Over expression of toll-like receptors in peripheral blood and synovial fluid monocytes of enthesitis related arthritis category of juvenile idiopathic arthritis (JIA-ERA) patients contributes to secretion of inflammatory mediators

Background

TLRs 2, 4 and 9 have been implicated in murine models and human patients of arthritis, but the other TLRs are not well-investigated. Thus, we studied TLR expression and signaling and effect of TLR ligand stimulation in peripheral blood (PB) and synovial fluid (SF) monocytes (MC) of ERA patients.

Methods

Levels of TLR2, TLR4 and TLR9 were measured by flow cytometry in ERA PBMC (n = 26), paired SFMC (n = 13) and healthy PBMC (n = 19) Real time PCR was done for TLRs 1-9 and their adaptors IRAK1, IRAK4, TRIF, TRAF3, TRAF6. PBMC and SFMC were stimulated with ligands for TLR1 (pam3-cys), 2 (peptidoglycan), 3 (polyI:C), 4 (LPS), 5 (flagellin) and 6 (zymosan). Levels of IL-6, IL-8 and MMP3 (ng/ml) were measured in the culture supernatants.

Results

ERA PBMC had higher MFI of TLR2 [295.5(48.1-598) vs 179(68.7-442); p < 0.05] and TLR4 [448(178-2581) vs 402(229-569); p < 0.05] compared to controls. Intracellular TLR9 expression showed no significant difference between both groups. In paired samples, SFMC had higher MFI of both TLR2 [485(141-1683) vs 353(180-598); p < 0.05] and TLR4 [1016(42.4-3159) vs 513(193-2581); p < 0.05] compared to PBMC. Difference in TLR9 expression was not significant [1]. Patient PBMC (compared to healthy control) and SFMC (compared to corresponding PBMC) had higher RNA expression of TLRs1, 2, 3, 4, 5 and 6 and downstream adaptors. Patients PBMC produced significantly higher IL-6 (13.51 vs 6.54) and MMP3 (61 vs 32.9) as compared to controls on stimulation by LPS. With peptidoglycan also IL-6 (30.58 vs 10.84) and MMP-3 (102.54 vs 49.45) was higher than controls. Patient PBMCs produced more IL-6 and IL-8 compared to healthy PBMCs on stimulation with Pam3-cys, poly I:C, flagellin and zymosan. In paired samples, SFMCs showed a trend towards higher IL-6 and IL-8 production compared to PBMCs (Table 1)

Table 1 Production of IL-6 and IL-8 [median (range) ng/ml] by PBMCs and SFMCs upon TLR ligand stimulation.

Conclusion

Increased TLR expression and signaling on PBMC and SFMC from JIA-ERA patients may exacerbate disease by upregulating IL-6, IL-8 and MMP-3 in response to microbial/ endogenous ligands. TLR pathway is a potential therapeutic target in these patients.