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 (Mendonça 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<0.05). Confocal immunofluorescence microscopy indicated an augmented expression of the ASC adaptor protein and ASC speck formation in unstimulated SCA monocytes (N=3), compared to CON monocytes (N=2). Accordingly, Western blotting showed a higher ASC expression in pooled SCA monocyte total cell extract (TCE, N=4), compared to that of pooled CON monocyte TCE (N=4); furthermore, increased cleaved caspase-1 (20 KDa) was identified in pooled SCA monocyte TCEs, compared to CON monocyte TCEs. Consistent with this observation, significant pro-caspase-1 co-precipitation, together with immunoprecipitated ASC, was achieved from pooled SCA monocyte lysates, while this pro-caspase-1/ASC association was almost non-detectable in CON monocyte lysates. Finally, IL-1β release by SCA monocytes (5x105 cell/ml, 37°C during 2h, N=8) was significantly abrogated by both NLRP3 inhibition (5µM MCC950) (decreased from 50.4± 14.4 to 30.3± 11.2 pg/ml released IL-1β; P<0.05) and caspase-1 inhibition (40 µM YVAD-FMK) (decreased to 20.8± 8.3 pg/ml IL-1β; P< 0.05). Interestingly, the inhibition of NFκB (10 µM BAY117082, an inhibitor of IκBα phosphorylation) also significantly reduced IL-1β release from SCA monocytes in 2h (9.64±2.66 pg/ml; N=8, P<0.01). As such, the inflammasome assembled in SCA monocytes is apparently of the NLRP3 type, and appears to involve NF-κB activated transcriptional regulation of IL-1β processing in these cells, most likely at the priming stage. Our data indicate that monocytes of SCA individuals present elevated ASC expression and oligomerization, mediated by the NLRP3 PRR sensor, culminating in augmented pro-caspase-1 recruitment by ASC and its cleavage to active caspase-1. As such, this canonical inflammasome assembly in SCA monocytes appears to contribute to the expressive quantities of IL-1β that are released by SCA monocytes; these cells may contribute to the elevated IL-1β processing that occurs in SCA, possibly representing a therapeutic target for the disease.

Disclosures

Conran: Bayer AG: Research Funding.

Author notes

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Asterisk with author names denotes non-ASH members.