Early cellular interactions and immune transcriptome profiles in human factor VIII‐exposed hemophilia A mice

Abstract

Background Developing factor VIII (FVIII) inhibitory antibodies is the most serious complication in hemophilia A treatment, representing a significant health and economic burden. A better understanding of the early events in an immune response leading to this outcome may provide insight into inhibitor development. Objective To identify early mediators of FVIII immunity and to detail immune expression profiles in the spleen and liver. Methods C57Bl/6 F8 E16 knockout mice were infused with 5-20 μg (2000-8000 IU kg ) of recombinant FVIII. Spleens were frozen at various time-points post-infusion and stained for FVIII and cellular markers. Splenic and liver RNA expression analysis was performed 3 h post-infusion of 0.6 μg (240 IU kg ) FVIII by nCounter technology using a 561-gene immunology panel. Results FVIII localization in the spleen did not change over 2.5 h. We observed significantly higher co-localization of FVIII with MARCO cells compared with SIGLEC1 and SIGNR1 in the splenic marginal zone. FVIII exhibited little co-localization with CD11c dendritic cells and the macrophage mannose receptor, CD206. Following FVIII infusion, the splenic mRNA profiling identified genes such as Tnfaip6 and Il23r, which are implicated in chemotaxis and a proinflammatory Th17 response, respectively. In contrast, an upregulation of Gfi1 in the liver suggests an anti-inflammatory environment. Conclusions FVIII co-localizes predominantly with marginal zone macrophages (MARCO ) in the murine spleen following intravenous infusion. Targeting pathways that are implicated in the early FVIII innate immune response in the spleen may lead to therapeutic interventions to prevent inhibitor formation.

The endothelial cell receptor stabilin-2 regulates VWF-FVIII complex half-life and immunogenicity

“…As LSECs express stabilin-2, MHC II, and costimulatory molecules, although at relatively low levels, there is a potential that they may function as APCs capable of presenting VWF-FVIII peptides to cognate CD4 + T cells (57,58). However, it is more likely that stabilin-2 expressed on the splenic sinusoidal endothelium acts as a key mediator in the directed shuttling of FVIII antigen in a VWF-dependent manner through marginal zone macrophage and marginal B cell compartments of the spleen (11,59,60). Furthermore, engagement of stabilin-2 initiates signaling through extracellular signal-regulated kinases 1 and 2 (ERK1/2), and can induce NF-κB activation of proinflammatory gene expression that can, in turn, enhance the likelihood of an immune response (61).…”

Advances in knowledge of inhibitor formation in severe haemophilia A

“…On the other hand, glycans may be protective, acting to shield underlying FVIII epitopes. The presence of glycans shields overlying FVIII protein epitopes and incomplete glycan coverage of the FVIII protein is hypothesised to result in antibody complexes and enhanced immunogenicity (Lai et al , ,b). Although there is a lack of evidence suggesting which hypothesis is more clinically relevant, a recent study proposes that glycans primarily drive the differences in immunogenicity between pdFVIII and rFVIII products even in the presence of its chaperone von Willebrand Factor (VWF) (Jankowski et al , ).…”

“…Recently, it was demonstrated that opsonisation via complement protein C3 plays an important role in enhancing the efficiency of FVIII uptake and the subsequent development of an immune response (Rayes et al , ). Alternatively, antibody‐mediated opsonisation of FVIII via immunoglobulin (Ig)‐M has been observed to increase inhibitor development (Lai et al , ,b), potentially acting through a complement receptor 1‐ or 2‐dependent process (Ferguson et al , ). The binding of IgG to FVIII similarly enhances its uptake but through Fcγ receptor mechanisms (Pincetic et al , ).…”

The national blueprint for future basic and translational research to understand factor VIII immunogenicity: NHLBI State of the Science Workshop on factor VIII inhibitors

“…been implicated in initial FVIII uptake in lymphoid organs. 7,8 How these innate factors and cell types (including innate immune and antigen presenting cells [APCs]) interact during the transition from initial innate immune signalling to the adaptive immune response remains to be defined.…”

“…For example, bacterial lipopolysaccharide (LPS) functions as an adjuvant and enhances FVIII inhibitor formation in haemophilic mice. Certain types of macrophages and marginal zone B cells have also been implicated in initial FVIII uptake in lymphoid organs . How these innate factors and cell types (including innate immune and antigen presenting cells [APCs]) interact during the transition from initial innate immune signalling to the adaptive immune response remains to be defined.…”