Session Title: Plenary Session III: Discovery 2012
Session Type: Plenary Sessions
Background/Purpose: This study was performed to determine whether expression of TLR5 is associated with Rheumatoid Arthritis (RA) disease activity as well as to examine the role of TLR5 ligation in the pathogenesis of RA.
Methods: Expression of TLR5 was determined in RA and normal (NL) PB monocytes and in vitro differentiated macrophages by real-time RT-PCR and/or flow cytometry. Next, linear regression analysis was employed to correlate expression of TLR5 with levels of TNF-a and DAS28 score in RA monocytes from 43-48 patients. Finally, the mechanism by which TLR5 ligation mediates RA pathogenesis was determined by endothelial chemotaxis and tube formation.
Results: We performed microarray studies to identify differentially regulated genes in RA synovial fluid macrophages from active patients and identified Toll like receptor (TLR)5 as one of the most highly upregulated genes in RA synovial fluid macrophages compared to normal macrophages. Using real-time RT-PCR and FACS analysis we confirmed that expression of TLR5 is significantly elevated in RA synovial fluid macrophages (35 fold) and RA monocytes (7 fold) compared to normal counterpart cells. Interestingly, we found that blockade of TLR5 on RA peripheral blood (PB) monocytes greatly reduces RA synovial fluid mediated TNF-a transcription by 80% suggesting that there are endogenous TLR5 ligands expressed in the RA synovial fluid that are crucial for joint TNF-a modulation. Since TNF-a stimulation is also capable of upregulating TLR5 levels there is a positive feedback modulation in RA monocytes between TNF-a and TLR5 ligation and expression. We found that patients with higher expression of TNF-a expressed elevated levels of TLR5 (R2=0.79, p=3.6×10-7) in RA monocytes and the concentrations of TLR5 and TNF-a strongly correlated with increased disease activity as determined by examination of 28 defined joints (DAS28) (correlation of TLR5 with DAS28; R2=0.75) (correlation of TNF-a with DAS28; R2=0.58). Since our previous studies demonstrated that TLR5 expression is elevated on RA synovial tissue endothelial cells compared to control tissue we asked whether ligation of this receptor induces angiogenesis and if TLR5 endogenous ligands present in RA synovial fluid play a role in this process. We found that when endothelial cells were exposed to a dose response of flagellin, a TLR5 agonist, migration of endothelial cells was induced at concentrations ranging from 0.1 to 100 ng/ml (p<0.05). Further, incubation of endothelial cells with neutralizing antibody to TLR5 significantly suppressed RA synovial fluid endothelial migration and tube formation suggesting that the RA synovial fluid contains TLR5 endogenous ligands that are chemotactic for TLR5+ endothelial cells.
Conclusion: Our observations highlight that there is a strong correlation between TNF-a and TLR5 expression with disease activity in RA monocytes suggesting that TLR5 may be a TNF-a responsive gene that is linked to RA progression through induction of angiogenesis.
N. D. Chamberlain,
O. M. Vila,
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/expression-of-tlr5-strongly-correlates-with-levels-of-tnf-a-and-das28-in-ra-monocytes-and-ligation-of-tlr5-induces-angiogenesis-in-ra/