Installing FVIII-Specific Tolerance in Hemophilia Via Engagement of the Aryl Hydrocarbon Receptor By Tryptophan Derivatives

Abstract Background: The development of inhibitors is the most serious complication a patient with hemophilia can experience on treatment with clotting factor concentrates. In a cohort of 100 severe hemophilia A patients, we have recently found that the inhibitor-positive status was associated with dysfunctional indoleamine 2,3-dioxygenase 1 (IDO1)1. The mechanisms whereby IDO1 promotes regulatory effects include production of tryptophan catabolites, collectively known as kynurenines. Some of those tryptophan derivatives are endogenous in nature and act as activating ligands for the Aryl hydrocarbon receptor (AhR), a transcription factor that belongs in the family of basic helix-loop-helix transcription factors2. Specifically, in dendritic cells (DCs), tryptophan-catabolite interactions with AhR promote the transcriptional induction of anti-inflammatory cytokines and the emergence of T regulatory (Treg) cells. Here, we report on the potential of tryptophan-related AhR ligands for inhibiting the formation of anti-FVIII antibodies in hemophilic (F8 KO) mice, a finding that could be important in developing novel strategies to prevent or eradicate FVIII inhibitors. Methods: Four different endogenous AhR ligands have been selected and used in these experiments. All molecules were administered in vivo to F8-deficient mice at incremental doses and by different routes (orally or intravenously), either concomitantly or after rhFVIII immunization. In specific experiments, AhR knockout mice and co-administration of AhR antagonist were used to analyze the impact of AhR deficiency. To co-deliver AhR ligands and FVIII antigens to APCs in vivo and induce antigen-specific Tregs, we administered molecules via a nanostructured delivery system3. Specifically, we engineered four types of nanoparticles (NPs), 60 nm in diameter, that were stabilized by a thiol-polyethyleneglycol (PEG) layer. Overall, we used 1) unloaded NPs, 2) NPs loaded with AhR ligands, 3) NPs loaded with rhFVIII, and 4) NPs loaded with AhR ligands and rhFVIII. Results: Administration of one of the four AhR ligands prevented the generation of anti-FVIII antibodies in nearly 80% of F8 KO mice. Similar effects were obtained with both the NPs-based approach and the oral delivery of ligands. Of note, the protective effect was negated by co-administration of the AhR antagonist CH-223191. Conclusions: Our results suggest that AhR is a possible molecular partner whereby tryptophan catabolites will control the immune response to FVIII. These findings might lead to novel interventions for preventing or eradicating inhibitors in hemophilia A patients. Bibliography: 1.Matino D., et al. IDO1 suppresses inhibitor development in hemophilia A treated with factor VIII. J Clin Invest. 2015 Oct 1;125(10):3766-81. 2. Yeste A, Nadeau M, Burns EJ, Weiner HL, Quintana FJ. Nanoparticle-mediated codelivery of myelin antigen and a tolerogenic small molecule suppresses experimental autoimmune encephalomyelitis. Proc. Natl Acad. Sci. USA 109(28), 11270-11275 (2012). 3. Bessede A, et al. Aryl hydrocarbon receptor control of a disease tolerance defence pathway.Nature. 2014;511(7508):184-190. Disclosures No relevant conflicts of interest to declare.

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