In HIV-exposed seronegatives (HESN), multiple and repeated exposure to HIV do not result in seroconversion.1,2 The variability in susceptibility to HIV infection has been intensively studied and was found to correlate with a variety of viral, genetic, immunological, and socio-behavioral variables that influence host cellular gene expression leading to increased antiviral activity.3 Examples of such correlates of protection include the expression of particular HLA class I molecules, mutation in the gene for the human chemokine receptor 5 (CCR5), and the presence of single-nucleotide polymorphisms in genes such as ERAP-2 and MX-2.4–7 Nevertheless, none of the factors associated with the HESN phenotype so far is fully capable of clarifying the precise mechanism whereby natural resistance to HIV infection is achieved, and the overall understanding of the molecular scenario underlying resistance to viral infection remains elusive. As a deeper knowledge of this phenomenon may enable exploitation of its basic mechanism(s) for the design of novel vaccines and therapies, we investigated a relatively new area of gene expression control mechanisms: microRNAs (miRNAs).
MiRNAs are small, single-stranded noncoding RNAs of 20–24 nucleotides in lengths that modulate gene translation of mRNAs by binding to complementary sequences generally located in the 3′-untranslated region (UTR) of target transcripts.8–10 Their biogenesis has been thoroughly described elsewhere and is mainly controlled by 2 enzymes known as Drosha and Dicer.11–13 Most cell types actively release miRNAs into the extracellular environment; these miRNAs are either encapsulated into vesicles or associated within protein complexes that protect them from RNAses. Once in the blood stream, miRNAs can be absorbed by cells and can actively control gene expression,14 resulting in a very effective paracrine control over nearby cells.15 Hence, serum/plasma miRNA concentration profile has been proposed as a biomarker to develop miRNA-based therapies and diagnostic tools.16–20 Not unimportantly, the great potential of using miRNA profiling is also because of their stability compared with messenger RNAs.21
MicroRNAs are well known to play essential functions in cellular differentiation, proliferation and apoptosis, and they are also involved in human diseases, in the modulation of innate and adaptive immunity, and in the modulation of host–virus interaction.8,10,22 Several studies examined the effects of HIV infection on miRNAs. Results showed that miRNAs can directly and indirectly modulate the HIV replication process through their interaction with cellular genes that are involved in the replication mechanisms. In particular, 5 cellular miRNAs (miR-28, miR-125b, miR-150, miR-223, and miR-382) were shown to target the 3′ long terminal repeat (LTR) region of the HIV genome, thus contributing to the maintenance of latency in resting CD4+ T cells and monocytes.23 More recently, 5 miRNAs with putative target sites in the HIV-1 RNA genome (miR-92a, miR-133b, miR-138, miR-149, and miR-326) were demonstrated to decrease HIV-1 infection.24 Among miRNAs that also indirectly target HIV-1 replication, miR-198,25 miR-27b, miR-29b, miR-150, and miR-22326 inhibit cyclin T1 expression in resting CD4+ T lymphocytes, whereas 6 other miRNAs (miR-15a, miR-15b, miR-16, miR-20a, miR-93, and miR-106b) target the message encoding the Tat cofactor Pur−α (purine-rich element binding protein α), resulting in a decreased susceptibility to HIV-1 infection in monocytes.27 The downregulation of miR-150 and miR-223 was instead shown to target the HIV-1 RNA genome “restricting” virus expression.28 Finally, analyses performed in HIV-infected individuals identified 62 miRNAs that are differentially modulated in peripheral blood mononuclear cell (PBMC) of HIV/AIDS patients with different CD4 T cell counts and viral loads.24,29
Although recent studies have described cellular and plasma miRNA profiles in a number of cohorts of HIV-infected and exposed subjects,14,28,30–35 additional studies are essential to examine more deeply the role of miRNAs in the field of HIV resistance and progression.
Hence, we profiled the expression of 84 miRNAs with a known antiviral and/or immunological activity in PBMC, plasma, and cell culture medium of HIV-1-infected cells from a well-characterized HESN cohort.36 Results demonstrated that the HESN phenotype is associated with a specific miRNA profile.
Blood samples were collected from 30 HESN and 30 HIV-1-positive individuals who are part of a serodiscordant cohort of heterosexual couples recruited at the S. Maria Annunziata Hospital in Florence, Italy, that has been followed since 1997.36 Thirty age-matched healthy controls (HC), without known risk factor for HIV infection, were also included in the study. The whole group is Italian of Caucasian origin. Inclusion criteria for HESN have been previously reported.36 All individuals were longitudinally followed for >4 years before the study by the Department of Obstetrics and Gynecology of the S. M. Annunziata Hospital. This allowed us to exclude from the study HESN and HIV-1-infected patients in whom sexually transmitted diseases or other pathologies were reported during that time period. The range of CD4 cell counts in HIV-1-infected patients was 71–927 cells per milliliter, and viral loads were <50–>750,000 copies per milliliter. All the patients were receiving antiretroviral therapy at the time of the study (Supplemental Digital Content, http://links.lww.com/QAI/A831).
The study was designed and performed according to the Helsinki declaration and was approved by the Ethics Committee of the participating units. All subjects provided written informed consent to participation in this study and none of them reported risk behaviors (eg, drug or alcohol abuse) that could alter miRNA expression.
No CCR5Δ32-homozygous subjects were included in the study.
HIV-1 Infection Assay
Whole blood was collected by venipuncture in vacutainer tubes containing EDTA (Becton Dickinson, Franklin Lakes, NJ), and basal PBMC were separated on Lympholyte-H separation medium (OrganonTeknica; Malvern, PA). PBMC from HESN and HC were infected with 0.5 ng/1 × 106 cells HIV-1Ba-L virus as previously described.5 One and 7 days postinfection 1 × 106 PBMC were analyzed for miRNA expression, whereas p24 antigen was analyzed on 7-days postinfection cell culture medium by HIV-1 p24 ELISA assay kits (XpressBio, Frederick, MD) in accordance with the manufacturer's protocol. MiRNA expression was analyzed on 7-days postinfection cell culture medium as well. Before any analyses, cells were counted and cell viability was assessed by an automated cell counter ADAM-MC (Digital Bio, NanoEnTek Inc., Seoul, Korea). Only samples showing at least 95% viability were used for further analyses.
Total RNA Extraction and miRNA Isolation From PBMC, Plasma and Cell Culture Medium
Total RNA was extracted as previously described.5 MicroRNAs were isolated from 600 μL of plasma and 300 μL of cell culture medium by NucleoSpinMiRNA Plasma Kit (MACHEREY-NAGEL, Düren, Germany) in accordance with the manufacturer's protocol.
MicroRNA Reverse Transcription and Real-Time PCR Array Analysis
One microgram of RNA was reverse-transcribed into first-strand cDNA in a 20 μL final volume at 37°C for 60 minutes using miScript II RT Kit (Qiagen, Venlo, the Netherlands) in accordance with the manufacturer's protocol. Expression level of 84 miRNAs with antiviral and/or immunological function was evaluated in unstimulated PBMC and plasma using a miRNA PCR Array (MIHS-111Z) (Qiagen). Experiments were run on all of the subjects included in the study pooled into a unique HESN, HC and HIV+ sample. Thus, the results represent the mean value of the different analyzed targets in HESN, HC, and HIV+ subjects. The arrays were performed on CFX ConnectTM Real time PCR system (BIO RAD, Hercules, CA). Undetermined raw CT values were set to 35. Expression profile was analyzed using the PCR Array Gene Expression Analysis Software (SABiosciences, Frederick, MD). For each miRNA, Ct values were transformed into relative quantities using as normalization factor the arithmetical mean of the references available in the arrays (SNORD68, SNORD72, RNU6-2 for basal PBMC and Cel_miR-39_1, miR-93 and RNU6-2 for plasma). Fold regulation of ±2.5 was considered significant.
Validation of Array Results by Individual Real-Time PCR
Targets showing marked differences among HESN, HC, and HIV+ patients in both unstimulated PBMC and plasma were retested by RT-PCR on each individual sample (PBMC, plasma, and cell culture medium). Samples were amplified using the miScript SYBR Green PCR Kit. The primers (Qiagen) used were: hsa-miR-15a-5p, hsa-miR-16-5p, hsa-miR-17-5p, hsa-miR-21-5p, hsa-miR-26a, has-miR-28-5p, hsa-miR-29a-3p, hsa-miR-29b-3p, hsa-miR-29c-3p, hsa-miR-34a-5p, hsa-miR-92a-3p, hsa-miR-125b-5p, hsa-miR-138-5p, hsa-miR-146a-5p, hsa-miR-150-5p, hsa-miR-155-5p, hsa-miR-190b, hsa-miR-223-3p, hsa-miR-326, hsa-miR-382-5p. Endogenous controls used to normalize the relative miRNA expression were: small RNAU6 for PBMC, miR-425 and miR-93 for plasma, and cell culture medium as used by others.25,37,38 Synthetic cel-miRNA-39 was also used as quality control of miRNA extraction for the measurement of extracellular miRNAs.37
Experiments were performed in duplicate for each sample and fold changes were calculated by the equation 2−ΔΔCt.
Gene Expression Analyses
One microgram of RNA from unstimulated PBMC was reverse-transcribed into first-strand cDNA as previously described.5 Real-time PCR was performed for Dicer, Drosha, and GAPDH using a SYBR Green PCR mix (Bio-Rad, Hercules, CA). Results were calculated relative to GAPDH as a housekeeping gene and expressed as 2−ΔΔCt.
Data were analyzed using Student's t test by GRAPHPAD PRISM version 5 (GraphPad Software, La Jolla, CA), and P-values of 0.05 or less were considered to be significant.
Expression of miRNAs in Unstimulated PBMC
Expression of 84 miRNAs thought to exert immunomodulant and/or antiviral properties was measured by real-time PCR in unstimulated PBMC of 30 HESN, 30 HIV+ and 30 HC subjects; individual samples were grouped together in these initial assays to create 3 different pools. Results showed that whereas the expression of 50 miRNAs was comparable in the 3 groups, (1) 10 miRNAs (miR-26b, miR-29b, miR-29c, miR-34, miR-146, miR-148a, miR-155, miR-195, miR-326, and miR-365b) were significantly increased in both HESN and HIV+ individuals compared with HC; (2) 9 miRNAs (miR-9a, miR-17, miR-18a, miR-20b, miR-21, miR-106b, miR-126, miR-130b, and miR-182) were upregulated and 3 miRNAs (miR-138, miR-150, and miR-190) were downregulated only in HIV+ compared with HC; and (3) the expression of 12 miRNAs (miR-15a, miR-16, miR-26a, miR-28, miR-29a, miR-92, miR-99a, miR-125b, miR-128, miR-142, miR-223, and miR-423) was significantly augmented in HESN alone compared with HC (Fig. 1A).
MicroRNAs in Plasma
Plasma concentration of the same miRNAs was measured next in the same individuals, once again grouped into 3 pools. Whereas no differences were seen in the expression of 53 miRNAs, the concentration of 31 miRNAs distinguished the 3 groups. Thus: (1) the concentration of 13 miRNAs (miR-23b, miR-28, miR-29a, miR-29b, miR-29c, miR-30c, miR-146, miR-150, miR-155, miR-190, miR-346, miR-326, and miR-382) was similarly increased in HESN and HIV-infected individuals compared with HC; (2) 4 miRNAs (miR-16, miR-17, miR-34, and miR-92) were increased and 2 miRNAs downregulated (miR-210 and miR-31) only in HIV+ patients compared with HC; and (3) 12 miRNAs (miR-15a, miR-21, miR-25, miR-26a, miR-98, miR-125b, miR-138, miR-139, miR-147a, miR-184, miR-214, and miR-223) were augmented in plasma of HESN alone compared with HC (Fig. 1B).
Validation of miRNA Expression in Unstimulated PBMC by Individual Real-Time PCR
The expression of the 20 miRNAs that were augmented in unstimulated PBMC and/or in plasma of HESN and HIV-infected individuals was validated next by individual real-time PCR. Results obtained in unstimulated PBMC confirmed that all these molecules are differently expressed in HESN and HIV-infected individuals compared to HC, with statistically significant differences being observed in 5 cases (miR-29a, miR-138, miR-150, miR-190, and miR-223). Notably, among these 5 molecules, the expression of 3 miRNAs (miR-138, miR-150, and miR-190) that play a role in HIV-1 latency was significantly reduced in HIV-infected patients alone. The expression of the 2 remaining miRNA (miR-29a and miR-223) was significantly increased exclusively in HESN (P < 0.01 for all comparisons) (Fig. 2).
Validation of miRNAs expression in Plasma by Individual Real-Time PCR
Plasma concentration of the same 20 miRNAs was validated as well by individual real-time PCR. Results confirmed that the concentration of a group of miRNAs (miR-28, miR-29a, miR-29b, miR-29c, miR125b, miR-146, miR-150, miR-155, miR-190, and miR-382) is increased in both HESN and HIV+ individuals compared with HC, indicating the presence of a “retroviral exposure signature” that is shared between these 2 groups of individuals, and occurs even in the absence of a productive infection. Plasma expression of miR-138 and miR-223, however, was increased in HESN alone compared with either HIV+ patients (miR-138 and miR-223: P < 0.05) or HC (miR-138: P < 0.05; miR-223: P < 0.0001) (Fig. 3). As the PCR normalizers used in the array and in the individual PCR are different, the replication of the results strengthens the key role played by these miRNAs in natural resistance to HIV-1 infection.
HIV-1 p24 and miRNA Expression Analyses After in Vitro HIV-1 Infection of PBMC
Cellular expression of the same 20 miRNAs was analyzed next after in vitro HIV-1 infection. PBMC from 30 HESN individuals and 30 HC were infected with HIV-1Ba-L virus and cultured for 7 days. HIV-1 p24 ELISA suggested that PBMC from HESN display a better control of HIV-1 infection (Fig. 4A). No differences in miRNA expression were observed 1-day postinfection in HESN compared with HC (data not shown). Seven days postinfection, however, all the 20 analyzed miRNAs were downregulated to various extents in HESN compared with HC; these differences reached statistical significance for miR-28 (P < 0.001), miR-29a (P < 0.05), miR-125b (P < 0.05), miR-150 (P < 0.05), and miR-223 (P < 0.05) (Fig. 4B).
Differential miRNA Expression in Cell Culture Medium From HIV-1-Infected PBMC
To verify whether miRNAs are indeed released in the extracellular milieu by HIV-1-infected cells, the expression level of the 20 selected miRNAs that were seen to be downregulated in in vitro HIV-1-infected PBMC (see above) was analyzed next in cell culture medium from the same PBMC. Results confirmed our hypothesis as the expression level of all the analyzed miRNAs was higher 7-days postinfection in cell culture medium of HESN compared with those of HC. These differences reached statistical significance for miR-28 (P < 0.05), miR-29a (P < 0.05), miR-125b (P < 0.05), miR-150 (P < 0.05), and miR-223 (P < 0.05) (Fig. 4C). These results suggest that after HIV-1 infection, these miRNAs are released in the extracellular milieu where they could display a paracrine control over neighboring cells.
Analysis of Dicer and Drosha Expression Level
As increased expression levels of miRNAs could be secondary to higher levels of Dicer and Drosha, 2 essential enzymes in miRNA biogenesis, these enzymes were analyzed in unstimulated PBMC of all of the subjects included in the study. Results did not show any appreciable difference in the expression level of Dicer and Drosha in PBMC of HESN compared with HC and HIV+ individuals (Fig. 5).
Eighty-four miRNAs that are mainly expressed by lympho-monocytes and involved in the regulation of immune responses and/or in host–virus interaction13,38 were analyzed to verify whether a particular miRNA profile could be identified in HESN; results were validated by analyses on 20 of these miRNAs by individual real-time PCR. The decision to study miRNA profile in PBMC was based on their accessibility and on the fact that PBMC include the major circulating targets of HIV-1 infection and therefore represent the logical first step toward characterizing cell subtype expression. Findings herein show that a number of miRNAs is upregulated both in HESN and HIV-infected individuals, indicating that exposure to HIV-1 impairs the miRNA profile independently of the establishment of overt infection. Additional results indicated that the expression of miR-29a and miR-223 was significantly augmented in plasma, basal PBMC, and cell culture medium from HIV-1-infected PBMC of HESN alone. The plausible mechanisms by which the increased expression of these 2 miRNAs could exert a protective role in resistance to HIV-1 infection are more than a few. Both miRNAs have been reported to be modulated in other retroviral infections such as HTLV-2, SIV, and HERV-L.39–42 MicroRNA-29a has been shown to play a crucial role in thwarting HIV-1 replication because it is able to bind both the 3′ UTR sequence and the HIV-1 encoded protein Nef, hence preventing viral replication.43 Furthermore, reduced miR-29a expression in HIV-1-infected patients has been associated with the presence of a secondary structure within the miR-29a binding region in HIV-1 LTR.44 Accordingly, reporter assay by transfection of miR-29 and ectopically expressed pri-miRNA resulted in a strong reduction of p24 levels, whereas knockdown of endogenous miR-29a/b by anti-miR transfection led to enhanced HIV-1 infection.44
MiR-223, on the other hand, is part of a selected group of cellular miRNAs known to play an active role in modulating the ability of HIV-1 to infect resting CD4+ T lymphocytes.23 This effect depends on the ability of miR-223 to bind the 3′ UTR of viral mRNA through complementary sequence. Notably, activation of resting CD4+ T cells, a process known to trigger HIV-1 replication and to enhance HIV-1 susceptibility, is associated with the downregulation of this miRNA. Two other independent studies confirmed miR-223 to be downregulated upon differentiation from monocyte to macrophages, thus shedding light on the mechanism associated with the productive HIV-1 infection taking place in macrophages.45,46 Finally, miR-223 was also reported to indirectly modulate cyclin T1 expression, resulting in lower HIV-1 replication,26 and to play a critical role in systemic cholesterol regulation by coordinated post-transcriptional control of multiple genes in cholesterol metabolism.47 Indeed, miR-223 overexpression is associated with a rise in ABCA1 mRNA and activity, resulting in an increased cholesterol efflux from cells47 and, potentially, a reduction of HIV-1 replication47–49 also via this mechanism. Notably, our results are in conflict with previous data showing a downregulation of miR-223 in resting CD4 T cells from HESN.28 However, because our PBMC samples have not been fractionated into cellular subset, we reasoned that some of the differences in miRNA signature could be explained by in/out flux of miRNAs from different cell types.
The above findings allow us to speculate that an initial exposure to subinfectious amounts of HIV-1 is sufficient to modify the expression of specific miRNAs in individuals in whom subsequent exposures to the same virus will not result in infection. Alternatively, it is possible that higher levels of these 2 miRNAs are “naturally” present in HESN before exposure, and that their augmented expression is protective or indicative of the presence of alternate mechanisms of defense. To further investigate these hypotheses, we next analyzed miRNA expression profiles after in vitro HIV-1 infection of PBMC from HESN and HC. Unexpectedly, the expression of all the analyzed miRNAs was downregulated to various extents in HESN compared with HC after in vitro infection, and differences reached statistical significance for miR-28, miR-29a, miR-125b, miR-150, and miR-223. We wondered how to reconcile the reduced susceptibility to HIV-1 infection in HESN PBMC with the observed decreased expression of anti-HIV-1 miRNAs. Advances in the understanding of miRNA physiology, showing that miRNAs can be released upon cellular activation, offer a plausible explanation for these results. In fact, recent data indicate that the release of miRNAs in association with exosomes, or with protein complexes, is not a passive phenomenon, but is actually a regulated active process. In fact, exosomal RNA content is not a mere reflection of what is found within the intracellular milieu and quantitative analyses show a selective enrichment of some miRNAs when exosomes are compared with cells.50–52 These data suggest that the controlled release of miRNAs into exosomes represents an additional layer of post-transcriptional regulation for miRNAs that, in this form, can have a rapid and effective paracrine effect on target genes of neighboring cells.
Our working hypothesis, indeed, is based on the assumption that when the immune system is triggered upon viral exposure, activated cells would release a detectable quantity of miRNAs into the bloodstream. In turn, these miRNAs would modulate gene expression in paracrine cells, thus controlling HIV-1 infection and replication. MicroRNA release after lymphocyte activation is dependent on the magnitude of immune response.53 Our cohort of HESN is characterized by an immune-activated phenotype;36,54 therefore, it is possible to speculate that increased miRNA release by HESN cells could be the logical consequence of a greater responsiveness of their immune system to viral exposure. Notably, the expression of the key miRNAs was increased in both cell culture medium of HIV-infected PBMC and plasma of HESN. Actually, as Fabbri et al55 demonstrated that secreted miRNAs (mainly miR-29a) may act as paracrine agonists of TLRs and TLR activation has been associated with resistance to HIV-1 infection,54 it is tempting to hypothesize that these miRNAs could be internalized by neighboring cells and modify their susceptibility to infection.
The differences observed in miRNA expression when in vivo and in vitro infected PBMC of HESN were analyzed could be due to the viral load used in the experimental procedure. Although the viral burden used in the in vitro assay was relatively low, cells were exposed to concentrations of HIV-1 that likely are much higher than those encountered upon natural exposure; this could have speeded up the release of those miRNAs that might have a protective effect in controlling HIV-1 infection. An alternate possible explanation is that, whereas PBMC were infected in vitro to an HIV-1 lab strain (HIV-1Ba-L), HESN are exposed to primary HIV strains whose virulence is likely different.
Finally, results showing that the expression level of Dicer and Drosha, two enzymes playing essential roles in miRNA biogenesis, was comparable in HIV-infected patients, HESN and HC suggest that exposure to HIV-1 changes miRNA profiles through mechanisms that are independent of Dicer and Drosha. Yet, in the light of recent acquisitions suggesting that Drosha protein level and nuclear localization can be altered by molecular mechanisms such as ubiquitination, acetylation56 and phosphorylation,57 without affecting mRNA levels, we cannot rule out that differences in miRNA expression observed in our cohort are dependent on these mechanisms. Further analyses are needed to elucidate this aspect.
The mechanism for miRNA upregulation in HESN remains elusive. Nevertheless, if HIV-1 exposure truly affects miRNA transcription and maturation in particular subsets of PBMC, such knowledge might be useful for the design of novel HIV-1 therapies and for diagnosis, prognosis, and treatment response parameters.
HIV-1Ba-L was provided through the EU programme EVA centre for AIDS Reagents NIBSC, UK. Thanks to healthy blood donors at Santa Maria Annunziata Hospital in Florence included in the study as healthy controls.
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