Indoleamine 2,3-Dioxygenase and Peripheral Tolerance to Exogenous Factor VIII: A Multi-Centre Pilot Study

Clinically relevant antibodies neutralizing the hemostatic effect of fVIII occur in approximately 20–25% of patients with hemophilia A on replacement therapy, and they are associated with significant mortality, morbidity, and a poorer life quality. Many factors predisposing to inhibitor formation have been advocated, but the reasons why some patients develop a neutralizing antibody response to FVIII remains unclear. Evidence suggests that hemophilia-associated mutations (resulting in the absence or severe truncation of the FVIII protein) are associated with the highest risk of inhibitor formation on replacement therapy, as those mutations may have prevented the early development of physiologic tolerance to human fVIII. Recent evidence suggests a role for professional antigen-presenting cells (APC) in the initiation of immunogenic versus tolerogenic responses to fVIII. In particular dendritic cells (DC) actively establish and maintain both central and peripheral tolerance to self. Several mechanisms contribute to DC tolerogenicity and the tryptophan catabolic pathway involving indoleamine 2,3-dioxygenase (IDO) may be at work. IDO is responsible for multiple regulatory effects on immune cells, including inhibition of activated T and B cell proliferation, apoptosis, and lymphocyte differentiation towards T or B cell regulatory phenotypes. The role of IDO and tryptophan catabolytes in immunoreactivity versus tolerance to self prompted us to investigate whether IDO has a role in peripheral tolerance to exogenous fVIII. Objectives: The goal of this multi-centre study was to examine IDO expression and function in peripheral blood mononuclear cells (PBMC) from hemophilic subjects to test the hypothesis that IDO is involved in the modulation of fVIII-specific responses and, particularly, that inhibitor-positive patients might have impaired IDO induction/activity and therefore higher propensity to develop anti-fVIII responses. Methods: Blood was collected from 21 severe (<0.01 IU/mL) hemophilia A patients who provided written informed consent and consisted of 9 inhibitor-positive patients (≥5 Bethesda Units) and 12 inhibitor-free patients (with undetectable inhibitor levels). PBMC were isolated by Ficoll-Paque Plus gradient centrifugation with standard procedures. IDO induction in PBMC was assessed by immunoblot analysis (WB) and Real-Time PCR (mRNA), while IDO activity was analyzed through HPLC evaluation of kynurenine production (the first breakdown product of tryptophan catabolism by IDO). Results: 11 out of 12 patients without inhibitor displayed normal IDO expression and function, while only 1 out of 9 patients with inhibitor had unimpaired IDO competence. Consistent results were obtained by i) immunoblot analysis of protein expression (densitometry units; inhibitor-positive patients, 0.78 ± 0.57; inhibitor-free patients 4.52 ± 2.9; p = 0.002), by ii) RT-PCR for IDO mRNA (mRNA fold change over control; inhibitor-positive patients 1.11 ± 0.54; inhibitor-free patients 3.18 ± 1.85; p = 0.007), and by iii) kynurenine production (kynurenine fold change over control; inhibitor-positive patients 0.86 ± 0.34; inhibitor-free patients 2.3 ± 0.87; p = 0.0002) (fig.1). Conclusions: These preliminary data show that the immunoregulatory enzyme IDO could be involved in peripheral tolerance to factor VIII. Studies with a larger number of patients (combined with studies of IDO gene polymorphism) and the implementation of suitable animal models (currently underway in our laboratory) might yield further insight into any possible relationships between defective IDO function and inhibitor development in hemophilic patients. Ultimately, the level of IDO expression could represent an additional prognostic factor in hemophilic patients on replacement therapy when combined with fVIII gene mutation (high- vs. low- risk) assessment and, possibly, HLA class II molecule expression profiling.

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No relevant conflicts of interest to declare.