GUT HOMING and HIV-1 PERMISSIVENESS ARE SPECIFICALLY REGULATED by RETINOIC ACID in CCR6+ T-CELLS

Background: The majority of HIV-infected CD4+ T-cells are localized in gut-associated lymphoid tissues (GALT). The integrin β7, CCR6, and CCR9 mediate the gut-homing and their expression is induced by retinoic acid (RA), a metabolite of vitamin A produced by the GALT dendritic cells. We previously identified CCR6 as a marker for memory CD4+ T-cells permissive to HIV and demonstrated that RA selectively increased HIV replication in CCR6+ T-cells by interfering with yet uncharacterized mechanism at entry and/or post-entry levels. In search for the identification of new molecular determinants of HIV permissiveness, we performed a genome-wide analysis of gene expression in RA-treated CCR6+ versus CCR6- T-cells. Methodology: CD4+ T-cells were sorted from PBMCs by negative selection using magnetic beads (Miltenyi). Memory (CD45RA-) CCR6+ and CCR6- T-cells were sorted by flow cytometry (BDAriaII). Cells were stimulated via CD3/CD28 and cultivated in the presence or absence of ATRA (10 nM) for 4 days. Total RNA was extracted for microarrays analysis (HT 12v4 BeadChip, Illumina; >46,000 probe sets per chip) and real-time RT-PCR quantification. In parallel, cells were used for flow cytometry validation of gene expression. Finally, a fraction of cells was exposed to HIV. Viral replication and integration was measured by HIV-p24 ELISA and real-time PCR, respectively. Results: Among 15,303 “present calls”, 1,538 and 1,285 probe sets were modulated by RA in CCR6- and CCR6+ T-cells, respectively, with only 466 probe sets being regulated by RA in both subsets (p-value <0.05). Gene classification using Gene Ontolog revealed that RA-treated CCR6+ T-cells expressed a specific transcriptional signature that included known HIV permissiveness factors, while RA-treated CCR6- T-cells expressed genes linked to HIV resistance. A number of transcripts were validated by real-time PCR and flow cytometry as being differentially expressed in RA-treated CCR6+ (e.g., RARESS3, KLF2, CCR5, CCR9, CXCR6) and CCR6- T-cells (e.g., ABCA1). Conclusions: We identified a molecular signature associated with HIV permissiveness in RA-treated CCR6+ T-cells. This signature includes RARESS3 or RA-induced gene 1 (RIG-I); the transcription factor KLF2 that regulates CCR5 transcription; CCR5, a major HIV co-receptor; CXCR6, a minor HIV co-receptor also involved in cell-to-cell transmission of HIV; CCR9, a chemokine receptor essential for the migration in lamina propria. At the opposite, RA-treated CCR6- T-cells upregulated expression of ABCA1, a known HIV restriction factor. Our studies reveal that transcriptional changes induced by RA in CCR6+ T-cells are associated with HIV permissiveness and gut-homing potential thus, providing a molecular mechanism for preferential HIV replication in GALT CD4+ T-cells.