The Characteristics And Mechanism Of Autophagy In CD4+T Cell During Hepatitis B Virus Chronic Infection

BackgroundCD4+T cells play important roles in the process of chronic HBV infection, with different lineages exerting diverse immune-regulation. In our early researches, Treg/Th17 ratio decreased along with the enhanced liver injury and fibrosis level in CHB patients, and furthermore, activated hepatic stellate cells (HSCs) modulated the differentiation and plasticity of CD4+T cells. On the other hand, T lymphocyte can also be modulated by itself, which are mainly executed by programmed cell death pathway like apoptosis and autophagy. Autophagy is defined as a process that intracellular membranes wrap part of cytoplasm, domestic organelles and proteins, which are to be degraded, to form autophagosome, and then lysosome and autophagosome fuse to be autolysosome, finally leading to degradation of their parcels material, in order to supply cell metabolic needs and organelle update. T lymphocytes and their subsets can achieve immune homeostasis through autophagy. Till now, the characteristics of autophagy have been demonstrated in parenchyma cells in tumorigenesis, autoimmune disease, viral infection, and so on. Recently, the phenomena of peripheral CD4+T cells has been found in HIV infection, however, it is not clear that whether the changes of CD4+T cells’autophagy exist in the process of chronic HBV infection and the possible factors and related signaling pathways which may be involved in the occurrence of autophagy.Methods1. The autophagy of peripheral CD4+T cells exist in the process of chronic HBV infectionClinical parameters, blood and liver biopsy specimens were collected from HBV carrier, CHB patients and liver cirrhosis (LC) patients. Peripheral blood mononuclear cells (PBMC) were isolated and purified CD4+T cell were collected by flow cytometry. Autophagosome ultrastructure was observed by using electron microscopy and the expression and localization of LC3B in CD4+T cells were detected by using immunofluorescence. The quantitative autophagy expressions of CD4+T cells were analyzed by multi-color flow cytometry using antibodies (CD4 and LC3B), and furthermore, the expressions of assosiated autophagy proteins including LC3B II/LC3I, Atg5, Atg7, Beclin-1 and p62 were tested by western blot.2. The related factors in modulating the autophagy of CD4+T cells in vitroSerum and hepatic HMGB1 expression in the process of chronic HBV infection were tested by ELISA and immunohistochemistry. The variant autophagy of CD4+T cells were evaluated by flow cytometry and electron microscopy after HBcAg and rHMGB1 stimulation to mimic viral and inflammatory risk factors in the process of chronic HBV infection. Based on the intricate relationship between autophagy and apoptosis, we also test the apoptosis of CD4+T cells by AnnexinV-PI staining.3. The related signal pathway in HMGB1-induced autophagy of CD4+T cellsWith or without HMGB1 stimulation, the protein expressions of HMGB1 receptors including TLR2, TLR4 and RAGE on CD4+T cells were analyzed by flow cytometry. And then, the possible intracellular downstream signaling of HMGB1 was further tested by adding related receptor neutralizing antibody.Results1. The autophagy of peripheral CD4+T cells existed in the process of chronic HBV infectionFirst, by using electron microscopy, autophagosome ultrastructure was observed in peripheral CD4+T cells of patients with chronic HBV infection, as well as the expression and localization of LC3B in CD4+T cells by immunofluorescence. Peripheral LC3/CD4 ratio, defined as the ratio of autophagy activity occurring in CD4+T cells, significantly increased during the chronic process of HBV infection, however, the ratio decreased in LC patients when compared with which in CHB patients. In consistent with the flow cytomety results, the expressions of associated autophagy protein including LC3 II/LC3I, Atg5, Atg7 and Beclin-1 enhanced along with inflammation and fibrosis progression in CHB patients and were down-regulated in LC patients, whereas autophagic degradation protein p62 presented the opposite trend.Further subgroup analysis found high HBV viral load and degree of inflammation indicated enhanced autophagy activity of CD4+T cell, while autophagy didn’t change significantly with HBeAg expression in spite of suggesting viral replication to some extent. Moreover, the autophagy of CD4+T cell increased with advanced stages/grades classifying hepatic inflammation and fibrosis degree in chronic hepatitis. However, there is no statistically significant increase between S2～3 and S4. And in LC patients, the autophagy of peripheral CD4+T cells also enhanced with deteriorated Child stages classifying liver function.2. Exogenous HMGB1 enhanced autophagic activities of CD4+T cellsIn CHB patients, there existed a significantly positive correlation between serum HMGB1 levels and the autophagy of CD4+T cells, suggesting that HMGB1 may be an important factor affecting autophagy. TEM detection and flow cytometry results showed HBcAg or HMGB1 alone stimulation could enhanced the autophagy of CD4+ T cells and the latter may be more potent, furthermore, the sequential stimulation with HBcAg and HMGB1 presented the superimposed effects on the autophagy. Besides, there were no significant differences of apoptotic activity between control and each stimulation group.3. RAGE-dependent JNK/ERK signaling was involved in HMGB1 induced autophagy of CD4+T cellsThe expressions of TLR2、TLR4 and RAGE on CD4+T cell surface from CHB patients at diverse fibrosis stages did not change significantly, however, after simulation with HMGB1, the expression of RAGE increased significantly. Pretreated with RAGE neutralizing antibody, HMGB1 enhanced autophagy activity of peripheral CD4+T cells decreased. Moreover, HMGB1 enhanced intracellular signaling expression of phosphorylated JNK/ERK but did not influence the expressions of p38MAPK and AKT, and the pretreatment with RAGE neutralizing antibody also inhibited the enhanced effect of HMGB1 on the expressions of JNK/ERK.ConclusionsThe autophagy of peripheral CD4+T cells existed in the process of chronic HBV infection by observing autophagosome ultrastructure using TEM and the expression and localization of LC3B using immunofluorescence. The autophagy activity of peripheral CD4+T cells exacerbated along with the enhanced level of serum viral replication, grades of inflammation and stages of fibrosis in CHB patients, and meanwhile, exacerbated along with the enhanced Child-pugh grades in LC patients. Both HBV itself (HBcAg) and inflammatory factors (HMGB1) can efficiently enhance the autophagy of peripheral CD4+T cells in CHB patients, especially the latter, and present the synergistic effects but show no significant effect on the apoptosis. Furthermore, RAGE-dependent JNK/ERK signal pathway was involved in HMGB1-induced autophagy of peripheral CD4+T cells in CHB patients.