Mechanisms of Iron Clearance from the Joint in FVIII-Deficient Mice after Induced Hemarthrosis

Introduction

Hemarthrosis in hemophilia causes toxic iron accumulation in the joint, which contributes to synovitis. This study aimed to explore mechanisms and timing of iron clearance from the joint space in mouse models of induced hemarthrosis.

Methods

Hemarthrosis was induced by sub-patellar puncture in FVIII-deficient mice and in hypocoagulable BALB/c (^HypoBALB/c) mice treated with 10 µg/ml warfarin for 7 days and 0.25 mg/kg anti-FVIII antibody 2 hours before knee puncture. Warfarin was reversed on day 2 post-injury with 100 IU/Kg 4-factor prothrombinase complex concentrate and the hematocrit determined in all mice as a measure of joint bleeding. Joint tissue was harvested at baseline and 2 or 4 weeks post-injury for analysis by histology. Ferric iron (Fe³⁺) was detected by Prussian Blue staining either during or after joint decalcification. Macrophages and macrophage-like synoviocytes were detected in joint tissue by immunohistochemistry with an anti-CD68 antibody. Synovial tissue was harvested from FVIII-deficient mice on day 3 and 2 weeks post-injury for gene expression studies by RNA sequencing (single-end; 75 bp) on an Illumina NextSeq500 platform. The limma-voom method (R BioConductor) was used for differential expression analyses.

Results

Knee injury caused substantial and comparable hemarthrosis in FVIII-deficient and ^HypoBALB/c mice (mean day 2 hematocrit: 27 % and 28 %, respectively). Post-decalcification Prussian Blue staining detected ferric iron accumulation in FVIII-deficient mice at week 4 only (5.3-fold increase compared to baseline, p=0.003). No ferric iron was detected in ^HypoBALB/c mice despite similar bleed volumes. In FVIII-deficient mice, Prussian Blue staining during decalcification was more sensitive and preserved detection of extracellular ferric iron, revealing a significant increase in ferric iron at 2 weeks post-injury relative to baseline (38-fold, p=0.005), which persisted at 4 weeks (23-fold, p=0.03). These findings coincided with increased CD68 staining at 2 weeks (36-fold, p=0.0002) and 4 weeks (8-fold, p=0.1). CD68-positive cells were dispersed throughout synovium at 2 weeks but appeared more clustered at 4 weeks and co-localized with iron staining, suggesting migration and iron uptake between 2 and 4 weeks post-injury. In ^HypoBALB/c mice, CD68 staining increased at 2 weeks (11-fold, p=0.008) but to a lesser extent than in FVIII-deficient mice, and was comparable to baseline at 4 weeks. Together, this suggests an altered mechanism of iron clearance in hemophilia. RNA sequencing revealed differential expression of 11/57 genes relating to iron transport in synovium on day 3, persisting somewhat at 2 weeks. Upregulated genes on day 3 included heme-oxygenase-1 (heme-degrading enzyme; 31-fold, p=3x10^-6), lipocalin-2 (iron-binding protein; 10-fold, p=0.001) and solute carrier family (slc) 11 member 1 (macrophage iron transporter; 3-fold, p=0.0004). Down-regulated genes on day 3 included ceruloplasmin (efflux of cellular iron; 17-fold, p=5x10^-5) and its homolog hephaestin (5-fold, p=0.002), CD163 (macrophage scavenging receptor for hemoglobin; 5-fold, p=0.03) and slc22 member 17 (lipocalin-2 receptor; 2-fold, p=0.03). Gene expression changes revealed key players involved in scavenging, degradation and transport of iron in synovium after hemarthrosis, and may expose mechanisms of impaired iron clearance in hemophilia pending further studies.

Conclusions

Iron handling after hemarthrosis, including uptake and transport in synovium and/or delivery to plasma transferrin, may be impaired in hemophilia and contribute to the evolution of hemophilic arthropathy. Unbiased RNA sequencing created several hypotheses that can be tested to further to elucidate mechanisms and timing of aberrant iron handling.