Fibroblast-activating protein as a biomarker for blood-induced joint inflammation in hemophilia

Abstract Introduction: Bleeding in patients with hemophilia A or B typically affects large joints such as elbows, knees and ankles. Bleeding into joints triggers an inflammatory reaction of the synovia that, if not treated appropriately, can result in painful and disabling arthritis. Exposure of the synovia to radical iron compounds released from red blood cells is thought to be an important element in the pathogenesis of arthritis, which in addition to inflammation includes induction of fibrosis, angiogenesis, chondrocyte apoptosis and osteoclast activation. As blood and wound debris are cleared by macrophages during wound healing, we hypothesized that chronic joint inflammation in hemophilia is a function of increased bleeding as well as deregulated wound healing. This concept was confirmed by our earlier work, showing that wound infiltration of macrophages and subsequent red blood cell clearance is impaired in hemophilia patients and that this process goes hand in hand with an amplified acute-phase response during bleeding. Here, we demonstrate that blood-induced joint inflammation represents a strong pro-fibrotic stimulus that can be visualized with 68Ga-FAPI PET/CT. Methods: To assess the effect of joint bleeding on inflammation and fibrosis, we induced a needle-puncture injury of the right knee in transgenic hemophilia A mice using a 32-gauge needle or injected the right knee of wild type mice with 500 µL anticoagulated blood. To assess the effect of monocyte-derived macrophages (MDM) on the clearance of blood, we eliminated monocytes by injecting wildtype mice intravenously with 200 µL clodronate liposomes prior to the injection of anticoagulated blood into the right knee joint. Wildtype mice injected with saline and/or saline liposomes served as controls. The joint diameter was measured using a digital caliper. Hemorrhage, synovitis and hemosiderin were visualized in midsagittal paraffin sections using hematoxylin and eosin (H&E) and fibrosis was visualized using Sirius Red. Immunohistochemistry was performed for the detection of macrophages (CD68), neutrophils (MPO), activated fibroblasts (FAP) as well as IL-1β, IL-6 and TGFβ. PET/CT scans of hemophilia patients were acquired 1 h after intravenous injection of 68Ga-FAPI, a highly specific tracer for FAP-expressing cells. Results: Needle puncture of the right knee of hemophilia mice resulted in an excessive hematoma of the synovia and the surrounding soft tissue, which reached its maximum extension on day 1, maintained its size until day 7 and was fully absorbed by day 28. Hemosiderin deposits formed between days 7 and 28 and remained in place until the end of the observation period on day 56 indicating that blood clearance was overwhelmed in hemophilia mice. Clearance of blood from the joints of blood-injected wildtype mice, on the other hand, was considerably more efficient unless wildtype mice were treated with clodronate liposomes that significantly reduced the capacity of macrophages to absorb injected blood. Delayed hematoma clearance in knee joints of hemophilia mice correlated with a delayed influx of macrophages and resulted in an acute inflammatory reaction of the synovia that was defined by enhanced neutrophil infiltration and a strong expression of IL-1β on days 1 and 7 after knee puncture. In parallel, we detected large amounts of FAP-positive fibroblasts in hematomas of punctured knees from hemophilia mice while there was no FAP expression in joints of wildtype mice at any time point. Infiltration of FAP-positive fibroblasts was accompanied by robust upregulation of IL-6 and TGF-beta in hemophilia joints and resulted in a fibrotic transformation with collagen deposition and extensive synovia hyperplasia. Paralleling our preclinical data, we detected a strong signal of FAP positivity in a hemophilia patient undergoing a 68Ga-FAPI PET/CT Scan of his elbow target joint while there was no specific FAP signal in a control patient with stable hemophilic arthropathy. Conclusion: We demonstrate that hemarthrosis in hemophilia represents a relevant inflammatory stimulus that leads to fibrotic joint degeneration. Blood-induced joint inflammation in hemophilia is amplified by impaired blood clearance and correlates strongly with FAP expression as well as IL-6 upregulation. Based on these data, we propose to further investigate 68Ga-FAPI PET/CT Scans in combination with plasma IL-6 levels as biomarkers for acute blood-induced synovitis in hemophilia.