Non-Canonical MYD88/TIRAP-Dependent Anti-Inflammatory Function of Pro-Coagulant Factor VIII

Context : FVIII is involved in the coagulation pathway. In the model developed by Hoffman in 1995, FVIII (with FIX) is at the center of coagulation. FVIII deficiency leads to a disease called hemophilia A. A deficiency of FVIII leads to inadequate coagulation and increases the risk of bleeding. Bleeding in hemophilic patient occurs mainly into large joints such as knee, elbow or ankle. Severe recurrence of hemarthrosis leads to irreversible arthropathy called hemophilic arthropathy (HA) characterized by a progressive complete destruction of the joint with permanent pain, musculoskeletal disability and poor quality of life. These patients have an increased inflammatory pattern. Prophylactic treatment of hemophilia A patients with the FVIII protein efficiently prevents bleeding events and preserves joint function. Recently, evidence for novel functions for FVIII across multiple systems, including the cardiovascular system, angiogenesis and maintenance of bone health has emerged. However, no study has shown the involvement of FVIII in inflammation. Here, we demonstrate that FVIII has an anti-inflammatory role and impacts the NF-κB pathway, independently from coagulation pathway.

Materials and methods : Human fibroblast-like synoviocytes (FLS) were isolated from synovial tissues from five different HA patients and two non-HA patients at the time of knee or ankle joint arthroscopic synovectomy after informed consent. Primary FLS were used between the third and the ninth passage. We also used commercial normal and rheumatoid arthritis FLS, as well as THP-1. In particular, THP1-Blue NK-κB cells allow monitoring the NF-κB signaling transduction pathway. Different commercial recombinant FVIII were tested following dialysis. We are also tested recombinant FIX and vWF. FLS (5x10⁵ cells) and THP1 (1x10⁷ cells) were activated or not (medium) with LPS from Salmonella abortus equi (1 µg/mL) in the presence or absence of FVIII for 6 hr. Alternatively, THP1-Blue (5x10⁵ cells) were activated or not (medium) with different PRR (Pattern Recognition Receptors) agonists for 24 hours: TLR2 (peptidoglycans), TLR4 (LPS) and TLR5 (flagellin). Cell supernatants were analyzed by ELISA and an ELISPOT were performed with a panel of 105 different cytokines.

Results :

Anti-inflammatory effect of FVIII

• After LPS stimulation and incubation with FVIII, cells expressed a unique profile of cytokine secretion, with a decrease in pro-inflammatory cytokines IL-1, IL-6 and TNF-alpha. This anti-inflammatory effect was not found when other coagulation factors, in particular vWF and FIX, were tested.

• A transcriptome analysis was performed on FLS from 3 different patients, normal FLS and THP1 cells and incubated alone or with FVIII. Transcriptomic results correlated with the inflammatory protein expression pattern, irrespective of the cell type.

FVIII is involved in the NF-κB signaling pathway through MYD88/TIRAP interaction

• Incubation of THP1 cells with the different TLR agonists resulted in an increase in the NF-KB signaling pathway. In the presence of FVIII, a decrease in NF-KB signaling was observed with TLR-2 and TLR-4 agonists but not with the TLR-5 agonist. NF-KB translocation in the nucleus, performed by AMNIS, was decreased when cells were incubated with FVIII (but not with vWF or FIX) after TLR-2/TLR-4 activation.

• Analysis of differences between TLR-2/TLR-4 and TLR-5 pathways showed that TIRAP is required only in TLR-2/TLR-4 to interact with MyD88 while TLR-5 pathway is required only for MyD88 activation.

• After silencing TIRAP by specific siRNA approach, no anti-inflammatory effect on NF-kB pathways was observed after FVIII incubation and TLR2/4 activation.

• Silencing TIRAP using specific siRNA abrogated the inhibitory effect of FVIII on TLR2/4-triggered NF-kB signaling. The results suggest that FVIII blocks the interaction between TIRAP and MyD88 and impairs the NF-kB pathway.

In conclusion, our study suggests a new potential role for FVIII in the control of inflammation through the NF-kB pathway. Further investigations are ongoing with animal models to confirm these data and clinical study will be performed.