Dataset Information

Toll-like Receptor Activation Induces Degeneration of Human Intervertebral Discs.

ABSTRACT: Toll-like receptors (TLR) are activated by endogenous alarmins such as fragmented extracellular matrix compounds found in the degenerating disc. TLRs regulate cytokine, neurotrophin, and protease expression in human disc cells in vitro, and thus control key factors in disc degeneration. However, whether TLR activation leads to degenerative changes in intact human discs is unclear. Nucleus pulposus (NP) cells isolated from non-degenerating discs increase IL-1? and nerve growth factor gene expression following treatment with Pam2CSK4 (TLR2/6 agonist) but not Pam3CSK4 (TLR1/2 agonist). Challenging NP cells with Pam2CSK4 or 30?kDa fibronectin fragments (FN-f, an endogenous TLR2 and TLR4 alarmin) increased secretion of proinflammatory cytokines. We then investigated the effect of TLR activation in intact, non-degenerate, ex vivo human discs. Discs were injected with PBS, Pam2CSK4 and FN-f, and cultured for 28 days. TLR activation increased proteoglycan and ECM protein release into the culture media and decreased proteoglycan content in the NP. Proteases, including MMP3, 13 and HTRA1, are secreted at higher levels following TLR activation. In addition, proinflammatory cytokine levels, including IL-6, TNF? and IFN?, increased following TLR activation. These results indicate that TLR activation induces degeneration in human discs. Therefore, TLRs are potential disease-modifying therapeutic targets to slow disc degeneration.

SUBMITTER: Krock E

PROVIDER: S-EPMC5719358 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Toll-like Receptor Activation Induces Degeneration of Human Intervertebral Discs.

Krock Emerson E Rosenzweig Derek H DH Currie J Brooke JB Bisson Daniel G DG Ouellet Jean A JA Haglund Lisbet L

Scientific reports 20171207 1

Toll-like receptors (TLR) are activated by endogenous alarmins such as fragmented extracellular matrix compounds found in the degenerating disc. TLRs regulate cytokine, neurotrophin, and protease expression in human disc cells in vitro, and thus control key factors in disc degeneration. However, whether TLR activation leads to degenerative changes in intact human discs is unclear. Nucleus pulposus (NP) cells isolated from non-degenerating discs increase IL-1β and nerve growth factor gene express ...[more]

PMID: 29215065

Similar Datasets

Project description: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.

Project description:BackgroundExtracellular matrix stiffness is emerging as a crucial mechanical cue that drives the progression of various diseases, such as cancer, fibrosis, and inflammation. The matrix stiffness of the nucleus pulposus (NP) tissues increase gradually during intervertebral disc degeneration (IDD), while the mechanism through which NP cells sense and react to matrix stiffness remains unclear. In addition, mitochondrial dynamics play a key role in various cellular functions. An in-depth investigation of the pathogenesis of IDD can provide new insights for the development of effective therapies. In this study, we aim to investigate the effects of matrix stiffness on mitochondrial dynamics in IDD.MethodsTo build the gradient stiffness model, NP cells were cultured on polystyrene plates with different stiffness. Western blot analysis, and immunofluorescence staining were used to detect the expression of mitochondrial dynamics-related proteins. Flow cytometry was used to detect the mitochondrial membrane potential and intracellular Ca2+ levels. Apoptosis related proteins, ROS level, and TUNEL staining were performed to assess the effect of substrate stiffness on NP cells.ResultsStiff substrate increased phosphorylation of dynamin-related protein 1 (Drp1) at Ser616 by activating extracellular signal-regulated kinase 1/2 (ERK1/2) pathway, which promoted mitochondrial fission and apoptosis in NP cells. Furthermore, Piezo1 activation was involved in the regulation of the post-translational modifications of Drp1 and mitochondrial fission caused by matrix stiffness. Inhibition of Piezo1 and ERK1/2 can effectively reduce stiffness-induced ROS elevation and apoptosis in NP cells.ConclusionsOur results revealed that stiff substrate causes Piezo1 activation and Ca2+ influx, results in ERK1/2 activation and phosphorylation of Drp1 at S616, and finally leads to mitochondrial fission and apoptosis in NP cells. These findings reveal a new mechanism of mechanotransduction in NP cells, providing novel insights into the development of therapies for treating IDD.

Dataset Information

Toll-like Receptor Activation Induces Degeneration of Human Intervertebral Discs.

Publications

Toll-like Receptor Activation Induces Degeneration of Human Intervertebral Discs.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets