L'ESPOSIZIONE AD HIV-1 INFLUENZA LA RISPOSTA IMMUNITARIA CD4-MEDIATA ALTERANDO IL PROFILO D'ESPRESSIONE DEI MICRORNA CELLULARI

MicroRNAs (miRNAs) are small single-strand non-coding RNAs that repress gene expression by inhibiting translation. MiRNAs are known to inhibit HIV-1 expression interfering with viral mRNAs. In order to better understand how HIV-related alterations in cellular physiology and immunologic control function, 3 classes of patients (éLTNP, Naive and multiply exposed to HIV-1 uninfected subjects, named MEU) were enrolled, from whom blood CD4+ cells were purified. In this cellular subtype the expression levels of 377 miRNAs were analyzed by TaqMan real-time PCR-based arrays. Only miRNAs expressed in 70% of patients of at least one class and of at least 1 log10 variation from healthy controls were selected. A similar analysis had also been performed in primary culture of CD4+T cells and monocyte-macrophages infected by R5 strains and in CD4+ cells exposed to recombinant and natural gp120 molecules in vitro. In all experiments, expression levels of Dicer and Drosha, two enzymes involved in miRNA biogenesis, were evaluated by real-time PCR. Similar miRNA profiles were detected in the CD4+ cells from éLTNP and Naive, although these infected patients had different parameters such as viral and proviral loads, infection time and CD4+ T cell number. Moreover, a complex down-regulation of miRNA was observed in the CD4+ cells from MEU subjects. This indicates that the only HIV-1 antigen exposure can modulate cellular programming process of CD4+ cells. Three up-regulated (miR-203, 449a, 502-5p) and five down-regulated (miR-329, 337-5p, 379, 503, 518d-3p) miRNAs, modulated in all patient classes, defined a HIV-1 signature. By hierarchical clustering of the miRNA profile, éLTNP clustered with Naive patients, whereas all MEU subjects grouped together; this suggests that miRNA expression may discriminate between infected and uninfected individuals. By statistical analysis, 16 miRNAs significantly differentiated éLTNP and Naive from MEU, while only the miR-155 discriminated éLTNP from Naive. Of these, 9 were involved in viral replication and/or in immune response. Computational studies suggested that target genes of altered miRNAs are involved in a lot of cellular processes and in binding/catalytic activity. According to the Dicer and Drosha expression, that was down-regulated in all patient classes, a correlation was observed between miRNA and the two enzymes expression. In contrast, no differences were observed in CD4+ cells infected in vitro or exposed to gp120 molecules. This suggests that there is a complex regulation of miRNA expression after HIV exposure, not only due to the miRNA biogenesis. The miRNA expression analysis in CD4+ cells exposed to gp120 molecules showed a modulation of 66 miRNAs; only 42% of these showed the same levels of expression, suggesting that the remaining percentage of miRNAs was modulated by other gp120 epitopes. When the gp120 was neutralized with mAbs, only 1/3 of altered miRNAs were reverted to the expression of non exposed controls. This indicates the existence of epitopes (other than the CD4 binding domain) capable of inducing a miRNA modulation. Finally, the miRNA expression analysis highlights that 5 miRNAs (miR-34c-p, 518f, 452, 202 and 487b) could have a role in HIV-CD4+ cell binding. In conclusion, in light of these results, it can be supposed that the altered miRNA profile is due to the constant HIV exposure of CD4+ cells through a bystander phenomenon rather than a direct effect. Therefore it can be assumed that only the exposure to gp120 molecules can leave a signature in immune cells.