Influence of Hydroxyurea on Neutrophil Microparticles: A SCA Model

Introduction: MPs are stable units that express on their surface specific proteins of the cells that were originated. In this sense, neutrophils MPs have a singular importance since they present in their surface myeloperoxidase (MPO), an enzyme that are involved with vascular inflammatory conditions. Studies have shown that the use of HU, currently the only pharmacological drug approved to treat SCA patients, leads to reduction in count neutrophils, monocytes and reticulocytes, and the slight increase in total hemoglobin concentration. However, some studies have shown that MPs levels are not affected by HU treatment. Recently, our group showed that nod-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome and interleukin (IL)-1β could contribute to the inflammatory condition in SCA patients. This study tested the hypothesis that neutrophils challenged with serum of sickle cell anemia (SCA) patients, treated with hydroxyurea (HU), decrease neutrophils microparticles (MPs) production. Therefore, the aim of this study was to evaluate the inflammatory state of neutrophils from healthy volunteers by NLRP3 and IL1B gene expression and of SCA patients treated or untreated with HU and to measure the production of MPs derived from neutrophils challenged with SCA patient's serum, treated or not with HU.

Methods: We enrolled 10 SCA patients (all in steady state with age of 8.0 ± 1.9 years), from the Fundação de Hematologia e Hemoterapia do Estado da Bahia (HEMOBA). Neutrophils were isolated from one healthy donor as previously described (Pitanga et al, 2014). The protein concentration of neutrophils MPs was evaluated using the BCA method (Thermo Scientific,Waltham, MA, USA) according to the manufacturer's protocol and described by Pitanga et al (2014). NLRP3 and IL1Bgene expressions genes were performed by real time PCR with SYBR Green assays from neutrophils RNA in trizol reagent. Relative expression was estimated through the 2^ddct method, with GAPDH and ACTB genes. Serum biochemical analysis was evaluated using commercially available biochemical kits. This study was conducted in accordance and approved by the Research Ethics Committee of the Fundação Oswaldo Cruz - FIOCRUZ, Brazil; and also with the Helsinki Declaration of 1975, and its revisions. The informed consents were signed by patients or official responsible.

Results: Our findings showed that NLRP3 (6.95 ± 1.77)and IL1B (6.46 ± 3.31) genes are highly expressed in neutrophils of SCA patients comparing to healthy volunteers (2.43 ± 0.73, p=0.0286; 2.65 ± 1.30, p=0.0081 respectively). Additionally, neutrophils from SCA patients treated with HU decrease NLRP3 gene expression (4.06 ± 1.12; p= 0.0357), but it was not observed for IL1B gene expression (6.03 ± 3.13; p=0.3472). We demonstrated that serum from SCA patients increased neutrophils MPs production (181.90 ± 53.00) comparing to healthy volunteer's serum (37.63 ± 13.06; p=0.0011). However, HU treatment decreased this scenario (30.33 ± 13.70; p=0.0022).

Conclusions: This study highlighted that HU acts decreasing NLRP3 gene expression and neutrophils MPs production suggesting that NLRP3 protein could participate of cellular activation and production of MPs, contributing to the maintenance of inflammation as well as vascular activation.