Increased Serum Levels of sCD206 Are Associated with Adverse Prognosis in Patients with HBV-Related Decompensated Cirrhosis

Background HBV-associated decompensated cirrhosis (HBV-DeCi) is attracting considerable attention due to disease acceleration and substantial mortality. Macrophages regulate the fibrotic process in DeCi. Soluble CD206 (sCD206) is primarily expressed by macrophages. We aimed to investigate whether sCD206 predicts mortality in patients with HBV-DeCi. Materials and Methods A total of 382 patients were enrolled between February 2020 and February 2021 and divided into nonsurviving and surviving groups according to 28-day, 3-month, and 6-month outcomes. Cox regression analysis was performed to confirm the independent prognostic factors of HBV-DeCi, and Kaplan–Meier analysis was performed to draw survival curves of sCD206. The predictive value of sCD206 was assessed at three time points according to the AUROC. Results The serum sCD206 level was significantly higher in deceased patients than surviving patients. Multivariate analysis showed that the level of sCD206 was related to an increased risk of 28-day, 3-month, and 6-month mortality (HR = 3.914, P < 0.001; HR = 3.895, P < 0.001; and HR = 4.063, P < 0.001, respectively). Patients with higher sCD206 levels had a worse prognosis than those with lower sCD206 levels. The best separation between the decedents and survivors was obtained by using the sCD206 level (AUROC: 0.830, 0.802, and 0.784, respectively) at 28 days, 3 months, and 6 months. Conclusion The macrophage-related marker serum sCD206 was associated with mortality in HBV-DeCi patients. High levels of serum sCD206 indicated a poor prognosis in these patients. Serum sCD206 has great predictive value for short-term and midterm mortality compared with the Child-Turcotte-Pugh (CTP) and model for end-stage liver disease (MELD) scores.


Introduction
Hepatitis B virus (HBV) infection is a serious global health problem; approximately 257 million people worldwide are chronic HBV surface antigen (HBsAg) carriers, of whom 78 million reside in China [1]. HBV infection is related to acute and chronic liver hepatitis, liver cirrhosis, and hepatocellular carcinoma (HCC) [2]. Decompensated cirrhosis (DeCi) is a common and serious liver disease that develops mainly in chronic hepatitis B (CHB) patients in Asia and is a disease state in which patients with cirrhosis develop variceal bleeding, ascites, hepatic encephalopathy (HE), or jaundice [3]. Once decompensation occurs, cirrhosis becomes a systemic disease with multiorgan dysfunction [4]. The median survival time drops from more than 12 years for the compensated state to approximately 2 years for the decompensated state due to these events [5]. The only curative therapy for DeCi is liver transplantation [6]. However, the shortage of inadequate social support and donor livers has limited the clinical application of liver transplantation. Therefore, it is crucial to screen patients with a high mortality risk as early as possible, which can help tailor the treatment strategy, early intervention, or even early transfer to an intensive care unit.
Activated macrophages are associated with the pathogenesis of liver fibrosis [7]. Macrophages can be activated under the stimulation of chronic and acute inflammatory reactions, and they are polarized into M1 and M2 macrophages [8]. CD206, a mannose receptor, is primarily expressed on the surface of macrophage cells, where it acts as a pattern recognition receptor [9]. Soluble CD206 (sCD206) is the soluble form of CD206, and the plasma concentration of sCD206 is increased in liver cirrhosis [10,11]. An increase in the soluble levels of CD206 has been confirmed to be a circulating marker of monocyte/macrophage activation in cirrhosis patients [11], and the shedding of sCD206 is promoted by pathogen-associated molecular patterns (PAMPs), including lipopolysaccharide [12]. In this study, we tested serological levels of sCD206 in HBV-DeCi patients and assessed whether serum sCD206 had an association with the mortality of such patients.

Study Population.
We assessed 382 patients diagnosed with HBV-DeCi at the First Affiliated Hospital of Nanchang University between February 2020 and February 2021. All patients were hospitalized for acute decompensation events. The research was approved by the Ethics Committee of our institution and was consistent with the Declaration of Helsinki. Written informed consent was obtained from all patients. The inclusion criteria were as follows: patients (1) were more than 18 years old, (2) were hepatitis B surface antigen positive for longer than 6 months, (3) were diagnosed with decompensated cirrhosis, and (4) had available follow-up data. The exclusion criteria were as follows: (1) patients with alcoholic liver disease, drug-induced liver injury, autoimmune hepatitis, or other viral infections (hepatitis A, C, or E or HIV infections); (2) complications with hepatocellular carcinoma; (3) complications with other malignancies; and (4) no specific test results.

2.2.
Definitions. The diagnosis of CHB was based on HBsAg positivity or detectable HBV DNA for more than six months. The diagnosis of DeCi was defined by radiologic evidence, biochemical results, or biopsy, including complications of cirrhosis, such as ascites, gastrointestinal bleeding, HE, spontaneous bacterial peritonitis (SBP), or hepatorenal syndrome. The Child-Turcotte-Pugh (CTP) score was calculated based on prothrombin time (PT), ascites, levels of albumin and serum bilirubin, and HE [13]. The model for end-stage liver disease (MELD) score was computed based on the formula: MELD = 9:6 × ln ½ serum creatinine ðScrÞ ðmg/dlÞ + 3:8 × ln ½total bilirubin ðmg/ dlÞ + 11:2 × ln ½prothrombin time ðinternational normalized ratio ðINRÞÞ + 6:43 × ðetiology : 0 if cholestatic or alcoholic, 1 otherwiseÞ [14]. The diagnosis and grading of ascites were defined according to the criteria proposed by the International Ascites Club [15]. HE was classified according to the practice guidelines of the European Association for the Study of the Liver (EASL) and American Association for the Study of Liver (AASLD) [16].
2.3. Determination of Plasma sCD206 Levels. Serum samples were stored at −80°C after collection from centrifuged peripheral blood. The sCD206 levels were determined using an enzyme-linked immunosorbent assay (ELISA) kit (Ray-Biotech, Norcross, GA, USA) according to the manufacturer's instructions.

Study Protocols.
Physical examination, laboratory test, demographic, treatment history, and radiological examination data were acquired from electronic medical records. All blood tests were measured within 24 hours of admission. The sCD206 results were collected from a datasheet generated by laboratory professional staff. Overall survival (OS) was defined as the duration from admission to death or the last follow-up time (6 months).

Management of Patients.
DeCi resulting from HBV was immediately treated with nucleoside analogs (entecavir alone 0.5 mg, lamivudine alone 100 mg, telbivudine alone 600 mg, or lamivudine 100 mg plus adefovir 10 mg daily). Patients with acute variceal bleeding were managed according to the Baveno VI consensus guidelines [17]. Patients with bacterial infection were immediately treated with empirical antibiotic therapy,   A multivariable Cox regression model was applied to identify predictive factors for overall survival, including both correlated predictive factors at univariable analysis (P < 0:050) and clinically relevant variables. The survival curves were calculated using the "rms," "survminer," and "survival" packages in the R software, of which the ggsurvplot function was used to perform the K-M survival curve. The ROC curves were calculated using the "survminer," "survival," "timeROC," and "pROC" packages in the R software. The Delong test was used to compare the AUROCs by the Med-Calc software. The reported statistical significance levels were all two-sided, with significance set at 0.050.

Demographic and Clinical
Features. As depicted in Figure 1, according to the criteria set at the beginning of our study, we finally included 382 patients who were diagnosed with HBV-DeCi. The demographic and clinical features of the included patients are shown in Table 1 Table 2. HBV-DeCi patients were distributed into nonsurviving and surviving groups according to their 28-day, 3month, and 6-month outcomes. The two groups significantly differed in bilirubin level, WBC count, PT, INR, levels of creatinine, ALT, AST, albumin, GGT, and ALP, CTP score, MELD score, and sCD206 level at all time points (all P < 0:050). Compared to the surviving group, the nonsurviving group had a higher bilirubin level, WBC count, PT, INR, level of creatinine, ALT, AST, GGT, and ALP, CTP score, MELD score, and sCD206 level and a lower albumin level (all P < 0:050). No significant differences in platelet counts or levels of MAP, PO 2 /FiO 2 , or serum sodium were observed at any follow-up time (P > 0:050).

Disease Markers
albumin, and sCD206, were significantly associated with overall survival at all follow-up times. All the significant factors found in the univariate analysis were included in the multivariable Cox regression analysis; the result revealed that the four variables, i.e., creatinine, ALP, albumin, and sCD206, were identified as independent prognostic factors of HBV-DeCi at any time point (Table 3, Figure 2). Figure 3, Kaplan-Meier analysis was performed to evaluate sCD206 in HBV-DeCi patients, and patients with high sCD206 expression had a poorer prognosis than those with low sCD206 expression (P < 0:001). The cutoff value of sCD206 was 0.621 according to the data analysis.    Disease Markers (Table 4, Figure 4). As shown in Table 5, the predictive performance of sCD206 was significantly higher than that of the CTP and MELD scores at all time points (P < 0:050). The comparisons of the AUROC between sCD206 and the two scores are shown in Figure 5.

Discussion
Our study demonstrated that the serum sCD206 level was increased significantly in HBV-DeCi patients and that it helped distinguish nonsurvivors from survivors. sCD206 was an independent risk factor for the prognosis of such patients, and it has better performance than the CTP and MELD scores for predicting outcomes in HBV-DeCi patients.
As described previously, DeCi is marked by the development of overt clinical signs, the most frequent of which are bleeding, ascites, jaundice, and encephalopathy. Once these signs occur, the disease usually progresses more rapidly toward liver transplantation (LT) and even death. Therefore, the grave prognosis in HBV-DeCi patients mandates the identification of better prognostic indicators. At present, many prognostic models have been used to predict the outcome of DeCi patients, such as the CTP score and MELD score. The CTP score is easy to calculate but includes two subjective variables, namely, HE and ascites. A previous study showed that the MELD score had good accuracy in predicting mortality in DeCi patients [18]; however, these scores do not contain markers that reflect disease progression or pathogenesis, so identifying new markers that can effectively predict outcome is necessary.
Liver macrophages represent >80% of the total macrophage population and play a key role in the development and progression of liver inflammation and fibrosis [19,20]. Macrophage activation by pathogen-associated molecular   7 Disease Markers patterns (PAMPs) and damage-associated molecular patterns (DAMPs), interferon-γ (IFN-γ), and the cytokine interleukin-12 (IL-12) are known to produce a proinflammatory response in macrophages, resulting in the release of IL-1β, IL-6, IL-12, tumor necrosis factor (TNF), and reactive oxygen species [21,22]. Moreover, the presence of an IFNL3-IFNL4 haplotype resulting in the production of IFN-γ3 is considered a promoter of fibrosis progression and hepatic inflammation [23]. CD163 was the hemoglobin-haptoglobin scavenger receptor highly expressed by macrophages; our previous study had confirmed that the plasma soluble CD163 had well predictive value in predicting outcome in DeCi patients [24]. As the other scavenger receptor, CD206 is also highly expressed by macrophages and is known as the mannose receptor [25]. CD206 is the soluble form and exists in culture media from human dendritic cells, human macrophages, and human serum [26]. As a  Disease Markers macrophage activation marker, sCD206 mediates a series of immune responses that recognize, phagocytose, and clear pathogens. A recent study showed that the levels of serum sCD206 stemmed from liver macrophages [26]. sCD206 may play important roles in the immune process of serious disease. Zou et al. found that the macrophage activation marker sCD206 was associated with the mortality of idiopathic pulmonary fibrosis patients [27]. Recently, it was confirmed that sCD206 was elevated in patients with acute-on-chronic liver failure (ACLF) and related to disease severity [28]. Previous studies have shown that sCD206 is significantly elevated in patients with cirrhosis compared to healthy individuals and increases with increased liver disease severity, and among patients with alcoholic cirrhosis, the sCD206 level predicts portal hypertension [12]. If portal pressure exceeds a certain threshold, the patient is at risk of developing life-threatening bleeding from varices. In addition, a previous study showed that CD206 expresses amphiregulin to promote regulatory Tcell activity and subsequently restrain CD8 T-cell-mediated antiviral function in a mouse HBV model, which may accelerate disease progression [29]. Importantly, sCD206 is stable during freezing and thawing and easily obtainable, and both commercial and house ELISAs are available, which makes them ideal biomarkers for use in daily clinical practice. Our study has several limitations. First, a validated cohort was lacking to confirm the findings. Second, the dynamic changes in serum sCD206 should be determined during the progression of HBV-DeCi. Third, multicenter studies with large sample sizes are required to confirm the current findings.
In conclusion, serum sCD206 levels are significantly elevated in nonsurviving HBV-DeCi patients, and sCD206 expression may be a crucial prognostic indicator in such patients. In addition, sCD206 has better discriminative power than MELD or Child-Pugh score for assessing short-term and midterm mortality. The present findings collectively suggest that the sCD206 level may be a predictor of prognosis in patients with HBV-DeCi.

Data Availability
The data that support the findings of this study are available from the corresponding author, X.Z., upon reasonable request.