EVALUATION OF IMMUNE MECHANISMS INVOLVED IN THE LACK OF CD4+ T CELL RECOVERY IN HIV-INFECTED PATIENTS NON RESPONDER TO ANTIRETROVIRAL THERAPY

INTRODUCTION HIV infection is characterized by a profound impairment of CD4+ T cell functionality with an immunological unbalance toward Th2 response during progression to AIDS. Antiretroviral therapy HAART suppresses viral replication and leads to the recovery of CD4+ T lymphocytes. 15-30% of HIV-infected HAART-treated patients are immunological non responder (INR) to therapy. Several factors are involved in this lack in CD4+ T cell recovery, our attention was focused on the excessive depletion of CD4+ T cells. Recently, hydroxychloroquine (HCQ), an anti-malarian drug, demonstrates to have antiviral and immune modulating effects. Aim of this study was to identify immune mechanisms involved in the lack of CD4+ T cell recovery observed in INR patients and to verify if HCQ might reduce immune activation typical of HIV infection thus leading to a recovery in CD4+ T cells. MATERIAL AND METHODS In the first part of this study, 67 HAART-treated HIV-infected patients were enrolled and stratified into two groups of the basis of CD4+ T cell counts (<500cells/µl and >500cells/µl) while in the second part of this study, analyses were conducted on 20 HAART-treated HIV-infected patients with an absolute CD4 count less than 200 cells/µl during the last 12 months of therapy. Patients were treated with 400mg/die of HCQ and analyses were conducted at baseline, 6 months of therapy and 2 months after HCQ suspension. Immune suppression, immune activation and apoptosis were evaluated in flow cytometry. To identify microbial translocation, plasma LPS was measured with immunoenzymatic assay and TLR signaling pathway was analyzed in Real-Time PCR. Analyses were conducted in both unstimulated and stimulated (HIV-, CMV-, LPS-, ssRNA-specific) conditions. RESULTS AND DISCUSSION Patients characterized by CD4+ cell counts less than 500cells/µl compared with individuals characterized by CD4+ cell counts more than 500cells/µl showed: 1) increase percentage of Treg cells subpopulations and 2) apoptosis, 3) higher percentage of immune suppressive cytokine-expressing CD4+ T cells, 4) a higher percentage of KI67+ CD4+ T cells in both unstimulated and stimulated conditions. 5) an increased percentage of TLR expression on Treg cells and 6) a higher concentrations in plasma LPS. Data herein indicate that defective CD4+ cell counts recovery observed in patients characterized by CD4+ cell counts less than 500cells/µl is associated with lower CD4+ nadir, gut microbial translocation and immune activation, augmented percentage and activity of Treg cells, and higher susceptibility to apoptosis. In the second part of the study, data showed a reduction in immune activation of CD4+ T cells, CD8+ T cells and monocytes. which was coupled with a reduction in plasma LPS concentration and of IL6 pro-inflammatory cytokines production. Moreover it has been shown the pivotal role played by plasmacytoid dendritic cells in the control of infection. Immune activation and immune suppression are strictly correlated, presence of immune suppressor state was well studied thanks to the analyses of both naïve and activated Treg lymphocytes, and TLR-expressing Treg cells: these populations were increased in percentage after 6 months of therapy. Several studies put in evidence HCQ effectiveness on TLR signaling pathway which influences immune activation. Flow cytometric analyses elucidate the reduction in TLR expressions by monocytes even in the presence of TLR4 and TLR7/8 agonists (LPS and ssRNA respectively). TLR signaling pathway was studied following PBMC stimulation with specific TLR agonists by Real-Time PCR: 6 months of HCQ treatment resulted in a reduction of TLR responsiveness and the effect was maintained also 2 months after HCQ suspension. CONCLUSIONS The lack in the recovery of CD4+ T lymphocytes characterizing immunological non responder patients to HAART is associated with a low CD4+ nadir, with immune activation due to an increase in microbial translocation and with immune suppression conducted by Treg cells through the induction of apoptosis mechanism and immune suppressor environment. The augment in Treg population might play a pivotal role in reducing immune activation and the increase of TLR-expressing Treg cells supports the effectiveness of a strong immune suppressive activity in controlling immune activation. Preliminary data suggest that HCQ plays a positive effect on CD4+ T cell recovery in immunological non responder patients to HAART and a control of immune activation, sustaining an application of this drug as immune modulating agent, in the treatment of HIV infection.