Canonical Inflammasome Formation in Monocytes of Sickle Cell Anemia Patients

Abstract Sickle cell anemia (SCA) is now recognized as a chronic vascular inflammatory disease, in which inflammatory mechanisms drive the vaso-occlusive process. The inflammasome complex, responsible for the maturation and release of the IL-1β and IL-18 cytokines, is formed by pattern recognition receptors (PRRs), such as the NLRP3 protein, which recognize damage (or pathogen) associated molecular patterns (DAMPs). Following recruitment of the ASC adapter protein by the PRR, caspase-1 is activated by proximity-dimerization on the inflammasome and processes pro-IL-1β and pro-IL-18 into their bioactive forms. Elevated plasma levels of IL-1β and IL-18 may play a significant role in driving SCA inflammatory processes and it is important to identify the cellular sources and molecular mechanisms that contribute to the processing of these cytokines in SCA. Elevated caspase-1 activity and IL-1β processing has previously been reported in the neutrophils of SCA individuals (Mendonca et al., ASH Abstract 2016) and we, herein, aim to determine whether inflammasome assembly also occurs in the SCA monocytes. Peripheral blood samples and monocytes, separated by percoll gradients, were obtained from healthy control individuals (CON, total N=20) and SCA patients (total N= 20, of which 10 patients were on 15-30 mg/kg/day hydroxyurea therapy). No significant differences in data obtained from patients on/off hydroxyurea therapy were observed; thus, findings from these two groups were combined. Caspase-1 activity was determined in phenotypically-characterized monocyte populations by flow cytometry (Fam-FlicaTM kit, Immunochemistry Technologies). IL-1β release was quantified by ELISA. The percentage of CD14+CD16+ inflammatory monocytes presenting caspase-1 activity was significantly augmented in SCA, when compared to CON individuals (10.6 ± 1.3, 6.5± 1.1 % caspase-1 positivity, N=20 and 13 respectively; P Disclosures Conran: Bayer AG: Research Funding.