Hemophilia A Inhibitor Subjects Show Unique PBMC Gene Expression Profiles That Include Up-Regulated Innate Immune Modulators

Ahmad Faisal Karim,Anthony R Soltis,Nadia P Ewing,Kathleen P Pratt,Matthew D Wilkerson,Christoph Königs,Clifton L Dalgard,Gauthaman Sukumar

doi:10.3389/fimmu.2020.01219

Abstract

Formation of pathological anti-FVIII antibodies, or “inhibitors,” is the most serious complication of therapeutic FVIII infusions, affecting up to 1/3 of severe Hemophilia A (HA) patients. Inhibitor formation is a classical T-cell dependent adaptive immune response. As such, it requires help from the innate immune system. However, the roles of innate immune cells and mechanisms of inhibitor development vs. immune tolerance, achieved with or without Immune Tolerance Induction (ITI) therapy, are not well-understood. To address these questions, temporal transcriptomics profiling of FVIII-stimulated peripheral blood mononuclear cells (PBMCs) was carried out for HA subjects with and without a current or historic inhibitor using RNA-Seq. PBMCs were isolated from 40 subjects in the following groups: HA with an inhibitor that resolved either following ITI or spontaneously; HA with a current inhibitor; HA with no inhibitor history and non-HA controls. PBMCs were stimulated with 5 nM FVIII and RNA was isolated 4, 16, 24, and 48 h following stimulation. Time-series differential expression analysis was performed and distinct transcriptional signatures were identified for each group, providing clues as to cellular mechanisms leading to or accompanying their disparate anti-FVIII antibody responses. Subjects with a current inhibitor showed differential expression of 56 genes and a clustering analysis identified three major temporal profiles. Interestingly, gene ontology enrichments featured innate immune modulators, including NLRP3, TLR8, IL32, CLEC10A, and COLEC12. NLRP3 and TLR8 are associated with enhanced secretion of the pro-inflammatory cytokines IL-1β and TNFα, while IL32, which has several isoforms, has been associated with both inflammatory and regulatory immune processes. RNA-Seq results were validated by RT-qPCR, ELISAs, multiplex cytokine analysis, and flow cytometry. The inflammatory status of HA patients suffering from an ongoing inhibitor includes up-regulated innate immune modulators, which may act as ongoing danger signals that influence the responses to, and eventual outcomes of, ITI therapy.

Highlights

Hemophilia A (HA) is caused by mutations in the gene encoding coagulation factor VIII (FVIII), with disease severity characterized by the resulting delayed plasma clotting time compared to normal human plasma
The present study investigates mechanisms of the human immune response to FVIII, analyzing blood samples from subjects in the following categories: (A) HA with a past inhibitor that resolved either following ITI or spontaneously; (B) HA with a current inhibitor; (C) HA with no inhibitor history and (D) non-HA healthy controls
The temporal gene expression profiles of the 4 groups were distinct: the HA and HA groups showed up-regulated genes at t = 4 h post-FVIII stimulation, while all groups show up- and down-regulated genes at the subsequent time points, with the up-regulated genes at t =16 h pronounced for the non-HA control group

Summary

Introduction

Hemophilia A (HA) is caused by mutations in the gene encoding coagulation factor VIII (FVIII), with disease severity characterized by the resulting delayed plasma clotting time compared to normal human plasma. Gene therapy approaches to correct HA are showing great promise [7], this is not yet an option for the pediatric population. Despite these advances, achieving and maintaining immune tolerance to FVIII remains a strong priority, even for patients on these alternative therapies, as the vast majority could still greatly benefit from FVIII therapy or supplementation to achieve hemostasis, whether prophylactically, on-demand, or in settings of trauma or surgery [8, 9]

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