Recent reports have indicated the miRNA-29 family as a putative regulator of key processes against HIV-1 infection. Thus, we compared the expression profile of miRNA-29a, miRNA-29b and miRNA-29c in HIV-1-infected patients and healthy donors, and evaluated whether and how differences in expression could influence the clinical progression of HIV-1 infection and the antiviral immune response.
Interestingly, we found a significant positive correlation between miRNA-29b levels and the age of healthy donors but not with that of HIV positive patients, suggesting an age-dependent regulation of miRNA-29b expression in healthy individuals. In agreement with this observation, Zhang et al. reported that mechanisms of transcriptional regulation of miRNA transcripts and altered expression of Argonaut proteins contribute to age-related changes in the expression of some miRNA and miRNA* strands [25]. The loss of correlation between age and miRNA-29b levels observed in the HIV-infected patients could be explained as a consequence of viral pathogenesis: HIV-1 infection in vivo is expected to exert physiologic effects on T-cell function which could be reflected in significant miRNA changes. Directly related to this, we found that levels of miRNA-29b expression were significantly higher in HIV-1-infected patients compared to the control group. No significant correlation was found between miRNA-29a and miRNA-29c expression levels and the age of either healthy donors or HIV-1 positive patients. We also failed to detect any significant difference between miRNA-29a and miRNA-29c transcript levels measured in healthy donors and HIV-1 positive patients, although we did observe a trend toward higher levels of both miRNAs in HIV-1-infected patients. This could be explained by the fact that although the three mature members of the miRNA-29 family share a common seed region sequence and are predicted to target largely overlapping sets of genes, they exhibit differential regulation in several cases and different subcellular distribution [26]. In addition, their stable expression levels are likely to depend on expression of the two clusters, alternative splicing of primary RNA and differential decay, all factors which may vary in a cell-specific manner [6].
In line with our results, it was recently reported that HIV-1-infected patients are characterized by an altered miRNA expression profile compared to healthy donors. Witwer and coworkers described an altered PBMC miRNA profile in elite suppressors and untreated viraemic patients compared to uninfected controls. However, they found that among miRNAs with significant expression changes expression levels were more often lower in viraemic individuals. For example, control PBMC had higher mean levels than viraemic PBMC of all miRNA-29 family members [13]. Furthermore, Houzet and colleagues profiled miRNA expression in PBMC from 36 HIV-1 seropositive individuals categorized into four classes based on their CD4+ T cell counts and viral loads. They found that specific miRNA signatures, including miRNA-29s, can be observed for each class [12]. Having observed that HIV-1 infection is associated with altered patterns of miRNA-29 expression, we tried to evaluate whether this phenomenon would affect the plasma viral load and CD4+ T cell count. In a first analysis, we did not find any significant association between miRNA-29 expression levels and plasma HIV RNA levels. This observation is in agreement with Witwer et al., who found no correlations with viral load in the viraemic group, although miRNA-29a has been reported to silence HIV-1 in vitro [7-9]. We also failed to find any significant association between miRNA-29s expression and CD4+ T cell count, whereas Witwer et al.’s study found both negative and positive correlations between several miRNAs, including miRNA-29a, and CD4+ T cell counts. However, this correlation was only present when elite suppressor, not included in our study population, and viraemic patients were grouped together. To further analyze the relationship between miRNA-29s expression and the viro-immunological markers of HIV-1 infection, we grouped the chronically HIV-1-infected patients on the basis of their plasma HIV RNA and CD4+ T cell count, to represent different stages of the infection. Although there was no significant difference in expression levels of miRNA-29a and miRNA-29b between patients with more or less severe HIV-1 infection, interestingly we found that miRNA-29c levels were related to both viral load and CD4+ T cell count. Indeed, patients expressing lower levels of miRNA-29c also had higher levels of viraemia and lower levels of CD4+ T cell count, suggesting a strong relation between miRNA-29c and these clinical markers of HIV-1 infection. This relation may imply that baseline lower levels of miRNA-29c expression in some individuals could negatively influence the progression of HIV-1 infection. Alternatively, the presence of HIV-1 or the host response against HIV-1 may reduce miRNA-29c expression, by inducing CD4+ T-cell depletion.
Several studies reported that the onset of HIV-1 latency could be influenced by cellular miRNA [27], but the role of miRNA-29s in controlling this phenomenon has not been well characterized. For the first time to our knowledge, our results show that miRNA-29c expression levels are significantly and negatively correlated with levels of integrated HIV-1 DNA. Thus, in our study population, patients with lower levels of miRNA-29c not only showed higher levels of plasma viraemia and a lower CD4+ T cell count, but also had higher levels of HIV-1 DNA, confirming the association between low levels of expression of miRNA-29c and poor prognosis. Probably, the higher rate of proliferation observed in patients with a lower CD4+ T cell count led to a larger overall reservoir size. On the other hand, we cannot exclude that by suppressing HIV-1 production miRNA-29a could regulate viral gene expression, modulating the viral life cycle and promoting the onset and maintenance of latency. Moreover, given that we found the same significant trend in miRNA-29a levels, miRNA-29a and miRNA-29c could influence HIV-1 latency by a common mechanism. However, miRNA-29a-c and HIV-1 DNA integrated levels were quantified only in total PBMCs, and may have missed differences in various cell types, such as resting central memory T cells and translational memory T cells, which serve as major sites for HIV-1 latency [28].
Interestingly, in this study we also found that miRNA-29a/b/c signature was similar in both CD4+ T lymphocytes and CD14+ monocytes to that previously observed in total PBMC of naive HIV-1 infected patients. Furthermore, we observed that the amount of miRNA-29a-c measured in CD4+ T lymphocytes and CD14+ monocytes were highly variable and not different in these cellular subsets. These findings indicate that both CD4+ T lymphocytes and CD14+ monocytes can actively contribute to the production of miRNA-29 a/b/c during HIV-1 infection and suggest that CD4+ T cell alteration does not explain all differential expression of miRNA-29a/b/c recorded in HIV-1 patients. In agreement, Witwer KW et al. reported that some cellular miRNAs that have been found at high levels across PBMC subsets or are even enriched in CD4+ T-cell subsets [29]-and would therefore be expected to decline along with CD4+ T cells were, to the contrary, negatively correlated with CD4+ T cell count [13]. Further studies are needed to characterize the effects of HIV-1 on miRNA-29s expression in PBMC and specific cell types both in vivo and, as far as possible, in primary culture ex vivo.
Our study is also the first to evaluate the influence of miRNA-29b on the expression of different isoforms of a novel anti-HIV cytokine, namely IL-32, during chronic HIV-1 infection. Given that six isoforms of IL-32 have been identified, we chose to detect IL-32α and the other IL-32 isoforms (β, γ, δ, ε, ζ) separately, using a real time PCR assay previously developed in our laboratory [17]. We found a weak inverse correlation between miRNA-29b and IL-32nonα levels in PBMC collected from HIV-1-infected patients, whereas no significant correlation was found with IL-32α levels, suggesting that miRNA-29b specifically targets other isoforms of IL-32. Our data are in agreement with those reported by Li and coworkers, who showed that miRNA-29b and IL-32 mRNA levels were negatively correlated in PBMC samples from HBV-infected patients [18]. However, further analyses will be needed to confirm the presence of these relationships with a larger sample size of HIV-1 infected patients and to identify which specific isoform/isoforms of IL-32 is/are regulated by miRNA-29b.
One of the tools used by IL-32 to fight viruses, including HIV-1, is its ability to induce MxA expression [17]. We previously demonstrated that IL-32γ can significantly induce expression of IFN stimulating genes (ISGs), including MxA, in PBMC collected from healthy donors, although to a lower extent than IFNα2b [17]. Moreover, recent reports showed that IL-32 exerts its antiviral activity through the induction of IFN-λ1 and IFN-β expression, which in turn act by inducing ISGs [18,19]. Thus, we evaluated the influence of miR-29b expression on the transcript levels of MxA in HIV-infected patients. As expected, patients expressing higher levels of miRNA-29b showed lower levels of MxA, confirming that MxA induction is regulated by IL-32, both directly and through a type I and III IFN-mediated pathway. Interestingly, we also found that CD4+ T lymphocytes as well as CD14+ monocytes were capable of producing miRNA-29b, IL-32 isoforms and MxA indicating that these cellular subsets were involved in the activation of the IL-32- and IFN-mediated antiviral response during HIV-1 infection. Taken together, these data suggest that the up-regulation of miRNA-29b observed in HIV-infected patients has a strong negative impact on the antiviral immune response and that the virus may take advantage of the change in the normal cell miRNA profile. On the other side, considering the complex and to some extent controversial role played by IFN-α/β in HIV-1 disease [30], our results indicated that miRNA-29b would contribute to the regulation of the rate of IFN activation by suppressing the IL-32 nonα isoforms levels during HIV-1 infection. However, several cellular pathways are regulated by both miRNA-29 and IFN subtypes highlighting the complexity of phenomenon analyzed [31-33]. In this regards, miRNA-29 can regulate and activate T-box transcription factors and IFN-γ production in helper T cells [31]. Furthermore, epigenetic changes mediated by miRNA-29 can induce IFN-λ1 production during viral infection and the suppression of the IFN-α receptor expression can be mediated by miRNA-29 in thymic epithelium to increase the threshold for infection-associated thymic involution [32,33].
In conclusion, we showed that transcription levels of all mature members of the miRNA-29 family are highly variable in HIV-1-infected patients and that the miRNA-29b expression pattern is altered in this population compared to healthy individuals. We also found that miRNA-29c expression is closely correlated with markers of HIV-1 clinical outcome, such as plasma viral load and CD4+ T cell count, and that both miRNA-29a and miRNA-29c could affect the HIV-1 proviral load. In addition, we demonstrated that miRNA-29b levels influence the rate of IL-32nonα and MxA expression, highlighting the role of the miRNA-29 family as a double-edged sword during in vivo HIV-1 infection.