We report here for the first time that the serum OPN level correlated with hepatic inflammation and fibrosis in heavy alcohol drinkers. The hepatic OPN expression level strongly correlated with hepatic neutrophils accumulation, the pro-fibrogenic factor TGFβ and hepatic fibrosis. Expression of OPN in adipose tissue correlated with hepatic fibrosis reinforcing the concept that adipose tissue was also inflamed in ALD. The serum level of OPN was an accurate independent factor in estimating significant liver fibrosis in both the estimation and validation cohort.
OPN expression detected in adipose tissue and in the liver of alcoholic patients was enhanced by the presence of hepatic fibrosis. The relative contribution of these two tissues in circulating OPN is difficult to estimate. While hepatic OPN expression correlated with the serum OPN level in our patients, further studies will be required to determine the relative role of these two tissues in modulating circulating OPN concentrations. Although the cellular origin of the increased hepatic OPN expression has not been precisely determined, increased OPN gene expression has been reported in macrophages, Kupffer cells, stellate cells, biliary epithelial cells and in inflammatory cells of the necrotic areas in rodent liver fibrosis models 
. The observation that hepatic inflammation was associated with elevated serum OPN levels is in favor of a role for OPN in liver inflammation. Recent studies have highlighted the role of OPN in inflammatory liver diseases such as alcoholic and non alcoholic liver diseases and T cell-mediated hepatitis 
. In rodent models of alcoholic liver diseases, neutrophils accumulation in the liver was mediated by OPN 
. In agreement with these reports, we showed in a small number of patients that hepatic and systemic OPN expression correlated with neutophil infiltration in the portal space. OPN also facilitated the infiltration and accumulation of macrophages at sites of injury during the initial steps after carbon tetrachloride intoxication 
. The recruitment and activation of the inflammatory and immune cells by OPN could enhance hepatic inflammation, which in turn may activate hepatic stellate cells and fibrogenesis. In agreement with this mechanism, the invalidation of the OPN gene decreased hepatic inflammation and consequently fibrosis in mice fed a choline-methione-deficient diet 
. Furthermore, it has been suggested that OPN could have fibrogenic properties. OPN expression was increased in activated hepatic stellate cells 
and was required for myofibroblast differentiation 
. OPN was regulated by the hedgehog pathway and directly promoted pro-fibrogenic responses 
. The upregulation of OPN has been reported in the liver of patients with ALD-cirrhosis but also with NASH-cirrhosis, primary biliary cirrhosis, autoimmune hepatitis and primary sclerosing cholangitis, which suggested that OPN induction is a conserved response to chronic liver injury 
. Excess weight was as an independent risk factor for fibrosis and cirrhosis in patients with ALD 
. More recently, Naveau et al.
demonstrated that adipose tissue inflammation correlated with the severity of pathological features in the liver 
. The increase in the production of OPN in adipose tissue could result from the activation of adipose tissue macrophages by LPS and cytokines such as TNFα and IL6. The latter were strongly increased in serum and adipose tissue of heavy alcohol drinkers 
. OPN plays an important role in the infiltration and accumulation of macrophages in the early stages of obesity. Indeed, we recently reported that elevated OPN expression in adipose tissue paralleled with adipose tissue macrophage infiltration and both phenomena were reversed after weight loss in obese patients 
. While further investigation is necessary to determine the molecular mechanism of adipose tissue inflammation, adipose tissue OPN expression could lead to enrichment of adipose tissue in macrophages and to increased adipose tissue inflammation. The OPN level in adipose tissue could be related more to inflammation than adipose tissue gain. Indeed, OPN expression in adipose tissue did not correlate with BMI, leptin expression and leptinemia.
We have shown for the first time that the serum OPN level was an independent factor that estimates significant fibrosis in two independent cohorts of patients with ALD. A larger group of alcoholic patients with all the data allowing to repeat the univariate and multiple regression modeling will be done in the future. Since circulating OPN could increase in obese individuals and that obesity is also associated with a higher propensity to fibrosis and progression of chronic liver diseases, we analyzed 60 alcoholic patients with available BMI to determine an independent association between OPN and fibrosis. In these patients (39% with a significant fibrosis (F≥2)), serum OPN correlated with hepatic fibrosis (rs
60) but not with BMI (rs
60). Using the univariate analysis, OPN but not BMI was associated with F≥2 (Table S2
). In a multivariate analysis including the OPN, γGT and BMI, OPN was the only independent variable when F≥2 was the judgment criterion (Table S3
). The serum OPN level could thus be considered as an independent factor estimating significant liver fibrosis. However, additional validations on a large number of alcoholic patients, controls subjects and morbidly obese group included patients with the full range of liver fibrosis with available BMI will be required. Liver biopsy remains the gold standard for assessment of liver fibrosis. However, problems with liver biopsy include sampling error and inter-observer variability. Fibroscan and the currently available blood-algorithm tests (Fibrotest®, FibroMeter®) or direct biomarker (Hyaluronate) can differentiate between mild and advanced disease 
. In the case of borderline results two or more methods can be combined. However, identification of novel markers is needed to improve blood-algorithm tests leading to quantification of fibrosis and to monitor the dynamic nature of fibrosis.
Interestingly, the OPN and FibroMeter® levels were the only independent variables when the F≥2 was the judgment criterion in a multivariate analysis including OPN, alkaline phosphatase, albumin, hemoglobin and FibroMeter® levels. Furthermore, OPN, FibroMeter® and hyaluronate estimated significant fibrosis with the same accuracy. OPN was a good marker in both retrospective (AUROC
0.89 (0.81, 0.94)) and prospective (AUROC
0.88 (0.79, 0.93)) studies. Moreover, the serum OPN levels accurately estimated advanced fibrosis (F≥3: AUROC
0.91 (0.83, 0.95)) and cirrhosis (F
0.91 (0.80, 0.96)) in alcoholic patients (from the estimation group).
Circulating levels of OPN were also modified in patients with liver complications related to the hepatitis C and B viruses. An elevated circulating OPN level was an excellent predictor of cirrhosis in patients with a hepatitis B infection 
. The circulating levels of OPN were also modified in patients with liver complications induced by a hepatitis C infection. Indeed, we report here that the serum OPN in patients with chronic viral hepatitis C correlates with hepatic fibrosis, as previously reported for plasma of hepatitis C virus infected subjects 
. OPN enhanced tumor development and metastases, since OPN was highly expressed in hepatocellular carcinoma (HCC) and correlated with worse prognosis 
. However, the role of OPN in these diseases is still poorly understood. It was recently proposed that OPN could be a potential target for the control of HCC 
In summary, circulating levels of OPN estimate, with good accuracy, significant fibrosis in heavy alcohol drinkers. Hepatic OPN also correlated with serum OPN and with hepatic inflammation, neutrophils infiltration, fibrosis and TGFβ expression. Infiltration of liver by parenchymal neutrophils is a prominent feature of Alcoholic Hepatitis 
. Up-regulation of OPN in liver could contribute to this infiltration and the severity of Alcoholic Hepatitis. Multiple factors stimulate parenchymal and nonparenchymal cells to produce this chemokine including LPS, IL6 and TNFα. Adipose tissue of alcoholic patients was further inflamed, as evaluated by OPN expression. OPN adipose tissue level also correlated with hepatic fibrosis. Since TNFα and IL6 were strongly increased in adipose tissue and serum of heavy alcohol drinkers 
, cytokines secreted by adipose tissue could enhance the inflammation and up-regulation of OPN in liver. The recruitment and activation of the inflammatory and immune cells by OPN could also enhance hepatic inflammation, which in turn may activate hepatic stellate cells and fibrogenesis. Furthermore, it has been suggested that OPN could have fibrogenic properties 
. Studies focusing on the role of OPN in liver and adipose tissue function will be appropriate approaches to acquire more insight into the pathogenesis of human ALD.