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S. Allali et al.
to promote a catch-bond formation between endothelial cell E-selectin and neutrophil L-selectin via tetrasaccharide sialyl Lewisx (sLex) expressed on L-selectin.44 This interac- tion triggers the activation of high-affinity β2-integrins, which leads to the formation of shear-resistant bonds with ICAM-1 on inflamed endothelium. Rivipansel, a syn- thetic pan-selectin antagonist previously shown to improve blood flow and survival in SCD mice, effectively blocks E-selectin recognition of sLex on L-selectin, thereby inhibiting neutrophil adhesion.44,45 In a prospective multi- center, randomized, placebo-controlled, double-blind, phase II trial of 76 SCD patients, those in the rivipansel arm showed a non-significant trend toward reduced time to res- olution of VOC, and a significant 83% reduction in mean cumulative intravenous opioid analgesic use.46 These results have supported an ongoing phase III trial of rivipansel for SCD VOC (#NCT02187003). However, the most encourag- ing results were obtained with another selectin antagonist specifically targeting P-selectin. In a randomized, placebo- controlled, double-blind, phase II trial of 198 SCD patients, treatment with crizanlizumab, a humanized monoclonal antibody directed against P-selectin, significantly reduced the median rate of VOC per year by 45%.47 A phase III trial with crizanlizumab is currently in progress (#NCT03814746) and this drug was recently approved by the US Food and Drug Administration. Among other poten- tial therapeutic agents targeting neutrophils, sevuparin, a novel drug candidate derived from heparin, was found to inhibit adhesion of RBC and leukocytes from SCD patients to human umbilical vein endothelial cells and prevented vaso-occlusion in sickle mice via a multimodal mechanism of action, including P- and L-selectin binding.48,49 Therefore, a phase II clinical trial of sevuparin for preventing VOC in SCD patients is ongoing (#NCT02515838). Intravenous immunoglobulin could also inhibit RBC–neutrophil interac- tions and neutrophil adhesion to endothelium in sickle mice by inhibiting Mac-1, and no adverse effects were observed in a phase I clinical trial of intravenous immunoglobulin in SCD patients.50 A phase II trial is currently ongoing (#NCT01757418).
An important point is that activated platelets as well as endothelial cells express P-selectin, which binds to PSGL-1 on neutrophils, thereby enhancing the formation of platelet-neutrophil aggregates.51 In SCD mice, such platelet-neutrophil aggregates have been observed in pul- monary arteriole microemboli, with resolution after selec- tive platelet P-selectin inhibition, which suggests the potential therapeutic interest of targeting P-selectin to pre- vent ACS.52 In the crizanlizumab phase II trial, the ACS rate did not differ between the active-treatment group and the placebo group, but this finding may be explained by the rarity of ACS in this trial, and further studies are need- ed to determine whether P-selectin blockade prevents ACS in SCD patients.47 The platelet–neutrophil association is also mediated by interactions between glycoprotein Ibα (GPIbα) on platelets and Mac-1 on neutrophils, which is positively regulated by the serine/threonine kinase isoform AKT2 in neutrophils during vascular inflammation.53 Here again, specific inhibition of AKT or GPIbα was recently found to attenuate in vitro neutrophil-platelet aggregation in SCD patients’ blood, thereby opening new therapeutic per- spectives for the prevention and treatment of VOC.54,55
Besides selectin-dependent interactions, adhesion of neutrophils to activated endothelium is modulated by dif- ferent mediators, such as endothelin-1, with elevated plas-
ma levels in SCD patients. In SCD mice, endothelin-1 appears to upregulate TNF-α–induced Mac-1 expression on neutrophils. Blocking endothelin receptors, especially the endothelin B receptor, on neutrophils strongly attenu- ates their recruitment, as demonstrated by intravital microscopy of SCD mice and microfluidic microscopy of SCD human blood.56
Another major point is that SCD patients, like SCD mice, display very high proportions of aged neutrophils, which have been positively correlated with endothelial adhesion, Mac-1 expression and the formation of neu- trophil extracellular traps (NET).57 Neutrophil aging appears to be mediated by microbiota via TLR/Myd88 sig- naling, and depletion of gut microbiota with antibiotics in SCD mice led to a significant reduction in the number of aged neutrophils, along with improved blood flow and increased survival. A reduced number of aged neutrophils has been reported in SCD patients receiving penicillin, which suggests an additional positive impact of prophy- lactic antibiotic treatment, mediated by microbiota deple- tion and reduction in the number of aged neutrophils.57 Moreover, bone marrow from SCD mice showed an accu- mulation of aged neutrophils, possibly impairing osteoblast function; thus, by reducing the number of aged neutrophils, microbiota depletion may improve osteoblast function and bone loss in SCD.58
In both SCD mice and patients, elevated plasma heme levels during VOC were found to promote the formation of NET, which are decondensed chromatin with granular enzymes released by activated neutrophils.59 In SCD mice, the presence of NET in the lungs contributes to acute lung injury and is associated with hypothermia and death, which can be prevented by clearing NET with DNAse I or by scavenging heme with hemopexin.59 Together with heme being able to trigger ACS in SCD mice, these results suggest that NET induced by heme may be involved in the pathogenesis of ACS.60 Heme may also contribute to a sus- ceptibility to infections in SCD patients by inducing HO- 1 expression during neutrophilic differentiation, thereby impairing the ability of neutrophils to mount a bactericidal oxidative burst. Indeed, a novel neutrophil progenitor sub- set expressing high levels of HO-1 was recently identified in the bone marrow of SCD children but not healthy con- trols.61
The main findings on the involvement of neutrophils in SCD pathophysiology are summarized in Figure 2.
Eosinophils
Independently of parasitic infections, eosinophils are more numerous in steady-state SCD patients than in healthy controls and display an activated phenotype.62,63 An increase in absolute eosinophil count appears to result from an increased level of GM-CSF in SCD.64 In vitro, adhe- sion of circulating eosinophils to fibronectin was found to be enhanced in SCD patients and mediated by α4β1 (VLA-4), lymphocyte function-associated antigen 1 (LFA- 1) and Mac-1 integrins.62 Subsequently, eosinophils from SCD patients were found to demonstrate greater sponta- neous migration and release higher levels of peroxidase, eosinophil-derived neurotoxin and reactive oxygen species (ROS) than eosinophils from healthy controls.63 Hydroxyurea treatment seems to reduce absolute eosinophil count and eosinophil adhesion and degranula- tion, which suggests an additional beneficial effect of hydroxyurea in SCD.63
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haematologica | 2020; 105(2)