STUDIO DELLA TRASLOCAZIONE DI MACROMOLECOLE MICROBICHE E DEI RESERVOIR VIRALI COME POSSIBILI MECCANISMI PATOGENETICI DI MANCATO RECUPERO DI CD4 IN SOGGETTI HIV-POSITIVI

Study of the translocation of bacterial bioproducts and HIV reservoirs as pathogenetic mechanisms behind the lack of CD4 T-cell reconstitution in HIV-infected subjects. BACKGROUND Up to 30% of HIV+ subjects starting HAART become Immunological Non responders (INRs) with incomplete CD4 recovery despite viral suppression. The mechanisms behind the lack of CD4 recovery still remain unclear, although many factors including older age, hepatitis coinfection, HIV residual viremia and CD4 nadir are involved. In particular, INRs feature higher immune activation, not completely reverted by HAART, possibly supported by microbial translocation (MT) and persistence of HIV viral reservoirs. The relationship between MT and reservoirs is not fully understood, more studies are needed to clarify their role in the setting of HIV-infection. Based on these premises, we aimed to investigate the role of MT and viral reservoir in the pathogenesis of INRs. In particular, we studied: Part 1: the translocating microflora composition. Part 2: the effect of Toll-like receptors (TLRs) stimulation. Part 3: the persistence of HIV viral reservoir METHODS & RESULTS (part 1) 44 HIV+ patients (pts) with CD4≤200/mmc were followed for 12 months (T12) following HAART introduction. At T12, pts were divided in: 15 Immunological Non Responders (INRs, CD4<200/mmc; HIV-RNA<60cp/mL) and 29 Partial Immunological Responders (PIRs, CD4≥250/mmc; HIV-RNA<60cp/mL).13 HIV-negative healthy subjects were enrolled as controls. Analyses: quantification of LPS and sCD14 plasma levels (ELISA); identification of 16S rRNA gene (PCR and sequencing analysis). We showed that PIRs and INRs presented similar elevated plasma levels of lipopolysaccharide (LPS) and its ligand sCD14 that were not lowered by virologically suppressive therapy. Bacterial 16S rRNA gene amplification and sequencing resulted in a highly polymicrobic peripheral blood microbiota both prior and after 12-month HAART. Several differences in bacterial composition were shown between patients’ groups, mainly the lack of probiotic Lactobacillaceae both prior and after therapy in INRs (p=.05). METHODS & RESULTS (part 2) PBMCs from HIV+ naive (NA; 12 with High Counts, HC: CD4>500; 11 with Low Counts, LC; CD4≤500), HAART-treated (HT; HIV-RNA <40cp/mL; 20 Full Responders, FR: CD4>350; 15 Immunological Non Responders, INR: CD4<350), and 10 HIV-negative (HIV-) were matured into Monocyte-Derived Macrophages (MDMs). PBMCs/MDMs were cultured with CD3/CD28, LPS, LTA, PGN, ssRNA, IFNg. HLA-DR/CD38 on CD8/CD4 and HLA-DR/CD69 on CD14+MDMs were analyzed (flow cytometry). HIV-1 p24 and sCD14 were measured in supernatants (ELISA) upon LPS, ssRNA and CD3/28 stimulation. TLR stimulation in HAART-naive pts did not result in significant changes upon T-cell activation. No differences were detected between NA and HIV- in CD69+MDMs; accordingly, MDM activation and p24 production were not affected by microbial challenge in HC and LC. However, p24 production from PBMCs increased significantly after ssRNA in HC alone (p=.031). Among HT, while INRs showed significant increases in CD38+CD8+ following broad microbial challenge (LPS,p=.0008; LTA,p=.009; PGN,p=.037; ssRNA,p=.0003; IFNg,p=.0042; CD3/28,p=.00041), FRs increased CD38+CD8+ only after ssRNA exposure (p=.0006). Of note, ssRNA challenge was the only one to account for a significant rise in CD69+MDMs in both INRs (p=.02) and FRs (p=<.01). ssRNA stimulation accounted for a significant p24 release from PBMCs both in INR and FR (p<.01 and p=.03 respectively), but resulted in increased p24 from MDMs in FRs alone (p=.01). METHODS & RESULTS (part 3) 30 HIV+ pts were enrolled: 14 Complete Responders, CRs (CD4≥500/mm, HIV-RNA<40cp/mL), 12 Partial Responders, PRs (CD4<500/mmc, HIV-RNA<40cp/mL) and 4 Long Term Non Progressors, LTNPs (CD4>500/mmc since at least 7 years, in absence of cART). On total PBMC, we measured activation (CD38+HLA-DR+), differentiation (CD45RA+CCR7+), proliferation (Ki67+), Th1, Th2, Th1-Th17, Th17, Treg, Tfh, PD-1, CD25 and CD127 expression (flow cytometry), plasma IL-2 and IL-7 (ELISA). Isolated CD4 were cultured with CD3/CD28, IL-7, SAHA and Prostratin. HIV-RNA was measured in supernatants (Real Time PCR). Reservoir size (total and integrated HIV-DNA, 2-LTR circles) were quantified by Real-Time PCR. LTNPs displayed higher activation on CD8 T-cells as compared to CRs (p=.017) and PRs (p=.043), whereas no differences were observed among the three study groups in all the other phenotypes. LTNPs showed higher reservoir size as compared to CRs and PRs, despite not reaching statistical significance. Accordingly, HIV-RNA production is higher in LTNPs as compared to HAART-treated patients (UNST p=.168 SAHA p=.088, CD3/CD28 p=.034) and these differences were maintained even comparing LTNPs to CRs (UNST p=.156; SAHA p=.056; CD3/CD28 p=.022) and PRs (UNST p=.066; SAHA p=.047; CD3/CD28 p=.016). Interestingly following stimulation only CRs and PRs were able to increase viral production following CD3/CD28 stimulation (p=.0037 ; p=.008; respectively). CONCLUSIONS Failure to control microbial translocation on HAART is associated with a polymicrobic flora circulating in peripheral blood that is not substantially modified by therapy in those patients starting therapy with very severe immune-depression. Broad microbial challenge accounts for T-lymphocyte activation in treated HIV+ pts with severe CD4 depletion, whereas viral challenge alone seems to most potently activate MDMs in HAART pts irrespective of CD4 reconstitution. Overall, microbial-derived bioproducts appear to enhance cellular activation, but impact HIV production only partially. Similar ability to reactivate HIV from latently infected CD4 T-cells pts reflected similar HIV reservoir sizes, with HAART-treated displaying the ability to reactivate HIV production following CD3/CD28 exposure, irrespectively of CD4 recovery. Despite we did not observe a clear relationship between MT and HIV reservoirs, it is evident that both of them in different ways could play a role in the pathogenesis of INRs, being key factors of immune activation. For this reason, we need to further investigate both MT and HIV reservoir as therapeutic targets, possibly evaluating new therapeutic approaches in addition to classical HAART. We could not exclude that the treatment for one of them may have beneficial effects on the other one.