Variation And Significance Of CD4~+ CD25~+ Regulatory T Cells In Chronic Hepatitis B Patients Complicated With Hepatic Steatosis

Objective: The role of CD4+ CD25+ regulatory T cells (Tregs) in chronic hepatitis B (CHB) patients who complicated with hepatic steatosis has not been clarified. In this study, Tregs in the peripheral blood as well as in the liver were detected, and the correlation between circulating CD4+ CD25+ Tregs frequency and serum HBV DNA levels, liver inflammation was evaluated. All the data were compared with those in CHB and normal controls, further more, to investigate the effect and significance of hepatic steatosis to Tregs in CHB patients. Together, the results would indicate a new idea for immune pathogenesis and treatment of CHB effectively.Methods: 30 patients in our hospital from April 2008 to December 2008 were enrolled in this study, including 10 patients with CHB plus hepatic steatosis and 20 with simple CHB. Pathologic analysis of liver tissue was performed in all the patients, and no one had received anti-HBV agent or immunoregulant 6 months before sampling. The diagnoses were complied with the diagnostic criteria of the 2000 Xi'an Viral Hepatitis Management Scheme issued by the Chinese Society of Infectious Diseases and Parasitology, and hepatic steatosis was confirmed by liver biopsy. 19 healthy individuals were chosen as controls.Serum ALT was measured using an Olympus AU2700 auto-biochemical analyzer. HBV markers, anti-HAV, anti-HCV, anti-HDV, anti-HEV and anti-HIV were detected by commercial enzyme immunoassay kits respectively.Peripheral heparinized blood samples were collected from all the enrolled individuals. For staining of CD4+ CD25+ Tregs, FITC-anti-CD4 and PE-anti-CD25 were used. After all the red blood cells were dissolved, 0.02M phosphate buffer saline (PBS) was used to wash the blood samples, then the remaining cells were fixed in paraformaldehyde-PBS and the frequency of CD4+ CD25+ T cells were detected by flow cytometric analysis, gated from lymphocytes.Haematoxylin-eosin (HE) and Masson staining were used for the general pathologic observation of liver and hepatic fibrosis respectively. Immunohistochemical staining (Power VisionTM method) was used to measure the expression of Foxp3. Positive expression was brownish yellow. For the enumeration of positive cells, Foxp3+ cells was counted in 5 high-power fields (×400) for each sample, then, the average value was taken.All data were analyzed by SPSS version 11.5 for Windows software. Kruskal-Wallis H test or Mann-Whitney U test was used for comparison between groups. Chi-square test was used for the comparison of constituent ratio between groups. Spearman correlation analysis was performed between the frequency of CD4+ CD25+ Tregs and HBV DNA load.Results: 1 ALT was raised up in CHB patients who complicated with hepatic steatosis and simple CHB patients. ALT and HBV DNA were similar between the two groups (P>0.05); ALT in the control group was normal (<40U/L), and serum HBV DNA load was negative (<1×103copies/ml). 2 Circulating CD4+ CD25+ T cell frequency in CHB plus hepatic steatosis patients had not been described previously. We observed that the circulating CD4+ CD25+ T cell frequency in these patients was lower than CHB patients (U=36.0, P<0.01), but higher than normal controls (U=36.0, P<0.01); CD4+ CD25+ T cell frequency in high ALT group (ALT≥80U/L) was higher compared with low ALT group (ALT<80U/L), U=61.0, P<0.05. 3 Pathologic analysis of liver tissue were performed in 30 patients. The inflammatory grade (G) of hepatic tissue in 14 patients was G2, other 16 patients was G3. The stage of fibrosis (S) in 9 patients was S1, 11 patients was S2, other 10 patients was S3. The liver tissue of CHB patients presented hepatic cells degeneration and necrosis in the form of apoptotic bodies and spotty foci of necrosis surrounded by inflammatory cells. Piecemeal necrosis even bridging necrosis also could be observed in CHB patients. The portal tracts were expanded, with inflammatory cell infiltration and fibrosis. Except for this, there was different degree of steatosis in the liver of the CHB plus hepatic steatosis patients. Steatosis in cells surrounding the central veins was most obvious. The hepatocytes were enlarged and full of lipid droplets, and their nucleuses were off-normal. The inflammatory grade and stage of fibrosis in patients who had CHB plus hepatic steatosis were similar to CHB group (P>0.05). The infiltrating Foxp3 positive Tregs mainly accumulated near the portal tract, and some were distributed intra-lobar part with inflammation. In the normal liver, we could not find any positive cells. Foxp3 positive Tregs in the group of CHB plus hepatic steatosis were less than those in simple CHB group (U=19.0, P<0.01), but more than control group (U=0, P<0.01). G3 group exhibited a dramatic increased infiltration of Foxp3 positive cells compared with G2 group (U=32.0, P<0.01). 4 The CD4+ CD25+ T cell frequency was not correlated with HBV DNA level (rs=0.081, P>0.05).Conclusions: 1 The circulating CD4+ CD25+ T cell frequency of CHB patients was significantly higher compared with normal controls, and CD4+ CD25+ T cells in high ALT group were more than those in low ALT group. Foxp3 positive Tregs in G3 group were more compared with G2 group. All the evidence indicated that CD4+ CD25+ Tregs had a close relation with hepatic inflammation. 2 ALT, HBV DNA, inflammatory grade and stage of fibrosis in patients who had CHB plus hepatic steatosis were similar to CHB group, under this circumstance, both the circulating CD4+ CD25+ T cell frequency and the number of Foxp3 positive Tregs in the liver were lower in CHB plus hepatic steatosis group compared with CHB group. The decreased Tregs may lead to a lower suppression of inflammatory response, further more, the progression of CHB would be accelerated. 3 The circulating CD4+ CD25+ T cell frequency was not correlated with HBV DNA level. It indicated that Tregs might have no effect on HBV DNA clearance.