PB0224 - Inflammatory cytokine expression after hemarthrosis differs with recombinant FVIII and FVIII-Fc fusion replacement in Factor VIII-deficient mice

Background: Systemic inflammatory cytokine response after hemarthrosis in relation to FVIII therapy is unknown. FVIII-Fc Fusion protein (FcFVIII) may modulate inflammation and immune responses via the Fc-portion of immunoglobulin.

Aims: To explore expression of inflammatory cytokines after hemarthrosis in Factor VIII-deficient mice when treated with recombinant human factor VIII (rhFVIII) compared to murine Fc-FVIII (mFcFVIII).

Methods: Hemarthrosis was induced by sub-patellar puncture in FVIII-deficient mice treated with saline, 100 IU/kg mFcFVIII (Fc species specificity) or 120 IU/kg rhFVIII (accounting for t1/2 differences). Plasma samples were collected at baseline, 1 day, 3 days, 1, 2 and 4 weeks after hemarthrosis (n=4-6 per group). Cytokine abundance was quantified with ELISA-based mouse cytokine inflammation 14-multiplex chemiluminescent assay (Quansys Biosciences) and log transformed. For each cytokine, undetectable samples were removed and cytokines with fewer than 50% detection across all samples were removed from analysis.

Results: Knee injury in FVIII-deficient mice resulted in hemarthrosis, which was prevented by rhFVIII and mFcFVIII prophylaxis. Hemarthrosis induced a decline in the majority of inflammatory cytokine abundance compared to baseline. At days 1 and 3, both pro-inflammatory (IL-1β, IL-2, IL-6, TNF-α) and anti-inflammatory cytokine (IL-4, IL-6) abundances decreased with rhFVIII but increased with mFcFVIII compared to saline. Except for IL-6, cytokine abundance was generally regulated concordantly by both FVIII-preparations between weeks 1 to 4. Abundances increased at week 1 and mostly decreased at 2 and 4 weeks compared to saline, although the magnitude of effect was notably less with mFcFVIII at 2 weeks. IL-6 discordantly decreased with rhFVIII but increased with mFcFVIII at 2 weeks (Figure).

Conclusion(s): Our findings suggest a temporal FVIII-preparation specific effect on systemic cytokine abundances after hemarthrosis especially at early (days 1, 3) and later (2 weeks) timepoints. This observation deserves further exploration to determine potentially unique effects of the Fc-portion of FcFVIII on inflammation modulation.