Acute-on-chronic liver failure (ACLF) is a clinical syndrome characterized by acute deterioration of liver function, marked by an extremely high short-term mortality rate, presenting significant challenges in disease management. Thus, comprehensive exploration of the pathophysiological characteristics of ACLF and precise evaluation of its prognosis are crucial for enhancing disease management. The Asian Pacific Association for the Study of the Liver (APASL) defines ACLF as an acute exacerbation of liver function in patients with pre-existing liver disease or cirrhosis, highlighting the significance of elevated bilirubin and coagulopathy in the diagnosis of ACLF. This contrasts with the European Foundation for the Study of Chronic Liver Failure (CLIF), which uses systemic organ or system failure as the primary diagnostic criterion (Sarin and Choudhury, 2016; Sarin et al., 2019). In the Asia-Pacific region, the onset and exacerbation of ACLF are closely linked to HBV infection, with chronic hepatitis and cirrhosis caused by HBV being the primary underlying conditions leading to ACLF in this area (Li et al., 2016). The liver pathology of HBV-ACLF patients demonstrates distinctive features, mainly showing as submassive hepatic necrosis with cholestasis and/or canalicular bilirubin stasis (sepsis), in contrast, alcohol-related ACLF patients primarily exhibit inflammation and bilirubin stasis (Mookerjee et al., 2011; Li et al., 2015). Hence, HBV-ACLF warrants distinct consideration and discussion.

Patients with cirrhosis are at a higher risk of developing ACLF compared to those without cirrhosis (Asrani et al., 2013; Hsu et al., 2023). Additionally, HBV infection correlates with the onset of various autoimmune pathologies, such as systemic lupus erythematosus, aplastic anemia, antiphospholipid syndrome, rheumatoid arthritis, type 1 diabetes, multiple sclerosis, and thyroid diseases (Maya et al., 2008). This indicates that HBV contributes to the disordering of the autoimmune system. During HBV infection, the apoptosis and necrosis of infected hepatocytes, driven by T cells and NK cells, alongside mitochondrial dysfunction and hepatic stellate cell activation, result in ongoing chronic inflammation in the liver (Piconese et al., 2018; Loureiro et al., 2023; You et al., 2023). Against the backdrop of autoimmune disarray, this inflammation and the subsequent leakage of cellular proteins can trigger the immune system to generate systemic autoantibodies. Autoantibodies are primarily classified into IgM and IgG types. IgM antibodies are considered to be involved in the clearance of cellular debris and degrade over a short term; IgG antibodies, associated with the breach of central immune tolerance, can persist and proliferate, often correlating with pathological processes (Tsokos et al., 2013). Recent studies on the role of autoantibodies in the diagnosis and progression of tumors and neurological diseases are increasing, confirming their clinical diagnostic value (Ezzikouri et al., 2015; Zhang et al., 2020; Do et al., 2021; Gövert et al., 2023). Hence, we hypothesized that the disruption of the autoimmune system, particularly through cirrhosis, may lead to the production of specific IgG autoantibodies, contributing to the progression of the disease. To validate the clinical utility of these antibodies, this study investigates the role of autoantibodies in HBV-related cirrhotic ACLF from an autoimmune perspective. The aim is to unveil new insights into the mechanisms of this complex disease and to identify potential biomarkers. The approach involves using immunoprecipitation (IP) and Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) technologies to screen for potential autoantibodies in the serum of patients with HBV-related cirrhosis, examining their correlation with the prognosis of ACLF.

Human AGO proteins, especially AGO2, are central to the RNA silencing complex, playing key roles in gene regulation and cellular processes like apoptosis and differentiation (Jo et al., 2015). AGO2, highly expressed in various cells (Fagerberg et al., 2014), has unique endonuclease activity (Meister et al., 2004; Cheloufi et al., 2010) and significantly impacts immune responses and inflammation (Höck and Meister, 2008; Nakanishi, 2022). In viral hepatitis, AGO2 enhances HBV replication by interacting with miR-146a (Ji et al., 2020) and aids HCV RNA stability through miR-122 (Shimakami et al., 2012). AGO2-linked miRISC complexes help stabilize miRNAs, providing diagnostic insights detectable by anti-AGO2 antibodies (Geekiyanage et al., 2020). This underscores the potential of circulating AGO2-Abs as biomarkers for disease diagnostics.

In this study we explored autoimmune responses by employing IP combined with iTRAQ, identifying a spectrum of proteins. Subsequent bioinformatics analyses honed in on the autoantigens AGO1-3. Utilizing CBA and IP, we confirmed the genuine presence of corresponding AGO1-3 autoantibodies in serum. CBA revealed positive AGO1-3 antibodies in the mixed serum of the exploratory cohort. IP assays indicated AGO2-Abs were consistently present, whereas AGO1-Abs and AGO3-Abs were intermittently undetected due to their low abundance or the sensitivity limits of the assays. Quantitative analyses in the test cohort specifically revealed that AGO2-Abs levels were significantly associated with disease progression, unlike AGO1-Abs and AGO3-Abs. This phenomenon is likely a result of intermolecular epitope spreading, caused by the high degree of homology (approximately 80%) among AGO proteins (Jakymiw et al., 2006), leading to a low-level antibody response to AGO1 and AGO3 in patient serum. Further analysis indicated that AGO2-Abs were pinpointed as not only markers of liver function deterioration but also as independent predictors for short-term mortality in ACLF, highlighting their crucial prognostic value.

Current scoring systems like Child-Pugh, MELD, and AARC are widely used for mortality and disease progression predictions in liver cirrhosis patients (Choudhury et al., 2017). However, these scores rely on variables such as biochemical markers, coagulation profiles, and clinical complications, making them vulnerable to fluctuations caused by symptomatic treatments like albumin supplementation, bilirubin reduction, and ascites management. These interventions may temporarily modify clinical parameters but typically do not change outcomes, underscoring the necessity for stable biomarkers in patient evaluation.

Autoantibody levels, unaffected by these symptomatic treatments, provide such stability. Our analysis showed that AGO2-Abs have similar predictive efficacy to traditional scores in assessing short-term mortality in ACLF. We observed a distinct gradient between optimal cutoff values for 28-day and 90-day mortality (146.54μg/ml vs 99.14μg/ml), not observed in the Child-Pugh, MELD, and AARC systems. This indicates a strong link between AGO2-Abs levels and the progression of ACLF, reflecting their potential for enhancing clinical management precision. Moreover, the incorporation of AGO2-Abs into these traditional scoring systems significantly increased diagnostic accuracy ( Table 4 ). This enhancement is likely tied to the symptomatic treatments received before hospital admission, which, while altering biochemical indicators, do not substantially affect overall inflammation or prognosis. Therefore, using AGO2-Abs, which remain stable despite symptomatic treatment, alongside these scores yields a more accurate prediction of mortality, proving advantageous for early prognostic assessments.

Furthermore, we observed significantly elevated AGO2-Abs levels in patients with hepatic encephalopathy (HE). While current theories like ammonia toxicity and neurotransmitter imbalances offer partial explanations for HE’s pathogenesis, they fall short of a comprehensive understanding, with evidence suggesting an autoimmune imbalance connection (Manzhalii et al., 2022). AGO2-Abs, previously detected in systemic autoimmune conditions and chronic Hepatitis C without specific disease association (Vázquez-Del Mercado et al., 2010; Satoh et al., 2013), have recently been found in autoimmune neurological disorder patients, indicating their potential as markers of neurological autoimmunity (Do et al., 2021). This association prompts speculation on the role of AGO2-Abs in HE development, highlighting the need for further investigation to validate their diagnostic utility for HE and to clarify AGO2’s involvement in HE pathogenesis.

We conducted preliminary investigations into the mechanistic link between AGO2 and the deterioration of liver function, identifying a band-like concentration of AGO2 expression around the periportal areas of the liver tissue. This pattern may lead to increased deposition of antigen-antibody complexes in the region, affecting the functionality of the periportal area, contributing to disturbances in hepatic microcirculation and bilirubin excretion, potentially exacerbating liver damage under a cirrhosis backdrop. Additionally, the significant correlation between TBIL levels and AGO2-Abs suggests a potential relationship between AGO2-Abs and bilirubin excretion. However, the specific mechanisms of injury require further study for validation.

Although AGO2-Abs shows promise as a biomarker for predicting mortality in ACLF patients, its utility is constrained due to its production by immune cells, making it less suitable for those on long-term corticosteroids and immunosuppressants, unlike the Child-Pugh, MELD, and AARC scoring systems. This study, limited to a single center, focused on HBV-associated ACLF, omitting an examination of AGO2-Abs in liver failures from diverse etiologies, thereby narrowing its scope. Given that autoimmune activation is prevalent in many forms of liver failure (Bernal et al., 2007), the prognostic potential of AGO2-Abs may extend to other liver failures, the prognostic potential of AGO2-Abs may extend to other liver failures, including those caused by HCV, drug-induced factors, or autoimmune liver diseases. However, due to the use of corticosteroids and immunosuppressants in patients with drug-induced liver injury and autoimmune liver diseases, the accuracy of AGO2-Abs in predicting the prognosis of these types of liver failure may not be as reliable as traditional scoring systems. Future studies should employ a multicentric approach to broaden the scope and enhance the generalizability of findings. Moreover, further research into the mechanistic role of AGO2 and its autoantibodies in liver disease is imperative to enhance our understanding of disease progression and facilitate the development of novel therapeutic strategies.

In patients with HBV-related cirrhotic ACLF, the presence of AGO2-Abs significantly predicts short-term mortality independently. Integrating AGO2-Abs with established scoring systems like Child-Pugh, MELD, and AARC substantially improves the predictive accuracy for short-term mortality. Hence, AGO2-Abs not only affirm their role as promising biomarkers but also introduce an innovative strategy to refine the management precision of ACLF patients.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The studies involving humans were approved by Ethics Committee of the Second Affiliated Hospital of Chongqing Medical University. The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study.

YW: Investigation, Data curation, Formal analysis, Writing – original draft, Writing – review & editing. YH: Investigation, Writing – original draft. JL: Investigation, Writing – original draft. HM: Validation, Visualization, Writing – original draft. ZS: Visualization, Writing – original draft. CW: Resources, Writing – original draft. YL: Resources, Writing – original draft. ZL: Resources, Writing – original draft. HS: Writing – review & editing. YY: Project administration, Methodology, Writing – review & editing. XS: Conceptualization, Funding acquisition, Resources, Supervision, Writing – review & editing.