Patrolling Monocytes Scavenge Endothelial Adherent Sickle Red Blood Cells in Sickle Cell Disease
Authors: Liu Yunfeng , Zhong Hui , Bao Weili , Mendelson Avital , An Xiuli , Shi Patricia , Chou Stella T. , Manwani Deepa , Yazdanbakhsh Karina 
Published: October 2019

Abstract
Sickle cell disease (SCD) is characterized by hemolytic anemia and increased entrapment of sickle red blood cells (RBCs) via attachment to the underlying activated vascular endothelium, resulting in vaso-occlusive crisis (VOC), marked by severe pain. The endothelial scavenging patrolling monocytes (PMos) expressing high levels of the heme oxygenase 1 (HO-1), a heme degrading enzyme, were recently shown to protect against vaso-occlusion in SCD, although their ability to scavenge endothelial-attached sickle RBCs was not tested. Here, we found that circulating PMos from SCD patients showed roughly 5% ± 0.5% engulfed GPA+ or Band3+ RBC specific material as compared to 0.7% ± 0.04% in healthy donor (HD) PMos or 0.85% ± 0.07% in SCD classical monocytes (CMos) as detected by flow as well as imagining flow cytometry, suggesting that PMos uptake RBCs in SCD. To further investigate this, RBCs purified from HDs (to mimic transfused cells) or SCD patients were labelled with CFSE, and co-cultured with purified monocytes without or with human microvascular endothelial cells (HMVEC). We found 11% ± 0.5% CFSE+ PMos in co-cultures with SCD RBCs in presence of HMVEC as compared to 2.7% ± 0.4% when cultured with HD RBCs, indicating that PMos engulf sickle RBCs, but not HD RBCs. Low levels of CFSE+ PMos (2-3%) were detected in cocultures with either sickle or control RBCs in the absence of HMVEC, implicating that PMos preferentially uptake endothelial cell (EC)-attached sickle RBCs. In contrast to PMos, CMos always had minimal CFSE+ reactivity (2-3%) when cultured with sickle RBCs or HD RBCs with or without HMVEC, supporting a role for PMos, but not CMos, as scavengers of sickle RBCs in the vasculature. Further analysis revealed significantly increased (two-fold) levels of annexin V+ apoptotic marker on sickle RBC engulfed PMos (CFSE+PMos), but also higher levels of HO-1 ((50% ± 3%) as compared to non-engulfed (CFSE-) PMos (1.3% ± 0.4%), suggesting that induction of HO-1 upon uptake of sickle RBCs may counteract the cytotoxic effects of engulfed RBC breakdown products in PMos. To test this hypothesis, monocytes were pre-treated with tin protoporphyrin IX (SnPPIX) to inhibit HO-1 activity prior to coculture with sickle RBCs and HMVEC. We found increased apoptosis in PMos from SnPPIX-treated (18% ± 0.8%) as compared to untreated cultures (6.6% ± 0.6%), consistent with a cytoprotective role of HO-1 induction in sickle RBC-engulfed PMos. Antibody blocking studies identified ICAM-1, VCAM-1, CD11a, CD18 as well as CD16 as key molecules involved in PMo-HMVEC-sickle RBC interactions, but not CD11b, CD11c, CD31, PSGL-1, CD32, CD64, Fc a/m receptor and phosphatidylserine. Interestingly, HMVEC activation induced by heme treatment resulted in significantly higher CFSE+ PMos (17.8% ± 0.9%) when cultured with sickle RBCs, but not HD RBCs (3.5% ± 0.6%), indicating that PMos preferentially uptake sickle RBCs bound to heme-treated ECs. To formally test this, DiI labelled mouse sickle or control RBCs were transfused to heme-treated Nr4a1-GFP mice which express GFP on their circulating PMos, followed by perfusion to remove non-EC attached cells. Using confocal microscopy, we detected DiI+ sickle but not control non-sickle RBCs, engulfed by GFP+ PMos in the vasculature of perfused recipients, confirming uptake of EC-attached sickle RBCs by PMos. Consistent with increased EC-attached sickle RBCs during VOC, SCD patients experiencing acute VOC (n=12) showed increased frequency of GPA+ PMos (13% ± 0.1% vs 5% ± 0.2%, p<0.01) but still only 1.8% ± 0.3% GPA+ CMos. However, total numbers of PMos including HO-1hi PMos were more than two-fold lower in these patients as compared to patients at steady state. Altogether, these data demonstrate for the first time that in SCD, PMos scavenge EC-attached sickle RBCs, but not transfused HD RBCs, through interactions involving manly integrins, resulting in HO-1 upregulation to counteract the cytotoxic effects of engulfed RBC breakdown products. With increased adherence of sickle RBCs to vasculature as a result of damage to ECs such as during VOC, PMos scavenge more RBCs, but their numbers become limiting with implications for beneficial effects of transfusions and potential for PMos as therapeutic targets against VOC in SCD.

Disclosures
No relevant conflicts of interest to declare.