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E. Lombardi et al.
tion in blood flow in the microcirculation that generally characterizes the early phase of an acute vaso-occlusive crisis.1,5,61-63 It is thus conceivable that our flow-based methodology could have a positive impact on monitoring therapy for SCD. Both FH and its FH19-20 segment nor- malized the transit of sickle RBC across the TNF-a-acti- vated vascular endothelial surface, abolishing the “stop- and-go” behavior of sickle RBC. This effect is of great importance because the transition time of sickle RBC in the microcirculation is critically related to the HbS poly- merization time and the generation of dense, dehydrated RBC, which contribute to the development of the acute clinical manifestations of SCD.1,10 It is important to note that FH appears to exert a non-canonical function in pre- venting cell adhesion. This complement regulator typical- ly inhibits complement activation via the AP by accelerat- ing the decay of C3 convertases and by mediating the degradation of C3b to iC3b and C3dg via the plasma pro- tease factor I. Since our experiments were performed using purified cells in the absence of plasma or serum, fac- tor I and the components involved in convertase forma- tion cannot have been involved in the observed effects. Rather, FH appears to interfere directly with cell-cell inter- action events between sickle RBC and endothelial cells.
To further elucidate the mechanism of complement opsonin-mediated adhesion and the role of FH as an anti- adhesive molecule for SCD RBC, we pre-coated vascular endothelial cells with either anti-P-selectin or anti-Mac-1 antibodies. Both surface molecules are expressed on acti- vated vascular endothelial cells and have been associated with opsonin interactions. Mac-1 is well-established as a functionally important receptor for iC3b which con- tributes to phagocytosis and cell activation. Whereas, the interplay of complement with P-selectin is less well described, several studies have shown interactions between C3b and P-selectin.56,64,65 We found that the adhe- sion of sickle RBC could be prevented by either anti-P- selectin or anti-Mac-1 antibody, indicating that both mol- ecules contribute to the adhesion of sickle RBC to the vas- cular endothelium. In our studies, blocking Mac-1 had a more pronounced effect on adhesion than did impairing P- selectin activity. The beneficial impact of interfering with Mac-1 in SCD has been supported by the reduction of RBC-neutrophil interactions in SCD mice treated with anti-Mac-1 antibody51 or in Mac-1-deficient SCD mice.57 Thus, our data indicate that complement is involved in the interaction between sickle RBC and the endothelium, pointing to a new additional mechanism contributing to the biocomplexity of acute events in SCD.
Our study therefore has potential implications for the
clinical management of SCD. Current treatments in devel- opment are focused on the role of selectins in the patho- genesis of SCD. For example, an anti-P-selectin antibody (crizanlizumab) that has been used to treat SCD has been reported to affect the period of time between acute pain crises in SCD patients.52,66,67 Similarly, the small molecule- based pan-selectin inhibitor, rivipansel, currently undergo- ing phase III clinical trials, is able to reduce the time required to resolve vaso-occlusive crises with a reduction in opioid treatment.68 Our findings suggest that targeting complement opsonization and/or opsonin-mediated cell adhesion could provide an alternative strategy. Whereas the use of exogenous full-length FH as a therapeutic tool is associated with some challenges, several smaller variants of the regulator have shown promise in preclinical trials for complement-mediated diseases such as paroxysmal nocturnal hemoglobinuria. Given the importance of FH domains 19-20 in interfering with RBC adhesion, mini-FH constructs containing this domain pair may be considered, since they may affect both AP activity and the adhesive function of existing opsonins.69,70 Alternatively, blocking opsonization itself at the level of C3 activation is also expected to impair complement-mediated adhesion.
In conclusion, we have first shown that complement activation on sickle RBC participates in the adhesion of sickle erythrocytes to the TNF-a-activated vascular endothelium (Figure 5B). We then further demonstrated that the FH19-20 segment is as efficient as FH in prevent- ing the adhesion of sickle RBC, and results in normaliza- tion of sickle RBC transit across the vascular endothelial surface. We suggest that chronic hemolysis may require high levels of FH to prevent RBC adhesion and entrap- ment in the microcirculation. Finally, our data indicate that FH might act as a multimodal molecule, preventing the opsonization of sickle RBC with C3 opsonins and target- ing the interaction of sickle RBC with the endothelium through the adhesion molecules P-selectin and Mac-1 (Figure 5B). Our findings provide a rationale for consider- ing FH-based inhibitors and other modulators of the AP as potential new therapeutic options in SCD.
Acknowledgments
This work was supported in part by grants FUR 2016-2017 to LDF and by NIH grant AI068730 to JDL We thank Dr. Sara Ugolini, Dr. Monica Battiston and Dr. Francesco Agostini for technical support, Ing. Leoardo Buscemi for LB software writing and Ing. Vincenzo Insalaca for video editing. We also thank Dr. Letizia Delmonte and Dr. Elisa Vencato for their contribution in preliminary experiments and Dr. Deborah McClellan for editing the manuscript.
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