Activation of the Acute-Phase Response in Hemophilia

Abstract

To identify recurrent inflammation in hemophilia, we assessed the acute-phase response in the blood of patients with hemophilia A and B. Compared to age- and weight-matched controls, blood levels of interleukin-6 (IL-6), C-reactive protein (CRP), and LPS-binding protein (LBP) were significantly elevated in the entire cohort of hemophilia patients but exhibited a particularly pronounced increase in obese hemophilia patients with a body mass index (BMI) ≥30. Subgroup analysis of the remaining nonobese hemophilia patients (BMI: 18-29.9) revealed a significant spike of IL-6, CRP, and LBP in connection with a de-novo increase of soluble IL-6 receptor α (sIL-6Rα) in patients with bleeding events within the last month. Hemophilia patients who did not experience recent bleeding had IL-6, CRP, and sIL-6Rα blood levels similar to healthy controls. We did not find increased IL-6 or acute-phase reactants in hemophilia patients with arthropathy or infectious disease. The role of IL-6 as a marker of bleeding in hemophilia was confirmed in hemophilia patients with acute bleeding events as well as in transgenic hemophilia mice after needle puncture of the knee, which exhibited an extensive hematoma and a 150-fold increase of IL-6 blood levels within 7 days of the injury compared to needle-punctured control mice. Notably, IL-6 blood levels shrunk to a fourfold elevation in hemophilia mice over controls after 28 days, when the hematoma was replaced by arthrofibrosis. These findings indicate that acute-phase reactants in combination with sIL-6Rα could be sensitive biomarkers for the detection of acute and recent bleeding events in hemophilia.

Fig. 1

Inflammatory signature in hemophilia . (A–C) , IL-6, CRP, and LBP concentrations in the plasma of hemophilia patients (H, hemophilia; n  = 59) compared to healthy controls (C, control; n  = 54). (D–F) IL-6, CRP, and LBP plasma levels in patients with severe (S; n  = 39) and moderate-to-mild hemophilia (M; n  = 20) compared to controls (C). * p  < 0.05, *** p  < 0.001, **** p  < 0.0001. Horizontal bars denote the mean. CRP, C-reactive protein; IL-6, interleukin-6; LBP, LPS-binding protein; n.s., nonsignificant.

Fig. 2

The effect of age and BMI on the inflammatory signature in hemophilia. (A–C) IL-6, CRP, and LBP levels in hemophiliacs (H) with ages 18–29, 30–49, and ≥ 50 years compared to age-stratified controls (C). (D–F) Plasma concentrations of IL-6, CRP, and LBP in a BMI-stratified cohort of hemophiliacs (H) with normal weight (Norm), overweight (Over), or obese compared to weight-stratified controls (C). * p  < 0.05, ** p  < 0.01, *** p  < 0.001; **** p  < 0.0001. Horizontal bars denote the mean. BMI, body mass index; CRP, C-reactive protein; IL-6, interleukin-6; LBP, LPS-binding protein.

Fig. 3

Bleeding affects the inflammatory signature . Plasma IL-6 (A) , CRP (B) , IL-6Rα (C) , and LBP levels (D) in hemophilia patients with BMI <30 and recent bleeding events occurring <1 month (<1) compared to hemophilia patients with BMI <30 and bleeding episodes >1 month (>1) and weight-stratified controls (C). * p  < 0.05, ** p  < 0.01, *** p  < 0.001; **** p  < 0.0001. Horizontal bars denote the mean. CRP, C-reactive protein; IL-6, interleukin-6; LBP, LPS-binding protein; n.s., nonsignificant; sIL-6Rα, soluble IL-6 receptor α.

Fig. 4

IL-6 as a marker of acute bleeding in hemophilia patients . Plasma IL-6 was measured longitudinally in hemophilia patients following an acute bleeding episode until the bleeding was resolved with coagulation factor replacement therapy. Courses of plasma IL-6 levels are shown in a patient with severe hemophilia B and a traumatic hemarthrosis of the knee (A) and five patients with severe hemophilia A, two presenting with traumatic joint bleeds (B , C) , one with a bleed following a tooth extraction (D) , one with a minor bleed 4 weeks after septoplasty surgery (E) , and one with an extensive traumatic bleed of the right latissimus dorsi muscle (F) . IL-6 values at time points 0, −1, and −2 are based on blood testing during routine visits prior to bleeding. Grey arrows symbolize bleeding onset. IL-6, interleukin-6.

Fig. 5

Plasma IL-6 correlates with intraarticular bleeding in transgenic hemophilia mice . (A) Representative images of transgenic factor VIII knockout B6;129S-F8 ^tm1kaz /J mice before (day 0) and day 1, 7, and 28 after needle puncture of the right knee joint compared to the nonpunctured left knee. (B , C) Panels depict whole mounts of the right knee joint before and after needle puncture injury (left, tibial plateau; right, femoral head) stained with H&E (B) or Sirius red (C). Black arrows indicate bleeding/hematoma (B, d1, and d7) and arthrofibrosis (C, d28; red). Scale bar: 500 µm. (D) High magnification images of the synovia. Black arrows show synovia hyperplasia and the thickening of the synovia layer over time. Scale bar: 50 µm. (E) Joint ratio (diameter of the punctured knee divided by the nonpunctured knee) presented as an average per treatment group and time point (H, hemophilia, black circles; C, control, grey squares; ea. n  = 6–10). (F) Intraarticular bleeding score (0, no bleeding; 1, some bleeding; 2, severe bleeding) in the punctured knee of hemophilia mice (H ^punctured , white diamonds) compared to the nonpunctured knee (H ^nonpunctured , black triangles). (G) Thickness of the synovia layer was measured according to the image scale in the joints of hemophilia mice with knee puncture (H ^punctured , white diamonds) or without knee puncture (H ^nonpunctured , black triangles). (H) Hemophilic synovitis was calculated using the Valentino scoring system (0–10 points). With knee puncture (H ^punctured , white diamonds) and without knee puncture (H ^nonpunctured , black triangles). (I , J) IL-6 and TNFα were measured over time in blood isolated from hemophilia (H) and control (C) mice. *** p  < 0.001, **** p  < 0.0001. IL-6, interleukin-6; TNFα, tumor necrosis factor α.