Characterization of Natural Anti-FVIII Antibodies and the Regulatory Role of the Gut Microbiota in FVIII Immunogenicity

Abstract

Patients with hemophilia A bleed spontaneously into the soft tissues and joints due to deficiencies in the coagulation protein Factor VIII (FVIII). One-third of patients develop neutralizing antibodies to standard intravenous replacement therapy, rendering it ineffective. A significant area of research focuses on identifying risk factors associated with the development of FVIII inhibitors and investigating mechanisms of inhibitor pathogenesis. Translating this knowledge into effective strategies or therapeutics to prevent FVIII inhibitor development is highly desired. Here, we have investigated the role of the gut microbiota and natural anti-FVIII antibodies in modulating one’s risk of developing FVIII inhibitors. The gut microbiota is an expansive network of microorganisms that line the gastrointestinal tract and has been shown to modulate the immune system in mice and humans. We first sought to determine if the gut microbiota plays a role in regulating FVIII inhibitor development by developing a hemophilia A mouse model of antibiotic-induced gut perturbation. Gut perturbation was associated with an enhanced FVIII immune response, decreased short-chain fatty acid (SCFA) production, and an altered transcriptomic environment in the spleen and draining mesenteric lymph nodes. We further characterized the effects of SCFAs on immune cells and found that only butyrate was able to potently inhibit B cell function ex vivo. Attempts to endogenously increase SCFA production and inhibit FVIII inhibitor formation through a high-fiber diet yielded subtle yet significant changes in the immune response and blood metabolome, revealing complex relationships between gut bacteria and host. Lastly, natural anti-FVIII antibodies are found in healthy human donors but appear to enhance immunogenicity to the FVIII protein in patients with hemophilia A. We characterized this natural antibody repertoire and found striking differences in the peripheral blood between mice and humans. We discuss how these findings fit into our current understanding of FVIII inhibitor development and shed light on how natural antibodies may modulate peripheral tolerance to the FVIII protein. Ultimately, this work explores two unknown factors related to the FVIII immune response and highlights the diversity of influences contributing to one’s FVIII inhibitor risk profile.