Study On B Lymphocyte Counts And Expression Of Toll-like Receptor 7 And 9 MRNA In HIV-1 Infection

ObjectiveKnown as the most crucial participants that mediate anti-viral humoral responses, B lymphocytes demonstrate numerous abnormalities in HIV-infected patients,which represents the pathogenic characteristics of HIV infection.Remarkably,It is recently reported that B lymphocytes express impotant natural pattern recognition receptors, toll-like receptors(TLRs),such as TLR7 and TLR9.TLR7 mainly recognizes single-stranded RNA while TLR9 is involved in the recognition of CpGoligodeoxynucleotides which exist in the genome of microorganism.Activation of the signaling pathway of toll-like receptor potentially influences the immunologic function of B lymphocytes.Up to date,it is not clear that how B lymphocytes in peripheral blood from HIV-infected patients changes.There is no report on the counts of B lymphocytes from Chinese HIV-infected patients.It is unknown the expression levels of TLR7 and TLR9 in B lymphocytes from HIV-infected patients are and to what extent they correlate with the disease progression of HIV infection.In our study,we examined the counts of peripheral blood B lymphocytes and the expression of TLR7 and TLR9 mRNA from Liaoning untreated HIV-infected patients in China,which should be evaluated in HIV infection progression,thus contributing to understanding HIV pathogenesis and revealing potential new targets for therapy.Material and Methods1.Study subjectsHIV-infected patients were recruited from Liaoning province of China.All HIV-infected subjects(N=27) were confirmed to be HIV-1 antibody positive as determined by western blot test and was antiretroviral therapy naive before blood sample collection.Informed consents for collection of demographics,behavioural data and blood samples were obtained from all study subjects.According to the course of progression and CD4⁺T counts,two groups of subjects were classified:chronic HIV-infected subjects(HIV group) and AIDS(AIDS group).The individuals who have been infected with HIV-1 for more than 5 years with CD4⁺T cells more than 200 per microlitre and without defined symptoms were classified as HIV group.Those with CD4⁺T cell counts less than 200 per microlitre or with defined symptoms were classified as AIDS group.Sixteen HIV negative healthy donors were involved randomly without diseases.2.T lymphocytes and B lymphocytes counts and ratiosT lymphocytes or B lymphocytes counts were measured using 20μl of TriTEST CD4/CD8/CD3 or CD3/CD16+56/CD45/CD19 reagent with 50μl anti-coagulated whole blood into the bottom of the TruCOUNT Tube using reverse pipetting and incubate for 15 minutes in the dark at room temperature,add 450μI FACS lysing solution to the tube,incubate for 15 min in the dark at room temperature and analyze the samples on the flow cytometer,using FACS MULTISET software to acquire T lymphocyte and CD19⁺B lymphocytes count and corresponding ratios.3.Separation of PBMCs by density centrifugationPeripheral blood mononuclear cells(PBMCs) of subjects were isolated by standard density centrifugation.4.Sorting CD19⁺B lymphocytes by MACS methodCD19⁺B lymphocytes were sorted through magnetic activated cells sorting system using CD19-conjugated microbeads according to the manufacturer's instructions.The purity of B lymphocytes was more than 90%as determined by flow cytometry with anti-CD20 antibody.5.Preparation of cDNA of CD19⁺B lymphocytesPBMCs were extracted from EDTA anticoagulated blood of healthy controls and HIV-infected patients.Total RNA was extracted with RNeasy mini kit(Qiagen).The RNAs were reverse transcribed according to the ImProm-â…¡^TM Reverse Transcription System(Qiagen).6.Quantification of TLR7 and TLR9mRNA levels by Real-time PCR.Using ABI7500 Real-time PCR system,TLR7 and TLR9 were assessed normalizing to housekeeping gene GAPDH.First-strand cDNA were used in a 25μl reaction mixture contained 2×Power SYBR11 Green PCR Master Mix 12.5μl,1μl primer pair respectively,2μl cDNA template,8.5μl RNase-free water.Reaction condition:heat initiaton in 10 min at 95℃and 40 cycles of 95℃15 seconds followed by 60℃for 1 min.Data were collected and analyzed using Sequence Detection software.Using 2-^ΔΔCt qualification method,differences of TLR7 and TLR9 mRNA were compared between groups.7.Statistical analysisSPSS Version 16 software were obtained for data analysis.Data are depicted as means±SD.Differences among the groups were compared using the LSD test and correlation was used Pearson correlation analysis for unpaired and normality data. P＜0.05 were considered to be significant.Results1.Assessment of T and B lymphocyte counts in HIV/AIDS patients The CD4⁺T lymphocyte counts of HIV/AIDS patients were lower than that of healthy controls(P=0.000).B lymphocyte counts in HIV group and AIDS group were lower than that of healthy controls(P＜0.05)2.Association between B lymphocyte counts and CD4+T lymphocyte counts in HIV/AIDS patientsThe counts of B lymphocytes correlate positively with CD4⁺T lymphocytes count in HIV/AIDS patients(r=0.534,P=0.006)and don't correlate with CD4⁺T lymphocytes count(r=0.301,P =0.107).3.Comparison of TLR7 and TLR9 mRNA levels between groupsThe level of TLR9 mRNA expression in HIV/AIDS patients is significantly lower than that in health controls(P=0.023).There is no significant difference of TLR7 mRNA expression between groups(P＞0.05).4.Association between levels of TLR7 and TLR9 mRNA of B lymphocyte and CD4+T lymphocyte counts in HIV/AIDS patientsThe level of TLR9 mRNA expression in B lymphocytes correlate positively with CD4⁺T lymphocytes count in HIV/AIDS patients(r=0.390,P=0.040).The level of TLR7 mRNA expression in B lymphocytes don't correlate with CD4T lymphocytes count in HIV/AIDS patients.Conclusions1.The count of B lymphocytes in Chinese HIV/AIDS patients were significantly lower than that in healthy control,and correlate positively with CD4⁺T lymphocytes count of Chinese HIV/AIDS patients,indicating the changes of B lymphocyte count may be associated with HIV disease progression.2.The level of TLR9 mRNA of B lymphocytes in HIV/AIDS patients was significantly lower than that in health control,and correlates positively with CD4⁺T lymphocytes count of Chinese HIV/AIDS patients,indicating the expression of TLR9 mRNA may be associated with HIV disease progression.