Innate Immune Mechanism of Hemarthrosis in Hemophilia-a Mice
Authors: Kaminski Tomasz W. , Tutuncuoglu Egemen , Ragni Margaret V. , Gladwin Mark T , Sundd Prithu 
Published: November 2019

Hemarthrosis is a chronic arthropathy responsible for irreversible joint damage, disability, and acute joint pain in hemophilia patients. Hemarthrosis is caused by spontaneous or traumatic bleeding into joints, which when recurrent, leads to synovial inflammation and cartilage degeneration. Although release of erythrocyte-derived damage associated molecular pattern molecules (eDAMPs) is believed to promote sterile inflammation in the synovium, the innate immune mechanism of hemarthrosis remains poorly understood and the current therapy is limited to factor replacement and pain management. Here, we use factor 8 total knock-out (F8TKO) hemophilia A mice (C57BL/6J background) that manifest a complete deletion of the F8 coding region, expressing no detectable F8 mRNA and exhibiting a severe hemophilia phenotype. Right knee joint capsules of F8TKO mice were punctured with a 30-g needle below the patella between the anterior portions of the femur and tibia, followed by assessment of bleeding severity score and histological analysis one-week post injury. Intravital multiphoton excitation fluorescence microscopy of injured synovium was performed to assess the role of thrombo-inflammatory events in promoting hemarthrosis. Neutrophil, platelets, and blood vessels were visualized by intravenous administration of fluorescent anti-Ly6G mAb, anti-CD49b mAb, and dextran, respectively. Protein and mRNA levels of proinflammatory cytokines were measured in plasma, joint, synovium, and cartilage tissue using Real-Time PCR and ELISA, respectively. F8TKO but not wild-type (WT) control (C57BL/6J) mice manifested unresolved joint bleeding, cartilage degeneration and synovitis leading to impaired mobility and high bleeding severity scores. Significantly more recruitment of neutrophils and neutrophil-platelet aggregates as well as elevated IL-1b levels were observed in the synovium of F8TKO compared to WT mice. These results are the first to suggest that sterile inflammation contributing to hemarthrosis in hemophilia involves enhanced neutrophil-platelet recruitment to the synovium. Currently, experiments are underway to identify the role of eDAMPs (heme and hemoglobin) mediated activation of TLR4 and inflammasome pathway in promoting IL-1b generation, neutrophil-platelet aggregation, and progression of joint injury in F8TKO mice

Disclosures
Ragni: OPKO: Research Funding; Sangamo: Research Funding; Shire/Takeda: Other: Advisory Board, Research Funding; Spark Therapeutics: Other: Advisory Board, Research Funding; Alnylam/Sanofi: Other: Advisory Board, Research Funding; BioMarin: Other: Advisory Board, Research Funding; Bioverativ/Sanofi: Other: Advisory Board, Research Funding. Gladwin:Globin Solutions, Inc: Patents & Royalties: Provisional patents for the use of recombinant neuroglobin and heme-based molecules as antidotes for CO poisoning; Bayer Pharmaceuticals: Other: Co-investigator; United Therapeutics: Patents & Royalties: Co-inventor on an NIH government patent for the use of nitrite salts in cardiovascular diseases .