OC 33.1 - Neutrophil extracellular traps promote gasdermin-D-dependent joint damage in hemophilia

Background: Hemophilic arthropathy (HA) is the predominant pathophysiology resulting from recurrent joint bleeding in hemophilia patients leading to permanent joint damage. Growing evidence suggests that repeated bleeding promotes HA by triggering sterile inflammation. Neutrophil-extracellular-traps (NETs) are known to promote sterile inflammation in inflammatory disorders, however, their role in promoting HA remains poorly understood.

Aims: Determine whether gasdermin-D-(GSDMD)-dependent generation of NETs promotes HA.

Methods: Knee injury was induced in FVIII-total-knockout (F8TKO) or littermate-wild-type (WT) as controls. Venous blood samples were collected from hemophilia patients with HA or matched control subjects. Scoring of bleeding severity and histology of injured joints were conducted to assess joint injury in mice. Intravital-(in vivo)-microscopy of the synovium was conducted in mice to assess the role of thrombo-inflammation/NETs in joint damage. Imaging-flow-cytometry and ELISA-assay were used to evaluate the number of circulating NETs (cNETs) in hemophilia patients and mice. Circulating neutrophils from HA patients, F8TKO mice and matched controls were isolated and activation of GSDMD-pathway was assessed using qRT-PCR (gene expression) and western blotting (protein levels/cleavage).

Results: F8TKO but not control mice manifested knee-joint damage and bleeding severity at day 5 post-knee injury. Remarkably, cNETs were elevated in plasma of HA patients and injured F8TKO mice but absent in plasma of controls. Intravital microscopy revealed accumulation of neutrophil-platelets aggregates in the synovium of injured F8TKO mice. Several genes (mRNA levels) associated with GSDMD-pathway/NETs-generation were upregulated in neutrophils of HA patients and injured F8TKOmice compared to controls. Western blot analysis confirmed activation of GSDMD-pathway in circulating neutrophils of HA patients and injured F8TKOmice but not in respective controls.

Conclusion(s): These findings are the first to suggest that GSDMD-dependent NETs-generation contributes to pathogenesis of joint-damage in hemophilia. These findings are the first to suggest that sterile inflammation in joints promotes systemic inflammatory-milieu in hemophilia by contributing to release of cNETs into the systemic circulation.