Neutrophil-Dependent Immune Response Promotes Joint Damage in Hemophilia

Background: Hemophilic arthropathy (HA) is the predominant pathophysiology resulting from recurrent joint bleeding in individuals with hemophilia and leads to permanent joint damage, chronic pain and reduced quality of life. Although FVIII replacement therapy or FVIII mimetics like HEMLIBRA have significantly improved the disease outcome, joint bleeding continues to occur in severely hemophilic patients (FVIII<1%). HA starts at an early age and develops progressively over time, warranting the need for a therapy that can intervene the progression of HA. Unfortunately, such therapy does not exist and the etiological mechanism of HA remains poorly understood.

Aim: To determine whether neutrophil Gasdermin-D (GSDMD)-dependent generation of neutrophil extracellular traps (NETs) in in the joint promotes HA.

Methods: Knee joints in FVIII-total knockout (F8 ^TKO) or littermate WT male mice were injured using a needle . Scoring of the bleeding severity and histology of injured joints were conducted to assess the joint injury in mice.Imaging-flow-cytometry and ELISA assays were used to evaluate the number of circulating NETs in the blood samples of patients diagnosed with HA and mice with following knee injury. Neutrophils from HA patients, F8 ^TKO mice and matched controls were isolated, activation of GSDMD-pathway components was evaluated in neutrophils using qRT-PCR (gene expression) and western blotting (protein levels and cleavage).

Results: F8 ^TKO but not control mice manifested unresolved joint injury and severe bleeding 5-days post knee injury. Circulating NETs (cNETs) and cNETs markers llike neutrophil elastase or citrulinated histones were significantly elevated in the plasma of hemophilia patients diagnosed with HA and injured F8 ^TKO mice but not plasma of control humans or injured WT mice or uninjured F8 ^TKO mice. Major genes (mRNA levels) associated with GSDMD-mediated NETs generation were significantly elevated in neutrophils of HA patients and injured F8 ^TKO mice compared to matched controls. Finally, western blot analysis revealed activation of GSDMD pathway in neutrophils isolated from HA patients and F8 ^TKO mice but not matched controls.

Conclusions: These findings are the first to suggest that GSDMD-dependent generation of NETs contributes to the pathogenesis of HA