Toll-like receptor 2 (TLR2) induces migration and invasive mechanisms in rheumatoid arthritis

Abstract

IntroductionThis study investigates the role of Toll-like receptor 2 (TLR2) in the regulation of migratory and invasive mechanisms in rheumatoid arthritis (RA).MethodsInvasion, migration, matrix metalloproteinase (MMP)-1, -3 and tissue inhibitor of matrix metalloproteinase-3 (TIMP-3) expression, β-integrin binding, cytoskeletal rearrangement and Ras-related C3 botulinum toxin substrate 1 (Rac1) activation in response to a TLR2-ligand, Pam3CSK4 (1 μg/ml), in ex vivo RA synovial tissue explants, primary RA synovial fibroblasts (RASFC) and microvascular endothelial cells (HMVEC) were assessed by Transwell Matrigel™ invasion chambers, enzyme-linked immunosorbent assay (ELISA), multiplex adhesion binding assay, reverse transcription polymerase chain reaction (RT-PCR), F-actin immunofluorescent staining, matrigel synovial outgrowths, Rac1 pull-down assays/Western blot and zymography. β1-integrin expression in RA/control synovial tissue was assessed by immunohistology. The effect of Pam3CSK4 on cell migration, invasion, MMP-3 and Rac1 activation was examined in the presence or absence of anti-β1-integrin (10 μg/ml) or anti-IgG control (10 μg/ml). The effect of an anti-TLR-2 mAb (OPN301)(1 μg/ml) or immunoglobulin G (IgG) control (1 μg/ml) on RASFC migration and RA synovial tissue MMP activity was assessed by wound assays, ELISA and zymography.ResultsPam3CSK4 significantly induced cell migration, invasion, MMP-1, MMP-3, MMP-2 and MMP-9 expression and induced the MMP-1/TIMP-3 and MMP-3/TIMP-3 ratio in RASFC and explants (p <0.05). β1-integrin expression was significantly higher in RA synovial tissue compared to controls (p <0.05). Pam3CSK4 specifically induced β1-integrin binding in RASFC (p <0.05), with no effect observed for β2-4, β6, αvβ5 or α5β1. Pam3CSK4 increased β1-integrin mRNA expression, Rac1 activation, RASFC outgrowths and altered cytoskeletal dynamic through induction of filopodia formation. Pam3CSK4-regulated cell migration and invasion processes, but not MMP-3, were inhibited in the presence of anti-β1-integrin (p <0.05), with no effect observed for anti-IgG control. Furthermore, anti-β1-integrin inhibited Pam3CSK4-induced Rac1 activation. Finally, blockade of TLR2 with OPN301 significantly decreased spontaneous release of MMP-3, MMP-2 and MMP-9 and increased TIMP-3 secretion from RA synovial explant cultures (p <0.05). Incubation of RASFC with OPN301 RA ex vivo conditioned media inhibited migration and invasion compared to IgG control.ConclusionsTLR2 activation induces migrational and invasive mechanisms, which are critically involved in the pathogenesis of RA, suggesting TLR2 as a potential therapeutic target for the treatment of RA.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-015-0664-8) contains supplementary material, which is available to authorized users.

Toll-Like Receptor 2 Ligation Enhances HIV-1 Replication in Activated CCR6 + CD4 + T Cells by Increasing Virus Entry and Establishing a More Permissive Environment to Infection

“…In addition, we report that TLR2 engagement induces a cytoskeletal rearrangement by augmenting the degree of actin polymerization. Our findings are in agreement with a previous work, which has revealed that Pam3CSK4 alters cytoskeletal dynamics (68). However, it should be noted that this work was carried out in human fibroblasts and not in CD4 ϩ T cells, as shown here.…”

“…Although TLR2 triggering does not affect surface expression levels of CCR5 and CD4, we quantified the extent of virus entry because it has been shown recently that Pam3CSK4 induces rearrangement of the F-actin cytoskeletal architecture (68). We performed an entry test using NL4.3 Balenv viruses and quantified the intracellular p24 content by enzymelinked immunosorbent assay (ELISA) in CD3/CD28-costimulated CD4 ϩ T cells that were either left untreated or treated with Pam3CSK4.…”

Acute serum amyloid A is an endogenous TLR2 ligand that mediates inflammatory and angiogenic mechanisms

“…In addition, TLR2 is expressed in SM perivascular regions,50 and in vitro TLR2 activation induces VEGF/IL-8 expression in synovial fibroblasts and chondrocytes,51 52 and MMP-9 in corneal epithelial cells and THP-1 macrophages 53. Furthermore, we have demonstrated using whole-tissue RA synovial explant cultures that OPN301 significantly inhibits spontaneous release of pro-inflammatory cytokines IL-1β, TNF-α, IFN-γ and IL-8, 34 and MMP-3, MMP-2 and MMP-9 54. In addition, culture of RASFC with conditioned media from OPN301-treated RA explants inhibited RASFC migration and invasion compared with IgG control 54.…”

“…Furthermore, we have demonstrated using whole-tissue RA synovial explant cultures that OPN301 significantly inhibits spontaneous release of pro-inflammatory cytokines IL-1β, TNF-α, IFN-γ and IL-8, 34 and MMP-3, MMP-2 and MMP-9 54. In addition, culture of RASFC with conditioned media from OPN301-treated RA explants inhibited RASFC migration and invasion compared with IgG control 54. Therefore, the ability of TLR2 to mediate A-SAA-induced changes in vascularity, chemokine expression and adhesion further support the hypothesis that A-SAA engages TLR2 to act locally in the RA joint.…”

Role of Toll-Like Receptors and Their Endogenous Ligands in the Pathogenesis of Rheumatoid Arthritis: A Review of Literature

“…Активация ТПР2 в синовиальных фибробластах у больных РА приводила к увеличению продукции большого количе-ства провоспалительных цитокинов, включая ИЛ1β и ФНОα [20,21]. В исследованиях на культурах клеток бы-ла показана связь между активацией ТПР2 и повышенным синтезом ММП, обладающих выраженной катаболиче-ской активностью, а также белка RANKL (receptor activator of nuclear factor kappa-B ligand; лиганд рецептора, активи-рующего ядерный фактор каппа-В), активирующего остео-кластогенез [22,23]. Отмечена статистически значимая связь экспрессии ТПР2 и 4 с синтезом ИЛ12 и ИЛ18 в си-новиальной ткани пациентов с РА [19].…”