| Objective: Hepatitis B virus (HBV)-related acute-on-chronic liver failure (ACLF) is a sudden, life-threatening syndrome that is characterized by severe liver dysfunction and variant complications as a result of excessive hepatocytes necrosis and apoptosis due to HBV infection. It accounts for more than 80% of all etiologies of liver failure in China. The mechanisms of HBV-related ACLF remain extremely unclear. It is proposed that dysregulation of host immune responses due to interaction between the virus and the host immune systems may substantially play an important role in its pathogenesis. In the present study, we investigated the kinetic characteristics of circulating CD4+CD25+ regulatory T cells (Treg), the expression and the distribution of intrahepatic Foxp3+ regulatory cells, as well as serum levels of interferon-γ(IFN-γ) and interleukin-10 (IL-10) in patients with HBV-related ACLF, and we further explored the relationships between CD4+CD25+ Treg and the pathogenesis and the prognosis of HBV-related ACLF.Methods: Thirty-two patients with HBV-related ACLF, 44 patients with chronic hepatitis B (CHB) and 41 healthy blood volunteers were recruited consecutively. Moreover, liver tissues were obtained from 6 ACLF patients and 6 liver donors during liver transplantations. The diagnoses of HBV-related ACLF and CHB were according to the Consensus Recommendations on Acute-on-chronic Liver Failure issued by Asian Pacific Association for the Study of the Liver, the Diagnostic and Treatment Guidelines for Liver Failure (2006) and the Guidelines on Prevention and Treatment for Chronic Hepatitis B in China (2005) issued by the Chinese Society of Hepatology and the Chinese Society of Infectious Disease, respectively. Liver function parameters, coagulation function parameters, serum virology, and serum HBV DNA loads were determined. The peripheral frequencies of CD4+CD25+ Treg were detected by flow cytometry, and their dynamic changes in ACLF patients were also observed. Immunohistochemical staining was performed to illustrate the amounts and the distributions of Foxp3+ regulatory cells. The Foxp3 mRNA expressions in peripheral blood mononuclear cells (PBMCs) and liver tissues were detected by reverse-transcription polymerase chain reactions. The serum IFN-γand IL-10 concentrations were determined by enzyme-linked immunosorbent assays. Moreover, we investigated the differences and the dynamic changes of circulating CD4+CD25+ Treg between the survival group and the non-survival group, and we also explored the relationships between the circulating CD4+CD25+ Treg frequencies and the MELD scores and the prognosis of liver disease.Results:1 Demographic and clinical characteristics of the studied subjectsAll studied subjects were comparable for age and sex. The ACLF patients displayed significantly higher levels of alanine transaminase (ALT), aspartate transaminase, total bilirubin, direct bilirubin, and creatinine, and international normalized ratio (INR) than CHB patients and healthy controls (all P < 0.05). There were no significant differences in HBV DNA loads and percentage of positive HBeAg between ACLF patients and CHB patients. With respective to the occurrence of severe complications in total ACLF patients, there were 9 having hepatic encephalopathy, 11 having spontaneous bacteria peritonitis, 5 having hepatorenal syndromes, and 2 having variceal bleeding.2 Increased peripheral CD4+CD25+ Treg frequencies in patients with ACLFPatients with HBV related-ACLF had a significantly higher frequency of peripheral CD4+CD25+ Treg (6.33%±1.49%) than CHB patients (vs. 4.16%±1.31%) and healthy controls (vs. 2.20%±0.69%) (all P < 0.001). Similarly, there is a significant difference in circulating proportion of CD4+CD25+ Treg between CHB patients and healthy controls (P < 0.001). In addition, ACLF patients with complications (6.51%±1.50%) displayed higher levels of CD4+CD25+ Treg than patients without any complications (vs. 5.94%± 1.42%), but there was no statistical significance (P = 0.55).3 ACLF patients had enriched intrahepatic infiltration of Foxp3+ regulatory cellsThe highest mean frequency of Foxp3+ cells were detected in liver sections from the patients with ACLF (14.7±3.6/hpf) compared with CHB patients (vs. 4.6±1.4/hpf) and healthy controls (vs. 0.4±0.2/hpf), respectively (all P < 0.001). The distribution of Foxp3+ regulatory cells were predominantly seen in the portal areas, and occasionally scattered in the hepatic sinusoids, where exhibited lymphocytes accumulation and inflammation.4 Foxp3 mRNA expressions are up-regulated in both PBMCs and liver tissuesThe relative Foxp3 mRNA levels in PBMCs from ACLF patients, CHB patients, and healthy controls were 0.78±0.16, 0.41±0.12, and 0.21±0.06, respectively (all P < 0.05 for comparisons between groups); while the relative levels of Foxp3 mRNA in liver tissues were 1.55±0.15, 1.00±0.31, and 0.17±0.04, respectively (all P < 0.05 for comparisons between groups).5 Peripheral CD4+CD25+ Treg frequencies were positively correlated with HBV replication and the disease severity in ACLF patients In both ACLF patients and CHB patients, there was a positive correlation between circulating frequencies of CD4+CD25+ Treg and serum HBV DNA loads (ACLF patients: r = 0.36, P = 0.04; CHB patients: r = 0.62, P < 0.001). However, no correlation was found between circulating CD4+CD25+ Treg and serum ALT levels and HBeAg/anti-HBe status. Moreover, circulating CD4+CD25+ Treg frequencies in ACLF patients were positively correlated with INR (r = 0.64, P < 0.001) and the MELD sore (r = 0.37, P = 0.04).6 Imbalanced serum IFN-γ/IL-10 ratio and a positive correlation between IL-10 level and peripheral CD4+CD25+ Treg in ACLF patientsThe serum IFN-γand IL-10 levels in ACLF patients (0.96±0.37 pg/ml; 0.77±0.33 pg/ml) was significantly higher than CHB patients (vs. 0.40±0.17 pg/ml; 0.48±0.27 pg/ml, all P < 0.001) and healthy controls (vs. 0.37±0.17 pg/ml; 0.54±0.38 pg/ml, all P < 0.001), but no significant difference was found between CHB patients and healthy controls. The imbalanced ratio between IFN-γand IL-10 was found in ACLF patients, and was much higher than those in CHB patients and healthy controls. There was a positive correlation in ACLF patients between serum IL-10 level and peripheral CD4+CD25+ Treg (r = 0.41, P = 0.02).7 Kinetic changes of peripheral CD4+CD25+ Treg in ACLF patients with different outcomesAccording to the outcomes of 30-day observation, the studied ACLF patients were divided arbitrarily into survival group and non-survival group. The non-survival group had a initially higher circulating CD4+CD25+ Treg frequency compared with the survival group (6.68%±1.32% vs. 5.44%±1.18%; P < 0.05), and showed an increasing trend with mean CD4+CD25+ Treg frequencies of 7.23%±1.34%, 7.79%±1.20%, 8.82%±0.91%, and 8.91%±0.18% on day 7, 14, 21, and 28, respectively. In contrast, the survival group exhibited a decreasing trend with values of 5.53%±1.28%, 5.28%±1.03%, 4.45%±0.87%, and 3.47%±0.61% at above mentioned time points.Conclusions:1 The patients with HBV-related ACLF exhibited increased circulating CD4+CD25+ Treg frequencies and intrahepatic Foxp3+ regulatory cell accumulations.2 The peripheral CD4+CD25+ Treg have been associated with HBV replication and more severe degrees of liver disease. The baseline level of peripheral CD4+CD25+ Treg and their kinetic modes during treatment may serve as markers for the short-term prognosis of HBV-related ACLF.3 HBV-related ACLF patients showed imbalanced IFN-γ/IL-10 ratio, and serum IL-10 was positively correlated with peripheral CD4+CD25+ Treg frequencies.4 Immune-modulation treatment may provide potential strategies for the interventions of HBV-related ACLF.
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