The Relativity Study On Phenotypes And Function Of Peripheral Monocyte With Immune Dysfunction Of Chronic Hepatitis B Patients

Chronic Hepatitis B (CHB) is a kind of severe contagious diseases.There are about 30 million CHB patients in our country. Up to now, thereare no effective therapies to treat this disease. The mechanism of HBVcontinuing infection is very complicated and not fully explained. One ofthe main reasons is that the function of immune system is impaired whenthe host is infected by HBV. One feature of HBV lasting infection is thefunctional impairment of T cell. The activation of T cell needs two kindsof signals, both of which are provided by APCs (antigen presenting cell).Both of macrophage and DC belong to APC, however only DC can activate na(i|¨)veT cell. Many studies have confirmed that monocyte is the precursor ofmacrophage and DC. Recent study suggest that monocyte can influence thepolarization and compassion of lymphocytes, and it can also shape thereaction of primary and memorial T cell of human and mouse.Monocyte is not only important in acquired immunity, but also in innateimmunity. Monocytes can recognize pathogen patterns with variousToll-like receptors(ThRs),such as TLR2 and TLR4 on the surface, and TLR3inside the cell. The activation of TLRs can induce cellular signaling inMo and therefore promote the production and secretion of related cytokines,such as TNF-α,IL-6,IL-10和IL-12, to regulate the immune response. Asa reason of that, the study of monocyte in CHB pathogenesis is veryimportant. However, the systemic research materials about monocyte andHBV chronic infection are still limited.The aim of my research is to investigate the relationship betweenmonocyte and the insufficiency of immunity in HBV chronic infection. Thestudy will focus on the three main functions of monocyte, which are antigenpresenting, inflammation reaction and immune regulation. PARTⅠPhenotype and subsets of peripheral monocyte fromchronic hepatitis B patientsObjectiveTo investigate the association phenotype and subsets of monocyteswith chronic hepatitis B.Method20 chronic hepatitis B patients were included in the CHB group, andthe twelve healthy were included in the control group. The expression ofTLR2 and TLR4 of monocyte was subjected to flow cytometric analysis. Andthe subsets of monocyte were analysed through flow cytometric analysis.ResultsThe percentage of blood monocyte in CHB group were 8.53±2.39%, Whichis higher than 6.22±1.16% of the N group; The percentage of CD14lowCD16+subsets were higher in CHB group (14.56±7.31%), than that of Ngroup(10.47±4.80%) (p＜0.05);TLR2: The percentage of TLR2 in CHB group were 77.15±13.78%,and MFIwas 1358.12±612.49; the percentage of monocyte TLR2 expression in Ngroup were 77.02±8.31%,and MFI was 1322.15±251.02; There is nosignificant difference between the two groups (p＞0.05).TLR4: The percentage of monocyte in CHB group were 76.88±10.27%, MFIwere 700.9±428.75; the percentage of monocyte in N group were 77.84±6.25%, MFI were 614.0±235.64; There were no difference between the twogroups (p＞0.05).Conclusion1. The positive correlation was found between elevated of percentagesof monocyte and HBV chronic infection. 2. There were no obvious relationship between the expression of TLR2,TLR4 of Mo and the dysfunction of T cell. PARTⅡThe expression of PD-H1 and CD155 of monocyte ofchronic hepatitis B patientsObjectiveTo investigate the association of of mRNA levels of B7-H1 and CD155with the function of T cell.MethodsThe research consisted of two groups: chronic hepatitis B group(CHBgroup) and normal group( N group). There were 16 CHB patients in the CHBgroup. They have not accepted any anti-virus or immune modulationtherapies in 6 months. And they have no other liver diseases or autoimmunediseases. The N group contains 10 healthy people. Monocytes were extractedfrom the peripheral blood, then these monocytes were stimulated by LPS.Mo were collected on 0h,6h and 24h post-stimulation. The level of BT-H1and CD155 mRNA were detected by Real-Time PCR.Results1. The level of BT-H1 mRNA:1.1. The mRNA level of B7-H1 were the same between CHB group(0.85±0.14)and N group(1.35±0.28) before the stimulation of LPS(p＞0.05): ThemRNA level of B7-H1 in normal group were 3179±1015, higher thanthat of CHB group(1709±598.9) (p＜0.05); The mRNA level of B7-H1in normal group were 1716±535.84 6 hours after stimulation, higherthan that of CHB group (1271±492.3) (p＜0.05);1.2. The mRNA level of B7-H1 were higer on 24 hours post-stimulation thanthat on 6 hours post-stimulation, the two groups are the same (p＜0.05).2. The level of CD155 mRNA:2.1. The mRNA level of CD155 were the same between CHB group(1.52±0.49)and N group(1.34±0.30) before the stimulation of LPS(p＞0.05); The mRNA level of B7-H1 in normal group were 1.31±0.09, higher than thatof CHB group(1.97±0.41) 6 hours after the LPS stimulation (p＜0.05); The mRNA level of B7-H1 in normal group were 6.30±1.30, higherthan that of CHB group(3.56±0.14) 24 hours after the LPS stimulation(p＜0.05);2.2. The mRNA level of CD155 were higher on 24 hours post-stimulation thanthat on 6 hours post-stimulation, the two groups are the same (p＞0.05).Conclusion1. There were no obvious relationship between the expression ofBT-H1,CD155 and the dysfunction of T cell;2. After the stimulation, the reaction of BT-H1 were earlier thanCD155 in bouth group; These two inhibitory receptors maybe take effectat different time. PARTⅢThe function of monocyte after stimulatin of LPS inchronic hepatitis B patientsObjectiveTo investigate the function of monocyte in HBV chronic infection andthe effect on T cell.MethodsThere were two groups: CHB group and N group. The CHB group contains16 patients. They have not received any anti-virus or immune modulationtherapies in 6 months. These patients have no other liver diseases andautoimmune diseases, 10 healthy people were included in N group. Themonocyte were extracted from blood. The protein level of TNF-α,IL-6,IL-10 and IL-12 were detected with ELISA.ResultsBefore the LPS stimulation, the concentration of TNF-αwas 38.83±7.69 pg/ml in CHB group, and 35.40±7.72 pg/ml in N group(p＞0.05);6 hours after the stimulation, the concentration of TNF-αwereelevated, and N group were 10320±51.05 pg/ml, higher than that of CHBgroup(5654±1350 pg/ml) (p＜0.05); 24 hours after the stimulation, theconcentration of TNF-αwere 14770±780.0 pg/ml, higher than that of CHBgroup(6384±175.9 pg/ml) (p＜0.05);Before the LPS stimulatin, the production of IL-6 in CHB group 1.804±0.60 pg/ml)was just the same as that of N group(1.103±0.76 pg/ml); Sixhours after the stimulation, the concentration of IL-6 higher in Ngroup(1069±26.93 pg/ml), higher than that of CHB group(1133±68.91pg/ml) (p＜0.05);Before the LPS stimulatin, the concentration of IL-10 were the samein both groups, N group were 13.14±4.39 pg/ml and CHB group were 4.61±2.01 pg/ml;After the stimulation, the concentration of IL-10 were higher on 6 hour, but there were the same between the two groups(p＞0.05).The N group were 861.4±157.8pg/ml, and CHB group were 380.4±66.4pg/ml. The level were 2214±557.2pg/ml in N group 24 hourspost-stimulation, the same as that of the CHB group(4241±914pg/ml) (p＞0.05).Before the LPS stimulatin, the production of IL-12 were the same inboth groups, N group were 9.78±0.65pg/ml and CHB group were 8.87±1.70pg/ml; After the stimulation, the production of IL-12 were higher on 6hour, but the same between the two groups(p＞0.05).The N group were 21.70±13.23pg/ml, and CHB group were 21.40±2.96pg/ml. The concentrationwere 16.80±1.56pg/ml in N group 24 hours post-stimulation, the same asthat of the CHB group(11.03±0.37pg/ml) (p＞0.05).Conclusion1. The monocyte can produce TNF-α,IL-6 and IL-10 after thestimulation of LPS, and the concentration were elevated during timecourse.2. The function of monocyte were impaired when HBV were chronicallyinfected, TNF-αand IL-6 were reduced after the stimulation of LPS whencompared with healthy group;3. There were no changes of IL-10 and IL-12 when HBV were chronicallyinfected, this suggest that the function of T cell were not influencedby monocyte through the production of cytokines IL-10 and IL-12.