The Study On Expression And Dynamic Changes Of The Frequencies Of Regulatory T Cells And The Levels Of Cytokines In HBV-related Acute-on-chronic Liver Failure Patients

ObjectiveThrough studyting expression and dynamic changes of the frequencies of regulatory T cells(Treg cells) in peripheral blood and the levels of proinflammatory cytokines and anti-inflammatory cytokines in serum in HBV-related acute-on-chronic liver failure (ACLF) initial therapy patients, so as to investigate clinical significance of Treg cells in peripheral blood and the levels of proinflammatory cytokines and anti-inflammatory cytokines in serum in HBV-related ACLF initial therapy patients progression.Method44 cases of HBV-related ACLF patients,46 cases of chronic hepatitis B patients and 20 cases of healthy control people were enrolled into their respective groups. Flow cytometry was applied to detect the frequencies of Treg cells, CD39+Treg cells, and CTLA-4+Treg cells in peripheral blood. ELISA(enzyme linked immunosorbent assay) was used to detect the levels of IL-1β, IL-6, IL-10, TGF-β1, and TNF-a in serum, and clinical parameters were investigated in the same time. Immunohistochemical method was adopted to detect intrahepatic Foxp3+ Treg cells expression in 5 cases of HBV-related ACLF patients,6 cases of chronic hepatitis B patients and 3 cases of healthy control people. The previous 44 cases of HBV-related ACLF patients were given a three weeks follow-up, the change trends of the frequencies of Treg cells, CD39+ Treg cells, CTLA-4+Treg cells in peripheral blood and the levels of IL-1β, IL-6, IL-10, TGF-β1, TNF-a in serum were observed dynamically.ResultsIn HBV-related ACLF group, the frequencies of Treg cells(5.69%±1.83%), CD39+ Treg cells(49.55%±25.01%) and CTLA-4+Treg cells(35.27%±12.28%) in peripheral blood were all higher than those in chronic hepatitis B group(4.56%±1.59%, P<0.01; 36.10%±20.65%, P<0.01; 28.99%±10.59%, P<0.01) and in healthy control group(2.64%±0.42%, P<0.01; 19.67%±9.09%, P<0.01; 24.52%±7.52%, P<0.01). In HBV-related ACLF group, intrahepatic Foxp3+ Treg cells expression in portal area (19.53hpf±9.47hpf) and hepatic lobular area(9.93hpf±2.51hpf) were both higher than those in chronic hepatitis B group(3.39hpf±1.12hpf, P<0.01; 0.72hpf±0.65hpf, P<0.01) and in healthy control group(0.22hpf±0.19hpf, P<0.05; 0.11hpf±0.19hpf, P<0.01). In HBV-related ACLF group, the levels of IL-1β(9.33pg/ml±6.85pg/ml), IL-6 (20.90pg/ml±23.44pg/ml), IL-10(8.48pg/ml±6.42pg/ml), TGF-β1 (426.23pg/ml±296.36pg/ml) in serum were all higher than those in chronic hepatitis B group(5.27pg/ml±5.45 pg/ml, P<0.01; 3.79pg/ml±4.09 pg/ml, P<0.01; 4.34pg/ml±2.50pg/ml, P<0.01; 181.91pg/ml±82.23pg/ml, P<0.01) and in healthy control group (2.20pg/ml±1.11pg/ml, P<0.01; 1.35pg/ml±1.55pg/ml, P<0.01; 2.83pg/ml±1.86pg/ml, P<0.01; 212.12pg/ml±108.24pg/ml, P<0.01), the levels of TNF-α(12.19pg/ml±10.32pg/ml) in serum were higher than those in healthy control group (6.67pg/ml±4.32pg/ml, P<0.05). The frequencies of CD39+ Treg cells in peripheral blood and the levels of IL-1βin serum in HBV-related ACLF interim group (53.83%±26.62%, P<0.05; 11.41pg/ml±7.64pg/ml, P<0.01) and late group (52.88%±23.24%, P<0.05; 9.08pg/ml±6.03pg/ml, P<0.05) were all higher than those in early group(25.44%±8.73%; 3.58pg/ml±2.55pg/ml). The levels of TGF-β1 in serum in HBV-related ACLF interim group(534.91pg/ml±347.19pg/ml) were all higher than those in early group (220.67pg/ml±86.88 pg/ml, P<0.05). In HBV-related ACLF group, the frequencies of Treg cells were positively correlated with the levels of Cre(r=0.333, P<0.05) and MELD score(r=0.388, P<0.01), the frequencies of Treg cells were negatively correlated with the levels of IL-1β(r=-0.382, P<0.01), the frequencies of CD39+ Treg cells were positively correlated with the frequencies of CTLA-4+Treg cells(r=0.487, P<0.01) and the levels of TGF-β1(r=0.325, P<0.05), the levels of IL-6 were positively correlated with the levels of IL-10(r=0.367, P<0.05), TBIL(r=0.470, P<0.01), Cre(r=0.454, P<0.01), MELD score(r=0.520, P<0.01) and MELD-Na score (r=0.506, P<0.01), the levels of IL-6 were negatively correlated with Na+(r=-0.348, P<0.05), the levels of IL-10 were positively correlated with CHE (r=0.388, P<0.01) and MELD-Na score(r=0.438, P<0.01), the levels of IL-10 were negatively correlated with Na+(r=-0.523, P<0.01), the levels of TGF-β1 were positively correlated with CHE(r=0.320, P<0.05). In HBV-related ACLF incomplete observed group(n=13), the frequencies of Treg cells(6.72%±1.42%), the levels of IL-6 (38.43pg/ml±31.84pg/ml), MELD score(34.9±7.1) and MELD-Na score (40.62±10.89) were all higher than those in improved group(n=14) (5.39%±2.15%, P<0.05; 15.61pg/ml±16.87pg/ml, P<0.05; 24.2±4.3, P<0.01; 22.49±6.18, P<0.01) and deteriorated group(n=13) (4.57%±0.93%, P<0.01; 13.00pg/ml±11.99pg/ml, P<0.01; 24.1±2.5, P<0.01; 20.06±7.02, P<0.01). The baseline of the frequencies of Treg cells, CD39+ Treg cells, CTLA-4+Treg cells in peripheral blood, the levels of IL-1β, IL-6, IL-10, TGF-β1, TNF-αin serum, MELD score and MELD-Na score were not statistically significant difference in improved group and deteriorated group(P average >0.05). We had a dynamic observation in this short-term follow-up, in improved group, MELD score and MELD-Na showed gradually decreasing trends during improved condition with patients, the frequencies of Treg cells showed a gradually rising trend, the levels of IL-6 and TGF-β1 showed gradually decreasing trends, the levels of TNF-a showed a shortly rising and gradually decreasing trend. But in deteriorated group, MELD score and MELD-Na showed gradually rising trends during deteriorated condition with patients, the frequencies of Treg cells showed a shortly rising and gradually decreasing trend, the levels of IL-6 showed a gradually rising and shortly decreasing trend, the levels of TGF-β1 showed a gradually decreasing and shortly rising trend. In improved group and deteriorated group, the frequencies of CD39+Treg cells and CTLA-4+Treg cells in peripheral blood, the levels of IL-1βand IL-10 in serum were not show significant fluctuations.ConclusionHBV-related ACLF patients suffered from immune imbalance and dysfunction of proinflammatory cytokines and anti-inflammatory cytokines. The frequencies of Treg cells, CD39+ Treg cells, CTLA-4+Treg cells in peripheral blood and the levels of IL-1β, IL-6, IL-10, TGF-β1, TNF-αin serum had a significant increase. Baselines of the frequencies of Treg cells in peripheral blood, the levels of IL-6, IL-10 in serum were correlated with severity of HBV-related ACLF patients. High baselines of the frequencies of Treg cells in peripheral blood, the levels of IL-6 in serum of HBV-related ACLF patients had poor prognosis. Dynamic changes of the frequencies of Treg cells in peripheral blood and the levels of IL-6, TGF-β1, TNF-αin serum were correlated with prognosis of HBV-related ACLF patients. The expression and dynamic changes the frequencies of Treg cells in peripheral blood and the levels of proinflammatory cytokines and anti-inflammatory cytokines in serum can be used as additional tools in disease progression of clinical observation and short-term prognosis prediction in HBV-related ACLF patients.