Toll-like receptor 3 activation promotes joint degeneration in osteoarthritis

Josef Stolberg-Stolberg,Meike Sambale,Michael Raschke,Sina Stuecker,Jessica Bertrand,Annika Boettcher,Joanna Sherwood,Thomas Pap

doi:10.1038/s41419-022-04680-5

Abstract

Osteoarthritis (OA) is characterized by cartilage degradation that is induced by inflammation. Sterile inflammation can be caused by damage-associated molecular patterns that are released by chondrocytes and activate pattern recognition receptors. We evaluate the role of toll-like receptor-3-activating RNA in the pathogenesis of OA. Toll-like receptor 3 (TLR3) was detected by semiquantitative reverse transcriptase PCR, western blotting and microscopy. Rhodamine-labelled poly(I:C) was used to image uptake in chondrocytes and full-thickness cartilage. The production of IFNβ in chondrocytes after stimulation with poly(I:C) as well as in the synovial fluid of OA patients was measured using ELISA. Chondrocyte apoptosis was chemically induced using staurosporine. Immunohistochemistry was performed to examine TLR3 expression and apoptosis in human and murine OA cartilage. RNA in synovial fluid was quantified by RiboGreen assay. Destabilisation of the medial meniscus was performed in TLR3−/− and wildtype mice. OA was assessed after eight weeks using OARSI score. TLR3 expression was confirmed by western blot and RT-PCR. Poly(I:C) was internalised by chondrocytes as well as cartilage and caused an increase of IFNβ production in murine (11.46 ± 11.63 (wo) to 108.7 ± 25.53 pg/ml; N = 6) and human chondrocytes (1.88 ± 0.32 (wo) to 737.6 ± 130.5 pg/ml; N = 3; p < 0.001). OA cartilage showed significantly more TLR3-positive (KL0 = 0.22 ± 0.24; KL4 = 6.02 ± 6.75; N ≥ 15) and apoptotic chondrocytes (KL0 = 0.6 ± 1.02; KL4 = 9.78 ± 7.79; N ≥ 12) than healthy cartilage (p < 0.001). Staurosporine-induced chondrocyte apoptosis causes a dose-dependent RNA release (0 ng/ml = 1090 ± 39.1 ng/ml; 1000 ng/ml=2014 ± 160 ng/ml; N = 4; p < 0.001). Human OA synovial fluid contained increased concentrations of RNA (KL0-2 = 3408 ± 1129 ng/ml; KL4 = 4870 ± 1612ng/ml; N ≥ 7; p < 0.05) and IFNβ (KL0-2 = 41.95 ± 92.94 ng/ml; KL3 = 1181 ± 1865ng/ml; N ≥ 8; p < 0.05). TLR3−/− mice showed reduced cartilage degradation eight weeks after OA induction (OARSI WT = 5.5 ± 0.04; TLR3−/− = 3.75 ± 1.04; N ≥ 6) which was accompanied by gradually decreasing levels of TUNEL-positive cells (WT = 34.87 ± 24.10; TLR3−/ = 19.64 ± 7.89) resulting in decreased IFNβ expression (WT = 12.57 ± 5.43; TLR3−/− = 6.09 ± 2.07) in cartilage (p < 0.05). The release of RNA by apoptotic chondrocytes thus activating TLR3 signalling is one possible way of perpetuating inflammatory cartilage changes. The inhibition of TLR3 could be a possible therapeutic target for OA treatment.

Highlights

Osteoarthritis (OA) is a progressive joint disease, which is associated with severe pain and impairment of movement which results in a significant reduction of the quality of life
Different factors, including cytokines, growth factors and Wnts have been identified as being involved in OA disease progression. Innate inflammatory signals such as damage-associated molecular patterns (DAMPs) have been described to activate pattern-recognition receptors (PRRs), which induce the expression of matrix metalloproteinase (MMP) as well as disintegrin and metalloprotease with thrombospondin motif (ADAMTS), resulting in cartilage thinning with progressive loss of proteoglycans and collagen [1, 2]
The data shows that Toll-like receptor 3 (TLR3) is expressed in human and murine cartilage and that intra-articular release of ds nucleotides by apoptotic chondrocytes is one possible pathway causing TLR3 activation, subsequent IFNβ release and osteoarthritic changes within the joint

Summary

Introduction

Osteoarthritis (OA) is a progressive joint disease, which is associated with severe pain and impairment of movement which results in a significant reduction of the quality of life. Different factors, including cytokines, growth factors and Wnts have been identified as being involved in OA disease progression. Innate inflammatory signals such as damage-associated molecular patterns (DAMPs) have been described to activate pattern-recognition receptors (PRRs), which induce the expression of matrix metalloproteinase (MMP) as well as disintegrin and metalloprotease with thrombospondin motif (ADAMTS), resulting in cartilage thinning with progressive loss of proteoglycans and collagen [1, 2]. Focuses mainly on the role of TLR2 and 4 in OA [4, 5] These TLRs have been studied in cartilage and chondrocytes extensively, not much is known about the role of nucleic-acid binding TLRs such as TLR3 and TLR7-9

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