Increased Serum Levels of LIGHT|[sol]|TNFSF14 in Nonalcoholic Fatty Liver Disease: Possible Role in Hepatic Inflammation

Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease, with an estimated prevalence of about 20%.1 NAFLD ranges from simple fat deposition to nonalcoholic steatohepatitis (NASH), characterized by steatosis, inflammation, and progressive fibrosis, ultimately leading to cirrhosis and end-stage liver disease.1, 2 NAFLD is frequently associated with obesity, dyslipidemia, and insulin resistance, disorders that constitute metabolic syndrome.3 Although enhanced oxidative stress, inflammation, and metabolic disturbances, potentially representing interacting mechanisms, have been implicated in the pathogenesis of NAFLD, the mechanisms that underlie the initiation and progression of this disorder are still not clear.4, 5, 6 LIGHT (homologous to lymphotoxin, exhibits inducible expression, and competes with HSV glycoprotein D for herpes virus entry mediator (HVEM), a receptor expressed by T lymphocytes) is a 29 kDa type 2 transmembrane protein belonging to the tumor necrosis factor (TNF) superfamiliy (TNFSF).7 LIGHT/TNFSF14 is primarily expressed on T cells and dendritic cells, but has also been found on platelets, monocytes, and granulocytes, being involved in innate and adaptive immune responses, as well as in the regulation of cell survival and proliferation.7, 8 LIGHT binds to the HVEM (HVEM/TNFRSF14), and is also a shared ligand with membrane-bound lymphotoxin αβ for LTβR/TNFRSF3.7 Studies in animal models and some clinical studies indicate that LIGHT may be crucial for the development of various autoimmune and inflammatory disorders,9, 10 and has also been implicated in the pathogenesis of atherosclerosis.11, 12 In addition, LIGHT has been found to promote hepatic inflammation in two independent experimental hepatitis models (i.e., hepatitis induced by concanavalin A and Listeria monocytogenes),13 and has been reported to modulate lipid homeostasis through interaction with LTβR, at least partly through inhibition of hepatic lipase expression in hepatocytes.14 Based on its role in inflammatory disorders such as atherosclerosis, we hypothesized a possible role for LIGHT in NAFLD, as similar pathogenic interactions between inflammation and lipid metabolism are involved. In the present study, this hypothesis was tested through various experimental approaches including in vivo studies in NAFLD patients as well as in vitro studies in hepatocytes. In the study of hepatic expression of LIGHT/HVEM/LTβR, we included another 14 patients (age: 48.1 (±9.7) years; BMI: 29.8 (±2.1) kg/m2; ALT 82 (37–273) U/l) with biopsy-verified simple steatosis (n=8) or NASH (n=6). Seven patients who underwent hepatic resection for liver metastasis from colorectal cancer (n=6) or carcinoid (n=1) served as controls (age: 62.7 (±7.5) years; BMI: 26.6 (±3.3) kg/m2; ALT: 22 (14–36) U/l). In the controls, the liver specimens were carefully evaluated for cancer cell infiltration both macroscopically and microscopically. However, although the control specimens showed few infiltrating lymphocytes, we cannot totally exclude that these specimens contain tumor-infiltrating T cells with low expression of LIGHT.17 Written informed consent for participation in the studies was obtained from all individuals. The regional Ethical Committee approved the studies, which were conducted according to the Declaration of Helsinki. Serum samples for the study were collected and stored as described previously.15 There are a few studies on increased LIGHT levels in obesity associated with elevated levels of triglycerides and impaired glycemic control.18, 19 In the present study, however, we found no correlation between LIGHT levels and BMI in either patients (r=−0.13, P=0.30) or controls (r=−0.16, P=0.56). Moreover, within the NAFLD group, there was no difference in LIGHT levels between those with BMI above (n=34) and below 30 kg/m2 (n=32) (mean (s.d.): 72.2 (49.4) vs. 84.4 (57.8) pg/ml, P=0.36, above and below 30 kg/m2, respectively). In the present study, we show that NAFLD patients are characterized by increased serum levels of the TNFSF member LIGHT as well as enhanced hepatic expression of its receptors, HVEM and LTβR, with no significant differences between simple steatosis and NASH. Our in vitro findings in Huh7 hepatocytes suggest that LIGHT induces an increased release of the inflammatory chemokine IL-8 in these cells, with enhancing effects when coincubated with H2O2. Our findings suggest that LIGHT-mediated inflammation could be involved in NAFLD pathogenesis, potentially involving interaction with enhanced oxidative stress. It could be argued that in contrast to serum levels, LIGHT levels were not increased in the liver from NAFLD patients. However, the hepatic expression of the LIGHT receptors were significantly upregulated in NAFLD, indicating a possibility for LIGHT-mediated pathology in NAFLD even if LIGHT itself is not upregulated within the liver. Several inflammatory cytokines have been implicated in the pathogenesis of NAFLD including TNF-related molecules such as TNF/TNFSF2,20 Fas ligand (TNFSF6),21 TNF-related apoptosis inducing ligand (TNFSF10),22 and osteoprotegerin (TNFRSF11b).23 Experimental studies have previously shown that LIGHT could promote hepatic inflammation and metabolic disturbances within the liver.13, 14 This cytokine has also been linked to the pathogenesis of various inflammatory and autoimmune disorders such as inflammatory bowel disease, nephritis, and rheumatoid arthritis.10, 24, 25, 26 However, to the best of our knowledge, this is the first report of increased serum levels of LIGHT, as well as increased hepatic expression of its receptors, HVEM and LTβR, in patients with NAFLD. Celik et al.27 have reported elevated LIGHT levels in patients with hepatitis C virus infection and in patients with rheumatoid arthritis, and we have previously shown elevated LIGHT levels in patients with coronary artery disease.28 However, although raised LIGHT levels are not specific for NAFLD, LIGHT could still have a pathogenic role in this disorder. In fact, a common feature of several inflammatory cytokines is that they are elevated and have a pathogenic role in a magnitude of disorders with inflammation as a common feature. Unfortunately, we have no data on the cellular source of LIGHT in fatty liver. In general, LIGHT is strongly expressed by activated T cells, and is also found in granulocytes, immature dendritic cells, and platelets.7, 8 In the liver, LIGHT+ T cells have been shown to inhibit hepatic lipase,14 and hepatic NK1.1+ T cells have been shown to produce LIGHT during experimental hepatitis.13 However, as soluble LIGHT may be rapidly cleaved from its membrane-bound form, mediating LIGHT-driven inflammation, the cellular source of LIGHT can be difficult to prove. Nonetheless, it is not inconceivable that the cellular source of LIGHT in NAFLD may be infiltrating T cells. Whereas several LIGHT effects seems to be mediated by its membrane-bound form, in particular on T cells (e.g., metabolic effects within the liver),7, 10, 14 soluble LIGHT has been found to promote hepatic inflammation.13 In the present study, we show that soluble LIGHT is a potent inducer of IL-8 production and release in Huh7 hepatocytes. Moreover, in line with studies in experimental hepatitis,13 the LIGHT-mediated inflammatory response in Huh7 hepatocytes seemed to be mediated through LTβR, as HVEM was expressed at very low levels in these cells. Thus, while HVEM is prominently expressed in T cells, LTβR is expressed in stromal cells or non-lymphoid hematopoietic cells including hepatocytes.7, 13 Yet, although HVEM was scarcely expressed in Huh7 hepatocytes, we cannot exclude an inflammatory interaction between LIGHT and HVEM during hepatic inflammation through T- cell-related mechanisms. However, even in experimental hepatitis, abundant with T cells and NK-T cells, the LIGHT-mediated hepatic inflammation seems primarily to be mediated through LTβR.13 Enhanced oxidative stress has been implicated to have an important role in the development and progression of NAFLD.4 In the present study, we show that the reactive oxygen species H2O2, in addition to promote IL-8 release in itself, enhanced the LIGHT-induced production and release of IL-8. IL-8 is a potent inflammatory cytokine that could promote generation of reactive oxygen species in leukocytes,29 and has also been linked to development of NAFLD.30 If IL-8 is operating in vivo within the liver, the interaction between LIGHT, H2O2, and IL-8 could potentially be part of a pathogenic loop during NAFLD progression. Moreover, although there was no difference between the expression of LIGHT and LTβR between simple steatosis and NASH, H2O2 level is presumable higher in NASH, potentially resulting in increased LIGHT-mediated effects. On the other hand, LIGHT has also been shown to promote liver regeneration31 and to counteract the TNF-induced apoptosis in hepatocytes,32 and the role of LIGHT in NAFLD is far from clear. The present study has some limitations such as relatively low number of patients in particular in the analyses of LIGHT expression in the liver, and the number of patients with simple steatosis and NASH could have been too low to detect differences between these two NAFLD subgroups. In addition, the control samples for liver specimens were not ideal and we lack data on the cellular source of LIGHT. However, we suggest that our findings indicate that LIGHT-mediated inflammation could be operating in NAFLD, involving interactions between IL-8 and oxidative stress, potentially representing a pathogenic loop in the deleveopment and progression of NAFLD (Figure 6). Our findings should encourage further studies on the role of LIGHT in NAFLD development and progression. Guarantor of the article: Kari Otterdal, MSc, PhD. Author contributions: K.O., contributions to the conception and design of study, performing the in vitro experiments, performing analyses and interpretation of data, drafting the manuscript; JWH/IPG/ZK, contributions to the conception and design of study, collection of patient and control material; AY/SH/FMS, performing analyses and interpretation of data; TBD, culturing the hepatocytes, performing analyses and interpretation of data; JKD/BH, contributions to the conception and design of study, involved in interpretation of data; PA, contributions to the conception and design of study, drafting the manuscript. All the authors have read and approved the manuscript. Financial support: The Norwegian Research Council, University of Oslo, the Southern and Eastern Norway Regional Health Authority and Inven2. Potential competing interests: None. We thank Ellen Lund Sagen for excellent technical assistance.