P-Selectin Blockage Potentiates the Inhibiting Effects of Inflammasome Cytokine Neutralization on Leukocyte Recruitment in an In Vivo Model of Acute Sickle Cell Vaso-Occlusion

The vaso-occlusive (VO) processes that characterize sickle cell disease (SCD) pathophysiology result from a complex interplay between altered blood rheology, inflammatory mechanisms, pan-cellular activation and adhesive interactions between red blood cells, leukocytes (WBCs), platelets and the endothelium. Inhibition of the binding of the P-selectin (CD62P) adhesion molecule, expressed on activated endothelium and platelets, in the form of crizanlizumab monoclonal antibody (mAb) therapy, has demonstrated documented success for preventing VO crisis (VOC) in SCD patients (N Engl J Med (2017) 376(5):429-39), leading to marketing authorization by numerous agencies. In turn, blockade of interleukin (IL)-1β activity, in the form of canakinumab administration, was well tolerated in SCD patients and associated with inflammatory marker reduction in a multicenter phase 2a study (Blood (2022)139(17):2642-52), with in vivo studies suggesting a role for the inflammasome-processed cytokines, IL-1β and IL-18, in amplifying vaso-occlusion in SCD mice (Blood Abstract: (2021) 138 (S1):856).

As targeting multiple mechanisms may enhance VOC prevention in SCD, we evaluated the combined effects of P-selectin blockage, together with IL-1β and/or IL-18 neutralization, on WBC recruitment during tumor necrosis factor-α (TNF)-induced VO processes in SCD mice. Townes mice (5-mo old) received (i.p.) either saline, anti-murine IL-1β mAb (01BSUR IgG2a; 200 µg/mouse), and/or anti-murine IL-18 (SK113AE-4 IgG1; 500 µg/mouse), and/or anti-murine CD62P (RB40.34 IgG1, BD; 30 µg/mouse), or an IgG1 antibody (iProt105125 or A110-1, BD). Previous dosing intravital microscopy (IM) protocols determined the optimal concentrations of antibodies in TNF-stimulated Townes mice (doses tested in µg/mouse; 100, 200 anti-IL-1β [N=3; 4]; 250, 500 anti-IL-18, [N=3; 4]; 10-60 anti-CD62P [N=4-7 each]; data not shown). VO-like processes were induced in mice at 30 min after anti-CD62P treatment or 21h post anti-IL1β / anti-IL-18 administrations by TNF injection (0.5μg, i.p.). At 3h after TNF, the cremaster muscles of the anesthetized mice were surgically exposed, and the rolling, adhesion and extravasation of WBCs in 6-10 venules each were observed using IM. As expected, anti-CD62P administration significantly inhibited the rolling of leukocytes along venule walls of TNF-induced SCD mice, but did not significantly affect WBC adhesion (Fig. 1A). In contrast, the administration of anti-IL-1β mAb significantly inhibited both WBC rolling and adhesion, when compared to saline (Fig 1B); likewise, IL-18 neutralization significantly reduced WBC rolling and adhesion at the venule walls, when compared to IgG1 (Fig. 1C). As previously demonstrated, the combined administration of anti-IL-1β and anti-IL-18 did not further reduce TNF-induced WBC rolling or adhesion in the microvasculature, compared to anti-IL-18 alone (Fig. 1D). Importantly, the co-administration of anti-CD62P, together with either anti-IL1β or anti-IL-18, further inhibited WBC rolling (Fig. 1B and C) and amplified the effect of IL-18 neutralization on WBC adhesion (Fig. 1C). Finally, CD62P inhibition in association with the neutralization of both IL1β and IL-18 further reduced WBC rolling in a non-significant manner and incurred a further significant decrease in the adhesion of WBCs (Fig. 1D). As regards WBC extravasation from cremaster venules, when evaluating the effect of CD62P inhibition together with either anti-IL1β or anti-IL-18, the additive effect observed was not significant (P>0.05; N=4-5); however, CD62P inhibition further reduced the extravasation of WBC in mice subjected to both IL1β and IL-18 neutralization (reduced by 71.1±8.3%, P=0.002; N=4-5).

In conclusion, P-selectin blockage potentiates the inhibiting effects of IL-1β and IL-18 neutralization on TNF-induced WBC adhesion to the microvasculature of a murine model of SCD. This potentiating effect may be mediated by further inhibition of WBC rolling and tethering to the endothelium, but concurrent inhibition of platelet-mediated cellular interactions by P-selectin inhibition should not be ruled out. Given that WBC recruitment to blood vessel walls is associated with the initiation of VO processes, we suggest that the combined use of biological therapies may boost these approaches’ individual beneficial effects on SCD VOC. Financial support: Novartis Pharma AG.

Gotardo:Novartis Pharma AG: Other: Reserch grant recipient, Research Funding. Kovarik:Novartis Pharma AG: Current Employment. Bruederle:Novartis Pharma AG: Current Employment. Millholland:Novartis Pharma AG: Current Employment. Vitaliti:Novartis Pharma AG: Current Employment. Costa:Global Blood Therapeutics: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria; Global Survey HCP Steering Committee for Global Blood Therapeutics (GBT): Consultancy. Conran:Novartis Pharma AG: Research Funding.