Expression of toll-like receptors in cartilage endplates cells: a role of toll-like receptor 2 in pro-inflammatory and -catabolic gene expression

Expression of toll-like receptors in cartilage endplates cells: a role of toll-like receptor 2 in pro-inflammatory and -catabolic gene expression | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Expression of toll-like receptors in cartilage endplates cells: a role of toll-like receptor 2 in pro-inflammatory and -catabolic gene expression Tamara Mengis, Laura Bernhard, Nick Herger, Irina Heggli, Jan Devan, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4424045/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Introduction The vertebral cartilage endplate (CEP), essential for intervertebral disc health, is susceptible to degeneration, which can be associated with chronic low back pain, disc degeneration, and Modic changes. Although it has been established that intervertebral disc cells express toll-like receptors (TLRs), which can recognize diverse pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs), triggering an immune response, it is unknown whether CEP cells (CEPC) share this trait. The CEP exhibits a significantly higher cell density compared to the disc, potentially heightening the relevance of CEPC in this context. This study aimed to identify TLRs on CEPC and explore their role in activating pro-inflammatory and catabolic gene expression. Methods Gene expression of TLR1-10 was measured with quantitative real-time polymerase chain reaction in human CEPs and expanded CEPC. Additionally, CEPC were stimulated with tumor necrosis factor alpha and interleukin 1 beta, specific TLR2/6, TLR2/1 and TLR4 agonist (Pam2csk4, Pam3csk4 and lipopolysaccharide) and with the 30 kDa N-terminal fibronectin fragment, a representative DAMP. TLR2 signaling was inhibited with TL2-C29. TLR2 protein expression was measured with flow cytometry. Results An ex-vivo analysis of CEP tissue found all 10 TLRs expressed while cultured CEPC lost TLR7 and TLR8 expression. TLR2 was the only TLR whose expression significantly increased after pro-inflammatory stimulation. Stimulation of the TLR2/6 heterodimer with Pam2csk4 upregulated TLR2 protein expression. After 48 hours of stimulation, all applied ligands upregulated expression of pro-inflammatory genes and the matrix metalloproteases 1 (MMP1), MMP3, and MMP13. TLR2 inhibition was able to specifically inhibit the upregulated genes. Conclusion The expression of TLR1-10 in CEPC indicates that the CEP is susceptible to PAMP and DAMP stimulation. TLR2 expression in CEPC is enhanced under inflammatory conditions and its stimulation has pro-inflammatory and pro-catabolic consequences. Therefore, TLR2 signaling in CEPC might play an important role in disc degeneration and Modic changes. Toll-like receptors cartilage endplate cells cartilage endplate disc degeneration Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction The vertebral cartilage endplate (CEP) is a thin hyaline cartilage structure separating the intervertebral disc from the vertebra. Its intactness is critical for the health of the disc [ 1 ], [ 2 ]. CEP degeneration has been associated with chronic low back pain (CLBP) [ 3 ], disc degeneration (DD) [ 4 ], [ 5 ], [ 6 ], [ 7 ], [ 8 ], and Modic changes (MC) [ 9 ], [ 10 ], [ 11 ]. Yet, the mechanisms linking CEP degeneration to DD and MC remain unclear. In-vivo and animal disc explant models showed that structural damage of the CEP can cause DD and MC [ 5 ], [ 12 ], [ 13 ]. Biochemical changes of the CEP, like dehydration, calcification, and fibrosis diminish its nutrient transport properties, leading to reduced nutrient availability in the disc, disc cell death, and DD [ 2 ], [ 14 ], [ 15 ]. While it has become clear that structural and biochemical changes of the CEP are important in DD and MC, the role of biological changes of the CEP have remained largely unexplored. In the intervertebral disc, the biological degenerative mechanisms are well studied, but in the CEP, which has an approximately 4-fold higher cell density than the intervertebral disc [ 16 ], these changes are poorly understood. Expression of matrix metalloproteinases (MMPs) and interleukins in cartilage endplate cells (CEPC) are suggested to play a role in CEP degradation [ 17 ]. In the intervertebral disc, expression of matrix proteases and pro-inflammatory cytokines is increased during degeneration [ 18 ], [ 19 ], [ 20 ], and expression of pro-inflammatory cytokines is even higher with adjacent MC [ 21 ], [ 22 ], [ 23 ]. Together, this leads to the resorption of the disc matrix, dehydration of the disc, and disc collapse. Disc cells also express various toll-like receptors (TLRs), with TLR2 being the only one that is responsive to inflammatory milieu such as interleukin-1β exposure. Additionally, TLR2 possesses the highest number of ligands, attributed to its capability to form diverse heterodimers [ 24 ]. The presence of TLRs conveys them the capacity to sense and respond to danger signals from damage-associated molecular pattern (DAMPs) and from pathogen-associated molecular patterns (PAMPs) [ 25 ]. For example, the 30 kDa N-terminal fibronectin fragment (FNf30 kDa), short hyaluronic acid fragments, and decorin are extracellular-matrix derived DAMPs that cause inflammatory and catabolic changes in disc cells by signaling through TLR2 and TLR4 [ 26 ], [ 27 ], [ 28 ]. This is important, because these fragments can be generated during DD and hence trigger a vicious inflammatory-catabolic loop within the disc [ 29 ], [ 30 ], [ 31 ]. Growing evidence also attest a role of intradiscal bacteria, mainly Cutibacterium acnes ( C. acnes ) in DD and MC [ 32 ], [ 33 ], [ 34 ], [ 35 ], [ 36 ], [ 37 ]. Disc cells sense C. acnes through TLR2 [ 38 ], [ 39 ], leading to inflammatory and catabolic changes in the disc [ 36 ], [ 40 ], which extend to the endplate and eventually cause marrow changes visible as MC [ 36 ]. Co-regulation of the expression of the TLR/MyD88/NFκB pathway in the disc and the bone marrow at levels with MC further support a role of TLRs in MC [ 23 ]. Taken together, these studies demonstrate a strong involvement of TLRs, and in particular of TLR2, in DD and MC, and hence targeting TLR2 is a discussed treatment for DD [ 41 ], [ 42 ]. In the CEP, understanding the role of TLRs, in particular of TLR2, is important because the higher cell density could cause strong local pro-inflammatory and catabolic changes. This could have detrimental consequences, because the function and stability of the thin CEP layer could quickly be impaired leading to DD, MC and ultimately LBP. Furthermore, sensing DAMPs and PAMPs from the disc could propel the inflammation from the disc to the bone marrow and assist in triggering MC. However, the expression and regulation of TLRs in CEPC is unknown to date. Therefore, the aims of this study were first to prove the expression of TLRs in CEPC, and second to address whether TLR2 signaling causes pro-inflammatory and pro-catabolic changes in CEPC. Methods CEP collection CEPs were collected from spinal fusion surgery patients that signed informed consent for further use of surgically removed biological material. The study was approved by the local Ethics Commission #2018 − 01486. Inclusion criteria for the selection of the patients were the absence of current or chronic systemic inflammatory or infectious diseases, cancer, as well as no prior lumbar fusion. The CEPs were collected in sterile tubes and processed within 1.5 hours after removal. RNA extraction from tissue A frozen CEP four-millimeter biopsy punch was cryo-milled in liquid nitrogen to extract RNA directly from CEP tissue. The resulting homogenized tissue was suspended in 350 µl of RLT buffer containing 1% β-Mercaptoethanol (Gibco, Reinach, Switzerland). Cell debris was removed by centrifuging followed by RNA isolation using RNeasy Mini Kit (QIAGEN, Hilden, Germany) according to manufacturer’s instructions, including the optional DNAse digestion step. CEPC isolation and culture CEPC were isolated according to harmonized protocols for disc tissue [ 43 ]. Briefly, CEPs were enzymatically digested overnight with 0.05% collagenase P (Roche, Basel, Switzerland) in Dulbecco’s modified eagle’s medium (DMEM) (Gibco, Reinach, Switzerland) supplemented with 10 % ftal calf serum (FCS), 50 U/ml penicillin streptomycin, 10 mM HEPES, 2 mM L-Glutamine and expanded to passage 1–2. Flow cytometry analysis confirmed that the cell population consisted of CEPCs and was free from monocytic cell contamination, showing CD34 − , CD45 − , and CD90 + expression (Supplementary Fig. 1).[ 44 ]. Culture of THP1 and HEK 293T cells Human embryonic kidney (HEK) 293T lenti-x cells (Tahara Cellosaurus) served as a negative control for TLR expression [ 45 ] and were cultured in DMEM with 10 % FS, 50 U/ml penicillin streptomycin, 2mM L-Glutamine and 6 g/L glucose prior to use. THP1 blue NfkB reporter cells (human leukemia monocytic cell line with NF-κB SEAP Reporter) (Invivogen) served as a positive control for TLR expression and were cultured according to the distributors instructions in RPMI 1640 supplemented with 2 mM L-glutamine, 10 mM HEPES, 10 % hat-inactivated FCS, Pen-Strep 100 U/ml, 1 mM sodium pyruvate, 4.5 g/L glucose (Gibco), 100 µg/ml Normocin (Invivogen), 200 µg/ml Zeocin (Invivogen) and 200 µg/ml G418 (Invivogen). Stimulation of cultured CEPC Prior to stimulation, all cells were treated with the endotoxin scavenger polymyxin B (10 ng/ml) (Invivogen, Toulouse, France) for two hours. To simulate the inflammatory milieu provided by the degenerating disc, CEPC were treated for 24 hours or 48 hours with 10 ng/ml tumor necrosis factor α (TNF-α) or 10 ng/ml interleukin 1 beta (IL-1β). To selectively activate the TLR2/1, TLR2/6, and TLR4, which are the most important receptors for extracellular-matrix derived DAMPs or bacterial cell-wall derived PAMPs, selective ligands were administered to the cell cultures for 24 and 48 hours. For TLR2/6 and TLR2/1 specific activation the synthetic ligands Pam2CysSerLys4 (Pam2csk4) and Pam3CysSerLys4 (Pam3csk4) (both Invivogen) were added, at 10 ng/ml and 1000 ng/ml, respectively. TLR4 specific activation was targeted with ultrapure E. coli lipopolysaccharide (LPS) (Invivogen) at 10 µg/ml and 50 µg/ml. In addition, the 30 kDa N-terminal fibronectin fragment (FNf30 kDa) (Sigma-Aldrich, Buchs, Switzerland) (2.5 µg/ml and 5 µg/ml) was used to represent a potential activation through extracellular matrix-derived DAMPs. To test for the specificity of Pam2csk4 signaling through TLR2, CEPC were pre-treated for 2 hours with 200 µM, 100 µM and 50 µM of the TLR2 inhibitor TL2-C29 (Invivogen) before 10 ng/ml Pam2csk4 was added. TL2-C29 blocks both TLR2/1 and TLR2/6 heterodimer signaling. RNA isolation from cells Cell lysis was conducted in RLT buffer containing 1% β-Mercaptoethanol (Gibco) and RNA isolation was carried out using the RNeasy Mini Kit (QIAGEN) following manufacturer's instructions, including the optional DNAse digestion step. Gene expression analysis Reverse transcription of 100 ng RNA was performed using the SensiFAST cDNA Synthesis Kit (Meridian Bioscience, United States). Relative mRNA levels were quantified with use of the SensiFAST SYBR No-Rox kit (Labgene, Châtel-Saint-Denis, Switzerland) on a magnetic induction real-time qPCR cycler (Labgene). Cycle conditions after initial denaturation at 95°C for 300 s were: 40 cycles of 5 seconds at 95°C, 20 seconds at 60°C, 10 seconds at 72°C, followed by melting curve analysis. Analysis was done with the ΔΔCq method and with normalization to the reference gene Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH). All used primer sequences are listed in Table 1 . Basal TLR expression was measured in unstimulated CEPC of 23 discs. In stimulation experiments, a total of 11 discs were used. Expression of TLR1, TLR2, TLR4, TLR6, of the pro-inflammatory genes IL6, IL8, CCL2 and of the matrix metalloproteases MMP1, MMP3, MMP9, MMP13 was measured in stimulation experiments. For TLR2 inhibition experiments, a total of 6 discs were used and IL6, TLR2, MMP1, MMP3, and MMP13 gene expression was measured. Table 1 Primers used for qPCR. Primers Forward Reverse CCL2 5'-CAG CCA GAT GCA ATC AAT GCC-3' 5'-TGG AAT CCT GAA CCC ACT TCT-3' GAPDH 5'-ATTCCACCCATGGCAAATTC-3' 5'-GGGATTTCCATTGATGACAAGC-3' IL6 5'-AGA CAG CCA CTC ACC TCT TCA G-3' 5'-TTC TGC CAG TGC CTC TTT GCT G-3' IL8 5'-GAG AGT GAT TGA GAG TGG ACC AC-3' 5'-CAC AAC CCT CTG CAC CCA GTT T-3' MMP1 5'-ATG AAG CAG CCC AGA TGT GGA G-3' 5'-TGG TCC ACA TCT GCT CTT GGC A-3' MMP3 5'-CAC TCA CAG ACC TGA CTC GGT T-3' 5'-AAG CAG GAT CAC AGT TGG CTG G-3' MMP9 5'- GCCACTACTGTGCCTTTGAGTC-3' 5'-CCCTCAGAGAATCGCCAGTACT-3' MMP13 5'-CCT TGA TGC CAT TAC CAG TCT CC-3' 5'-AAA CAG CTC CGC ATC AAC CTG C-3' TLR1 5'-CAGTGTCTGGTACACGCATGGT-3' 5'-TTTCAAAAACCGTGTCTGTTAAGAGA-3' TLR2 5'-GGCCAGCAAATTACCTGTGTG-3' 5'-AGGCGGACATCCTGAACCT-3' TLR3 5'-CCTGGTTTGTTAATTGGATTAACGA-3' 5'-TGAGGTGGAGTGTTGCAAAGG-3' TLR4 5'-CAGAGTTTCCTGCAATGGATCA-3' 5'-GCTTATCTGAAGGTGTTGCACAT-3' TLR5 5'-TGCCTTGAAGCCTTCAGTTATG-3' 5'-CCAACCACCACCATGATGAG-3' TLR6 5'-GAAGAAGAACAACCCTTTAGGATAGC-3' 5'-AGGCAAACAAAATGGAAGCTT-3' TLR7 5'-TTTACCTGGATGGAAACCAGCTA-3' 5'-TCAAGGCTGAGAAGCTGTAAGCTA-3' TLR8 5'-TTATGTGTTCCAGGAACTCAGAGAA-3' 5'-TAATACCCAAGTTGATAGTCGATAAGTTTG-3' TLR9 5'-GGACCTCTGGTACTGCTTCCA-3' 5'-AAGCTCGTTGTACACCCAGTCT-3' TLR10 5'-CTGATGACCAACTGCTCCAA-3' 5'-AGTCTGCGGGAACCTTTCTT-3' Flow cytometry Flow cytometry was utilized to assess the surface expression levels of TLR-2 (Biolegend) in unstimulated CEPC, 72 hours Pam2csk4-stimulated CEPC, and 72 hours Pam3csk4-stimulated CEPC. Cultured HEK293T cells were used as a negative and THP1 NfkB reporter cells as a positive control. The cells were blocked according to manufacturer’s instructions with 5 µl of True-Stain Monocyte Blocker™ for 10 minutes and stained for 45 minutes with either 10 µg/ml monoclonal PE anti-human CD282 (TLR2) antibody (isotype: Mouse IgG2a, κ) (Biologened). After washing, cells were stained for 10 minutes with 4',6-Diamidino-2-Phenylindole, Dilactate (DAPI) to differentiate live and dead cells. Washed cells were analyzed on a BD LSRFortessa (BD Biosciences, New Jersey, USA). Data was analyzed with FlowJo v10.8 software. Cell doublets and dead cells were excluded from the analysis. Statistical Analysis Ex-vivo -delta Cq values measured in CEP tissue were compared to the -delta Cq values measured in HEK and THP1 cells using one-way ANOVA, followed by Dunnett’s multiple comparisons testing. Statistical analysis for the stimulation and inhibition experiments were done by one-way analysis of variance (ANOVA) on the log2 fold change of ΔΔCq values, followed by Dunnett‘s multiple comparisons test. For flow cytometry experiments, differences in median fluorescence intensity were tested with one-way ANOVA followed by Dunnett’s multiple comparisons test. All statistical analyses were performed using GraphPad Prism V10.2.0. The significance level was α = 0.05, if not stated otherwise. Results Patient demographics The CEPs originated from the lumbar levels L3/4, L4/5 and L5/S1 and the corresponding discs had an average Pfirrmann grade of 3.8 (range 2–5). The age of the patients included ranged from 28 to 87 years. Both men and women were included. Different patients were used for each stimulation and inhibition experiments. For each experiment, there was no statistical difference in level, degree of disc degeneration, age, and sex (Supplementary Tables 1, 2 and 3) between test groups. TLR expression in CEP tissue Genes of all TLRS (TLR1-10) were expressed in ex-vivo CEP tissue (n = 3). Expression of all TLRs was significantly higher than in HEK cells, which were used as a negative control for TLR expression. Additionally, expression levels of TLR1, 3, 4, 9, and 10 in CEP tissue were significantly higher compared to the positive control THP1 cells. The other TLRs (TLR2, 5, 6, 7, and 8) had similar expression levels to THP1 cells (Fig. 1 ). Basal TLR expression in cultured CEPC Isolated and expanded CEPC expressed all cell surface TLRs (TLR1, TLR2, TLR3, TLR4, TLR6, TLR10). Expression of the intracellular TLR8 and TLR9 gene expression was not detected in cultured CEPC (Supplementary Fig. 2). Since TLR2 can dimerize with TLR1 and TLR6, correlation of TLR2 with these TLRs was tested (Fig. 2 A/B). TLR2 expression only correlated with TLR6 expression (R = 0.581, p = 0.004). Regulation of TLR1, 2, 4, and 6 expression under inflammatory conditions TLR1 was only increased upon 24 hours and 48 hours of IL-1β stimulation as well as 48 hours 10 ng/ml TNF-α stimulation (Fig. 2 C). TLR2 was significantly upregulated after 24 hours in response to 10 and 100 ng/ml Pam2csk4, 1000 ng/ml Pam3csk4, and 10ng/ml TNF-α (Fig. 2 D). After 48 hours almost all applied stimulations, including Fnf30 kDa induced a significant increase of TLR2 expression (Fig. 2 D). In contrast, TLR4 expression was not changed by any of the applied stimulations (Fig. 2 E). For TLR6 expression, only a 48-hour exposure to 10 ng/ml LPS and 10 ng/ml TNF-α induced a significant increase (Fig. 2 F). Inflammatory gene upregulation upon TLR activation Exposure to TLR ligands led to an inflammatory response, as indicated by the heightened expression of three key inflammatory genes – IL6, IL8, and CCL2. All three measured genes showed a similar upregulation pattern. After 24 hours, all experimental conditions induced a significant increase in response, except for FNf30 kDa and the lower dosage of 10 ng/ml Pam3csk4 (Fig. 3 ). However, after 48 hours, in addition to the other stimulations, the 5 µg /ml FNf30 kDa also resulted in a significant increase in IL6 expression (Fig. 3 ). Protease upregulation through TLR2/6 activation Stimulation with TLR agonists as well as inflammatory cytokines TNF-α and IL-1β upregulated several matrix proteases in CEPC after 24 hours. MMP1 and MMP3 were significantly increased through 10 ng/ml and 1000 ng/ml Pam2csk4, 1000 ng/ml Pam3csk4, 50 ng/ml LPS, 10ng/ml TNF-α, and 10ng/ml IL-1β (Fig. 4 A, B). This pattern persisted after 48 hours, with addition of FNf30 kDa, which after this time also increased MMP1 and MMP3 significantly (Fig. 4 A, B). For MMP9, the response pattern was similar to MMP1 and MMP3 with the difference that FNf30 kDa could not upregulate MMP9 expression (Fig. 4 C). After 24 hours, MMP13 exhibited a less pronounced response than the other MMPs. Only Pam2csk4, IL-1β, and TNF-α upregulated its expression. However, after 48 hours, MMP13 showed a significant increase in response to all applied stimulations, except for the lower dosage of Pam3csk4 and the lower concentration of FNf30 kDa (Fig. 4 D). Among all measured proteases, MMP3 demonstrated the strongest response. A 48-hour exposure to 1000 ng/ml Pam2csk4 increased expression by an average fold change of 856.3 ± 456.2. Stimulation of TLR2/6 heterodimer increases TLR-2 on CEPC cell surface Flow cytometry showed more TLR2 on CEPC compared to HEK cells (used as a negative control) and a lower number compared to the monocyte cell line THP1 (Fig. 5 A). A significant increase in surface TLR2 on CEPC (fold change = 1.4, p = 0.005) was observed after a 72 hour stimulation with 1000 ng/ml Pam2csk4 (Fig. 5 B), but not with Pam3csk4 (Fig. 5 C). TL-C29 inhibits TLR2 signaling The TLR-2 inhibitor TL2-C29 attenuated the Pam2csk4-mediated upregulation of pro-inflammatory and catabolic genes expression. The addition of 50 µM TL2-C29 was only sufficient to create a significant decrease in IL6 and MMP3 expression (Fig. 6 A/D). However, with the addition of 100 µM and 200 µM of the TL2-C29 inhibitor, the upregulation of IL6, TLR2, MMP1, MMP3, and MMP13 induced by Pam2csk4 were also significantly reduced (Fig. 6 A-E). Discussion This is the first systematic study showing that CEPC express all TLR1-10. Interestingly, TLR8 and TLR9 expression is lost when the cells are cultured and expanded. Expression of the TLR2 and TLR7 genes have previously been reported using micro arrays [ 46 ]. Single cell RNA sequencing and bulk RNA sequencing showed that CEPC are transcriptionally different from nucleus pulposus and annulus fibrosus cells of the intervertebral disc, and hence CEPC should be investigated separately [ 46 ], [ 47 ]. Disc cells express TLR1-6, 9, and 10 [ 25 ]. Therefore, expression of TLR7 seem to be specific to CEPC. In contrast to disc cells, expression of TLRs is independent of the degree of disc degeneration, indicating that regulation of TLR expression is different than in the disc. This underscores that CEPC should be investigated separately from disc cells. CEPC expressed all cell surface TLRs, i.e. TLR1, TLR2, TLR4, TLR5, and TLR6. Of those, TLR2 and TLR4 are of particular importance in DD and MC, because they can be activated by extracellular matrix fragments generated during DD and by C. acnes , a bacterium that has been found at increased concentrations in discs at MC levels, and that can trigger DD and MC in animal model [ 26 ], [ 36 ], [ 37 ], [ 40 ], [ 48 ], [ 49 ]. TLR2 signaling requires heterodimerization with TLR1 or TLR6 [ 26 ]. TLR4 homodimerizes for activating downstream signaling. To test the regulation of TLR1, 2, 4, and 6 under inflammatory conditions, CEPC were stimulated with the TLR1/2 and TLR2/6 specific ligands Pam3csk4 and Pam2csk4, with TLR4 specific ultra-pure LPS, and with IL-1β or TNF-α to test TLR-signaling independent regulation [ 50 ]. TLR2 expression was found to be upregulated under all these conditions. TLR4 and TLR6 were not affected at all, and TLR1 was only upregulated by IL-1β. On protein level, stimulation with Pam2csk4 but not with Pam3csk4 enhanced TLR2 expression, indicating a positive feedback loop of TLR2/6 stimulation with TLR2 expression. The correlation of TLR2 expression with TLR6 but not with TLR1 further supports the relevance of TLR2/6 signaling in CEPC. In intervertebral disc cells and chondrocytes from osteoarthritic joints, similar to our data, only TLR2 was upregulated after IL-1β stimulation [ 50 ], [ 51 ]. Together, this suggests that TLR2/6 signaling could be of importance in CEPC. Whether this affects sensitivity or intensity of TLR2/6 signaling in-vivo cannot be concluded from these experiments. Expression of pro-inflammatory cytokines and MMPs is enhanced after TLR2 and TLR4 signaling in the disc and in hyaline cartilage, leading to matrix resorption and impaired function [ 27 ], [ 28 ], [ 50 ], [ 51 ], [ 52 ], [ 53 ], [ 54 ]. Here, stimulation of TLR1/2, TLR2/6, and TLR4 enhanced expression of pro-inflammatory cytokines and of MMPs, suggesting a potential detrimental role of TLR2 and TLR4 signaling in endplate degradation [ 17 ]. Blocking TLR2 signaling abrogated the TLR2/6 enhanced expression of pro-inflammatory cytokines and MMPs. This proves that signaling indeed occurs through TLR2 and that the observed upregulation of cytokines and MMPs is functionally linked to TLR2 stimulation. TLR2 signaling could occur in CEPC through DAMPs or PAMPs that have been shown to be present in degenerating intervertebral discs. For examples, FNf30 kDa generated during DD activates TLR2 [ 27 ], [ 30 ], [ 31 ], and C. acnes engage TLR2 [ 38 ]. Which C. acnes factors activate TLR2 is unclear. In keratinocytes during acne vulgaris, Christie-Atkins-Munch-Petersen 1 factor (CAMP1) binds TLR2 [ 55 ], and di- and tri-acylated lipoproteins engage TLR1/2 and TLR2/6 heterodimers, respectively [ 56 ]. C. acnes also secretes hyaluronidase, which can cleave hyaluronic acid into fragments acting as DAMPs [ 28 ], [ 57 ], and sialidases, which can disrupt TLR-inhibitory mechanisms [ 55 ], [ 58 ]. While it can be expected that CEPC respond to C. acnes , this needs to be proven. A TLR2 response in the CEP may have other consequences than a TLR2 response in the disc because sensory nerve fibers end in the CEP [ 59 ], and hence pro-inflammatory and neurotrophic factors secreted by CEPC after TLR2 stimulation could directly act in a paracrine manner on these fibers and enhance pain sensitization. Importantly, nerve fiber density in CEPs is increased in MC and therefore TLR2 signaling in MC CEPs could affect pain sensitization even stronger [ 60 ], [ 61 ]. Overexpression of MMPs could also degrade the matrix of the anyway thin CEP and increase the risk for damaging with detrimental consequences like MC [ 62 ], avulsion-type herniations [ 63 ], Schmorl’s nodes [ 64 ], and endplate fractures [ 65 ]. Lastly, overexpression of pro-inflammatory cytokines and MMPs by CEPC may also affect the adjacent bone marrow and lead to inflammatory changes even before the CEP is damaged. This study is limited by the fact that the significance of TLR signalling in CEPC for the pathogenesis of DD and MC cannot be derived from the experiments performed Conclusion In conclusion, this study shows that CEPC express TLRs, that TLR2 is overexpressed under pathologically relevant inflammatory conditions, and that TLR signaling causes overexpression of pro-inflammatory cytokines and MMPs. This is important because the CEP has a critical function in maintaining disc health and, thus, preventing this function is associated with DD and MC. Declarations Ethics approval and consent to participate The study was approved by the local Ethics Commission #2018-01486; approved on 24 August 2018. The study was conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study. Consent for publication Not applicable Availability of data and material Data is provided within the manuscript or supplementary information files Competing interests No, I declare that the authors have no competing interests as defined by BMC, or other interests that might be perceived to influence the results and/or discussion reported in this paper. Funding This study was supported by a career research grant from the Foundation for Research in Rheumatology (FOREUM) (SD) and by a grant from the Swiss National Science Foundation (SD, Grant No. 207989). Acknowledgements We would like to thank the Swiss Center for Musculoskeletal Biobanking (SCMB), which worked together with us to provide the samples in the shortest time possible for further processing. Furthermore, we also thank the spine surgery team at the Balgrist University Hospital for providing us with fresh samples. Lastly, we would like to thank Rayana Daudovan for her contribution to the project by doing her high school (Matura) thesis with us. Authors' information (optional) Additional information No competing interests by any of the authors. References Roberts S, Urban JP, Evans H, Eisenstein SM. ‘Transport properties of the human cartilage endplate in relation to its composition and calcification.’, Spine (Phila Pa 1976) , vol. 21, no. 4, pp. 415–20, Mar. 1996. Wong J et al. 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Hulme PA, Boyd SK, Ferguson SJ. ‘Regional variation in vertebral bone morphology and its contribution to vertebral fracture strength.’, Bone , vol. 41, no. 6, pp. 946–57, Dec. 2007, 10.1016/j.bone.2007.08.019 . Additional Declarations No competing interests reported. Supplementary Files SupplementaryFigures.pdf SupplementaryTables.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4424045","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":307795801,"identity":"bc2a3af8-6e86-4535-9418-a07b1d5daaef","order_by":0,"name":"Tamara Mengis","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4ElEQVRIiWNgGAWjYLCDAwwVYJoNryoeVC1nSNXCwNhGhBZ79tOJnytqGKL5pdsvHvw577C9fAPzswd4beHJ3Sx55hhD7sw5ZwoO8247nNjYwGZugN9huRskG9iA5I2chMOM2w4nMDPwsEng1cL/dvPPhn8MufuBWg7+nHPYno2gFoncbZKNbUBbJNIPHOBtOMzYQ1DLjbfbLBv7JHJn3MhhOMxzLD1xBjObGV4t7P25m282fLPJ7Z+R/vjjjxpre/n25md4tUABSA0PNJyYiVAPs/AB8WpHwSgYBaNgRAEAaBBIjQV5B8YAAAAASUVORK5CYII=","orcid":"","institution":"University of Zurich","correspondingAuthor":true,"prefix":"","firstName":"Tamara","middleName":"","lastName":"Mengis","suffix":""},{"id":307795802,"identity":"6b4f37d7-6b26-4564-a345-6cd3495554b9","order_by":1,"name":"Laura Bernhard","email":"","orcid":"","institution":"University of Zurich","correspondingAuthor":false,"prefix":"","firstName":"Laura","middleName":"","lastName":"Bernhard","suffix":""},{"id":307795803,"identity":"03d579df-35b3-47b9-8b6b-b280156015d8","order_by":2,"name":"Nick Herger","email":"","orcid":"","institution":"University of Zurich","correspondingAuthor":false,"prefix":"","firstName":"Nick","middleName":"","lastName":"Herger","suffix":""},{"id":307795804,"identity":"45d82c94-e3d5-4bad-871e-47513bc7757f","order_by":3,"name":"Irina Heggli","email":"","orcid":"","institution":"University of Zurich","correspondingAuthor":false,"prefix":"","firstName":"Irina","middleName":"","lastName":"Heggli","suffix":""},{"id":307795805,"identity":"8311b675-d9eb-47e4-9811-ead1a956ae4e","order_by":4,"name":"Jan Devan","email":"","orcid":"","institution":"University of Zurich","correspondingAuthor":false,"prefix":"","firstName":"Jan","middleName":"","lastName":"Devan","suffix":""},{"id":307795806,"identity":"88560ff8-83d0-4121-902c-bb8d1e01682f","order_by":5,"name":"Roy Marcus","email":"","orcid":"","institution":"Universitätsklinik Balgrist","correspondingAuthor":false,"prefix":"","firstName":"Roy","middleName":"","lastName":"Marcus","suffix":""},{"id":307795807,"identity":"c43500ac-5117-4499-a04f-591896f6d911","order_by":6,"name":"Christoph Laux","email":"","orcid":"","institution":"Universitätsklinik Balgrist","correspondingAuthor":false,"prefix":"","firstName":"Christoph","middleName":"","lastName":"Laux","suffix":""},{"id":307795808,"identity":"ccf3f8f2-11dd-402f-8d25-65bc8da66331","order_by":7,"name":"Florian Brunner","email":"","orcid":"","institution":"Universitätsklinik Balgrist","correspondingAuthor":false,"prefix":"","firstName":"Florian","middleName":"","lastName":"Brunner","suffix":""},{"id":307795809,"identity":"e63a2f4a-0b14-48f4-9962-cdab47dba4b9","order_by":8,"name":"Mazda Farshad","email":"","orcid":"","institution":"Universitätsklinik Balgrist","correspondingAuthor":false,"prefix":"","firstName":"Mazda","middleName":"","lastName":"Farshad","suffix":""},{"id":307795810,"identity":"7f882d17-45e8-440e-a0a4-94f45f62d9ef","order_by":9,"name":"Oliver Distler","email":"","orcid":"","institution":"University Hospital of Zurich","correspondingAuthor":false,"prefix":"","firstName":"Oliver","middleName":"","lastName":"Distler","suffix":""},{"id":307795811,"identity":"4f8b01af-8fab-4bb1-8889-9589204e5300","order_by":10,"name":"Stefan Dudli","email":"","orcid":"","institution":"University of Zurich","correspondingAuthor":false,"prefix":"","firstName":"Stefan","middleName":"","lastName":"Dudli","suffix":""}],"badges":[],"createdAt":"2024-05-15 09:16:05","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4424045/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4424045/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":57368066,"identity":"f9a78e9c-8d81-4d6f-92aa-04c7d5c3dcb2","added_by":"auto","created_at":"2024-05-29 17:39:51","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":322045,"visible":true,"origin":"","legend":"\u003cp\u003eEx-vivo analysis of TLR gene expression in CEP tissue. TLR1-10 were measured with qPCR in RNA isolated from CEP tissue, HEK cells and THP1 cells. The graphs present the negative delta Cq values normalized to GAPDH. Significant differences in expression levels between CEP tissue, THP1 cells and HEK cells were tested using ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4424045/v1/5104c3d935641cc8e8ee8da7.jpg"},{"id":57368068,"identity":"3698fdd3-0963-44f5-b20f-4ba318fe25ed","added_by":"auto","created_at":"2024-05-29 17:39:51","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":871989,"visible":true,"origin":"","legend":"\u003cp\u003eIn vitro CEPC TLR expression and regulation. (A, B) The association between TLR2 and (A) TLR1 as well as (B) TLR6, both of which it has the potential to form dimers with. (C, D, E, F) Gene expression of TLRs in CEPC following 24-hour and 48-hour stimulation with varying concentrations of Pam2csk4, Pam3csk4 (10 ng/ml and 1000 ng/ml), LPS (10 ng/ml and 50 ng/ml), FN fragment 30 kDA (FNf 30kDa) (2.5 µg /ml and 5 µg /ml), TNF-α (10 ng/ml), and IL-1β (10 ng/ml) is depicted. The panels illustrate the expression levels of (C) TLR1, (D) TLR2, (E) TLR4, and (F) TLR6. Significance was tested on log2 fold change of delta delta Cq values by repeated measures one-way ANOVA, followed by multiple comparisons which compared each condition to the unstimulated condition at the respective timepoint. Dunnet statistical hypothesis testing was applied to correct for multiple comparisons. Asterisk signify significance: * p \u0026lt; 0.05, ** p \u0026lt; 0.001, *** p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4424045/v1/8aa424b0e7efc23b7dfe3c74.jpg"},{"id":57368071,"identity":"3d0d2a1e-1232-4ae9-988e-5e44bc2433fd","added_by":"auto","created_at":"2024-05-29 17:39:51","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":670023,"visible":true,"origin":"","legend":"\u003cp\u003eInflammatory gene expression of \u003cstrong\u003e(A)\u003c/strong\u003e IL6, \u003cstrong\u003e(B) \u003c/strong\u003eIL8 and \u003cstrong\u003e(C)\u003c/strong\u003e CCL2 in CEPC following 24-hour and 48-hour stimulation with varying concentrations of Pam2csk4, Pam3csk4 (10 ng/ml and 1000 ng/ml), LPS (10 ng/ml and 50 ng/ml), FN fragment 30 kDa (FNf30 kDa) (2.5 µg /ml and 5 µg /ml), TNF-α (10 ng/ml), and IL-1β (10 ng/ml) is depicted. Significance was tested on log2 fold change of delta delta Cq values by repeated measures one-way ANOVA, followed by multiple comparisons which compared each condition to the unstimulated condition at the respective timepoint. Dunnet statistical hypothesis testing was applied to correct for multiple comparisons. Asterisk signify significance: * p \u0026lt; 0.05, ** p \u0026lt; 0.001, *** p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4424045/v1/5630c0ab5615a92adb40597c.jpg"},{"id":57368070,"identity":"2d454a1b-3a24-4eca-85a5-2e7345a4a9fd","added_by":"auto","created_at":"2024-05-29 17:39:51","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":844515,"visible":true,"origin":"","legend":"\u003cp\u003eProtease gene expression of (A) MMP1, (B) MMP3, (C) MMP9 and (D) MMP13 in CEPC after either 24 or 48 hours of Pam2csk4, Pam3csk4 (10 ng/ml and 1000 ng/ml), LPS (10 ng/ml and 50 ng/ml), FN fragment 30 kDA (FNf) (2.5 µg/ml and 5 µg/ml), TNF-α (10 ng/ml), and IL-1β (10 ng/ml) stimulation. Significance was tested on log2 fold change of delta delta Cq values by repeated measures one-way ANOVA, followed by multiple comparisons which compared each condition to the unstimulated condition at the respective timepoint. Dunnet statistical hypothesis testing was applied to correct for multiple comparisons. Asterisk signify significance: * p \u0026lt; 0.05, ** p \u0026lt; 0.001, *** p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4424045/v1/5d71b51f60d9b5532c8465b7.jpg"},{"id":57368073,"identity":"007ab97f-e3c0-4ab5-ad65-6c1266ee9be3","added_by":"auto","created_at":"2024-05-29 17:39:51","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":277594,"visible":true,"origin":"","legend":"\u003cp\u003eProtein expression of TLR2. (A)Representative case of TLR2 measured on CEPC (orange), with HEK cells (red) serving as the negative control and THP1 cells (orange) as the positive control.(B/C) Effect of 72 hours of stimulation with (B) Pam2csk4 and (C) Pam3csk4 at concentrations of 10 ng/ml and 1000 ng/ml on TLR2 levels illustrated as percentage normalized to unstimulated CEPC. Significance was tested with one-way ANOVA on median fluorescence corrected for multiple comparisons using Dunnett multiple comparison test.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4424045/v1/01ab725df68660fa25881bd0.jpg"},{"id":57368072,"identity":"a97007d4-356f-43db-a082-eaffdc7c0f18","added_by":"auto","created_at":"2024-05-29 17:39:51","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":397641,"visible":true,"origin":"","legend":"\u003cp\u003eInhibition of TLR2 signaling with three different dosages (50 µM, 100 µM and 200 µM) of TL2-C29 added 2 hours prior to adding Pam2csk4. Genes that showed upregulation through Pam2csk4 stimulation were used to test if this upregulation could be inhibited by blocking TLR2. The inflammatory gene (A) IL6, the responding TLR (B) TLR2, as well as the proteases (C) MMP1, (D) MMP3 and (E) MMP13 were measured. Statistical significance was tested on log2 fold change of delta delta Cq values by repeated measures one-way ANOVA, followed by multiple comparisons which compared each condition to the unstimulated condition at the respective timepoint. Dunnet statistical hypothesis testing was applied to correct for multiple comparisons. Asterisk signify significance: * p \u0026lt; 0.05, ** p \u0026lt; 0.001, *** p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4424045/v1/14c8edac409632f61bc22d68.jpg"},{"id":59381429,"identity":"ddcf7f2a-f5a5-403c-9156-810a291c2179","added_by":"auto","created_at":"2024-07-01 05:41:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4070519,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4424045/v1/e016b416-9e48-4e29-8ded-3116dc95d7ec.pdf"},{"id":57368067,"identity":"78bfc439-37f7-4295-b40d-a4bdd83ca5f0","added_by":"auto","created_at":"2024-05-29 17:39:51","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":372442,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigures.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4424045/v1/fcbcf5ebc0803a6157d854cb.pdf"},{"id":57368069,"identity":"261fbbb0-794a-43d8-9006-50d98a6c5852","added_by":"auto","created_at":"2024-05-29 17:39:51","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":128070,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTables.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4424045/v1/31b670c976fe07085e8b6b0e.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Expression of toll-like receptors in cartilage endplates cells: a role of toll-like receptor 2 in pro-inflammatory and -catabolic gene expression","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe vertebral cartilage endplate (CEP) is a thin hyaline cartilage structure separating the intervertebral disc from the vertebra. Its intactness is critical for the health of the disc [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. CEP degeneration has been associated with chronic low back pain (CLBP) [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], disc degeneration (DD) [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], and Modic changes (MC) [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Yet, the mechanisms linking CEP degeneration to DD and MC remain unclear. In-vivo and animal disc explant models showed that structural damage of the CEP can cause DD and MC [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Biochemical changes of the CEP, like dehydration, calcification, and fibrosis diminish its nutrient transport properties, leading to reduced nutrient availability in the disc, disc cell death, and DD [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. While it has become clear that structural and biochemical changes of the CEP are important in DD and MC, the role of biological changes of the CEP have remained largely unexplored. In the intervertebral disc, the biological degenerative mechanisms are well studied, but in the CEP, which has an approximately 4-fold higher cell density than the intervertebral disc [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], these changes are poorly understood. Expression of matrix metalloproteinases (MMPs) and interleukins in cartilage endplate cells (CEPC) are suggested to play a role in CEP degradation [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn the intervertebral disc, expression of matrix proteases and pro-inflammatory cytokines is increased during degeneration [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], and expression of pro-inflammatory cytokines is even higher with adjacent MC [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Together, this leads to the resorption of the disc matrix, dehydration of the disc, and disc collapse. Disc cells also express various toll-like receptors (TLRs), with TLR2 being the only one that is responsive to inflammatory milieu such as interleukin-1β exposure. Additionally, TLR2 possesses the highest number of ligands, attributed to its capability to form diverse heterodimers [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The presence of TLRs conveys them the capacity to sense and respond to danger signals from damage-associated molecular pattern (DAMPs) and from pathogen-associated molecular patterns (PAMPs) [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. For example, the 30 kDa N-terminal fibronectin fragment (FNf30 kDa), short hyaluronic acid fragments, and decorin are extracellular-matrix derived DAMPs that cause inflammatory and catabolic changes in disc cells by signaling through TLR2 and TLR4 [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. This is important, because these fragments can be generated during DD and hence trigger a vicious inflammatory-catabolic loop within the disc [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Growing evidence also attest a role of intradiscal bacteria, mainly \u003cem\u003eCutibacterium acnes\u003c/em\u003e (\u003cem\u003eC. acnes\u003c/em\u003e) in DD and MC [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Disc cells sense \u003cem\u003eC. acnes\u003c/em\u003e through TLR2 [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], leading to inflammatory and catabolic changes in the disc [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], which extend to the endplate and eventually cause marrow changes visible as MC [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Co-regulation of the expression of the TLR/MyD88/NFκB pathway in the disc and the bone marrow at levels with MC further support a role of TLRs in MC [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Taken together, these studies demonstrate a strong involvement of TLRs, and in particular of TLR2, in DD and MC, and hence targeting TLR2 is a discussed treatment for DD [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn the CEP, understanding the role of TLRs, in particular of TLR2, is important because the higher cell density could cause strong local pro-inflammatory and catabolic changes. This could have detrimental consequences, because the function and stability of the thin CEP layer could quickly be impaired leading to DD, MC and ultimately LBP. Furthermore, sensing DAMPs and PAMPs from the disc could propel the inflammation from the disc to the bone marrow and assist in triggering MC. However, the expression and regulation of TLRs in CEPC is unknown to date. Therefore, the aims of this study were first to prove the expression of TLRs in CEPC, and second to address whether TLR2 signaling causes pro-inflammatory and pro-catabolic changes in CEPC.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCEP collection\u003c/h2\u003e \u003cp\u003eCEPs were collected from spinal fusion surgery patients that signed informed consent for further use of surgically removed biological material. The study was approved by the local Ethics Commission #2018\u0026thinsp;\u0026minus;\u0026thinsp;01486. Inclusion criteria for the selection of the patients were the absence of current or chronic systemic inflammatory or infectious diseases, cancer, as well as no prior lumbar fusion. The CEPs were collected in sterile tubes and processed within 1.5 hours after removal.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eRNA extraction from tissue\u003c/h2\u003e \u003cp\u003eA frozen CEP four-millimeter biopsy punch was cryo-milled in liquid nitrogen to extract RNA directly from CEP tissue. The resulting homogenized tissue was suspended in 350 \u0026micro;l of RLT buffer containing 1% β-Mercaptoethanol (Gibco, Reinach, Switzerland). Cell debris was removed by centrifuging followed by RNA isolation using RNeasy Mini Kit (QIAGEN, Hilden, Germany) according to manufacturer\u0026rsquo;s instructions, including the optional DNAse digestion step.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eCEPC isolation and culture\u003c/h2\u003e \u003cp\u003eCEPC were isolated according to harmonized protocols for disc tissue [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Briefly, CEPs were enzymatically digested overnight with 0.05% collagenase P (Roche, Basel, Switzerland) in Dulbecco\u0026rsquo;s modified eagle\u0026rsquo;s medium (DMEM) (Gibco, Reinach, Switzerland) supplemented with 10 % ftal calf serum (FCS), 50 U/ml penicillin streptomycin, 10 mM HEPES, 2 mM L-Glutamine and expanded to passage 1\u0026ndash;2. Flow cytometry analysis confirmed that the cell population consisted of CEPCs and was free from monocytic cell contamination, showing CD34\u003csup\u003e\u0026minus;\u003c/sup\u003e, CD45\u003csup\u003e\u0026minus;\u003c/sup\u003e, and CD90\u003csup\u003e+\u003c/sup\u003e expression (Supplementary Fig.\u0026nbsp;1).[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eCulture of THP1 and HEK 293T cells\u003c/h2\u003e \u003cp\u003eHuman embryonic kidney (HEK) 293T lenti-x cells (Tahara Cellosaurus) served as a negative control for TLR expression [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e] and were cultured in DMEM with 10 % FS, 50 U/ml penicillin streptomycin, 2mM L-Glutamine and 6 g/L glucose prior to use. THP1 blue NfkB reporter cells (human leukemia monocytic cell line with NF-κB SEAP Reporter) (Invivogen) served as a positive control for TLR expression and were cultured according to the distributors instructions in RPMI 1640 supplemented with 2 mM L-glutamine, 10 mM HEPES, 10 % hat-inactivated FCS, Pen-Strep 100 U/ml, 1 mM sodium pyruvate, 4.5 g/L glucose (Gibco), 100 \u0026micro;g/ml Normocin (Invivogen), 200 \u0026micro;g/ml Zeocin (Invivogen) and 200 \u0026micro;g/ml G418 (Invivogen).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eStimulation of cultured CEPC\u003c/h2\u003e \u003cp\u003ePrior to stimulation, all cells were treated with the endotoxin scavenger polymyxin B (10 ng/ml) (Invivogen, Toulouse, France) for two hours. To simulate the inflammatory milieu provided by the degenerating disc, CEPC were treated for 24 hours or 48 hours with 10 ng/ml tumor necrosis factor α (TNF-α) or 10 ng/ml interleukin 1 beta (IL-1β). To selectively activate the TLR2/1, TLR2/6, and TLR4, which are the most important receptors for extracellular-matrix derived DAMPs or bacterial cell-wall derived PAMPs, selective ligands were administered to the cell cultures for 24 and 48 hours. For TLR2/6 and TLR2/1 specific activation the synthetic ligands Pam2CysSerLys4 (Pam2csk4) and Pam3CysSerLys4 (Pam3csk4) (both Invivogen) were added, at 10 ng/ml and 1000 ng/ml, respectively. TLR4 specific activation was targeted with ultrapure E. coli lipopolysaccharide (LPS) (Invivogen) at 10 \u0026micro;g/ml and 50 \u0026micro;g/ml. In addition, the 30 kDa N-terminal fibronectin fragment (FNf30 kDa) (Sigma-Aldrich, Buchs, Switzerland) (2.5 \u0026micro;g/ml and 5 \u0026micro;g/ml) was used to represent a potential activation through extracellular matrix-derived DAMPs.\u003c/p\u003e \u003cp\u003eTo test for the specificity of Pam2csk4 signaling through TLR2, CEPC were pre-treated for 2 hours with 200 \u0026micro;M, 100 \u0026micro;M and 50 \u0026micro;M of the TLR2 inhibitor TL2-C29 (Invivogen) before 10 ng/ml Pam2csk4 was added. TL2-C29 blocks both TLR2/1 and TLR2/6 heterodimer signaling.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eRNA isolation from cells\u003c/h2\u003e \u003cp\u003eCell lysis was conducted in RLT buffer containing 1% β-Mercaptoethanol (Gibco) and RNA isolation was carried out using the RNeasy Mini Kit (QIAGEN) following manufacturer's instructions, including the optional DNAse digestion step.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eGene expression analysis\u003c/h2\u003e \u003cp\u003eReverse transcription of 100 ng RNA was performed using the SensiFAST cDNA Synthesis Kit (Meridian Bioscience, United States). Relative mRNA levels were quantified with use of the SensiFAST SYBR No-Rox kit (Labgene, Ch\u0026acirc;tel-Saint-Denis, Switzerland) on a magnetic induction real-time qPCR cycler (Labgene). Cycle conditions after initial denaturation at 95\u0026deg;C for 300 s were: 40 cycles of 5 seconds at 95\u0026deg;C, 20 seconds at 60\u0026deg;C, 10 seconds at 72\u0026deg;C, followed by melting curve analysis. Analysis was done with the ΔΔCq method and with normalization to the reference gene Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH). All used primer sequences are listed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eBasal TLR expression was measured in unstimulated CEPC of 23 discs. In stimulation experiments, a total of 11 discs were used. Expression of TLR1, TLR2, TLR4, TLR6, of the pro-inflammatory genes IL6, IL8, CCL2 and of the matrix metalloproteases MMP1, MMP3, MMP9, MMP13 was measured in stimulation experiments. For TLR2 inhibition experiments, a total of 6 discs were used and IL6, TLR2, MMP1, MMP3, and MMP13 gene expression was measured.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePrimers used for qPCR.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePrimers\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eForward\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eReverse\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCCL2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5'-CAG CCA GAT GCA ATC AAT GCC-3'\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5'-TGG AAT CCT GAA CCC ACT TCT-3'\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eGAPDH\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5'-ATTCCACCCATGGCAAATTC-3'\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5'-GGGATTTCCATTGATGACAAGC-3'\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eIL6\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5'-AGA CAG CCA CTC ACC TCT TCA G-3'\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5'-TTC TGC CAG TGC CTC TTT GCT G-3'\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eIL8\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5'-GAG AGT GAT TGA GAG TGG ACC AC-3'\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5'-CAC AAC CCT CTG CAC CCA GTT T-3'\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMMP1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5'-ATG AAG CAG CCC AGA TGT GGA G-3'\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5'-TGG TCC ACA TCT GCT CTT GGC A-3'\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMMP3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5'-CAC TCA CAG ACC TGA CTC GGT T-3'\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5'-AAG CAG GAT CAC AGT TGG CTG G-3'\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMMP9\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5'- GCCACTACTGTGCCTTTGAGTC-3'\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5'-CCCTCAGAGAATCGCCAGTACT-3'\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMMP13\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5'-CCT TGA TGC CAT TAC CAG TCT CC-3'\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5'-AAA CAG CTC CGC ATC AAC CTG C-3'\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTLR1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5'-CAGTGTCTGGTACACGCATGGT-3'\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5'-TTTCAAAAACCGTGTCTGTTAAGAGA-3'\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTLR2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5'-GGCCAGCAAATTACCTGTGTG-3'\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5'-AGGCGGACATCCTGAACCT-3'\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTLR3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5'-CCTGGTTTGTTAATTGGATTAACGA-3'\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5'-TGAGGTGGAGTGTTGCAAAGG-3'\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTLR4\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5'-CAGAGTTTCCTGCAATGGATCA-3'\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5'-GCTTATCTGAAGGTGTTGCACAT-3'\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTLR5\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5'-TGCCTTGAAGCCTTCAGTTATG-3'\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5'-CCAACCACCACCATGATGAG-3'\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTLR6\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5'-GAAGAAGAACAACCCTTTAGGATAGC-3'\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5'-AGGCAAACAAAATGGAAGCTT-3'\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTLR7\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5'-TTTACCTGGATGGAAACCAGCTA-3'\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5'-TCAAGGCTGAGAAGCTGTAAGCTA-3'\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTLR8\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5'-TTATGTGTTCCAGGAACTCAGAGAA-3'\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5'-TAATACCCAAGTTGATAGTCGATAAGTTTG-3'\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTLR9\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5'-GGACCTCTGGTACTGCTTCCA-3'\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5'-AAGCTCGTTGTACACCCAGTCT-3'\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTLR10\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5'-CTGATGACCAACTGCTCCAA-3'\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5'-AGTCTGCGGGAACCTTTCTT-3'\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eFlow cytometry\u003c/h2\u003e \u003cp\u003eFlow cytometry was utilized to assess the surface expression levels of TLR-2 (Biolegend) in unstimulated CEPC, 72 hours Pam2csk4-stimulated CEPC, and 72 hours Pam3csk4-stimulated CEPC. Cultured HEK293T cells were used as a negative and THP1 NfkB reporter cells as a positive control. The cells were blocked according to manufacturer\u0026rsquo;s instructions with 5 \u0026micro;l of True-Stain Monocyte Blocker\u0026trade; for 10 minutes and stained for 45 minutes with either 10 \u0026micro;g/ml monoclonal PE anti-human CD282 (TLR2) antibody (isotype: Mouse IgG2a, κ) (Biologened). After washing, cells were stained for 10 minutes with 4',6-Diamidino-2-Phenylindole, Dilactate (DAPI) to differentiate live and dead cells. Washed cells were analyzed on a BD LSRFortessa (BD Biosciences, New Jersey, USA). Data was analyzed with FlowJo v10.8 software. Cell doublets and dead cells were excluded from the analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eEx-vivo -delta Cq values measured in CEP tissue were compared to the -delta Cq values measured in HEK and THP1 cells using one-way ANOVA, followed by Dunnett\u0026rsquo;s multiple comparisons testing. Statistical analysis for the stimulation and inhibition experiments were done by one-way analysis of variance (ANOVA) on the log2 fold change of ΔΔCq values, followed by Dunnett\u0026lsquo;s multiple comparisons test. For flow cytometry experiments, differences in median fluorescence intensity were tested with one-way ANOVA followed by Dunnett\u0026rsquo;s multiple comparisons test. All statistical analyses were performed using GraphPad Prism V10.2.0. The significance level was α\u0026thinsp;=\u0026thinsp;0.05, if not stated otherwise.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003ePatient demographics\u003c/h2\u003e \u003cp\u003eThe CEPs originated from the lumbar levels L3/4, L4/5 and L5/S1 and the corresponding discs had an average Pfirrmann grade of 3.8 (range 2\u0026ndash;5). The age of the patients included ranged from 28 to 87 years. Both men and women were included. Different patients were used for each stimulation and inhibition experiments. For each experiment, there was no statistical difference in level, degree of disc degeneration, age, and sex (Supplementary Tables\u0026nbsp;1, 2 and 3) between test groups.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eTLR expression in CEP tissue\u003c/h2\u003e \u003cp\u003eGenes of all TLRS (TLR1-10) were expressed in ex-vivo CEP tissue (n\u0026thinsp;=\u0026thinsp;3). Expression of all TLRs was significantly higher than in HEK cells, which were used as a negative control for TLR expression. Additionally, expression levels of TLR1, 3, 4, 9, and 10 in CEP tissue were significantly higher compared to the positive control THP1 cells. The other TLRs (TLR2, 5, 6, 7, and 8) had similar expression levels to THP1 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eBasal TLR expression in cultured CEPC\u003c/h2\u003e \u003cp\u003eIsolated and expanded CEPC expressed all cell surface TLRs (TLR1, TLR2, TLR3, TLR4, TLR6, TLR10). Expression of the intracellular TLR8 and TLR9 gene expression was not detected in cultured CEPC (Supplementary Fig.\u0026nbsp;2). Since TLR2 can dimerize with TLR1 and TLR6, correlation of TLR2 with these TLRs was tested (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA/B). TLR2 expression only correlated with TLR6 expression (R\u0026thinsp;=\u0026thinsp;0.581, p\u0026thinsp;=\u0026thinsp;0.004).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eRegulation of TLR1, 2, 4, and 6 expression under inflammatory conditions\u003c/h2\u003e \u003cp\u003eTLR1 was only increased upon 24 hours and 48 hours of IL-1β stimulation as well as 48 hours 10 ng/ml TNF-α stimulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). TLR2 was significantly upregulated after 24 hours in response to 10 and 100 ng/ml Pam2csk4, 1000 ng/ml Pam3csk4, and 10ng/ml TNF-α (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). After 48 hours almost all applied stimulations, including Fnf30 kDa induced a significant increase of TLR2 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). In contrast, TLR4 expression was not changed by any of the applied stimulations (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). For TLR6 expression, only a 48-hour exposure to 10 ng/ml LPS and 10 ng/ml TNF-α induced a significant increase (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eInflammatory gene upregulation upon TLR activation\u003c/h2\u003e \u003cp\u003eExposure to TLR ligands led to an inflammatory response, as indicated by the heightened expression of three key inflammatory genes \u0026ndash; IL6, IL8, and CCL2. All three measured genes showed a similar upregulation pattern. After 24 hours, all experimental conditions induced a significant increase in response, except for FNf30 kDa and the lower dosage of 10 ng/ml Pam3csk4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). However, after 48 hours, in addition to the other stimulations, the 5 \u0026micro;g /ml FNf30 kDa also resulted in a significant increase in IL6 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eProtease upregulation through TLR2/6 activation\u003c/h2\u003e \u003cp\u003eStimulation with TLR agonists as well as inflammatory cytokines TNF-α and IL-1β upregulated several matrix proteases in CEPC after 24 hours. MMP1 and MMP3 were significantly increased through 10 ng/ml and 1000 ng/ml Pam2csk4, 1000 ng/ml Pam3csk4, 50 ng/ml LPS, 10ng/ml TNF-α, and 10ng/ml IL-1β (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, B). This pattern persisted after 48 hours, with addition of FNf30 kDa, which after this time also increased MMP1 and MMP3 significantly (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, B). For MMP9, the response pattern was similar to MMP1 and MMP3 with the difference that FNf30 kDa could not upregulate MMP9 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). After 24 hours, MMP13 exhibited a less pronounced response than the other MMPs. Only Pam2csk4, IL-1β, and TNF-α upregulated its expression. However, after 48 hours, MMP13 showed a significant increase in response to all applied stimulations, except for the lower dosage of Pam3csk4 and the lower concentration of FNf30 kDa (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Among all measured proteases, MMP3 demonstrated the strongest response. A 48-hour exposure to 1000 ng/ml Pam2csk4 increased expression by an average fold change of 856.3\u0026thinsp;\u0026plusmn;\u0026thinsp;456.2.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eStimulation of TLR2/6 heterodimer increases TLR-2 on CEPC cell surface\u003c/h2\u003e \u003cp\u003eFlow cytometry showed more TLR2 on CEPC compared to HEK cells (used as a negative control) and a lower number compared to the monocyte cell line THP1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). A significant increase in surface TLR2 on CEPC (fold change\u0026thinsp;=\u0026thinsp;1.4, p\u0026thinsp;=\u0026thinsp;0.005) was observed after a 72 hour stimulation with 1000 ng/ml Pam2csk4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB), but not with Pam3csk4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eTL-C29 inhibits TLR2 signaling\u003c/h2\u003e \u003cp\u003eThe TLR-2 inhibitor TL2-C29 attenuated the Pam2csk4-mediated upregulation of pro-inflammatory and catabolic genes expression. The addition of 50 \u0026micro;M TL2-C29 was only sufficient to create a significant decrease in IL6 and MMP3 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA/D). However, with the addition of 100 \u0026micro;M and 200 \u0026micro;M of the TL2-C29 inhibitor, the upregulation of IL6, TLR2, MMP1, MMP3, and MMP13 induced by Pam2csk4 were also significantly reduced (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-E).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis is the first systematic study showing that CEPC express all TLR1-10. Interestingly, TLR8 and TLR9 expression is lost when the cells are cultured and expanded. Expression of the TLR2 and TLR7 genes have previously been reported using micro arrays [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Single cell RNA sequencing and bulk RNA sequencing showed that CEPC are transcriptionally different from nucleus pulposus and annulus fibrosus cells of the intervertebral disc, and hence CEPC should be investigated separately [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e], [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Disc cells express TLR1-6, 9, and 10 [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Therefore, expression of TLR7 seem to be specific to CEPC. In contrast to disc cells, expression of TLRs is independent of the degree of disc degeneration, indicating that regulation of TLR expression is different than in the disc. This underscores that CEPC should be investigated separately from disc cells.\u003c/p\u003e \u003cp\u003eCEPC expressed all cell surface TLRs, i.e. TLR1, TLR2, TLR4, TLR5, and TLR6. Of those, TLR2 and TLR4 are of particular importance in DD and MC, because they can be activated by extracellular matrix fragments generated during DD and by \u003cem\u003eC. acnes\u003c/em\u003e, a bacterium that has been found at increased concentrations in discs at MC levels, and that can trigger DD and MC in animal model [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e], [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. TLR2 signaling requires heterodimerization with TLR1 or TLR6 [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. TLR4 homodimerizes for activating downstream signaling. To test the regulation of TLR1, 2, 4, and 6 under inflammatory conditions, CEPC were stimulated with the TLR1/2 and TLR2/6 specific ligands Pam3csk4 and Pam2csk4, with TLR4 specific ultra-pure LPS, and with IL-1β or TNF-α to test TLR-signaling independent regulation [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. TLR2 expression was found to be upregulated under all these conditions. TLR4 and TLR6 were not affected at all, and TLR1 was only upregulated by IL-1β. On protein level, stimulation with Pam2csk4 but not with Pam3csk4 enhanced TLR2 expression, indicating a positive feedback loop of TLR2/6 stimulation with TLR2 expression. The correlation of TLR2 expression with TLR6 but not with TLR1 further supports the relevance of TLR2/6 signaling in CEPC. In intervertebral disc cells and chondrocytes from osteoarthritic joints, similar to our data, only TLR2 was upregulated after IL-1β stimulation [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e], [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Together, this suggests that TLR2/6 signaling could be of importance in CEPC. Whether this affects sensitivity or intensity of TLR2/6 signaling in-vivo cannot be concluded from these experiments.\u003c/p\u003e \u003cp\u003eExpression of pro-inflammatory cytokines and MMPs is enhanced after TLR2 and TLR4 signaling in the disc and in hyaline cartilage, leading to matrix resorption and impaired function [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e], [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e], [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e], [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e], [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Here, stimulation of TLR1/2, TLR2/6, and TLR4 enhanced expression of pro-inflammatory cytokines and of MMPs, suggesting a potential detrimental role of TLR2 and TLR4 signaling in endplate degradation [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Blocking TLR2 signaling abrogated the TLR2/6 enhanced expression of pro-inflammatory cytokines and MMPs. This proves that signaling indeed occurs through TLR2 and that the observed upregulation of cytokines and MMPs is functionally linked to TLR2 stimulation.\u003c/p\u003e \u003cp\u003eTLR2 signaling could occur in CEPC through DAMPs or PAMPs that have been shown to be present in degenerating intervertebral discs. For examples, FNf30 kDa generated during DD activates TLR2 [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], and \u003cem\u003eC. acnes\u003c/em\u003e engage TLR2 [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Which \u003cem\u003eC. acnes\u003c/em\u003e factors activate TLR2 is unclear. In keratinocytes during acne vulgaris, Christie-Atkins-Munch-Petersen 1 factor (CAMP1) binds TLR2 [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e], and di- and tri-acylated lipoproteins engage TLR1/2 and TLR2/6 heterodimers, respectively [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. \u003cem\u003eC. acnes\u003c/em\u003e also secretes hyaluronidase, which can cleave hyaluronic acid into fragments acting as DAMPs [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e], and sialidases, which can disrupt TLR-inhibitory mechanisms [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e], [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. While it can be expected that CEPC respond to \u003cem\u003eC. acnes\u003c/em\u003e, this needs to be proven.\u003c/p\u003e \u003cp\u003eA TLR2 response in the CEP may have other consequences than a TLR2 response in the disc because sensory nerve fibers end in the CEP [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e], and hence pro-inflammatory and neurotrophic factors secreted by CEPC after TLR2 stimulation could directly act in a paracrine manner on these fibers and enhance pain sensitization. Importantly, nerve fiber density in CEPs is increased in MC and therefore TLR2 signaling in MC CEPs could affect pain sensitization even stronger [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e], [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. Overexpression of MMPs could also degrade the matrix of the anyway thin CEP and increase the risk for damaging with detrimental consequences like MC [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e], avulsion-type herniations [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e], Schmorl\u0026rsquo;s nodes [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e], and endplate fractures [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. Lastly, overexpression of pro-inflammatory cytokines and MMPs by CEPC may also affect the adjacent bone marrow and lead to inflammatory changes even before the CEP is damaged.\u003c/p\u003e \u003cp\u003eThis study is limited by the fact that the significance of TLR signalling in CEPC for the pathogenesis of DD and MC cannot be derived from the experiments performed\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, this study shows that CEPC express TLRs, that TLR2 is overexpressed under pathologically relevant inflammatory conditions, and that TLR signaling causes overexpression of pro-inflammatory cytokines and MMPs. This is important because the CEP has a critical function in maintaining disc health and, thus, preventing this function is associated with DD and MC.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch1\u003eEthics approval and consent to participate\u003c/h1\u003e\n\u003cp\u003eThe study was approved by the local Ethics Commission #2018-01486; approved on 24 August 2018. The study was conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study.\u003c/p\u003e\n\u003ch1\u003eConsent for publication\u003c/h1\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003ch1\u003eAvailability of data and material\u003c/h1\u003e\n\u003cp\u003eData is provided within the manuscript or supplementary information files\u003c/p\u003e\n\u003ch1\u003eCompeting interests\u003c/h1\u003e\n\u003cp\u003eNo, I declare that the authors have no competing interests as defined by BMC, or other interests that might be perceived to influence the results and/or discussion reported in this paper.\u003c/p\u003e\n\u003ch1\u003eFunding\u003c/h1\u003e\n\u003cp\u003eThis study was supported by a career research grant from the Foundation for Research in Rheumatology (FOREUM) (SD) and by a grant from the Swiss National Science Foundation (SD, Grant No. 207989).\u003c/p\u003e\n\u003ch1\u003eAcknowledgements\u003c/h1\u003e\n\u003cp\u003eWe would like to thank the Swiss Center for Musculoskeletal Biobanking (SCMB), which worked together with us to provide the samples in the shortest time possible for further processing. Furthermore, we also thank the spine surgery team at the Balgrist University Hospital for providing us with fresh samples. Lastly, we would like to thank Rayana Daudovan for her contribution to the project by doing her high school (Matura) thesis with us.\u003c/p\u003e\n\u003ch1\u003eAuthors\u0026apos; information (optional)\u003c/h1\u003e\n\u003ch1\u003eAdditional information\u003c/h1\u003e\n\u003cp\u003eNo competing interests by any of the authors.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eRoberts S, Urban JP, Evans H, Eisenstein SM. \u0026lsquo;Transport properties of the human cartilage endplate in relation to its composition and calcification.\u0026rsquo;, \u003cem\u003eSpine (Phila Pa 1976)\u003c/em\u003e, vol. 21, no. 4, pp. 415\u0026ndash;20, Mar. 1996.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWong J et al. 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Although it has been established that intervertebral disc cells express toll-like receptors (TLRs), which can recognize diverse pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs), triggering an immune response, it is unknown whether CEP cells (CEPC) share this trait. The CEP exhibits a significantly higher cell density compared to the disc, potentially heightening the relevance of CEPC in this context. This study aimed to identify TLRs on CEPC and explore their role in activating pro-inflammatory and catabolic gene expression.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGene expression of TLR1-10 was measured with quantitative real-time polymerase chain reaction in human CEPs and expanded CEPC. Additionally, CEPC were stimulated with tumor necrosis factor alpha and interleukin 1 beta, specific TLR2/6, TLR2/1 and TLR4 agonist (Pam2csk4, Pam3csk4 and lipopolysaccharide) and with the 30 kDa N-terminal fibronectin fragment, a representative DAMP. TLR2 signaling was inhibited with TL2-C29. TLR2 protein expression was measured with flow cytometry.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAn ex-vivo analysis of CEP tissue found all 10 TLRs expressed while cultured CEPC lost TLR7 and TLR8 expression. TLR2 was the only TLR whose expression significantly increased after pro-inflammatory stimulation. Stimulation of the TLR2/6 heterodimer with Pam2csk4 upregulated TLR2 protein expression. After 48 hours of stimulation, all applied ligands upregulated expression of pro-inflammatory genes and the matrix metalloproteases 1 (MMP1), MMP3, and MMP13. TLR2 inhibition was able to specifically inhibit the upregulated genes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe expression of TLR1-10 in CEPC indicates that the CEP is susceptible to PAMP and DAMP stimulation. TLR2 expression in CEPC is enhanced under inflammatory conditions and its stimulation has pro-inflammatory and pro-catabolic consequences. Therefore, TLR2 signaling in CEPC might play an important role in disc degeneration and Modic changes.\u003c/p\u003e","manuscriptTitle":"Expression of toll-like receptors in cartilage endplates cells: a role of toll-like receptor 2 in pro-inflammatory and -catabolic gene expression","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-29 17:39:46","doi":"10.21203/rs.3.rs-4424045/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9bbd5956-2e2c-4d3f-a8cb-ea6a4d3aa94e","owner":[],"postedDate":"May 29th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-07-01T05:33:17+00:00","versionOfRecord":[],"versionCreatedAt":"2024-05-29 17:39:46","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4424045","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4424045","identity":"rs-4424045","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}