Tolerance Breakdown in Hemophilia A Mice is Orchestrated by Bacterial Infection Modulating Antigen-Specific B and T cell Responses

Introduction: Hemophilia A is a hereditary bleeding disorder caused by a deficiency in coagulation factor VIII (FVIII). Despite FVIII replacement therapy, a significant proportion of patients develop inhibitory antibodies, rendering treatment ineffective [1]. To address this, a high-dose regimen known as immune tolerance induction (ITI) has been developed, aiming to prevent inhibitor formation [2]. In a recent study, our group identified FVIII-specific regulatory T cells (Tregs) as key players in ITI success [3]. However, infectious challenges, such as Staphylococcus aureus (S. aureus), have been implicated in ITI failure [4] [5]. Understanding the mechanisms underlying tolerance breakdown could lead to novel biomarkers for predicting ITI outcomes.

Method: To investigate the impact of infection on the FVIII-specific immune responses, hemophilia A mice were initially treated with 2UI FVIII biweekly to induce tolerance. Subsequently, one group was infected with S. aureus (USA300 lac). Spleens and blood were aseptically harvested for further analysis. We monitored changes in immune cell populations, antibody production, and FVIII levels via flow cytometry, ELISA, ELISPOT, and RNAseq. In vitro, mouse, and human B and T cell infection assays were performed to decipher the molecular mechanism of tolerance breakdown.

Results: A significant increase in FVIII-specific antibody and inhibitor formation was detected in vivo following infection of tolerized HemA mice. It was accompanied by an expansion of FVIII-specific B cells and a decline in FVIII-specific Treg function. S. aureus infection seems to operate directly and indirectly by interacting with FVIII-specific tolerized B cells and enhancing T helper cell activity, respectively, resulting in an elevated pro-inflammatory cytokine production. Furthermore, tolerized human FVIII-specific B cells can be reactivated in vitro upon infection. Thus, bacterial infections can restimulate formerly suppressed FVIII-specific B cell responses in mice and men.

Conclusion: These results suggest that S. aureus infection can undermine ITI by promoting a pro-inflammatory environment and the FVIII-specific immune response. By identifying infection-mediated risk factors, such as cytokines, we can potentially tailor treatment strategies and minimize complications.

Publication History

Article published online:
13 February 2025

• References

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