Volume 14 Supplement 1

Proceedings of the 8th Global Arthritis Research Network (GARN) Meeting and 1st Bio-Rheumatology International Congress (BRIC)

Open Access

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

  • Arpita Myles1 and
  • Amita Aggarwal1
Arthritis Research & Therapy201214(Suppl 1):P50

DOI: 10.1186/ar3651

Published: 29 February 2012

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.

Cultured with

IL-6

IL-8

 

Normal PBMC (n = 5)

ERA PBMC (n = 7)

ERA SFMC (n = 3)

Normal PBMC (n = 5)

ERA PBMC (n = 7)

ERA SFMC (n = 3)

Medium

4.4 (1.5-5.4)

7.6 (3-16.6) *

18 (9.3-24.2)

10 (4.3-12.6)

12.6 (8.1-35.7)

10 (4.3-12.6)

TNF

13.6 (9.6-14.8)

16 (12-35)

21(18-30)

30.8 (15.8-36.3)

34.4 (30.8-46.1)

31 (15.8-36.3)

Pam3cys

15.1 (13.3-19.6)

44 (26-62) **

53 (28-71)

37.1 (11.4-41.8)

106 (42.6-147.6) **

37.1 (11.4-42)

PolyI:C

13.1 (1.3-25.8)

28 (24-4) *

46 (45-65)

33.6 (31.1-56.3)

126(78-167) *

34(31.1-56.3)

Flagellin

14.1 (6.7-23.5)

34.9 (15-39.2) *

52.6 (40-53.8) #

35.2 (16.1-84.2)

115 (73-162) **

35 (16.1-84.2)

Zymosan

14.7 (8.8-36.3)

34 (28.4-39) *

48 (39.8-56.1) #

56 (12.6-89.6)

106 (103-163) **

56 (12.6-90)

p < 0.05** 0.01 ERA PBMC versus control PBMC p <#0.05 ERA SFMC versus ERA PBMC.

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.

Authors’ Affiliations

(1)
Department of Clinical Immunology, Sanjay Gandhi Postgraduate Institute of Medical Sciences Lucknow

References

  1. Myles A, Aggarwal A: Expression of Toll-like receptors 2 and 4 is increased in peripheral blood and synovial fluid monocytes of patients with enthesitis-related arthritis subtype of juvenile idiopathic arthritis. Rheumatology. 2011, 50: 481-488. 10.1093/rheumatology/keq362.View ArticlePubMedGoogle Scholar

Copyright

© Myles and Aggarwal; licensee BioMed Central Ltd. 2012

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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