Toll-like receptor 3 activation promotes joint degeneration in osteoarthritis

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.

Type I IFN in Glomerular Disease: Scarring beyond the STING

“…Instead, these TLRs are only activated in the event that extracellular DNA or RNA from damaged or dead bystander cells has been endocytosed and the nucleic-acid-containing endosome fuses with an acidified endosome containing the TLRs [ 78 ]. Indeed, there are several reports in the literature where extracellular mRNA or mitochondrial RNA derived from damaged cells has been engulfed by neighboring cells, resulting in TLR3 activation and sterile inflammation [ 63 , 79 , 80 ]. Highlighting a route for entry for nucleic acids, Bertheloot et al showed that RNA released from damaged cells can be detected by the receptor for advanced glycation end products (RAGE) to elicit their internalization and activate TLR7 and TLR8 [ 81 ].…”

Healthy and Osteoarthritis-Affected Joints Facing the Cellular Crosstalk

“…In vitro, it was revealed that TLR-3 is upregulated in chondrocytes of OA cartilage and reacts to apoptotic chondrocytes. The TLR-3 knockout mice were protected from OA-like cartilage breakdown [ 177 ]. It is also worth mentioning, that satellite glial cells of the dorsal root ganglia (DRG) express many TLRs.…”

Structural and Molecular Changes of Human Chondrocytes Exposed to the Rotating Wall Vessel Bioreactor

“…Afterwards, the fluorescence images were analyzed by cell counting using ImageJ for Windows. TUNEL-and DAPI-positive cells were put into relation ((TUNEL positive cells/total number of cells) *100) [36].…”