OC 20.1 - Neutrophil Extracellular Traps promote joint injury in hemophilia

Background: Hemophilic arthropathy (HA) is major morbidity affecting hemophilia patients. Epidemiological evidence suggests that recurring episodes of joint-bleeding contribute to the development of HA in 70-85%of hemophilic patients. Despite major advances in the treatment to prevent joint bleeding, HA continues to be major morbidity affecting hemophilia patients and the etiological mechanism contributing to the progression of HA remains elusive.

Aims: 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 by priming innate immune pathways in neutrophils. The aim of the study was to identify pathways contributing to the development of HA.

Methods: In the study, we used a model of puncture-induced knee joint injury in FVIII-total-knockout (F8TKO) mice and blood samples from hemophilia patients diagnosed with HA. Intravital multi-photon-excitation fluorescence microscopy imaging of injured joint cavity in living F8TKO or control mice was used to evaluate NETs formation within the joint structure. Imaging-flow-cytometry and ELISA assays were used to estimate the number of circulating NETs in 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 classify the joint injury in mice.

Results: 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 the 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 following knee-injury but not plasma of control humans or mice.

Conclusion(s): 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.