Neutrophil-Dependent Interferonopathy Promotes Hemophilic Arthropathy
Authors: Kaminski Tomasz , Katoch Omika , Ragni Margaret V. , Sundd Prithu 
Published: December 2024

Background: Recurrent joint bleeding (hemarthrosis) is the most frequent clinical manifestation of severe hemophilia A. If not properly managed, even subclinical hemarthrosis can progress to hemophilic arthropathy (HA), a debilitating condition marked by inflammation of the synovium, neo-angiogenesis and infiltration by neutrophils, eventually leading to joint-bone remodeling and permanent joint-damage. Recent evidence suggests that joint bleeding promotes the release of erythrocyte-derived damage-associated molecular pattern molecules (DAMPs) such as heme and hemoglobin that trigger sterile inflammation in the joints. However, the early events leading to the development of HA remain unclear.
Aim: To identify early inflammatory events contributing to the development of HA.
Methods: Knee joints in FVIII-total knockout (F8TKO) or littermate WT male mice were injured using the model of single puncture-induced hemarthrosis. Scoring of the bleeding severity and histology of joints were conducted to assess the joint injury in mice. Imaging-flow-cytometry and ELISA assays were used to evaluate the number of circulating neutrophil extracellular traps (NETs) in the blood samples of patients diagnosed with/without HA and mice with/without knee injury. Neutrophils from HA patients, F8TKO mice and matched controls were isolated, activation of inflammatory caspases such caspase-1/4/5 (Human) and caspase-1/11 (Mouse)-dependent signaling was assessed in neutrophils using qRT-PCR (gene expression) and western blotting (protein levels and cleavage). ELISA was used to assess type-I interferon (IFN-α) production in neutrophils and how it promotes NETs generation.
Results: F8TKO but not control mice manifested unresolved joint injury and severe bleeding 5-days post knee injury. Circulating fragments of NETs (cNETs) and NETs markers (neutrophil elastase and citrullinated histones H3) were significantly elevated in the plasma of hemophilia patients diagnosed with HA and injured F8TKO mice but not in plasma of control humans, injured WT mice or uninjured F8TKO mice. Type-I interferon signaling (IFN-I) is known to promote the expression of major inflammatory genes associated with NETs generation. Indeed, the levels of interferon-α were significantly elevated in neutrophils of injured F8TKO mice than injured WT mice. Next, western blotting analysis revealed the presence of cleaved (active) forms of Caspase-5 and Caspase-11 in neutrophils isolated from HA patients and F8TKO mice, respectively, but not in matched injured or uninjured controls. Principal genes (mRNA levels) associated with upstream type-IFN signaling and downstream caspase-5/11 mediated NETs generation were significantly elevated in neutrophils of HA patients and injured F8TKO mice compared to matched injured or uninjured controls.
Conclusions: These findings are the first to suggest that IFN-I signaling and activation of caspase-5/11 in neutrophils promotes generation of NETs that contribute to the pathogenesis of early stages of HA and promote systemic inflammation following a local joint injury.