Joint injury induces a robust proinflammatory cytokine response in hemophilia A mice: Implications for immune-mediated arthropathy

Abstract

Abstract Background: Hemophilic arthropathy, driven by repeated joint bleeds and unresolved inflammation, remains a major complication in patients with hemophilia A. Although the mechanical and hemostatic contributors are well recognized, the immunological responses following joint injury in the context of FVIII deficiency are poorly characterized. Objective: To define the systemic inflammatory cytokine profile triggered by joint injury in a murine model of hemophilia A. Methods: Plasma samples were collected from FVIII-deficient (F8-/-) mice 72 hours after needle-induced knee joint injury (n = 4) and from uninjured naïve F8-/- controls (n = 6). Cytokine profiling was performed using a 32-plex mouse cytokine array, and statistical analysis was carried out with unpaired t-tests or the Mann-Whitney U-test if the data did not pass the normality test. Results: Joint injury triggered a complex proinflammatory response, with several cytokines showing highly significant increases compared to naïve controls. The elevated cytokines were categorized into three groups: 1) The strongest upregulation (P < 0.001) was observed in IL-9 (65.96 ± 10.65 pg/mL vs. 16.29 ± 10.15 pg/mL) and IL-17 (4.61 ± 1.63 pg/mL vs. 0.35 ± 0.23 pg/mL), key mediators of Th2 and Th17 inflammation, respectively, indicating robust T-cell–mediated immune activation. 2) Moderate increases (P < 0.01) included TNF-α (75.57 ± 18.07 pg/mL vs. 3.01 ± 2.09 pg/mL), a central driver of synovial inflammation and joint destruction; LIF (0.72 ± 0.21 pg/mL vs. 0.19 ± 0.18 pg/mL), a cytokine involved in tissue remodeling and chronic inflammation; and MIP-1β (CCL4) (270.9 ± 187.4 pg/mL vs. 13.73 ± 16.92 pg/mL), which recruits monocytes and macrophages to inflammation sites. 3) Mild yet significant increases (P < 0.05) were seen in IL-10 (32.74 ± 20.44 pg/mL vs. 8.08 ± 6.31 pg/mL), an anti-inflammatory cytokine potentially reflecting a compensatory regulatory response; IL-12p70 (41.52 ± 20.15 pg/mL vs. 4.88 ± 5.24 pg/mL), a cytokine that promotes Th1 responses; IP-10 (CXCL10) (234.2 ± 213.4 pg/mL vs. 48.66 ± 33.72 pg/mL), and MIG (CXCL9) (24.91 ± 4.02 pg/mL vs. 17.00 ± 4.73 pg/mL), both involved in T-cell trafficking; and MIP-2 (CXCL2) (236.3 ± 166.5 pg/mL vs. 22.31 ± 34.71 pg/mL), a neutrophil chemoattractant involved in acute joint inflammation. Conversely, IL-13—a Th2 cytokine with anti-inflammatory and tissue repair roles—was significantly decreased (10.59 ± 3.12 pg/mL vs. 31.68 ± 20.43 pg/mL), indicating a shift away from regulatory Th2 responses. No significant differences were observed in the remaining cytokines assessed. Conclusion: These results reveal that joint injury in hemophilia A mice induces a hierarchically structured proinflammatory cytokine cascade, with marked Th17, Th1, and chemokine responses, and suppression of regulatory Th2 signals. The prominence of IL-17, TNF-α, and MIP-1β suggests key mediators driving hemophilic joint inflammation and provides rationale for targeting these pathways in efforts to mitigate arthropathy. Moreover, the suppression of IL-13 may contribute to the persistence of inflammation and impaired resolution. Together, these findings highlight potential biomarkers and therapeutic targets in hemophilia-related joint disease.