| Objective:Human immunodeficiency virus(HIV)infected patients with atypical disease progression are of particular importance because they can provide important information regarding HIV pathogenesis and therapy.The first evidence of long-term non-progressors(LTNPs)was reported in 1993,showing that 15% of individuals infected with HIV maintain a CD4 count >500 cells/μl[1,2].In 2005,study showed that approximately 1/300 HIV-infected patients had undetectable plasma HIV RNA loads without antiretroviral therapy(ART)[3].Described as “Elite Controllers”(ECs)in 2007,these patients maintained HIV RNA levels below 50 copies/ml for at least 1 year in the absence of ART[4-8].Although there is overlap between LTNPs and ECs,they are not identical.Most ECs exhibit minimal reductions in CD4+ T cells over time,although reductions are observed in some ECs.Furthermore,some LTNPs have ongoing viral production,while others do not [3,5,7,9-11].Transcriptomic analysis showed that ECs with higher CD4+T cell numbers were indistinguishable from HIV-1-negative individuals.In contrast,ECs with lower CD4+ T cell numbers were similar to ART-treated patients,but different from HIV-1-negative individuals[27].Alterations in T cell homeostasis predict the loss of immunological control in ECs [28].However,viral control differs among LTNPs and the reason for this difference is currently unknown.LTNPs maintaining high viral loads are prone to long-term disease progression,with reduced life expectancy compared to HIV negative individuals [12,13].Analysis of the underlying reason for the differing levels of viral control in LTNPs may facilitate an understanding of HIV pathogenesis and may provide for new approaches to immune intervention.The level of virus control is affected by immunologic and virologic factors.Micro RNAs(mi RNAs)may be a potential mechanistic factor involved in this process.It has been shown that mi RNAs play important roles in the regulation of immune responses and viral production.In HIV infection,mi RNAs can directly modulate viral production by targeting the HIV-1 genome or by indirectly modulating cellular genes that influence viral propagation [14-18].Dramatic advances have been made in understanding how mi RNAs regulate the development and function of immune cells [29,30],including CD8+T cells which are key players in the antiviral immune response[25,26].It is established that mi RNAs modulate the expression of numerous regulatory proteins required for the development,differentiation,and function of CD8+T cells[31]..Studies have shown that,in HIV infection,mi RNAs modulate the expression of key markers associated with T cell exhaustion or dysfunction,such as interleukin 10 and B lymphocyte-induced maturation protein-1 [21].We postulated that differential mi RNA profiles may contribute to the divergent control of viral load in LTNPs by affecting viral production and/or CD8+T cell function.Although previous studies have identified mi RNA profiles in HIV-infected ECs and LTNPs [14,32-35],the role of mi RNAs in the differential control of viral load in LTNPs has not been addressed.In this study,mi RNA expression profiles of LTNPs were assessed.The expression of mi R-19 b was found to be significantly increased in the peripheral blood mononuclear cells(PBMCs)of LTNPs with low(LTNP-Ls)compared to high levels(LTNP-Hs)of virus production.In addition,mi R-19 b promoted proliferation,and expression of interferon gamma(IFN-γ)and granzyme B,while inhibiting CD8+T apoptosis induced by anti-CD3/CD28 stimulation.The phosphatase and tensin homolog(PTEN)is a target of mi R-19 b regulating CD8+T cell function in HIV infection.Furthermore,we found that mi R-19 b directly inhibits viral production in HIV-infected T cells in vitro.Our results revealed a previously unknown mechanism of sustained viral control by mi R-19 b in a subtype of LTNPs,suggesting that mi R-19 b may be a novel target for immune intervention in HIV infection.Methods: 1.Study population In total,samples obtained from 27 LTNPs,six typical progressors(TPs),and four healthy controls(HCs)were analyzed.The LTNPs were HIV positive patients who maintained normal CD4+ T cell counts(CD4 >500 cells/μl)for >10 years(mean ± SD: 14.72 ± 1.79 years at the time of sample collection)without receiving ART(Supplemental Table 1).The TPs were ART naive HIV positive patients who progressed to CD4 counts <500 cells/μl at 2.53 ± 0.95 years(Supplemental Table 2).The initial 347-mi RNA array was performed in a training cohort,including nine LTNPs(age,mean ± SD: 41 ± 6 years;gender,eight males and one female),six TPs(age,mean ± SD: 30 ±15 years;gender,six males)and four HCs(age,mean ± SD: 37 ± 6 years;gender,four males).From the training cohort,mi RNAs differentially expressed in LTNPs with differing viral loads were detected in a subsequent validation group that included 18 LTNPs.Ethical approval was obtained from the First Hospital of China Medical University,Shenyang,China and written informed consent was provided by all participants.2.mi RNA array analysis Quantitative real time polymerase chain reaction(q RT-PCR)-based high-throughput mi RNA profiling was performed at Quanto Bio Biotechnology Co.Ltd.(Beijing,China).Briefly,total RNA extracted from peripheral blood mononuclear cells(PBMCs)was isolated using TRIzol?(Invitrogen,Carlsbad,USA).Escherichia coli poly(A)polymerase was used to add adenines to the 3’ end of RNA molecules lacking a poly(A)tail.After oligo d T annealing,a universal tag was attached to the 3’ end of c DNAs during c DNA synthesis using retrotranscriptase Superscript ⅡI(Invitrogen).With this universal tag,a SYBR?-based q RT-PCR was performed using mi RNA-specific forward primers and a reverse universal primer mix.Of note,U1 and U6 were used in the training cohort for normalization.The variation of change in the threshold cycle(CT,target-CT,and control)was evaluated and used as a relative qualitative value.3.RT-PCR quantification of mi RNA and m RNA We extracted mi RNAs from cells using the mi RNeasy Micro kit(Qiagen,Hilden,Germany).The RNA was reverse transcribed using a Primpscript? RT reagent kit(TAKARA,Dalian,China)according to the instructions provided by the manufacturer.Subsequently,RT-PCR was performed using a SYBR? Premix Ex Taq? Ⅱ(TAKARA).The levels of mi RNA were normalized to the U6 small nucleolar RNA and quantified through the relative quantification method(2-ΔΔCt),as previously described(31).Cellular total m RNA was isolated using the RNeasy Micro kit(Qiagen).The c DNA was generated using the Primpscript? RT reagent kit(TAKARA).The levels of m RNA were quantified through the SYBR? Premix Ex Taq? Ⅱ(TAKARA),normalized to GAPDH transcripts,and expressed using the relative quantification method(2-ΔΔCt).All primer sequences for the quantification of mi RNA and m RNA are listed in Supplemental Table 3. 4.Isolation of cells PBMCs were obtained by Ficoll–Hypaque density gradient centrifugation.If indicated,CD8+ or CD4+ T cells were further purified from isolated PBMCs by negative selection with magnetic beads using a CD8+ or CD4+ T cell Enrichment Kit(Cell purity >95%,Stem Cell Technologies,Vancouver,Canada).The following antibodies were used for immunostaining to isolate cell subtypes: FITC-CD3,APC-cy7-CD8,APC-CD4,PE-cy7-CD14 and 7-AAD(Biolegend,San Diego,CA,USA).CD4+ T cells(CD3+CD4+),CD8+ T cells(CD3+CD8+),and monocytes(CD3-CD14+)were selected from 7-AAD-negative live PBMCs using a FACSAria? flow cytometer(BD Biosciences,Franklin Lake,NJ,USA).5.Cell culture The Jurkat human leukemia T cells,Clone-X cells,and primary cells were maintained in RPMI1640 media(Hy Clone,Logan,UT,USA)supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin.The 293 T cells were maintained in DMEM media(Hy Clone)supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin.6.Transfection Transfection of mi RNAs to cell lines was achieved using Lipofectamine? 2000(Invitrogen).Briefly,20 μM mi R-19 b mimics or controls(Gene Pharma,Shanghai,China)were transfected to 293 T cells,Jurkat cells,or Clone-X cells according to the protocol provided by the manufacturer.In primary cells,Lipofectamine? RNAi MAX(Invitrogen)was used for the transfection according to the protocol provided by the manufacturer.Briefly,20 μM mi R-19 b mimics,inhibitors(Gene Pharma),or controls were transfected to isolated CD8+T or CD4+T cell-depleted PBMCs.In addition,isolated primary CD4+ T cells from healthy controls were transfected with 20 μM mi R-19 b mimics or controls.The forced reduction of phosphatase and tensin homolog(PTEN)was achieved by introducing 20 μM PTEN si RNA(Invitrogen)to isolated CD8+T cells.The si RNA control used in this experiment was non-specific Stealth RNAi? Negative Control Duplexes.The sequences of the mimics and inhibitors are listed in Supplemental Table 3.7.Proliferation assays After transfection(24 h),Jurkat cells and primary CD8+T cells were labeled with Cell Trace? Violet(Thermo Fisher Scientific,Waltham,USA)for 15 min at 37°C according to the instructions provided by the manufacturer,washed with complete medium,and cultured(1 × 106 cells/ml).Primary CD8+T cells were cultured in the presence of anti-CD3/CD28(3 μg/ml;Gibco,New York,NY,USA).After incubation for 5 days,the dividing cells were analyzed using a BD LSR Ⅱ flow cytometer and the Flow Jo software.8.Detection of apoptosis After transfection(72 h),Jurkat cells were stained with PE-conjugated anti-Annexin V and 7-AAD(Biolegend).After transfection(24 h),primary CD8+T cells were stimulated using anti-CD3/CD28(3 μg/ml).After stimulation(48 h),CD8+T cells were stained with PE-cy7-conjugated anti-CD3,APC-cy7-conjugated anti-CD8,PE-conjugated anti-Annexin V,and 7-AAD(Biolegend).The cells were analyzed using a BD LSR Ⅱ flow cytometer and the Flow Jo software.9.Cell cycle assay Cell cycle phases were determined using the BD Cycletest? Plus DNA Reagent Kit(BD Biosciences)according to the instructions provided by the manufacturer.In brief,Jurkat cells were cultured for 72 h after transfection.After transfection(24 h),primary CD8+T cells were cultured for 48 h under stimulation using anti-CD3/CD28(3 μg/ml).The distribution of DNA content was determined using a BD LSR Ⅱ flow cytometer and analyzed using the Flow Jo software.10.Intracellular staining of IFN-γ and granzyme B After transfection(24 h),primary CD8+T cells were stimulated using anti-CD3/CD28(3 μg/ml)for 24 h.The protein transport inhibitor(Golgi Stop;1 μl/ml,BD Biosciences)was added to the culture for the last 6 h.The cells were stained with PE-cy7-conjugated anti-CD3 and APC-cy7-conjugated anti-CD8(Biolegend).Subsequently,intracellular staining was performed by incubating the cells in 1X Perm/Wash Buffer for 15 min in the dark,followed by incubation with APC-conjugated anti-IFN-γ and FITC-conjugated anti-granzyme B for 30 minutes at 4°C.After staining,the cells were fixed in 1% formaldehyde.The intracellular expression of IFN-γ and granzyme B was determined using a BD LSR Ⅱ flow cytometer and data were analyzed using the Flow Jo software. 11.IFN-γ ELISPOT assay CD4+ T cells were depleted from PBMCs using anti-CD4 MAb-coated magnetic beads(Biolegend)as described in the instructions provided by the manufacturer.The Human IFN-γ ELISpot Kit(Mabtech,Nacka,Sweden)was used to detect the secretion of IFN-γ according to the instruction manual.After transfection(24 h),2 × 105 CD4+ T cell-depleted PBMCs were added per well(in duplicates)in a volume of 200 μl.The HIV-1 gag peptide pools(10 μg/ml,Sigma)were added for 20 h.Anti-CD3/CD28(3 μg/ml)were used as a positive control,and negative controls consisted of cells without stimuli.The number of IFN-γ-secreting cells was calculated by subtracting the negative control(medium only)values.A positive response was defined as >50 spot-forming units/106 PBMC.12.In-vitro infection Viral particles were produced by transfecting 293 T cells with HIV-1 p NL4-3 plasmids and vesicular stomatitis virus glycoprotein(VSV-G)plasmids.Transfection of mi R-19 b mimics,p NL4-3 plasmids,and VSV-G plasmids into 293 T cells was performed to detect the effects of mi R-19 on HIV production.The levels of p24 in the supernatants were measured by ELISA(Biomedical Engineering Center of Hebei Medical University,Hebei,China)2 days later.For the infection of Clone-X cells,the cells were transfected with mi R-19 b mimics for 24 h and subsequently infected with VSV-G pseudotyped HIV-1(NL4-3)virus.GFP+ cells were detected by flow cytometry 48 h after infection.Replication-competent HIV-1 isolate was used to test the effects of mi R-19 b in primary CD4+ T cells.Isolated primary CD4+ T cells from healthy controls were transfected with mi R-19 b mimics or controls.After transfection(24 h),the cells were stimulated using anti-CD3/CD28(3 μg/ml).A cryopreserved primary HIV-1 isolate – obtained by a co-culture using mixed PBMCs from an HIV-1-infected patient and a healthy donor – was thawed and added to the cells.The supernatant was collected after 3 days of infection and the levels of p24 in the supernatants were measured by ELISA.13.Statistical analysis Principal component analysis(PCA)was used(Origin 9.1 software)to analyze the distribution of mi RNAs in HIV-infected patients with differing disease progression.The non-parametric Mann–Whitney test was used to determine differences between LTNPs with a relatively high viral load(>1000 copies/ml)(LTNP-Hs)and LTNPs with relative control of viral load(<1000 copies/ml)(LTNP-Ls).A paired t-test was used to analyze differences in CD8+T cell function between groups.Data analysis was performed using the SPSS 21.0 and Graph Pad Prism Version 5.0 software packages.A P<0.05 was considered statistically significant.Results: 1.mi RNA profiles distinguish LTNPs with different virus levels A training cohort was formed including nine LTNPs,six TPs,and four HCs to identify the mi RNA profiles of LTNPs.Using q RT-PCR-based arrays,the expression levels of 347 mi RNAs were quantified.Based on an unsupervised PCA of all array data,the six TPs,nine LTNPs and four HCs were segregated into two groups.All the HCs were clustered in one group.Most of the TPs were clustered in the other group,except one TP with a relatively low viral load(<1000 copies/ml),indicating that HIV infection alters mi RNAs.This finding was consistent with those reported in previous studies(32-34).Interestingly,the nine LTNPs were divided into two groups,one of which was very close to the TPs(Group A,n=6)and another that was intertwined with the HCs(Group B,n=3).We subsequently sought to identify differences between the two groups of LTNPs.By comparison of clinical characteristics(i.e.,age,number of CD4+T cells,and viral loads),we found that viral load was the only significantly different parameter between the two groups of LTNPs(P=0.024).Six LTNPs in Group A,whose mi RNA profiles were similar to those of TPs,had a relatively high level of viral load(> 1000 copies/ml,hereinafter referred to as “LTNP-Hs”).Three LTNPs in Group B,whose mi RNA profiles were similar to those of HCs,had relative control of viral load(VL < 1000 copies/ml,hereinafter referred to as “LTNP-Ls”).The results of the unsupervised PCA suggest that the expression of mi RNAs can distinguish LTNP-Hs and LTNP-Ls,and may account for the differing viral loads observed in LTNPs.2.Expression of mi R-19 b is high in LTNP-Ls in comparison with that in LTNP-Hs Subsequently,the differential expression of mi RNAs in LTNP-Hs and LTNP-Ls was assessed.The mi RNAs were determined to be significantly differentially expressed in LTNP-Hs and LTNP-Ls with a Benjamini–Hochberg false discovery rate-adjusted P<0.05.We found that 78 mi RNAs were differentially expressed with >2-fold change between the three LTNP-Ls and six LTNP-Hs in the training cohort(adj.P<0.05).Among those,55 mi RNAs were upregulated and 23 were downregulated in LTNP-Ls compared with that in LTNP-Hs.Using an unsupervised clustering method,78 mi RNAs accurately distinguished and clustered LTNP-Hs and LTNP-Ls.A total of 75 mi RNAs with differential expression levels between LTNP-Hs or LTNP-Ls and HCs or TPs(P<0.05)were excluded to identify mi RNAs that can uniquely differentiate LTNP-Hs from LTNP-Ls.This exclusion was carried out because these mi RNAs may reflect differences caused by HIV infection or the stages of infection.Only three mi RNAs which were differentially expressed between LTNP-Hs and LTNP-Ls in the training cohort were selected,including mi R-15a(P=0.024),mi R-19b(P=0.048),and mi R-33(P=0.024).The expression levels of these three mi RNAs were assessed in a subsequent validation group,including ten LTNP-Hs and eight LTNP-Ls.In the validation cohort,only mi R-19 b was verified to be highly expressed in LTNP-Ls compared with LTNP-Hs(P=0.034).Subsequently,CD8+,CD4+ T cells,and monocytes from LTNPs were sorted to identify the cell subtypes in which the expression of mi R-19 b was altered.We found that the expression of mi R-19 b decreased in CD4+T cells and CD8 T cells in LTNP-Hs(n=10)compared with that observed in LTNP-Ls(n=9)(P=0.041;P=0.028).There were no differences observed in the expression of mi R-19 b in monocytes between the two groups.3.mi R-19 b promotes proliferation and expression IFN-γ,and inhibits the apoptosis of primary CD8 T cells As the most important immune effector cell population,CD8+T cells play a key role in anti-HIV immune responses.We hypothesized that mi R-19 b may contribute to the control of the virus in LTNP-Ls by regulating CD8 T cell function.This was the first study addressing this question.Initially,we overexpressed mi R-19 b in Jurkat cells by transfection with mi RNA mimics to investigate the effect of mi R-19 b on lymphocyte proliferation and apoptosis.After transfection(48 h),mi R-19 b was highly expressed.After 5 days,the proliferation of mi R-19b-overexpressing cells was significantly increased compared with that observed in the control group,suggesting that mi R-19 b promotes cell proliferation(P=0.013).Overexpression of mi R-19 b significantly accelerated the cell cycle(P=0.033).Furthermore,the percentage of Annexin V+7-AAD-apoptotic cells was lower following the overexpression of mi R-19b(P=0.005).These data indicate that mi R-19 b promotes proliferation and inhibits apoptosis of T cell lines.We subsequently assessed the role of mi R-19 b in regulating the function of primary CD8+T cells by transfection of mi R-19 b mimics into CD8+T cells obtained from HCs.We found that overexpression of mi R-19 b significantly promoted the proliferation of CD8+T cells after anti-CD3/CD28 stimulation(P=0.010).Similarly,forced expression of mi R-19 b significantly inhibited apoptosis of CD8 T cells after stimulation of the anti-CD3/CD28(P=0.009).CD8 T cells were sorted using magnetic beads to study the effect of mi R-19 b on the cytotoxic function of CD8 T cells.Overexpression of mi R-19 b in CD8+T cells significantly increased the intracellular levels of IFN-γ and granzyme B after stimulation of the anti-CD3/CD28(P=0.006;P=0.002,respectively).These results demonstrate that mi R-19 b promotes cell proliferation,and the expression of IFN-γ and granzyme B in CD8+T cells.Moreover,it inhibits the apoptosis of CD8+T cells after stimulation of the anti-CD3/CD28.4.mi R-19 b regulates CD8+ T cell function via expression of PTEN Cell signaling pathways involving mi R-19 b target genes were assessed through a bioinformatics analysis(http://diana.imis.athena-innovation.gr/)to further explore the molecular mechanistic basis of mi R-19 b regulation.The FOXO signaling pathway plays critical roles in cell cycle regulation and is the main cell signaling pathway in which mi R-19 b target genes are involved.Through the detection of several key molecules in the FOXO pathway using q RT-PCR,we found that overexpression of mi R-19 b in Jurkat cells significantly reduced the expression level of PTEN(P<0.001).The direct regulation of PTEN by mi R-19 b was demonstrated using a Luciferase reporter assay,q RT-PCR,and western blotting.Considering that PTEN is closely associated with cell proliferation and cell cycle,we sought to determine the effect of mi R19 b on the function of CD8+T cells by regulating the expression of the target gene PTEN.We reduced the expression of PTEN in primary HCs CD8+T cells using specific si RNAs.Cell proliferation was promoted and apoptosis was inhibited via the knockdown of PTEN in comparison with the negative control group(P=0.040;P=0.047,respectively).Moreover,the secretion of granzyme B by CD8 T cells was significantly increased(P=0.006)and that of IFN-γ showed an increasing trend(P=0.094)after stimulation of the anti-CD3/CD28,in response to the suppression of PTEN.These findings demonstrate that inhibition of PTEN,which is a potential target of mi R-19 b,exerts similar effects on CD8 T cell function to those observed following the overexpression of mi R-19 b.5.mi R-19 b regulates the function of CD8+ T cells from HIV-infected patients The function of mi R-19 b in CD8 T cells from 7 HIV-infected patients was also studied.Primary CD8+T cells from HIV-infected patients were sorted.Following the overexpression of mi R-19 b,CD8+T cells showed a significant increase in proliferation(P=0.010),and secretion of IFN-γ(P=0.010)and granzyme B(P=0.003)after stimulation of the anti-CD3/CD28.Apoptosis of CD8 T cells was significantly reduced in comparison with the controls(P=0.040).Furthermore,the expression of mi R-19 b was inhibited by transfection of mi R-19 b inhibitors into CD8+ T cells from HIV patients.Contrary to the mi RNA overexpression results,the proliferation of CD8 T cells was reduced(P=0.043),the secretions of IFN-γ(P=0.023)and granzyme B(P=0.049)were reduced,and the apoptosis of CD8+T cells was increased(P=0.035).Subsequently,the expression of PTEN in primary CD8+T cells was inhibited using si RNA to verify that PTEN was a mi R-19 b target gene involved in the regulation of CD8+T cell function in HIV infection.Consistent with the findings reported in healthy patients,inhibition of PTEN resulted in an increase in proliferation(P=0.005)and secretion of IFN-γ(P<0.001)and granzyme B(P=0.012),as well as a decrease in CD8+T cell apoptosis(P=0.006)in HIV-infected patients.Furthermore,si RNA was used to suppress the expression of PTEN in mi R-19b-low-expressing CD8 T cells to verify that mi R-19 b affects CD8 T cell function through regulation of PTEN.Following the downregulation of PTEN,there was no statistical difference detected in proliferation,expression of IFN-γ and granzyme B,or apoptosis compared with controls.These data suggest that downregulation of PTEN antagonizes the effect of mi R-19 b inhibitors on the function of CD8 T cells in HIV patients.An IFN-γ ELISPOT assay was performed to further confirm the effect of mi R-19 b on HIV specific CD8+T cells.We found that overexpression of mi R-19 b significantly increased the secretion of IFN-γ by gag peptide-stimulated CD8+T cells(P=0.041).Of note,the secretion of IFN-γ was significantly reduced in response to suppression of the expression of mi R-19 b by inhibitors(P=0.020).These data suggested that mi R-19 b augments the function of HIV specific CD8 T cells.6.mi R-19 b inhibits viral replication in in-vitro HIV-infected T cells Lastly,we hypothesized that mi R-19 b may play a role in the direct inhibition of HIV replication,besides its regulation of CD8 T cells.The levels of mi R-19 b in the plasma have been reported to be associated with CD4 T cell counts,indicating that mi R-19 b may be a biomarker for the monitoring of the HIV immune status.However,its direct effects on HIV viral production have not been reported.We found that overexpression of mi R-19 b by mimics reduced the production of HIV.Following the co-transfection of mi R-19 b mimics and the p NL4-3 plasmid into 293 T cells,the expression level of P24 in supernatants was lower than that in the control group after 48 h of culture(P=0.040).Furthermore,Clone-X cells were infected with the same titer of HIV pseudovirus,the percentage of HIV-positive cells overexpressing mi R-19 b was lower compared with that in the control group(P=0.007).Finally,we infected primary CD4+ T cells from normal controls using replication-competent HIV-1 virus isolates and found that overexpression of mi R-19 b inhibited the production of HIV(P=0.001).These data demonstrated that,besides its role in the regulation of CD8 T cells,mi R-19 b inhibits viral production,leading to lower viral levels.Conclusion: 1.mi R-19 b is highly expressed in LTNPs with sustained viral control.2.mi R-19 b plays an anti-apoptotic and pro-proliferative role in CD8 T cells 3.mi R-19 b increase the intracellular IFN-γ and Granzyme B of CD8 T cells 4.The forced reduction of PTEN antagonizes the effect of mi R-19 b inhibition 5.Overexpression of mi R-19 b leads to the lower levels of HIV replication. |
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