Sickle cell disease (SCD), the most common inherited hemoglobinopathy worldwide, results in the production of abnormal hemoglobin S. Polymerization of this hemoglobin causes red blood cell sickling and pathophysiological consequences. Vaso-occlusive crises (VOCs) are the most frequent clinical complication of SCD and these crises can be triggered by different events, such as cold, dehydration, stress, and bacterial or viral infections. Models to study the mechanisms of VOC in SCD mice do exist, but there is little information regarding infection-induced VOC.

As part of a larger study that aims to compare the mechanisms by which different physiological and molecular triggers cause VOC, in in vivo and ex vivo protocols, this study has the objective of delineating the cellular and molecular mechanism by which the influenza A viral capsid component, PB1-F2, can induce VOC in the Townes SCD mouse model.

Male Townes SS mice (20 weeks) were randomly distributed into two groups: Vehicle Control (intranasal vehicle administration, n = 6) or PB1-F2 (PB1-F2; 1 nmol, intranasal, 3 hours of incubation, n = 6). Optimal PB1-F2 dosing, administration times and route were previously determined in C57BL/6 mice. After administrations, anesthetized mice were submitted to laser Doppler perfusion monitoring (LDPM) to measure the perfusion and velocity of blood flow of the pelvic cutaneous microcirculation. Leukocyte recruitment in the cremaster microcirculation was evaluated by intravital microscopy (6 – 8 microvessels per mouse were filmed for quantification of leukocyte rolling and adhesion). Additionally, peripheral blood samples were collected for the analysis of neutrophil-platelet aggregates (CD45+/Ly6G+/CD41+) by flow cytometry. All animal procedures were approved by Commission of Ethics in Animal Experimentation (University of Campinas).

LDPM demonstrated that, compared to vehicle administration, a single intranasal administration of PB1-F2 significantly reduced the blood flow velocity and perfusion of the cutaneous microcirculation by approximately 18% and 15% respectively (p < 0.05). With regard to leukocyte recruitment in the cremaster microcirculation, PB1-F2 administration significantly increased the number of rolling leukocytes (p < 0.001), but did not increase the adhesion of leukocytes to the blood vessel wall. These microvascular alterations were associated with a modest increase in PB1-F2-induced neutrophil-platelet aggregate formation in the peripheral blood of the mice (p < 0.05).

In conclusion, the viral component, PB1-F2, when given in a single intranasal administration, can induce vaso-occlusion in a SCD mice model, significantly decreasing microcirculatory blood flow and perfusion. Our data suggest that the acceleration of leukocyte rolling movements along venule walls, and the formation of aggregates between neutrophils and platelets in the circulation may contribute to this vaso-occlusion. Our findings also highlight the importance of standardizing new models for the study of VOC and its mechanisms in pre-clinical models of SCD for testing specific pharmacological approaches for preventing or reversing virally-induced VOC.

#2021/11851-8, #2019/18886-1, São Paulo Research Foundation (FAPESP).