis and NASH (Bieghs, Van Gorp, Wouters, et al., 2012; Ishibashi et al., 1993; Ishibashi et al., 1994). Secondly, elimination of dietary cholesterol to LDLR / mice prevented the development of steatohepatitis compared to mice fed a cholesterolsupplemented diet, suggesting that cholesterol is the main trigger for NASH (Wouters et al., 2008). Thirdly, it was found that dietary cholesterol demonstrated an induction of genes involved in the inflammatory response, especially acute inflammation in the context of atherosclerosis in mice. These data were confirmed by biochemical measure-

3 ments of representative proteins for the acute inflammatory response (Vergnes et al., 2003). Consistently, the administration of increasing loads of dietary cholesterol to AP- OE*3-Leiden mice for 10 weeks displayed a marked systemic inflammatory response and the formation of early atherosclerotic lesions. Micro-array analysis in the livers of these mice demonstrated an upregulation of several inflammatory pathways, suggesting that high dietary cholesterol is correlated with liver inflammation (Kleemann et al., 2007). In line, increased dietary cholesterol intake also exacerbated inflammation in the livers of LDLR / mice (Subramanian et al., 2011). In conclusion, the derived observations from mouse and human research, consistently point towards nutritional cholesterol as the best documented risk factor for atherogenesis (Steinberg, 2002). Increasing evidence now demonstrates that, similar to atherosclerosis, dietary cholesterol can also be viewed as the initiating factor for the development of NASH. 3 Inflammatory Mechanisms Triggered by Cholesterol Macrophages are a certain type of immune cells that play a central role in the initiation, maintenance and resolution of inflammation within different tissues. Depending on the activation signals, tissue resident macrophages can be either classically activated (proinflammatory) or alternatively activated (anti-inflammatory) (Martinez & Gordon, 2014). During inflammation, macrophages initiate three important processes; antigen presentation, phagocytosis (= ingestion of cells, bacteria or particles), and production of several cytokines and chemokines (Bhargava & Lee, 2012; Fujiwara & Kobayashi, 2005). Since macrophages are the body s first line of defense and are therefore the most crucial immune cells during inflammation, we will in particular focus on the role of macrophages during atherosclerosis and NASH. 3.1 Foam Cell Formation As described above, LDL-cholesterol levels are elevated in the circulation during metabolic inflammation and cause injury to the endothelium and underlying smooth muscle cells. In order to protect the body against these harmful effects of excess plasma LDLcholesterol, macrophages are recruited into the vascular wall where they bind and take up LDL particles via the so-called scavenger receptors. Consequently, LDL starts to accumulate inside the macrophages leading to a foamy macrophage phenotype. While this process is initially intended to be protective, it is now shifted towards inflammation as the foamy appearance causes the macrophage to activate the inflammatory response through the secretion of pro-inflammatory mediators and matrix-degrading proteases (Moore et al., 2013). Macrophage-derived foam cells are the main characteristic of fatty streaks, one of the hallmarks of early-stage atherosclerosis in mice and humans and promote disease progression (Ross, 1999). The prolonged presence of these foamy macrophages inside the atherosclerotic plaque creates a cytotoxic environment due to endoplasmic reticulum and oxidative stress. These aberrations cause the foamy macrophages

4 to go into apoptosis, release their lipid contents and attract even more macrophages and other immune cells for phagocytic clearance, thus further propagating inflammation. These events lead to the formation and expansion of the pro-thrombotic necrotic core, hereby triggering plaque disruption and thrombosis (Moore et al., 2013). Similar to foam cell formation during atherosclerosis, LDLR / mice also displayed foamy Kupffer cells (KCs), the resident macrophage population in the liver, which correlated with hepatic inflammation (Wouters et al., 2008). These fat-laden KCs predominantly contained cholesterol and displayed a pro-inflammatory phenotype (Leroux et al., 2012). Additionally, by using agents that mimic cholesterol accumulation in macrophages, tumor necrosis factor-α production was induced in vitro (Iftakhar et al., 2009). In line, feeding the same mice a high-fat diet without cholesterol demonstrated reduced hepatic inflammation without swollen KCs (Wouters et al., 2008). Recent evidence in patients is consistent with the data obtained from cell culture and mice; whereas steatotic patients have normal KCs, in NASH patients the KCs transformed into foam cells (Ioannou et al., 2013). Inflammation triggered by cholesterol-rich foam cells, is a well-established mechanism in the field of cardiovascular disease and has been recognized as a significant parameter during atherosclerotic plaque formation. Increasing evidence now shows similar observations in mice and patients with NASH, suggesting a shared disease mechanism between NASH and atherosclerosis (Bieghs, Rensen, et al., 2012). Altogether, accumulation of LDL-cholesterol inside macrophages and the subsequent formation of foam cells is a trigger for both atherosclerosis and NASH. 3.2 Cholesterol Uptake via LDL- and Scavenger Receptors Foam cell formation has been shown to depend on the route of cholesterol uptake and therefore, a separation must be addressed between non-modified LDL and modified LDL cholesterol. Non-modified LDL is taken up by cells via receptor-mediated endocytosis, a mechanism by which cells recognize extracellular ligands by the LDLR and subsequently internalize these ligands by inward budding of the plasma membrane (Goldstein & Brown, 2009). Upon binding to the LDLR, LDL is internalized as clathrin coated vesicles that are then converted into endosomes. Here, a more acidic ph (4.5 to 5) exists, which leads to the dissociation of the ligand-receptor complex. The LDLR is recycled to the surface of the cell, and the endosomes combine with lysosomes, which contain acid hydrolases that can easily degrade all components of LDL. After hydrolysis, cholesterol is transferred into the cytoplasm via Niemann-Pick type C proteins where it can be further degraded to bile acids or secreted via cholesterol efflux transporters (Rosenbaum & Maxfield, 2011). The cholesterol derived from LDL in the lysosome was found to be responsible for the regulation of processes that are aimed at stabilizing the cholesterol content of the cell (Goldstein & Brown, 2009). First of all, transport of sterolregulated membrane-bound transcription factors, called SREBPs, to the Golgi complex is blocked. As a result, genes encoding for enzymes involved in cholesterol synthesis are suppressed (Horton et al., 2002). One of these enzymes includes 3-hydroxy- 3methylglutaryl coenzyme A reductase (HMG-CoA), the rate-limiting enzyme of cholesterol production (Brown & Goldstein, 1999). Next to that, LDL-derived cholesterol is

5 able to activate a cholesterol-esterifying enzyme, acyl CoA: cholesterol acyl-transferase (ACAT), thereby storing excess cholesterol as cholesteryl droplets in the cytoplasm (Brown et al., 1975). Most importantly, the LDLR is subject of negative feedback regulation in the presence of marked elevations of cholesterol, as cholesterol suppresses the transcription of the LDLR gene (Brown & Goldstein, 1999). Taken together, nonmodified LDL uptake via the LDLR activates several intracellular processes that are aimed at lowering the cholesterol content, i.e. by decreasing LDL internalization, by transportation of cholesterol out of the lysosome into the cytoplasm and by inhibition of cholesterol production. These intracellular mechanisms give rise to the concept that in case of LDLR-mediated cholesterol uptake, cells are protected from cholesterol accumulation and subsequent foam cell formation. Unlike the uptake of non-modified LDL, in vitro studies have shown that the uptake of oxidative modified LDL by macrophages can contribute to cholesterol accumulation and subsequent formation of foam cells (Griffin et al., 2005b; Jerome et al., 1998). These oxidized LDL (oxldl) fractions are taken up by scavenger receptors (SRs), present on macrophages. In contrast to the LDLR, SRs are not downregulated in response to an increase in cellular cholesterol content. These results suggest that SR-mediated LDL uptake is the main cause for foam cell formation (Kunjathoor et al., 2002b). The two main SRs responsible for the uptake of modified LDL by macrophages are scavenger receptor A (SR-A) and CD36 (Kunjathoor et al., 2002b). Unlike LDL and acetylated LDL (acldl), treatment with modified LDL elevated gene expression and protein levels of SR-A and CD36 in macrophages (Yimin et al., 2011; Yoshida et al., 1998). Literature describes distinct affinity for binding of oxldl between these two SRs. In contrast to CD36, SR-A binds and mediates uptake of oxldl to a lesser extent. Next to SRmediated uptake of modified LDL, but to a much lesser extent, the phagocytic uptake of aggregated LDL by macrophages is an additional proposed mechanism that contributes to foam cell formation (Khoo et al., 1988). In summary, whereas the processes involved in non-modified LDL uptake are tightly regulated and protect cells from becoming foam cells, the SR-mediated uptake of modified LDL was found to be responsible for the development of macrophage foam cell formation. Rather than non-modified cholesterol, these data suggest that modified cholesterol in particular is the substantial risk factor for foam cell formation. Since foam cells are a major source of pro-inflammatory cytokine production (Libby, 2002), modified LDL could drive inflammatory responses. 3.3 Oxidized LDL and Its Implications in Atherosclerosis and NASH The most common form of cholesterol modification described in literature, is oxidation. Minimally oxidized forms of LDL contain lipid oxidation products without extensive protein modification. These oxldl particles stay longer in the plasma and are therefore more prone for further oxidation. As modification proceeds, the highly oxldl particle now turns into a structure similar to pathogen-related epitopes and therefore will be removed from plasma through binding to SRs and uptake by macrophages (Itabe et al., 2011). Recent studies show that oxldl contributes to inflammatory processes through

6 interaction with immune cells and disturbed intracellular cholesterol trafficking. To date, an increasing amount of evidence implicates an important role for oxldl in obesity-related inflammatory disorders, such as atherosclerosis (Li & Mehta, 2005; Nishi et al., 2002) and cardiovascular disease (Fraley & Tsimikas, 2006; Holvoet et al., 2001). In this paragraph, we will evaluate current data that supports the view of oxldl as the inflammatory trigger for atherosclerosis and NASH. Oxidatively modified LDL has been implicated in the pathogenesis of atherosclerotic disease as it has been observed within atherosclerotic lesions (Yla-Herttuala et al., 1989). A mouse model for atherosclerosis lacking either CD36 or SR-A is protected from atherosclerotic development (Febbraio et al., 2000; Makinen et al., 2010; H. Suzuki et al., 1997) and demonstrates defective foam cell formation in vitro (Febbraio et al., 2000). In line, oxldl is associated with increased intima-media thickness in the carotid arteries of clinically healthy men (Hulthe & Fagerberg, 2002). Moreover, a study of patients undergoing angiography 6 months following coronary angioplasty indicated that levels of antibodies against oxidatively modified LDL may predict progression and regression of atherosclerotic lesions (Hulthe, 2004). It has been suggested that circulating oxldl may be considered a biochemical risk marker for coronary heart disease (Toshima et al., 2000) and an increase in the ratio of oxldl to LDL has been independently associated with the severity of coronary atherosclerosis in humans (Vasankari et al., 2001). Similar to atherosclerosis, oxldl was shown to be involved in the development of NASH. Recently, a novel mouse model for NASH has been developed by using a combination of oxldl and a high-fat diet. Administration of oxldl to wildtype highfat diet-fed mice displayed the entire pathology of NASH, i.e. steatosis, hepatic inflammation, fibrosis, and also lipid-laden macrophages, dyslipidemia and aggravated hepatic lipid peroxidation (Yimin et al., 2011). Like typical macrophages, KCs also express these SRs (Naito et al., 1991) and are thus capable of taking up modified cholesterol (Kunjathoor et al., 2002a). Haematopoietic deletion of SR-A and/or CD36 in hyperlipidemic mice resulted in decreased hepatic inflammation, indicating that SR-mediated uptake of modified cholesterol by KCs is the trigger for the development of steatohepatitis (Bieghs, Verheyen, et al., 2012; Bieghs et al., 2010). By specifically inactivating one of the two predominant SRs on macrophages, research has shown that contribution of each one of these two receptors to hepatic inflammation is similar. Altogether, internalization of modified lipids by SR-A and CD36 plays an important role during atherosclerosis and NASH. In line, loading bone marrow-derived macrophages of LDLR / mice with oxldl, hereby mimicking foam cell formation, showed to be more inflammatory than macrophages without oxldl loading (Bieghs, Van Gorp, Wouters, et al., 2012). Taken together, these data demonstrate the causal role of oxldl as a driver of the inflammatory response. 3.4 Disturbed Intracellular Cholesterol Trafficking From the data described above it is clear that SR-mediated macrophage uptake of ox- LDL is the leading trigger for foam cell formation and inflammation, however, the subsequent intracellular pathway has not been established. One proposed theory is a defec-

7 tive intrinsic mechanism of lipid trafficking inside macrophages, which is described as follows: Once internalized, oxldl is transported to the lysosomal compartment where it is poorly degraded or hydrolyzed and therefore accumulates in lysosomes. This is in contrast to native or acldl, which are normally degraded by lysosomal enzymes followed by relocation into the cytoplasm for further processing (Jerome et al., 2008b). Lysosomal trapping of oxldl, probably due to impaired cholesteryl ester hydrolysis or an alteration in lysosomal ph (Schmitz & Grandl, 2009), has the potential to damage and disrupt the lysosomal membrane. Since lysosomes are involved in a wide variety of biological processes, cholesterol-induced lysosomal damage can lead to inflammation by several mechanisms. Evidence showed that intracellular cholesterol crystals were formed by CD36-mediated uptake of oxldl. In turn, these crystals triggered the release of proteases and reactive oxygen species and induced lysosomal disruption, hereby activating the NLRP3 inflammasome and subsequent pro-inflammatory interleukin-1 production (Duewell et al., 2010; Hornung et al., 2008; Moore et al., 2013; Sheedy et al., 2013). A process closely connected to the inflammasome is a process called autophagy, a pathway whereby proteins are targeted to the lysosome for disposal. On the one hand evidence supports the view that autophagy controls inflammasome activation, on the other hand it has been demonstrated that components of the inflammasome itself can mediate autophagy (Byrne et al., 2013; Harris et al., 2011). Impairments in the autophagic pathway have been strongly associated with lysosomal storage disorders (Nixon, 2013). Moreover, mice deficient for macrophage-specific caspase-1 and -11, two essential inflammasome components, were protected from lysosomal cholesterol crystallization, autophagy and hepatic inflammation (Hendrikx et al., 2013). These data suggest that autophagy and the inflammasome play an important role in cholesterol-induced inflammation. Additional to inflammasome activation and autophagy, the toll-like receptor (TLR) signaling pathway has also been proposed to promote a cholesterol-induced inflammatory response. In Niemann-Pick type C1 (NPC1) cells, a model for lysosomal cholesterol accumulation, the TLR4 level was shown to be increased, particularly in the intracellular endosomal fraction. In turn, this could lead to enhanced cytokine secretion that contributes to the inflammatory phenotype present in NPC1 disease (M. Suzuki et al., 2007). Additional studies demonstrated that, depending on its extent of oxidation, oxldl is recognized by different TLRs, hereby leading to NF-κB activation as well as chemokine and cytokine production (Bae et al., 2009; Stewart et al., 2010). Macrophage-derived foam cells, as those present during atherosclerosis, predominantly contain enlarged lysosomes filled with cholesterol and cholesterol crystals, instead of cholesterol ester storage into the cytoplasm (Duewell et al., 2010; Griffin et al., 2005a). In accordance with atherosclerosis, accumulation of cholesterol and cholesterol crystals inside lysosomes of KCs were also observed in a mouse model representing NASH (Figure 1) (Bieghs, van Gorp, Walenbergh, et al., 2012; Bieghs, Verheyen, et al., 2012). In line with these data, hepatic inflammation was found to be associated with increased cholesterol storage inside lysosomes of KCs, providing evidence that lysosomal cholesterol accumulation in KCs is crucial for inflammation in the context of NASH (Bieghs, van Gorp, Walenbergh, et al., 2012; Bieghs, Verheyen, et al., 2012).

8 C so Ly al sm f at (A) yt op la sm ic fa t (B) Figure 1: Electron microscopy pictures of KCs from mice showing that lysosomal cholesterol accumulation is correlated with hepatic inflammation. Pictures of KCs were taken of LDLR / mice on an HFC diet for 3 weeks, i.e. these mice develop hepatic inflammation (A). The LDLR / mice on chow for 3 weeks do not develop hepatic inflammation (B). Altogether, mounting evidence demonstrate that NASH exhibits similar characteristics to atherosclerosis, including foam cell formation and cholesterol-filled lysosomes. Regarding the latter observation, it has been proposed that advanced stages of atherosclerosis are analogous to a modified form of lysosomal storage disorders (Jerome, 2006). Therefore, these results indicate that NASH can be considered as a modified form of a lysosomal storage disorder as well. In conclusion, unlike acldl or native LDL, oxldl is trapped inside lysosomes and is able to trigger inflammation during atherosclerosis and NASH. Remarkably, the observed lysosomal cholesterol trapping during these inflammatory diseases is similar to the pathogenesis of the so-called lysosomal storage disorders, whereby excessive amounts of cholesterol are stored inside lysosomes. One of the lysosomal storage disorders is the NPC1 disease, whereby dysfunctional NPC1 proteins are not able to transport cholesterol out of the lysosome into the cytoplasm (Frolov et al., 2013). 3.5 Intercellular Communication in the Liver during Cholesterol-induced Inflammation Besides KCs as the initial trigger for cholesterol-induced hepatic inflammation, other liver cells play an important role in mediating liver inflammation. Once KCs are activated, for example by uptake of modified cholesterol (Bieghs, Van Gorp, Wouters, et al., 2012), a wide range of inflammatory mediators and signaling molecules such as cytokines, reactive oxygen species, proteases and lipid mediators are released rapidly (Roberts et al., 2007). These mediators are important for further amplifying the inflammatory response through direct cell-cell communication either between KCs themselves

9 or between KCs and neighbouring hepatocytes (Hoebe et al., 2001), sinusoidial endothelial cells and hepatic stellate cells. KC-derived tumor necrosis factor-α and CCchemokine receptor 2 contribute to the elevated secretion of other cyto- and chemokines, facilitating activation and infiltration of neutrophils and macrophages into the liver (Miura et al., 2012; Roberts et al., 2007). In short, cholesterol-induced KC activation and concomitant inflammation enhances cytokine-driven hepatocellular signaling pathways, hereby rendering KCs to further augment inflammation through interaction with other cell types in the liver. Thus, cellular interactions between cells in the liver are crucial for hepatic inflammation. Interestingly, NPC1 might have an impact on hepatic intercellular communication. In the absence of tumor necrosis factor-α, a crucial cytokine that recruits macrophages, NPC1 mice demonstrated less foamy macrophages and hepatocyte apoptosis (Rimkunas et al., 2009). Moreover, NPC1 mice showed disrupted features of intercellular communication (Saez et al., 2013), which is known to play a role under inflammatory conditions in KCs (Eugenin et al., 2007) and hepatocytes (Gonzalez et al., 2002). On the other hand, NPC1 has been proven to be atheroprotective by contributing to intracellular cholesterol trafficking in macrophages (Zhang et al., 2008). Interestingly, besides that Cx43 protein expression, one of the main proteins involved in intercellular communication, is affected by cholesterol (Elmes et al., 2011; Zwijsen et al., 1992), it also has been shown to contribute to interleukin-1 release and inflammasome activation in macrophages (Ali et al., 2011). These data suggest that NPC1-mediated intercellular communication could also play an important role in cholesterol-induced liver inflammation. 4 Treatment Options against Cholesterol-induced Inflammation Simple lifestyle modifications, such as body weight management and appropriate nutritional counseling (with or without regular physical exercise and cognitive-behavior programs), are currently recommended to reduce risk for cardiovascular diseases and were proven to be, so far, the most effective therapy for NASH. Body weight reduction leads to a loss of adipose tissue as well as liver TGs, which further leads to improvements in peripheral and hepatic insulin sensitivity, and prevention of hepatic injury (Hammer et al., 2008; Satapathy & Sanyal, 2010). In addition to lifestyle changes, current therapies utilized weight loss drugs and possibly bariatric surgery (Hammer et al., 2008; Satapathy & Sanyal, 2010). Moreover, we will describe specific therapy options for cholesterol-induced inflammation that are currently used or are subject of further research to improve treatment options. 4.1 Lipid-lowering Therapies Given the fact that dyslipidemia, or high cholesterol levels, is a defining element of the metabolic syndrome, lipid-lowering agents are potential candidates for treatment options in the atherosclerosis field and for NASH. These lipid-lowering drugs include pol-

10 yunsaturated fatty acids (PUFAs), peroxisome proliferator-activated receptor (PPAR) agonists, statins and niacin. These drugs all possess anti-inflammatory properties and regulate metabolism (Bieghs, Van Gorp, Wouters, et al., 2012; Fu et al., 2001; Knopp, 1999; Lukasova et al., 2011; Wang et al., 2002; Zambon & Cusi, 2007). It was previously reported that PUFAs have a blood cholesterol-lowering effect and thereby reduce the risk of heart disease. Recent work reported a positive effect of PUFAs on lobular inflammation and ballooning of the liver in mice, as well as in human NASH, although the human study lacked a control group (Ishii et al., 2009; Tanaka et al., 2008). Therefore, it has been proposed that randomized controlled trials of adequate size are needed in the future to propose such PUFA treatment to NASH patients (Musso et al., 2010). Another widely used class of lipid-modifying agents are fibrates. Treatment with fibrates resulted in a substantial decrease in plasma TGs and is usually associated with a moderate decrease in LDL cholesterol and an increase in HDL cholesterol concentrations. Mechanistically, it was shown that fibrates are ligands for PPAR, thereby modifying lipid metabolism. However, the use of fibrates is still controversial. Fenofibrate administered to mice has been shown to ameliorate hepatic inflammation, while human studies demonstrated no difference in plasma liver enzymes or without changes in histological endpoints for NASH (Basaranoglu et al., 1999; Laurin et al., 1996; Shiri- Sverdlov et al., 2006). Although PPARα activation has been shown to improve metabolic syndrome parameters and liver tests in humans, it did not improve steatosis and effects on liver histology remained minimal and unclear (Fernandez-Miranda et al., 2008). Activation of the other members of the PPAR family (PPARβ/δ) should also be tested in long-term studies in humans, to confirm efficacy for the attenuation of the metabolic syndrome (Bensinger & Tontonoz, 2008; Karpe & Ehrenborg, 2009; Reilly & Lee, 2008). Besides fibrates, statins are widely known to be used as lipid-lowering drugs. Statins lower cholesterol levels by inhibiting the enzyme HMG-CoA reductase, which plays a central role in the production of cholesterol in the liver. Recent studies using statins showed significant improvements or even normalization in serum aminotransferase and lipid/cholesterol levels, reduction in liver steatosis at follow-up, as well as improvements in hepatic inflammation in patients with NASH (Ekstedt et al., 2007; Georgescu & Georgescu, 2007; Gomez-Dominguez et al., 2006; Kiyici et al., 2003). Interestingly, patients who received statins even demonstrated reduced oxldl, which could be relevant for NASH patients with increased plasma oxldl levels (Resch et al., 2006). Still, statin-treated NAFLD patients developed advanced fibrosis based on liver histology after a long-term follow-up period (Ekstedt et al., 2006; Hyogo et al., 2008). In conclusion, the beneficial effects of statins and fibrates on NASH are still debatable, due to clear limitations to monitor NASH. In atherosclerosis, current evidence suggests that statin and niacin therapy have pleiotropic effects on the vascular endothelium, plaque stability and inflammation and may benefit patients regardless of their cholesterol levels (Liao & Laufs, 2005; Ridker et al., 2008; Wu et al., 2010). It is important to note that the use of lipid-lowering drugs is controversial due to their potential hepatotoxicity in patients with underlying hepatic diseases (Ballantyne et al., 2001; Pyorala et al., 2004). Moreover, the combination of statins and fibrates may also raise the risk of myopathy and rhabdomyolysis (Jacobson & Zimmerman, 2006). The difference in bene-

11 ficial outcome after statin therapy could be explained by the fact that statins are directed at lipid lowering in general and are not directly related to oxldl. Therefore, future adequate and well-designed human intervention studies examining the effect of statins or fibrates on NAFLD/NASH should be conducted. 4.2 Anti-oxLDL Therapies As evidence was provided for the relevant role of oxldl in triggering inflammation, therapy options in which oxldl are targeted directly are promising. Oxidation structurally modifies the LDL particle, whereby the phosphorylcholine (PC) headgroups, one of the so-called oxidation-specific epitopes, can be found on the outer surface (Shaw et al., 2000). Oxidation-specific epitopes are viewed as damage associated molecular patterns (DAMPs) and therefore serve as ligands for immune recognition (Miller et al., 2011). Since these PC epitopes are also present on the capsular polysaccharide cell wall of Streptococcus pneumoniae (Briles et al., 1982), cross reactivity exists between PC epitopes from oxldl and this bacterium. Therefore, a protective effect against atherosclerosis and NASH upon active immunization with heat-inactivated S. pneumoniae in LDLR / mice was found. Immunized mice fed an HFC diet showed less foamy KCs, decreased hepatic inflammation and a reduction in the development of atherosclerosis, compared to mice without immunization (Bieghs, van Gorp, Walenbergh, et al., 2012; Binder et al., 2003). More importantly, reduced inflammation was associated with lower cholesterol oxidation and an increase of IgM autoantibody levels against oxldl in plasma (Bieghs et al., 2010). Thus, anti-oxldl antibodies of the IgM subtype are protective against atherosclerosis and steatohepatitis, supporting the view that oxldl plays an important role in the development of obesity-related inflammatory diseases. Targeting oxldl by using natural occurring antibodies could be further developed into a novel application to treat human NASH. 4.3 Intracellular Cholesterol Modulation Therapies As the involvement of cholesterol trapping inside lysosomes of macrophages was described in triggering inflammation, the usage of lysosomal cholesterol accumulation as a potential target for novel therapy options in atherosclerosis and NASH is highlighted. A possible way to elucidate the effect of lysosomal cholesterol accumulation is stimulating the transport of cholesterol from lysosomes into the cytoplasm. However, this is a challenging issue as it was previously shown that lysosomal cholesterol derived from ox- LDL is resistant to efflux (Dhaliwal & Steinbrecher, 2000; Lougheed et al., 1991; Yancey & Jerome, 2001). Moreover, it was shown that even though acldl-derived cholesteryl esters (CEs) are usually efficiently hydrolyzed and cleared, a 3-day pre-incubation of macrophages with oxldl impaired the subsequent ability of lysosomes to hydrolyze acldl-derived CEs (Jerome et al., 2008a). Besides, studies in both arteries and cells in culture suggest that the cholesterol in lysosomes is trapped and cannot be decreased simply by inhibiting further uptake of lipoproteins or by increasing efflux of extralysosomal cholesterol stores (Jerome & Lewis, 1990; Yancey & Jerome, 2001; Yancey et

12 al., 2002). Thus, to therapeutically target lysosomal cholesterol accumulations, unique new methods must be investigated. Potential mediators of intracellular cholesterol transport are liver X receptor (LXR) activating oxysterols such as 27-hydroxycholesterol (27HC). 27HC is an intermediate in bile acid synthesis and the major oxysterol present in the human circulation. In line with greatly reduced 27HC production in NPC1-deficient cells, incubation with 27HC was shown to dramatically reduce lysosomal cholesterol in NPC1 -/- fibroblasts (Frolov et al., 2003). Similarly, injecting LDLR / mice with 27HC during or after an HFC diet resulted in reduced lysosomal cholesterol accumulation in KCs and consequently reduced hepatic inflammation (Bieghs et al., 2013). Recently also 25-hydroxycholesterol (25HC), another oxysterol, was shown to correct the transport defect in NPC1 mutant cells (Ohgane et al., 2013). These data support the view that oxysterols can prevent and reduce lysosomal cholesterol accumulation in macrophages, thereby reducing the inflammatory response. Finally, it was recently demonstrated that TGs delivered to cultured macrophages as part of TG-rich particles (TRPs), dramatically reduced lysosomal CE accumulation and improves lysosomal function, possibly by decreasing lysosomal ph and restoring lysosomal CE hydrolysis (Ullery-Ricewick et al., 2009). Interestingly, it was shown that macrophages hydrolyze CE more efficiently when it is introduced into lysosomes of macrophages as a mixed CE and TG particle, compared to CE-containing particles alone (Mahlberg et al., 1990). Knowing that TRPs can be taken up by macrophage foam cells, thereby influencing the ability of foam cells to metabolize the overload of lysosomal CE, make these TRPs an interesting therapeutic option for multiple related disorders. Collectively, these findings are promising, since so far lysosomal cholesterol has been shown to be highly resistant to removal (Dhaliwal & Steinbrecher, 2000; Lougheed et al., 1991). 5 Conclusion This book chapter summarizes mouse and human studies that provide mechanisms by which (modified) cholesterol could affect inflammation. Moreover, it offers an updated overview from basic research work concerning cholesterol and liver inflammation to potential therapeutic targets. Appendix A List of Abbreviations 25HC: 25-hydroxycholesterol 27HC: 27-hydroxycholesterol ACAT: acyl CoA: cholesterol acyl-transferase acldl: acetylated LDL CE: cholesteryl ester DAMPs: damage associated molecular patterns

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