Angiogenesis in rheumatoid arthritis is directly fostered by TLR5 ligation and indirectly through IL-17 induction

Rheumatoid arthritis is a chronic autoimmune disease in which Toll like receptor (TLR) activated innate immunity plays an important role (1, 2). Previous studies document expression of TLR2 and TLR4 in RA peripheral blood, tissue and fluid (3–5) and further the significance of these receptors was demonstrated in the pathogenesis of acute as well as in the chronic models of experimental arthritis (6). This specific interest in TLR2 and TLR4 was initiated by identifying endogenous ligands for these receptors in RA which include fibrinogen, HSP60, 70 and 96 as well as EDA fibronection (7). Ligation of TLR2 and TLR4 has been associated with production of proinflammatory cytokines, chemokines, destructive matrix metalloproteinases and proangiogenic factors in RA specimen (1) suggesting that TLR2/4 activation can contribute to RA pathogenesis by inducing leukocyte migration, bone erosion and angiogenesis (1, 8, 9). We recently demonstrated for the first time that expression of TLR5 is elevated in RA and osteoarthritis (OA) compared to normal (NL) synovial tissue myeloid and endothelial cells (10). We documented that TLR5 endogenous ligand(s) are present in RA synovial fluid (SF) and that ligation of TLR5 in RA myeloid cells can promote production of a number of proinflammatory factors (10). Further significance of myeloid TLR5 expression in RA pathogenesis was validated by demonstrating a strong correlation between DAS28 score and concentrations of TLR5 and TNF-α in RA monocytes (10). Our previous study focused on examining the role of TLR5 in RA myeloid cells, however the impact of TLR5 on RA or CIA endothelial cell function is completely undefined. Angiogenesis is an early and a critical event in RA that promotes leukocyte infiltration and pannus formation thereby perpetuating chronic inflammation (11–13). Interestingly neovascularization mediated by TLR2 and TL4 ligation has been shown to be due to secretion of a number of proangiogenic factors such as IL-8, VEGF and TNF-α from RA synovial tissue fibroblasts and/or myeloid cells (9, 14). Others have shown that TLR2 induced angiogenesis and cell invasion is in part due to secretion of Ang-2 from RA synovial explants and its binding to Tie2 expressed on synovial sublining endothelial cells (15). There are other observations that demonstrate that LPS activation of NF-κB pathway is responsible for HIF-1α induced VEGF production in macrophages (16). These results suggest that angiogenesis triggered by TLR2 and TLR4 ligation is indirectly mediated via production of potent proangiogenic factors. We demonstrate for the first time that TLR5 ligation induces endothelial chemotaxis and tube formation through activation of PI3K/AKT1 pathway. We next show that like in RA, expression of TLR5 is elevated in CIA lining and sublining macrophages and endothelial cells. We document that post onset treatment with TLR5 agonist enhances CIA disease severity by promoting TH-17 cell differentiation as well as joint neovascularization. Lastly we show that differentiation of TH-17 cells and production of joint IL-17 is interconnected with TLR5 mediated angiogenesis as blockade of IL-17 cascade can significantly suppress endothelial chemotaxis in response to TLR5 agonist condition media. Our novel results suggest that TLR5 ligation mediates RA angiogenesis either directly or indirectly through TH-17 development. These results suggest that disruption of TLR5 function may suppress RA pathogenesis.