| Objective:Acquired Immune Deficiency Syndrome?AIDS?is a kind of serious infectious disease caused by the human immunodeficiency virus?HIV?.The occurrence of combined antiretroviral therapy?cART?significantly reduces the morbidity and mortality of HIV-infected induviduals,but there is a large difference after receiving treatment in the crowd.In most people,the function of the immune system is restored to a certain extent after 1-2 years treatment,effectively controlling the virus replication and the level of CD4+T cells is significantly increased,known as the Immunological Responders?IR?,But after antiviral treatment 1-2 years even longer time,some patients,the function of immune system does not effective recovery,CD4+T cell levels have not increased even reduce obviously,called Immunological Nonresponders?INR??The Immune abnormalities in INRs appeared more prominent and appeared,clarifing its immune response mechanism for therapy is significance topromote the recovery of diseases.The deterioration of the gut ePithelium and enlarged microbial translocation triggered by the rapid depletion of gastrointestinal CD4+T cells leads to a Persistent,systemic activation,and inflammation of HIV infections.Antiretroviral therapy?ART?extends the lifesPan and the quality of life of HIV-infected patients while completely eliminating chronic immune activation and inflammation does not occur.A comprehensive understanding of the molecular and cellular basis of the inflammatory status of INR allows these remaining challenges of HIV treatment to be addressed.Mononuclear macrophage system is the main source of inflammatory cytokines.Human monocytes are divided into three subgroups according to the surface markers:CD14 and CD16.Classical monocytes?CD14++CD16-?;intermediate monocytes?CD14++CD16+?;and nonclassical monocytes?CD14+CD16++?.Nonclassical and intermediate monocytes are collectively described as CD16+monocytes.Compared with classical monocytes,CD16+monocytes secrete higher levels of proinflammatory cytokines and are more proficient in presenting Ag.However,the distribution of monocyte subgroups in patients with different immune responses after HIV treatment is still unclear.The increased secretion of various activated media and inflammatory factors in plasma after HIV infection can not be recovered or close to normal level after ART treatment,which may affect the immune recovery.The immune response of monocytes requires a large amount of energy supply and the metabolic status of monocytes is transformed from aerobic oxidation to glycolysis.The abnormal metabolic status affects the progress and recovery of HIV disease.Glucose transporter 1?Glut1?is a main molecule in cell glucose metabolism.Because of monocytes in HIV infection in chronic activation state for a long time,after glucose intake increase,thus Glut1 is associated with monocytes of the immune response may affect the secretion of various cytokines and cause immune response after antiviral treatment.In this study,we examined the effect of HIV infection on the expression of Glut1 on classical,intermediate,and nonclassical monocytes.We found that,irrespective of IR or INR,HIV infection increases the frequency of Glut1 expressing proinflammatory CD16+monocytes and causes a marked increase in INR subgroup and the relationship between immune recovery and effects on inflammatory cytokines secretion.Interferon-inducible protein 10?IP-10?,has been recorded in HIV-infected subjects,among these,IP-10 is functionally categorized as an inflammatory chemokine which attributed to inflammatory disorders,immune dysfunction,and tumor development.In HIV infection,elevated blood levels of IP-10 are associated with rapid disease progression and persistent immune activation.Previous studies found that IP-10 decreased the function of T cells and NK cells and stimulated HIV replication.This indicates that the regulation of IP-10 expression in HIV infection influences the delay of inflammation,slowing down disease progression.However,the factors triggering the significant up-regulation of IP-10,especially at a post-transcriptional level in HIV infection have not been clearly elucidated.Methods:1.Patient selectionIn part 1,a total of 28 cases collected samples,people infected with HIV?n=19?,the treatment time for 18-24 months,baseline CD4+T cell count:200-500 cells/mL,screening of HIV infection after antiviral treatment immunological responders?IR,n=9?were defined as delta CD4 change from baseline>30%;Immunological Nonresponders?INR,n=10?were defined as delta CD4 change from baseline<20%,trends to decrease,or after treatment no obvious increase of CD4+T cells.Healthy control?HC,n=9?were derived from age matched healthy crowd.In part 2,32 treatment-naive HIV-infected patients and35 HCs participated in this study.The demographic information and clinical characteristics of the subjects are listed in Table 2.Peripheral blood mononuclear cells?PBMCs?were freshly isolated by Ficoll centrifugation,and plasma samples were collected.Ethical approval was obtained from the First Hospital of China Medical University and all participants were informed of the collection of blood samples and provided written consent prior to enrolment in the study.2.Primary cell selection and monocyte subgroup detection.Peripheral blood mononuclear cells?PBMCs?were freshly isolated by Ficoll centrifugation,Peripheral monocytes subsets were detected with Fluorescent antibody:CD14?PE-Cy7?,CD16?APC-Cy7?,Glut1?PE?and stained for 30 min at 4?in dark.Then samples were centrifuged with 350g,10min at room tempreture.The fluorescence was detected and analyzed using LSR?and FACS Diva software?Becton-Dickinson,USA?.BD FACS Aria flow cytometer were used for primary cell isolation.3.THP-1 and THP-1-MA cells cultureTHP-1 human acute monocyte leukemia cells were cultured in RPMI 1640suPPlemented with 10%FBS?HyClone?.To promote the differentiation of THP-1 to THP-1-MA cells,the THP-1 were treated with phorbol 12-myristate 13-acetate?PMA;100ng/m L;Sigma?for 48 h.4.siRNA deliveryThe transfection of siRNA-Glut1(20?M,Invitrogen were performed with HiperFect Transfection Reagent?Qiagen?.Briefly,20?M siRNA were transfected into THP-1 cells for 48 h.Subsequently,the THP-1 cells were stimulated using Lipopolysaccharide?LPS;1?g/mL;Sigma?and incubated for a further 24 h.The forced reduction process of ISG15was achieved by employing the 20?M ISG15 siRNA for 24h?Invitrogen?.This was followed by THP-1 cell treatment with PMA for 48 h,into which was transfected 20 nM mimics by HiperFect Transfection Reagent?Qiagen?.For the inhibition of ISG15 in THP-1 cells,ISG15 siRNA was transfected to THP-1 cells for 24h.The siRNA control was represented by non-specific Stealth RNAi?Negative Control Duplexes.5.miRNA mimics and inhibitors transfectionThe transfection of miRNA mimics,inhibitors and controls?Gene Pharma?were performed with HiperFect Transfection Reagent?Qiagen?.Briefly,20?M mimics or inhibitors were transfected into THP-1,THP-1-MA cells for 48 h.Subsequently,the THP-1 cells were stimulated using LPS?1?g/m L;Sigma?and incubated for a further 24 h.The cells were collected for RNA extraction,and the supernatants were collected for IP-10detection.Transfection of primary CD14+monocytes with mimics was performed with RNAiMAX?Invitrogen?according to the manufacturer's protocol.6.Reverse transcription and quantitative real-time PCRmiRNA was extracted from cells using the miRNeasy Micro kit?Qiagen?.Total RNA was isolated using the RNeasy Micro kit?Qiagen?,and the purified RNA was treated with DNase I reagent to eliminate genomic DNA contamination.The RNA was reversely transcribed using Primpscript?RT reagent kit?TAKARA?according to the instructions provided by the manufacturer.The real-time PCRs for the detection of miRNA and mRNA were performed with SYBR?Premix Ex Taq?II?TAKARA?.All the Primer sequences are listed in Table 3.The levels of miRNA expression were normalized to small nucleolar RNA?snRU6?,while mRNA expression was normalized to GAPDH.The relative expression levels of miRNA and mRNA were calculated based on the change in cycling threshold method as 2-??Ct.7.Bio-Plex Pro?Human 7 Cytokine AssaysDilution standard,beads,antibodies and SA-PE.Add 50ul diluted beads to each well of the assay.After washing the plate 2 times,add 50ul samples,standards,blank to each well.Incubate on shaker at 900 rpm at room temperature and protect from light for 1h.Then add 25ul antibodies to well and incubate for 1h.Washing the plate 3 times and adding50ul SA-PE,away from light 15 min,finally add 125 ul Assay buffer,shake the plate 900rpm for 30s,then read the plate and analyze the datas?Bio-Plex20000?.8.IP-10 detectionThe measurements of supernatant IP-10 Produced from cell culture and plasma from HIV-infected patients and HCs were performed using an ELISA kit?R&D Systems?.Concentrations of IP-10 were calculated using the standard curve and multiplied by the dilution factor.9.Luciferase activity assayAccording to the target prediction,the IP-10 3'UTR contained two appropriate sequences with miR-21 binding sites?nt 196-202 and 235-240?.The 3'UTR fragments?synthesized by GenePharma?,containing the putative binding sites for miR-21,were cloned into the GP-miRGLO Vector.The luciferase reporter assay was performed in 293T cells cultured in 48-well Plates.The 293T cells were co-transfected with the IP-10 3'UTR Reporter?wild type,mut-1,mut-2?,empty vector plasmid?GP-mi RGLO?,and appropriate miRNA mimics and inhibitors?20?M?,using the Lipofectamine 2000 reagent?Invitrogen?.At 24 h after transfection,firefly and renilla luciferase activities were measured using the Dual-Luciferase Report Assay?Promega?.10.Statistical analysisGraphPad Prism was used to conduct statistical analyses,and the comparison between the miRNA and IP-10 levels was performed using the student's t-test.The nonparametric Mann–Whitney test was used to determine the differences between HIV patients and HCs.The correlations between variables were evaluated using the Spearman's correlation coefficient,and all p values under 0.05 were considered statistically significant.Results:1.Differences in immune recovery after antiviral treatment of HIV-infected patients.Statistical analysis was conducted in the samples between IR and INR groups.There were significant differences after 12 months of treatment,12M?P=0.001?,18M?P=0.003?,24M?P=0.0003?.The CD4+T cells of HIV-infected individuals were analyzed and found that the CD4+T cell counts of IR group was significantly increased?P<0.0001?and there was no difference between treatment patients and HCs.However,in the INR group,the number of CD4+T cells after treatment was still significantly lower than the healthy control?P<0.0001?.IR group was significantly higher than that in the INR group?P=0.0003?.2.HIV infection is associated with an increased percentage of circulating CD16+monocytes expressing Glut1The analysis of the proportion of monocyte subgroups in HIV patients and healthy controls found that the proportion of[I]monocytes in the IR group and INR group was higher than that in the healthy control group?P=0.019,P=0.038?.In addition,there was no difference between[NC]and[I]monocytes in healthy control group,and the proportion of[I]monocytes in IR group and INR group was significantly higher than that of[NC]monocytes?P=0.008,P=0.020?,but there was no difference between the subgroups of IR and INR.Sorting out the monocyte subsets for detecting the expression of Glut1.We found that Glut1 in[NC]and the[I]monocytes in INR were higher than in IR group?P=0.016;P=0.008?and HC group?P=0.04,P=0.04?.The results showed that the subgroup ratio of monocytes was independent of the immune recovery state,while the surface expression of Glut1 was significantly increased in the INR group,which may be the key molecule to affect the immune recovery.3.The effect of Glut1 on cytokines secreted by monocytes.In order to explore the effect of Glut1 on the immune response of monocytes,the siRNA siRNA-Glut1 was deliveried to THP-1 and detected the secretion of cytokines.After successful knock-down Glut1,LPS stimulation for 24h.We found that the secretion of IL-6 and IP-10 in supernatant were obviously decreased?P=0.03,P=0.03?,prove that expression of Glut1 reductions would reduce the secretion of IL-6 and IP-10.4.The relationship between inflammatory cytokines in plasma and immune reconstruction.The plasma samples of 19 subjects were collected,including 12 plasma samples of HIV infection after antiviral treatment,including IR?n=6?,INR?n=6?and HCs?n=7?.The results showed that only IP-10 in INR group was significantly lower than the IR and the HCs?P=0.048;P=0.001?,IP-10 is a key factor in the immune response after antiviral treatment of HIV infection and increasing secretion may lead to the failure of immune response.5.Elevated IP-10 levels in HIV-infected patients are linked to disease progressionWe investigated levels of IP-10 secretion in the HIV-infected patients enrolled in our study.All of these patients had considerably greater IP-10 Plasma concentrations?458.0±329.5 pg/m L?than HCs?115.0±56.3 pg/m L??P<0.0001?.Then we studied the association of levels of IP-10 with disease progression.We found that IP-10 was negatively correlated with CD4+T-cell counts?r=-0.595,P=0.0003?and positively correlated with viral load?r=0.706,P<0.0001?.We then divided the patients into two groups according to CD4+T cell counts and viral load.IP-10 levels were significantly higher in the group of patients with seriously reduced CD4+T cell counts?<350 cells/?L;P=0.029?and the group with high viral loads?LogVL>4;P=0.002?.The correlation between IP-10 and disease progression confirmed that IP-10 is a good biomarker in HIV disease progression.6.Identification of individual miRNAs for IP-10 production regulationThree miRNA target prediction tools were employed to identify mi RNA candidates:TargetScan,miRanda,and DIANA.These online tools showed that six miRNAs possessed a conserved 6–7mer seed matching the IP-10 3'UTR.These were miR-15a,mi R-16,miR-21,miR-135a,miR-200c,and miR-503.IP-10 exhibited two binding sites for miR-21 and just one for the other mi RNAs.The THP-1 cells were overexpressed for the six miRNA candidates and stimulated with LPS to identify the miRNAs which could regulate IP-10production.IP-10 secretion was largely reduced by the overexpression of miR-21?P=0.003?and miR-200c?P<0.0001?compared to the control.mi R-21 was identified for further studies.7.miR-21 regulates IP-10 secretion through THP-1 cellsNext,we transfected miR-21 mimics or inhibitors to the THP-1 cells,finding that the transfection of mimics greatly inhibited the IP-10 protein released?P=0.041?and miR-21inhibitors efficiently enhanced IP-10 production in THP-1 cells after LPS stimulation?P=0.015?.Our results suggest that miR-21 altered the protein,but not mRNA expression level of IP-10.8.Identification of IP-10 as direct target of miR-21We explored the potential of miR-21 to directly target the 3'UTR of IP-10.Luciferase Report vectors carrying the full 3'UTR of wild-type?WT?IP-10,mutation site 1?mut-1,196-202?,or mutation site2?mut-2,235-240?were constructed.Reporters were transfected into 293T cells with either miR-21 mimics or inhibitors,and luminescence in miR-21mimic-treated cells was clearly less than in controls?P=0.005?.The results show that miR-21 can directly target the 3'UTR of IP-10 and affect subsequent transcription.Therefore,we also worked to identify the effective binding site between IP-10 and miR-21.According to our results,miR-21 can directly target IP-10 3'UTR through bind site 1?196-202?.9.miR-21 is downregulated in monocyte of HIV infected patientsMonocyte is a main source of IP-10,we sorted monocytes from HIV-infected individuals?n=6?as well as HCs?n=5?to detect the relative expression of miR-21.The levels of miR-21 in monocytes of HIV-infected patients were considerably lower?P=0.044?than those of HCs,whereas IP-10 levels in plasma exhibited the opposite tendency?P=0.029?.There was a trend of negative correlation between miR-21 expression in monocytes and plasma IP-10 concentrations?r=-0.502,P=0.057?.We found that overexpression of miR-21 in primary monocytes?P=0.002;?from HIV infected patients has the decreasing tendency of IP-10 production by LPS stimulation?P=0.057?.The results suggesting that miR-21 may contribute to the regulation of IP-10 production in monocytes in the context of HIV infection.10.Elevated expression of miR-21 and IP-10 in THP-1-MA cellsWe detected the alterations of miR-21 and IP-10 during the differentiation process from THP-1 to THP-1-MA cells.As illustrated in Fig.4A and B,the expression of miR-21and IP-10 was remarkably more enhanced in THP-1-MA than in THP-1 cells?P=0.002and P=0.037,respectively?.We subsequently measured the IP-10 levels of unstimulated and stimulated THP-1 and THP-1-MA cells.The results showed a similar trend of increase in IP-10 secretion within the supernatant of THP-1-MA cells over that in THP-1 cells?P=0.0008?,especially after LPS stimulation?P=0.0004?.11.IP-10 expression in monocytes can not be regulated by mi R-21To determine the potential of miR-21 to suppress IP-10 secretion in macrophages.we transfected miR-21 mimics or inhibitors to the THP-1 cells,but we observed that miR-21mimics or inhibitors did not play a significant role in IP-10 secretion of LPS-stimulated THP-1-MA cells.12.Elevated expression of ISG15 weakens the regulation of IP-10 by miR-21 in THP-1-MA cellsWe then explored the potential mechanisms controlling the differential effects of miR-21 on IP-10 production between monocytes and macrophages.We found that the successful inhibition of ISG15 by siRNA?P=0.017?did significantly decrease both IP-10 mRNA expression?P=0.039?and protein production?P=0.021?.Nevertheless,suppression of ISG15 in THP-1 cells still led to reduced IP-10 mRNA expression?P=0.014?and IP-10protein production?P=0.010?.These findings support the thesis that enhanced ISG15levels in THP-1-MA facilitate IP-10 production and therefore overcomes the inhibition of IP-10 by mi R-21,potentially accounting for the differential effect of miR-21 to IP-10expression in monocytes and macrophages.Conclusion:1.The analysis of the proportion of monocyte subgroups in HIV patients and healthy controls found that the proportion of intermediate monocytes in the IR group and INR group was higher than that in the healthy control group.But there was no difference between the subgroups of IR and INR.The results showed that the subgroup ratio of monocytes was independent of the immune recovery state,while the surface expression of Glut1 was significantly increased in the INR group,which may be the key molecule to affect the immune recovery.2.Target prediction tools were employed to identify miRNA candidates and IP-10exhibited two binding sites for miR 21.Bolstering miR-21 levels using mimics resulted in the obvious suppression of LPS-induced IP-10 in monocyte leukemia cells THP-1 and vice versa.The analysis of the primary monocytes of HIV patients revealed significantly less miR-21 than in healthy controls,this was opposite to the tendency of IP-10 levels in plasma.3.The secretion of IP-10 due to LPS stimulation was not affected by miR-21 modulation in the differentiated THP 1 macrophages?THP-1-MA?.We found a novel switch,IFN stimulated gene 15?ISG15?,which triggers the expression of IP-10 and is significantly upregulated during the differentiation of THP-1 into THP-1-MA.The inhibition of ISG15 can restore the regulation of IP-10 by miR-21. |
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