Background/Purpose:

TLR5 expression is highly elevated in RA and CIA lining and sublining macrophages and endothelial cells compared to non-arthritic controls. Additionally, expression of TLR5 in RA myeloid cells closely correlates with disease activity score, indicating that ligation of TLR5+ cells intensifies disease progression. Hence studies were conducted to determine whether dysregulation of TLR5 function can be utilized as a promising strategy for RA treatment.

Methods:

CIA mice were treated with IgG or anti-TLR5 antibody on days 23, 27, 30, 34, 37, 41, 44 and 48 and mice were sacrificed on day 49 post induction. CIA joint inflammation and bone erosion were assessed by measuring ankle circumference as well as H&E and TRAP staining. Joint myeloid cell recruitment and their phenotype were evaluated by F480 and iNOS immunostaining. Proinflammatory factors secreted from CIA joint were quantified by ELISA in the IgG and anti-TLR5 antibody treated mice. Remodeling of mouse bone marrow progenitor cells into M1 macrophages was examined following flagellin treatment by real-time RT-PCR and FACS analysis.

Results:

To uncover whether disruption of TLR5 ligation is a potential for RA therapy, CIA mice were treated with monoclonal anti-TLR5 antibody or IgG control. Results from these experiments demonstrate that CIA mice treated with anti-TLR5 antibody have markedly lower joint swelling starting on day 44 until day 48 post onset compared to the IgG group. Consistently, histological studies document that anti-TLR5 treatment was capable of reducing CIA synovial inflammation, joint lining thickness and bone erosion by 40% compared to the control mice. We next found that anti-TLR5 treatment impairs migration of circulating myeloid cells into the CIA joint. To better understand the mechanism by which blockade of TLR5 function relieves arthritis, joint myeloid cell phenotype was evaluated in CIA synovial tissues. Histological examination demonstrates that the frequency of iNOS+ M1 macrophages is 40% higher in the IgG treated CIA mice compared to anti-TLR5 group. Corroborating with this notion, M1 macrophage producing factors, IL-6 and CCL2, are significantly suppressed in the CIA joints following anti-TLR5 treatment compared to the control group. Confirming the findings in vivo, we established that flagellin ligation to TLR5 can transform naïve mouse myeloid cells into M1 macrophages. This differentiation process was assessed by transcription of TNF and IL-6 and frequency of CD80 staining which was dysregulated by blockade of TLR5. Moreover, we show that TRAP+ bone eroding osteoclasts are 30% higher in the control group compared to CIA joints treated with anti-TLR5. These findings are in agreement with our recent data, revealing that ligation of TLR5 plays a key role in osteoclast formation through a mechanism that is predominantly contingent on myeloid cells and their production of RANK and TNF-a.

Conclusion:

We conclude that TLR5 ligation promotes joint myeloid cell infiltration and can further remodel the newly recruited myeloid cells into M1 macrophages or fully mature osteoclasts suggesting that blockade of TLR5 can be employed as a promising new therapeutic target in RA.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/blockade-of-tlr5-ligation-is-a-novel-strategy-for-ra-therapy/