Assessment of EN-RAGE, sRAGE and EN-RAGE/sRAGE as potential biomarkers in patients with autoimmune hepatitis

Autoimmune hepatitis (AIH) is a liver disease characterized by the autoimmune-induced injury of hepatocytes which can lead to cirrhosis and hepatic failure. The diagnosis and disease management of AIH patients remain challenging due to the diversity of clinical phenotypes and the presence of confounders such as alcohol and viruses. Recently, EN-RAGE and sRAGEs have been implicated in inflammatory-immune response. Nonetheless, their natural behaviour and relationship to disease activity as well as clinical predictive values in AIH development or therapy-induced remission have not been reported. Sixty-seven AIH patients and thirty gender- and age-matched healthy controls (HC) were enrolled. The serum concentrations of EN-RAGE, sRAGE and their ratio (EN-RAGE/sRAGE) in these subjects were measured by ELISA. Besides, the correlations of three parameters with clinical features and therapeutic response were analyzed, respectively. Furthermore, their potential predictive values for monitoring the AIH progression and therapeutic response were also evaluated. Higher serum EN-RAGE, lower sRAGE and higher EN-RAGE/sRAGE value were observed in AIH patients. EN-RAGE and sRAGE as well as EN-RAGE/sRAGE were correlated with liver necroinflammation parameters, cirrhosis occurrence and therapeutic response. In addition, we identified that EN-RAGE/sRAGE, EN-RAGE and sRAGE had valuable predicting power for AIH patients, AIH patients with normal ALT and cirrhosis incidence, respectively. More importantly, EN-RAGE/sRAGE also exerted predicting power for the remission in AIH patients. AIH patients rendered distinct patterns of serum EN-RAGE, sRAGE or EN-RAGE/sRAGE compared to healthy controls. Moreover, these three parameters exhibited potentials as novel biomarkers for AIH diagnosis and prognosis evaluation.

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Background
Autoimmune hepatitis (AIH) is a liver-specific autoimmune disease characterized by hypergammaglobulinemia, increased circulating autoantibodies, transaminases, and histological evidence of interface hepatitis [1]. AIH occurs in all ethnicities and ages, with a female predominance. Intolerance to self-antigens followed by destruction of hepatic parenchyma by T lymphocyte-mediated response is identified as the key pathogenic mechanisms [2]. Untreated AIH can lead to liver cirrhosis and terminal failure [3]. So far, autoantibodies against nuclear antigens (ANA) or smooth muscle antibodies (ASMA), combination of liver kidney type 1 (LKM1) and other liver biochemical indexes such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma globulin and immunoglobulin G (IgG) are widely used as biomarkers for AIH diagnosis. However, the sensitivity and specificity of these markers for AIH diagnosis and evaluation of disease severity or therapeutic effect are still very limited, leading to misdiagnosis, inaccurate disease assessment and missing the treatable stage of AIH [4]. Therefore, unveiling new quantifiable biomarkers that are closely related to its pathogenesis as well as accurately reflects disease severity and therapeutic response is urgently needed.
The receptor for advanced glycation end products (RAGE), a receptor expressed on the cell surface, can be bound by multiple types of damage-associated molecular patterns (DAMPs), including S100/calgranulins, high-mobility group box 1 (HMGB1) and advanced glycation end products (AGEs), leading to the activation of the downstream cascades which is responsible for inflammatory-immune response [5]. Extracellular newly identified receptor for advanced glycation end products binding protein (EN-RAGE), also known as S100A12, is a calcium-binding proinflammatory protein predominantly secreted by activated granulocytes, macrophages and lymphocytes [6]. When secreted extracellularly, EN-RAGE, as one type of the DAMPs, induces persistent inflammatory-immune response by binding to RAGE followed by activation of intracellular signal cascades in lymphocytes and production of proinflammatory cytokines [7]. Increased serum EN-RAGE levels have been detected in multiple inflammatory-immune disorders [8,9]. However, it is still unclear how EN-RAGE participates in the inflammatory-immune process and whether it can serve as a useful indicator for AIH progression.
Soluble RAGE (sRAGE), a splicing variant of the fulllength receptor, is a soluble isoform containing only the RAGE extracellular domain formed by proteolytic cleavage. sRAGE counteracts DAMPs-RAGE interaction by sequestering and eliminating DAMPs [10]. Therefore, sRAGE can function as a "decoy" by binding to EN-RAGE and prevent the ligation between EN-RAGE and RAGE, which attenuate the inflammatory-immune response. Clinically, the serum levels of sRAGE are reduced in several types of auto-immune diseases, such as systemic lupus erythematosus [11], hashimoto's thyroiditis [12] and guillain-barré syndrome [13]. AIH is also an autoimmune disease with hepatocellular injury caused by liver-infiltrated T cells including T helper type 1 (Th1), Th2 and Th17 cells [1,14]. T lymphocyte activation and differentiation was reported to be regulated by RAGE ligation by an in vivo mouse study [15]. Recent studies showed a protective role of sRAGE during the process of hepatocellular injury [16,17]. However, the serum level of sRAGE in AIH and its underlying relationship with disease activity remain elusive.
The activation of RAGE by EN-RAGE and the antagonism by sRAGE results in inflammatory-immune responses augmentation and mitigation, respectively. Nonetheless, to date, the relationship of serum EN-RAGE or sRAGE levels with disease activity has not been well defined in AIH. In this study, we determined the serum levels of EN-RAGE and sRAGE as well as their ratio (EN-RAGE/sRAGE) and explored their correlation with clinical parameters in a welldefined cohort of AIH patients, aiming to explore whether EN-RAGE, sRAGE or EN-RAGE/sRAGE can be used as potential disease biomarkers during AIH progression.

Patients
Sixty-seven newly diagnosed AIH patients were prospectively recruited from the First and Second Affiliated Hospital of Chongqing Medical University from May 2016 to May 2018. Patients were not included if they were detected with other concomitant disease entities, including virus hepatitis, drug-induced hepatic injury, genetic and metabolic liver disease, hepatic malignancies, primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC) and other autoimmune diseases. AIH was diagnosed according to the simplified criteria suggested by the IAIHG [18]. Clinical characteristics of patients were recorded at the time of AIH diagnosis. The patients were treated with decreasing doses of azathioprine and prednisone. Remission was defined according to the international guidelines. Biochemical remission was defined as the normalization of alanine aminotransferase (ALT), aspartate aminstransferase (AST) and IgG levels after treatment. Additionally, thirty age and gender-matched healthy controls [Age median and interquartile range (IQR): 48 (18); female, n (%): 49 (73.2%)] who did not have evidence of liver diseases or other chronic disorders were enrolled as healthy controls (HCs). All serum samples from thepatients and HCs were stored at − 80° until use. The study was approved by the Institutional Ethics Committee for human studies at Chongqing Medical University, Chongqing, China. All the procedures were following the Declaration of Helsinki. Patient characteristics were summarized in Table 1.

Enzyme-linked immunosorbent assay
Serum samples were analyzed by commercial human EN-RAGE (Cusabio, China) and sRAGE enzyme-linked immunosorbent assay kits (JYM, China) used according to the manufacturer's instructions. Samples were run in duplicate.

Statistical analysis
Data were analyzed using SPSS 17.0. Mann-Whitney or Kruskal-Wallis test was performed to determine significance of EN-RAGE and sRAGE as well as their ratio (EN-RAGE/sRAGE) in AIH patients with various clinical and biochemical features. Correlation coefficients (r) were calculated using spearman correlation. Paired t test was used for EN-RAGE and sRAGE as well as EN-RAGE/sRAGE in AIH with remission analysis. Receiver operating characteristic (ROC) curves were generated to classify patients in different groups, as well as for the evaluation of predicting power for serum EN-RAGE and sRAGE as well as EN-RAGE/sRAGEvia calculation of the area under the ROC curve (AUC), sensitivity and specificity according to standard formulas. All the data represents the median and interquartile range (IQR); A p value < 0.05 was considered statistically significant.

Serum levels of EN-RAGE, sRAGE and their ratio (EN-RAGE/ sRAGE) in AIH patients
To assess whether serum levels of EN-RAGE and sRAGE are abnormally altered in AIH, we analyzed their levels in AIH and compare them to healthy controls (HC significant differences were observed among ANA group, ASMA group and ANA + ASMA group (Fig. 2g-i).

Correlations of EN-RAGE, sRAGE and EN-RAGE/sRAGE with hepatic biochemical indexes
Hepatic biochemical necroinflammation indexes such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), albumin (ALB), total bilirubin (TB) are crucial factors for reflecting liver functional state. We further analyzed the relationship between hepatic biochemical parameters and EN-RAGE, sRAGE or their ratio (EN-RAGE/sRAGE), respectively in AIH patients. Both serum EN-RAGE and the EN-RAGE/sRAGE ratio showed a positive correlation with ALT and AST but not ALB or TB in AIH patients (Table 2). In contrast, serum sRAGE was negatively correlated with ALT and AST but not with ALB and TB in AIH patients (Table 2).  (Fig. 3c).

Recovery phase of AIH patients possess lower EN-RAGE and EN-RAGE/sRAGE but higher sRAGE
To further investigate the relationship of between therapeutic effect and EN-RAGE, sRAGE or EN-RAGE/ sRAGE, twenty-one AIH patients who achieved disease remission were enrolled in this study. By monitoring serum EN-RAGE, sRAGE, EN-RAGE/sRAGE value and clinical parameters for these patients, we found that posttreatment group showed a striking reduction of ALT and AST but improved level of ALB, implying that there is a mitigation of liver damage and recovery of liver functions. Moreover, IgG level was decreased in the posttreatment group, suggesting that there is a disease remission upon the AIH therapy (Table 3). Interestingly, after the therapeutic treatment for AIH, serum levels of EN-RAGE were dramatically diminished while sRAGE were elevated (Fig. 4a, b). As expected, EN-RAGE/ sRAGE value was also decreased in the remission phase of AIH patients (Fig. 4c). More importantly, EN-RAGE, sRAGE, EN-RAGE/sRAGE were significantly correlated with ALT and AST during the whole process of therapy, respectively ( Fig. 4d-i).     identified EN-RAGE/sRAGE also has potential predicting power for the remission of AIH patients.

Discussion
AIH is an inflammatory liver disease characterized by liver immunotolerance failure leading to the destruction of hepatic parenchyma attacked by autoreactive T lymphocyte [14]. Elucidation of host inflammatory-immune response involved in AIH pathogenesis provides new perspectives for identification of biomarkers and therapeutic targets. EN-RAGE and sRAGE have recently gained interest because they function as important modulators in inflammatory immune response by acting on RAGEmediated downstream cascades [5,20]. Here, we assessed serum levels of EN-RAGE and sRAGE as well as their ratio EN-RAGE/sRAGE in AIH patients and analyzed their potential predicting power for disease progression and therapeutic response. Growing evidence revealed that EN-RAGE/RAGE signaling pathway-mediated inflammatory-immune response play an important role in infection, autoimmunity and cancer [10,21,22]. However, the role of EN-RAGE/RAGE signaling in AIH-associated inflammatory-immune response has not been well-understood. Our present study demonstrated AIH patients exhibited elevated EN-RAGE and reduced sRAGE levels along with increased RAGE/sRAGE value in a well-defined cohort, indicating that EN-RAGE and sRAGE may exert opposite functions on regulating the AIH pathogenesis. This finding supports previous studies showing high EN-RAGE or low sRAGE levels in other inflammatory-immune disorders [23][24][25]. Based on data from assessment of differentiating power of EN-RAGE, sRAGE and EN-RAGE/ sRAGE for AIH by ROC analysis, we identified EN-RAGE/sRAGE had the highest diagnostic efficacy.
The necroinflammation markers such as AST and ALT have been widely used for AIH diagnosis, prediction and monitoring disease activity [4]. Here, we observed significant correlations of EN-RAGE, sRAGE or EN-RAGE/ sRAGE with these necroinflammatory indexs, implying that EN-RAGE/RAGE system activation may be involved in AIH-associated liver necroinflammatory. However, AIH patients may display normal ALT level leading to the inaccurate assessment of disease severity or even misdiagnosis [19]. In this study, we found that EN-RAGE has a predicting power for AIH patients with normal ALT. Mechanistically, AIH-associated liver injury is caused by autoimmune response to liver autoantigens, which involves a variety of immune cells, cytokines, autoantibodies and complement-mediated cytotoxicity. Various cytokines have been reported to participate in the pathogenesis of AIH. Th1 cytokine induce macrophage and T-cell activation, Th2 cytokine induce the production of autoantibodies and Th17 cytokines produce pro-inflammatory cytokines, all of which ultimately contribute to hepatocyte necroinflammation [1]. One previous in vivo study demonstrated that RAGE activation was responsible for the Th1 cell activation and cytokine production in pathogenesis of autoimmune response [15]. Secreted EN-RAGE was also reported to induce inflammatoryimmune response-mediated proliferation and activation of mononuclear macrophage and lymphocyte by binding to RAGE [26]. Combining our present findings and previous studies, inflammatory-immune response triggered by EN-RAGE/RAGE ligation may participate in AIH-associated necroinflammatory activity. Asymptomatic autoimmune hepatitis can lead to cirrhosis without treatment. Clinically, around 30% of AIH patients showed histological evidence of cirrhosis [27], suggesting early diagnosis of AIH accompanied by cirrhosis is pivotal. We found high EN-RAGE but low sRAGE levels alone with high EN/RAGE/sRAGE value in AIH with cirrhosis. Simultaneously, our present data identified that sRAGE could efficiently discriminate AIH patients with cirrhosis from without cirrhosis, generating 89.74% sensitivity and 60.71% specificity. Mechanistically, persistent AIH-mediated necroinflammation induces activation and differentiation of quiescent hepatic stellate cell and portal fibroblast into myofibroblast, a major contributor for collagen synthesis and extracellular matrix proteins (ECM) deposition, leading to the fibrous scar and terminal cirrhosis [28,29]. Recently, elevated EN-RAGE and sRAGE were reported to involve diabetesrelated cystic fibrosis and lead to worsening lung function [30]. EN-RAGE was also proven to activate fibroblast and cause dermal fibrosis by binding RAGE [31]. Therefore, we speculate that EN-RAGE/RAGE system activation might also involve in AIH-associated cirrhosis by acting on hepatic stellate cells or portal fibroblasts.
Prednisolone alone or in combination with azathioprine is the standard treatment for autoimmune hepatitis, and this treatment improves clinical, biochemical and histological features and prolongs survival in AIH patients [32]. In our present study, AIH posttreatment showed substantial normalization of clinical parameters ALT, AST and IgG. Previous study demonstrated that EN-RAGE was associated with response to therapy in juvenile idiopathic arthritis [33]. Here, we also observed an association of EN-RAGE, sRAGE or EN-RAGE/ sRAGE with therapeutic effects. And a better predicting Nonetheless, several limitations are existed in this study. Firstly, we did not assess the specificity of serum EN-RAGE and sRAGE for AIH compared to other liver autoimmune disease entities such as primary biliary cirrhosis, primary sclerosing cholangitis or their overlap syndrome. Secondly, our study was conducted in Chinese patients who were type I AIH. Further studies are required to confirm these results in type II AIH patients which mainly distributed in Europe and the United States. Finally, the detailed molecular mechanism regarding EN-RAGE/sRAGE signaling activation in AIH pathogenesis and whether sRAGE can serve as a therapeutic target still need further clarification.

Conclusions
In conclusion, this study demonstrated that AIH patients rendered distinct patterns of serum EN-RAGE, sRAGE or EN-RAGE/sRAGE compared to healthy controls. In addition, three parameters were correlated with necroinflammation, cirrhosis incidence and therapeutic response. Furthermore, we identified EN-RAGE/sRAGE, EN-RAGE and sRAGE, had predicting power for AIH patients, AIH patients with normal ALT and cirrhosis incidence, respectively. More importantly, EN-RAGE/ sRAGE also exerted predicting power for therapeutic response in AIH patients. Taken together, our findings indicate that serum EN-RAGE, sRAGE or EN-RAGE/ sRAGE exhibited potentials as novel biomarkers for the diagnosis and prognosis evaluation in AIH patients, opening a very attractive field of research on these molecules in the pathology of AIH and identification of therapeutic targets.
Additional file 1: Figure S1. Serum levels of EN-RAGE and sRAGE as well as EN-RAGE/sRAGE in AIH patients with different genders.