Unique Inflammatory Signature in Hemophilic Arthropathy: Epigenetic Changes Due to Interaction between Blood and Fibroblast-like Synoviocytes

Context: Hemophilic patients bleeding occur 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. Physiopathology of blood joint disease remains unclear. Some data suggest that HA and rheumatoid arthritis (RA) may share similar pathogenicity including elevation of inflammatory cytokines, chronic proliferative synovitis and cartilage destruction. However, in these two diseases the origin of inflammation differs and is linked to autoimmunity in RA and bleeding in HA. HA is the unique model of arthropathy in which resident cells like fibroblasts-like synoviocytes (FLS) are directly in contact with blood. Therefore, we hypothesized that FLS in HA could have a unique inflammatory signature as a consequence of contact with blood.

Materials and methods: Human 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 were obtained from patients. Experiments with FLS cultures were performed between the third and the ninth passage. During that time, cultures were constituted of a homogeneous population of fibroblastic cells. THP-1 cells line has been used as a control.

Results: FLS from hemophilic patients have a singular inflammatory cytokines profile

- FLS (5x10⁵ cells) were activated or not (medium) with LPS from Salmonella abortus equi (1 µg/ml) for 6 h and activated-cells supernatants were analyzed by ELISA. ELISPOT were performed with a panel of 105 different cytokines. After LPS stimulation, HA FLS expressed a unique profile of cytokines secretion, which differs from non-HA FLS and THP-1 cell line including IL-1α, IL-6 and IL-8 involved in innate immunity, IFN-γ, IL-5, IL-17A and IL-22 involved in adaptive immunity and MCP-1, M-CSF, MIF and MIP-3α involved in macrophages polarization.

- Constitutive expression of IL-1 and IL-6 mRNA was detectable in all cells, but in contrast, as shown previously in normal FLS, no TNF-α mRNA was detectable. To investigate mechanisms responsible for the lack of IL-1 protein synthesis in LPS-activated HA FLS, cells were incubated with LPS for 3 h and then with 5 µg/ml actinomycin D. After 2h of actinomycin D treatment, IL-1 mRNA became lower in LPS-activated HA FLS versus LPS-activated non-HA FLS and THP-1 cells. These results indicate that de novo synthesized IL-1 mRNA has a short half-life in LPS-activated HA FLS.

- Since cytokines mRNA seems to be unstable, we then investigated microRNA involvement in the regulation of cytokines. A miRNOME (Transcriptome Profiling miRNA, AFFYMETRIX) analysis showed that miRNAs expression was different between HA and nonHA FLS. Five have a very high difference in expression (hsa-miR664b-5p, hsa-miR4516, hsa-miR4289, hsa-miR6719-3p, hsa-miR675-5p), which potentially target mRNA involved in inflammatory pathways (NFKB, MAPK, Wnt/beta-catenin).

In conclusion our study demonstrates for the first time that the presence of blood in contact with FLS may induce durable epigenetic changes that may participate to the pathophysiology of hemophilic arthropathy.