Epidemiological evidence suggests that recurring episodes of joint-bleeding contribute to the development of hemophilic arthropathy (H) in 70-85% of hemophilia patients. Despite major advances in the treatment to prevent joint bleeding, HA continues to be a major morbidity affecting hemophilia patients and the etiological mechanism contributing to the progression of HA remains poorly understood. Recent evidence suggests that the accumulation of blood in the joints may lead to the release of erythrocyte-derived DAMPs (eDAMPs) such as heme and hemoglobin that can promote sterile inflammation, however, the innate immune pathways contributing to this pathophysiology remain unknown.

In the study, we used a model of puncture-induced knee joint injury in FVIII-total knockout (F8TKO) mice and blood samples from hemophilia-A patients diagnosed with HA. Intravital multi-photon-excitation fluorescence intravital (in vivo) microscopy of injured synovium in live F8TKO or control mice was conducted to assess neutrophil-platelet aggregation and NETs generation in the knee-joint. Imaging-flow-cytometry and ELISA assays were used to estimate the number of circulating NETs in plasma of patients diagnosed with HA and mice after the knee-injury procedure. Scoring of the bleeding severity, histology, IHC and confocal imaging of joints were conducted to quantify the joint injury in mice.

F8TKO but not control mice manifested knee-joint injury and severity of bleeding 5-days post knee-injury. Progression of knee-joint injury was associated with increased neutrophil accumulation and NETs shedding within the synovium of F8TKO mice. Circulating NETs were significantly abundant in the plasma of hemophilia patients diagnosed with HA and F8TKO mice following knee-injury but not plasma of control humans or mice.

These findings are the first to suggest that NETs contribute to pathogenesis of HA in hemophilia. Currently, experiments are underway to identify the innate immune pathways that promote NETs shedding, leading to joint-damage in hemophilia.

Disclosures

Ragni: Takeda Therapeutics: Membership on an entity's Board of Directors or advisory committees; Bioverativ (Sanofi): Membership on an entity's Board of Directors or advisory committees; BioMarin Pharmaceutical: Membership on an entity's Board of Directors or advisory committees; Alnylam (Sanofi): Membership on an entity's Board of Directors or advisory committees; University of Pittsburgh: Research Funding; Spark Therapeutics: Membership on an entity's Board of Directors or advisory committees. Sundd: CSL Behring Inc: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Research Funding.

Author notes

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© 2021 by The American Society of Hematology
2021
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