Highly Active Antiretroviral Therapy Alters Inflammation Linked to Platelet Cytokines in HIV-1-Infected Patients

ToThe Editor—Chronic immune activation and persistent inflammation are critical features of human immunodeficiency virus type 1 (HIV-1) infection [1, 2]. For example, CD40 and its soluble ligand (sCD40L) are upregulated upon human immunodeficiency virus (HIV) infection, and their enhanced interaction inhibits production of interferon α by plasmacytoid dendritic cells [3]. Recently, Naranbhai et al reported that innate immune activation drives HIV acquisition, despite the use of a tenofovir microbicide gel [4]. They suggested that dampening inflammation may reduce primary infection. Among the numerous actors involved in immune activation and inflammation during HIV infection, activated platelets are usually underconsidered. However, platelets are the major source of circulating sCD40L, a master immune activator [5]. Platelets are activated by infectious pathogens, including HIV, because they sense infectious danger and secrete adapted panels of cytokines [6]. Levels of inflammatory platelet-derived products are elevated in the plasma of HIV-1 infected patients [7]. We assessed, in an in vitro model, platelet participation in inflammation in HIV-1–infected patients who were or were not receiving highly active antiretroviral therapy (HAART). A total of 41 HIV-positive patients without coinfection who had been receiving stable HAART for at least 1 year and 30 HIV-positive patients who, in accordance with World Health Organization recommendations (ie, their CD4+ T-cell count was >350 cells/µL), were not receiving HAART were recruited at our hospital. Written informed consent was obtained for inclusion into the study, which was approved by the French National Agency for the Safety of Medicines and Health Products (study B111011-40). Forty healthy blood donors were recruited as negative controls after signing the donation form, which states that donation of blood does not preclude use of the donation for nontherapeutic purposes (in accordance with the French Public Health Code). Clinical data on patients of different groups are presented in Table 1. Baseline Demographic Characteristics, Human Immunodeficiency Virus (HIV) Data, and Levels of Inflammatory Factors in Plasma and Released by Platelets Data are mean (interquartile range), unless otherwise indicated. P values were calculated by the t test or the χ² test for proportions. Abbreviations: GRO-α, growth-regulated oncogene–α; HAART, highly active antiretroviral therapy; NNRTI, nonnucleoside reverse transcriptase inhibitor; RANTES, regulated and normal T-cell expressed and secreted; sCD40L, soluble CD40 ligand; sCD62P, soluble CD62P; TRAP, thrombin receptor–activating peptide. aP< .05 vs HIV-negative healthy blood donors. bP < .05 vs HIV-positive patients with HAART. c Normalized for 3 × 108 platelets. dP < .05 vs unstimulated platelets of the same patient group. Baseline Demographic Characteristics, Human Immunodeficiency Virus (HIV) Data, and Levels of Inflammatory Factors in Plasma and Released by Platelets Data are mean (interquartile range), unless otherwise indicated. P values were calculated by the t test or the χ² test for proportions. Abbreviations: GRO-α, growth-regulated oncogene–α; HAART, highly active antiretroviral therapy; NNRTI, nonnucleoside reverse transcriptase inhibitor; RANTES, regulated and normal T-cell expressed and secreted; sCD40L, soluble CD40 ligand; sCD62P, soluble CD62P; TRAP, thrombin receptor–activating peptide. aP< .05 vs HIV-negative healthy blood donors. bP < .05 vs HIV-positive patients with HAART. c Normalized for 3 × 108 platelets. dP < .05 vs unstimulated platelets of the same patient group. Platelet-free and platelet-rich plasma (PRP) samples were prepared as described previously [6, 8]. Freshly isolated platelets were stimulated with thrombin receptor–activating peptide (TRAP)–SFLLRN peptide (50 µg/mL for 30 minutes; Sigma-Aldrich, Saint-Quentin Fallavier, France). Supernatants were cryopreserved at −80°C until later assay. Levels of soluble CD40L (sCD40L), growth-regulated oncogene (GRO)–α, regulated and normal T-cell expressed and secreted (RANTES), and soluble CD62P (sCD62P) were quantitated in plasma samples and PRP supernatants by Luminex technology (Merck-Millipore, Molsheim, France). Concentrations are expressed as mean values ± SD, and each sample was assayed in triplicate. The values were compared by the t test or χ² test for proportions. P < .05 was considered statistically significant. HAART recipients and untreated HIV-1–infected patients had comparable platelet counts, which were significantly lower than those in healthy controls (Table 1). Therefore, concentrations of factors released by platelets were normalized for 3 × 108 platelets. Consistent with other studies [7, 9], we observed that plasma levels of several platelet-associated inflammatory molecules were significantly increased in HIV-positive patients, which supports the participation of platelets in systemic immune activation during HIV infection. Moreover, we found that platelets displayed different responses to TRAP according to the HIV status of patients and whether HAART was implemented. Platelets from HAART-treated patients, and even more platelets from untreated patients, released significantly more sCD40L and GRO-α, spontaneously and upon TRAP-stimulation, than platelets from controls (Table 1). This suggests a hyperresponsive status of platelets during HIV infection, which may be dampened during HAART. The same observations were made for the release of RANTES and sCD62P by platelets from untreated subjects but not HAART recipients. Indeed, upon TRAP-stimulation, platelets from HAART recipients released significantly less RANTES and sCD62P than platelets from controls, reinforcing the notion that HAART limits the inflammatory response of platelets. However, platelets from HAART recipients, unlike those from other patients, were not able to release sCD62P in response to TRAP, suggesting platelet “exhaustion” for this factor. This possibility is supported by the fact that plasma sCD62P levels in HIV-infected HAART recipients are 2-fold and 3-fold higher than those in untreated HIV-infected patients and healthy controls, respectively. Therefore, the dramatic increase in sCD62P levels in HIV-infected patients likely resulted from the shedding of CD62P from the membrane of activated platelets, which is consistent with preserved platelet activity [10]. This finding suggests that, although hyperresponsive, stimulated platelets from HAART-treated HIV-infected patients release moderate amounts of RANTES and no additional sCD62P, they retain their capacity to release additional sCD40L and GRO-α, which could participate in activation of the immune system. Naranbhai et al suggested the use of strategies that combine antiretroviral and innate immune activation suppressants. Our present data support their proposal, using molecules targeting a platelet-inflammatory role in combination with antiretroviral molecules. Acknowledgments. We thank the staff and patients of the Infectious Diseases Department of the University Hospital of Saint-Etienne; anonymous, voluntary blood donors at the EFS Auvergne-Loire; and C. A. Arthaud and M. A. Eyraud, for invaluable technical assistance. Financial support. This work was supported by the Agence Nationale de Recherche sur le SIDA et les hépatites virales (SF/CP/N°160). Potential conflicts of interest. All authors: No reported conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.