Toll-Like Receptor 2 Induced Senescence in Intervertebral Disc Cells of Patients with Back Pain can be Attenuated by o-Vanilin

Backgroud: There are an increased level of senescent cells and Toll Like Receptor-1 -2 -4 and -6 (TLR) expression in degenerating intervertebral discs (IVDs) from back pain patients. However, it is currently not known if the increase in expression of TLRs is related to the senescent cells or if it is a more general increase on all cells. It is also not known if TLR activation in IVD cells will induce cell senescence. Methods: Cells from non-degenerate human IVD were obtained from spine donors and cells from degenerate IVDs came from patients undergoing surgery for low back pain. Gene expression of TLR-1,2,4,6, senescence and senescence-associated secretory phenotype (SASP) markers was evaluated by RT-qPCR in isolated cells. Matrix synthesis was veried with safranin-O staining and Dimethyl-Methylene Blue Assay (DMMB) conrmed proteoglycan content. Protein expression of p16 INK4a , SASP factors and TLR-2 was evaluated by immunocytochemistry (ICC) and/or by enzyme-linked immunosorbent assay (ELISA). Results: An increase in senescent cells was found following 48h induction with a TLR-2/6 agonist in cells from both non-degenerate and degenerating human IVDs. Higher levels of SASP factors, TLR-2 gene expression and protein expression was found following 48h induction with TLR-2/6 agonist. Treatment with o-Vanillin reduced the number of senescent cells, and increased matrix synthesis in IVD cells from back pain patients. Treatment with o-Vanillin after induction with TLR-2/6 agonist reduced gene and protein expression of SASP factors and TLR-2. Co-localized staining of p16 INK4a and TLR-2 demonstrated that senescent cells have a high TLR-2 expression. Conclusions: Taken together our data demonstrate that activation of TLR-2/6 induce senescence and increase TLR-2 and SASP expression in cells from non-degenerate IVDs of organ donors without degeneration and backpain and in cells from degenerating human IVD of patients with disc degeneration and backpain. The senescent cells showed high TLR-2 expression suggesting a link between TLR activation and cell senescence in human IVD cells. The reduction in senescence, SASP and TLR-2 expression suggest o-Vanillin as a potential disease modifying drug for patients with disc degeneration and backpain.


Background
Low back pain is a global health problem that has been associated with intervertebral disc (IVD) degeneration (1)(2)(3). It is experienced by approximately 80% of individuals at some time in their lifespan (4). Globally back pain is the number one cause of years lived with disability (4). The personal costs in reduced quality of life, as well as the economic cost to healthcare systems are enormous and exceeds $100 billion per year in the US alone (5). Current evidence suggests that changes in the biomechanical properties of degenerating discs is associated with matrix fragmentation, in ammation and pain (6). However, it is less clear how pain and degeneration are initiated and how they could be prevented. There is a growing interest in the accumulation of senescent cells in degenerating and ageing tissues. These senescent cells are viable cells that can no longer divide. Senescence can be induced due to the successive shortening of telomere length during replicative cycles (7). In addition, the number of senescent cells can also be increased by stressors including DNA damaging agents, oxidative stress, mitochondrial dysfunction, load induced injury and disruption of epigenetic regulation. This phenomenon is called stress-induced premature senescence and it is believed to be linked to the accumulation of senescent cell in degenerate IVDs (8,9). Furthermore, senescent cells release an array of in ammatory cytokines, chemokines, and proteases known collectively as the senescence-associated secretory phenotype (SASP) (10).
All senescent cells have common features, but they also possess distinct characteristics which are linked to the different types of senescence (replicative & stress-induced senescence), cell and tissue types (11,12). The in ammatory environment triggered by senescent cells prevents adjacent cells from maintaining tissue homeostasis (13,14) and it is proposed to induce senescence in a paracrine manner thus exacerbating tissue deterioration (15). Currently, conventional pharmacotherapy for IVD degeneration has both a high cost and many potential negative side effects, which has stimulated the interest in natural plant-based products with anti-in ammatory and regenerative properties, as an alternative or adjunct to conventional therapy. These products are being investigated for potential e cacy in a wide range of disorders with an in ammatory component, including osteoarthritis and cancer (16,17). Recently, there have been a number of synthetic and natural drugs described with a speci c mode of action to target and remove senescent cells, referred to as senolytics (18,19).
Senolytics target many different pathways such as interfering with the dependence receptors, which promote apoptosis when unoccupied by ligands. Targeting and blocking signaling pathways involved in cell survival regulation interferes with mitochondrial-dependent apoptosis (20). One natural senolytic, o-Vanillin, a metabolite of a Curcumin, has anti-in ammatory properties and potent senolytic activity with a very wide non-toxic window for non-senescent IVD cells (18). Treatment with o-Vanillin has previously been shown to increase proteoglycan production of nucleus pulposus (NP) cells pellet culture (18).

Furthermore, o-Vanillin interacts with a variety of cell surface receptors including Toll-like Receptors
(TLR), Vanilloid, Chemokine and Opioid receptors and could broadly reduce the levels of pro-in ammatory mediators and reduce matrix degradation, possibly preventing IVD degeneration (21,22).
Furthermore, studies using multiple cell lines proposed that TLR activation is associated with the induction of senescent cells and SASP factor release (26)(27)(28).
The present study investigates a possible link between the increase of TLRs and senescent cells in degenerate IVDs from patients undergoing surgery for low back pain. We show that a TLR-2/6 agonist increased the number of senescent cells from non-degenerate IVDs and in cells from degenerate IVDs. As well, we describe that TLR-2 has the highest expression and co-localization with senescent cells from degenerate IVDs from patients undergoing surgery for low back pain. Furthermore, treatment with o-Vanillin reduced the number of cells co-localized for TLR-2 and senescence markers. From this study, we propose that TLR-2 has a role in the increase of senescent cells found in degenerating IVDs and that o-Vanillin's senolytic and anti-in ammatory activity could be a disease modifying pharmaceutical for low back pain.

Methods
Tissue collection and cell isolation All procedures performed were approved by the ethical review board at McGill University (IRB#s A04-M53-08B and A10-M113-13B). Non-degenerate IVDs from humans with no history of back pain were obtained through a collaboration with Transplant Quebec. Degenerate IVDs were obtained from patients with chronic low back pain that received discectomies to alleviate pain. Donor information is presented in Supplementary Table 1. IVD cells were isolated, as previously described (29). Brie y, samples were washed in phosphate-buffered saline solution (PBS, Sigma-Aldrich, Oakville, ON, Canada) and Hank'sbuffered saline solution (HBSS, Sigma-Aldrich, Oakville, ON, Canada) supplemented with Primocin TM (InvivoGen, San Diego, CA, USA) and Fungiozone (Sigma-Aldrich, Oakville, ON, Canada). Then, the matrix was minced and digested in 0.15% collagenase type II (Gibco) for 16 hours at 37°C. Cells were passed through both a 100-μm lter and 70-μm lter, before being re-suspended in Dulbecco's Modi ed Eagle Media (DMEM, Sigma-Aldrich, Oakville, ON, Canada) supplemented with 10% fetal bovine serum (FBS, Gibco), Primocin TM , Glutamax (Oakville, ON, Canada), and maintained in a 5% CO 2 incubator at 37 °C.

In Vitro Cell Culture and Treatment
Monolayer culture: Experiments were performed with NP cells from non-degenerate IVDs and degenerate IVDs (NP and AF cells) within passage 1 to 2. 20,000 cells were seeded in 8-well chamber slides (Nunc™ Lab-Tek™ II Chamber Slide™ System) for immunocytochemistry experiments following treatment. 300,000 cells were seeded in 6-well plates (Sarstedt, TC plate 6-well, Cell+, F) for ELISA and RNA extraction following treatment. All cells were left to adhere for 12 to 24 hours and then serum-starved in DMEM with 1X insulin-transferrin selenium (ITS, Thermo Fisher, Waltham, MA, USA) for 6 hours prior to treatment. To examine the effects of different treatments, healthy cells were treated with either 100 ng/ml Pam2CSK4 (TLR-2/6 agonist, Invivogen), 100 ng/mL Pam3CSK4 (TLR-1/2 agonist, Invivogen) or 5μg/mL lipopolysaccharide (LPS) (TLR-4 agonist, Invivogen) for 6, 12, 24 and 48 hours. Cells were either left untreated (negative control) or treated with 100 ng/mL of Pam2CSK4 for 48 hours of which treatment with 100 μM o-Vanillin (Sigma-Aldrich, Oakville, ON, Canada) was initiated in the last 6 hours of incubation (18,23,30).
Pellet culture: 300,000 cells/tube were collected by centrifugation at 1500 rpm for 5 minutes. Pellets were incubated in 1mL DMEM, 2.25g/L glucose (Sigma-Aldrich, Oakville, ON, Canada), 5% FBS, 5 μM ascorbic acid, 1% GlutaMAX, 0.5% Gentamicin (Thermo Fisher, Waltham, MA, USA) at 37°C and 5% CO 2 . Pellets were left in DMEM for four days to form and stabilize (in pretreatment media) and then treated with 100 μM o-Vanillin (Sigma-Aldrich, Oakville, ON, Canada) for four days, meanwhile pellets in the control group stayed in DMEM with vehicle 0.01% DMSO (Sigma-Aldrich, Oakville, ON, Canada). Following the treatment period, pellets from both groups were cultured for 21 days and their culture media was collected every 4 days and pooled as post-treatment media. Triton X-100) for 10 minutes. Both healthy monolayer cultures and pellet samples were blocked with hydrogen peroxide for 10 minutes, washed three times, and saturated with 1% BSA, 1% goat serum, and 0.1% Triton X-100 for 10 minutes. All samples were incubated at 4°C overnight for p16 INK4a antibody (CINTec Kit, Roche) and PBS-T for negative control. The HRP/DAB Detection IHC Kit (Abcam, ab64264) was used for detection. Counting staining was applied with Meyer's hematoxylin (Sigma-Aldrich, Oakville, ON, Canada) for 2 minutes. Samples were rinsed with water (30s), 75% ethanol (15s), and 95% ethanol (15s) afterwards and coverslips were mounted with Permount TM Mounting Medium (Fisher Scienti c). Images were captured as described (18) for Safranin-O staining, and analyzed with Fiji Image J (version 2.1.0/1.53c).

Real-time Quantitative Polymerase Chain Reaction (RT-qPCR)
RNA was extracted using the TRIzol chloroform extraction method previously described (31). 500 ng of RNA was then reverse transcribed using a qScript cDNA Synthesis Kit ( Table 2. All reactions were conducted in technical triplicate, and fold-changes in gene expression were calculated by using the 2 −ΔΔCt method, after normalizing to actin and non-treated samples (32).

Protein analysis
To determine the concentration of NGF, IVD cells were cultured in monolayer (250,000 cells/sample) and then lysed using 300 μL of Cell Lysis buffer (RayBiotech, Norcoss, GA, USA). Cell lysates were incubated for 48 hours at room temperature and protein concentrations were determined using ELISA kits, according to the manufacturer's instructions (RayBiotech, Norcoss, GA, USA). Cell culture media from degenerate IVD cells cultured in monolayer and in pellets was used to assess the concentrations of IL-6, IL-8, IL-1b and TNF-a. 150 μL of monolayer culture media and pellet pre-treated and pooled post-treated media was used. ELISAs were performed as per the manufacturer's instructions (RayBiotech, Norcoss, GA, USA). Colorimetric absorbance was measured with a Tecan In nite M200 PRO (Tecan, Männedorf, Switzerland) spectrophotometer and analyzed with i-control 1.9 Magellan software (Tecan, Männedorf, Switzerland). Protein levels of the treated conditions and controls were then compared.

Dimethylmethylene Blue Assay
Dimethylmethylene Blue (DMMB) assays were conducted as previously described (18) to quantify sulfated glycosaminoglycans (sGAG) in the conditioned media of IVD pellets with or without o-Vanillin treatment. Chondroitin sulfate was used to generate the standard curve. Pooled post-treatment media samples from treated and untreated pellets were used. All samples were ensured to fall into the linear portion of the standard curve. Each sample was placed in triplicate into clear 96-well plates (Costar, Corning, NY, USA). DMMB dye was then added to the wells. The absorbance was measured immediately at room temperature using Tecan In nite T200 spectrophotometer (Mannedorf, Switzerland).

Statistical analysis:
Data was analyzed using Graph Prism 8 (Graph Pad, La Jolla, CA, USA). Analysis was performed using two-tailed Student's t test or Two-way ANOVA. Speci c tests are indicated in the gure legends with the corrections. A p-value <0.05 was considered statistically signi cant. Data are presented as mean ± SD.
O-Vanillin reduced the number of senescent cells and enhanced proteoglycan production in cell pellet cultures from degenerate IVDs O-Vanillin senolytic activity and effect on matrix production has never been assessed on cells from patients with back pain and degenerating IVDs. Here we evaluated o-Vanillin's senolytic activity in 3D pellet cultures of IVD cells back pain patients. The pellet cultures were treated with o-Vanillin (100mM) or vehicle (DMSO 0.01%) for 4 days. At the end of the treatment period, the pellets were maintained in standard culture media for 21 days with the post treatment media collection occurring every 4 days. The senolytic activity was evaluated by immunostaining for the senescence marker p16 ink4a (Fig. 2A). The percentage of p16 ink4a positive cells decreased signi cantly from 14.66% ± 2.758 in the untreated control to 6.38% ± 0.4973 in the o-Vanillin treated pellets (p < 0.05) (Fig. 2B). Safranin-O staining was used to evaluate proteoglycan content. A more intense red staining was observed, indicating higher proteoglycan content in the o-Vanillin treated IVD cell pellets compared to the control sample (Fig. 2C). Furthermore, a DMMB assay was performed to assesses sGAG content in the culture media (33). Pooled media from all post-treatment time points in the o-Vanillin treated cell pellets (1.62 mg/ml ± 0.4134) had signi cantly higher sGAG content then the untreated control pellets (0.33mg/ml ± 0.2876) (p < 0.05) (Fig. 2D). We then evaluated o-Vanillin's ability to reduce SASP factors (IL-1b, IL-8, IL-6 and TNF-a) that are commonly produced by senescent IVD cells (18). Using ELISA immunoassay, we compared the percent difference of the pooled post-treated media over the pre-treated media. A signi cant decrease was observed in all evaluated SASP factors measured in the media of o-Vanillin treated compared to the untreated controls. The percentage of difference in post compared to pre-treatment and measured in o-Vanillin and control groups were respectively for IL-1b (13.75 % ± 3.473 vs 30.63 % ± 3.279, p < 0.01) , IL-8 (38.38 % ± 12.903 vs 61.5 % ± 18.821, p < 0.05), IL-6 (13.38 % ± 5.867 vs 25.75 % ± 1.652, p < 0.05) and TNF-a (19.38 % ± 0.408 vs 46 % ± 0.750, p < 0.0001) (Fig. 2E).
o-Vanillin reduced the number of cells co-expressing TLR-2 and p16 ink4a in cells exposed to TLR-2/6 agonist Based on our ndings that exposure to TLR-2/6 agonist caused a signi cant increase in p16 ink4a and TLR-2 gene expression in both cell and pellet cultures from non-degenrate and degenerate IVDs, we investigated the possiblilty that senescent IVD cells have an elevated TLR-2 expression. Protein expression of TLR-2 and p16 ink4a was assessed by immunohistochemistry in IVD cells from patients with back pain and IVD degeneration following a 48 hrs exposure to TLR-2/6 agonist. (Fig. 5A). Quanti cation of TLR-2 and p16 ink4a was done by measuring the percent of cells positive from the total cell population for the two markers. Following TLR-2/6 activation it was found that there was a signi cant increase in the expression of TLR-2 (53.17% ± 8.684, p < 0.001) and p16 ink4a (47.19% ± 7.951, p < 0.001) when compared to untreated controls; TLR-2 (29.92% ± 9.448) and p16 ink4a (25.95% ± 6.071) (Fig. 5B-C). Furthermore, treatment with o-Vanillin for the nal 6 hrs signi cantly reduced this increase for TLR-2 (36.3% ± 8.057, p < 0.001) and p16 ink4a (31.07% ± 3.854, p < 0.001) (Fig. 5B-C). Finally, to verify the link between TLR-2 and cell senescence in IVD cells, we assessed the percentage of cells co-expressing p16 ink4a and TLR-2 by determining the percentage of senescent cells (p16 ink4a positive cell) that express TLR-2. In the untreated control, 26% ± 1.611 of the senescent cells expressed TLR-2 while following TLR2/6 exposure the percentage of senescent cells expressing TLR-2 increased signi cantly to 61.05% ± 6.946 (p < 0.001) (Fig. 5D). The most noteworthy nding was that o-Vanillin signi cantly reduced the number of senescent cells expressing TLR-2 to 27.57% ± 2.509 (p < 0.001) when exposed to TLR-2/6 agonist. (Fig. 5D). These ndings indicate a link between TLR-2 expression, cell senescence and SASP factor production that contribute to IVD degeneration and pain. This deleterious role of TLR-2 is blocked by the dual senolytic and anti-in ammatory effects of o-Vanillin.

Discussion
Several studies including our own have demonstrated that senescent cells accumulate in degenerating IVDs, and suggested that an elevated SASP factor release and increased expression of TLRs contribute to IVD degeneration (18,23). Here we have shown a potential link between the accumulation of senescent cells and TLR activation. As well, we show that o-Vanillin, a TLR antagonist and senolytic compound, has regenerative and anti-in ammatory effects on cells from degenerating IVDs (34).
In chondrocytes and IVD cells, TLRs are, in addition to molecules derived from pathogens, activated by exposure to intracellular proteins such as HSP60, HSP70, S100A8/9, HMGB1 released in response to stress and extracellular matrix fragments such as bronectin, aggrecan, biglycan and other by-products of tissue degeneration (35). As well, it has been reported that synthetic TLR-2 and 4 agonists can induce IVD degeneration, increase in ammatory environment and increase in expression of TLRs (23,30). The present study, demonstrates that TLR-2 activation, in addition to inducing an in amatory environment, caused IVD cells from non-degenerate IVDs to become senescent. We used cells of IVDs from organ donors with no signs of degeneration or history of back pain. These IVDs have a low number of senescent cells and low levels of SASP factor release compared symptomatic degenerating IVDs (18). Our results demonstrate that the synthetic TLR2 agonist (Pam2CSK4), caused the greatest increase in senescent cell number, TLR-2 expression and SASP factor release in cells from non-degenerate IVDs after 48 h exposure. Our previous study using TLR-1, 2, and 4 agonists found the cytokines (IL-1b, 6, 8), chemokines, proteases (MMP3, MMP13) and TLR-2 expression were greatest following exposure to the same TLR-2/6 agonist in NP cells of non-degenerate IVDs (23). Other studies have shown that continuous stimulation of TLR-4 promotes cellular senescence in mesenchymal stem cells (36).
Moreover, TLR-2 and 10 have been found to be key mediators of senescence in IMR90 cells, a human diploid broblast cell line (26).
We then veri ed that these ndings were also seen in cells isolated from degenerating IVDs of patients undergoing surgery to reduce low back pain (18,29). TLR-2 activation of cells from symptomatic IVDs induced expression of SASP factors (CCL-2,5,7,8,IL-6,8,GM-CSF,TNF-a,NGF,BNDF,CLCX-1,10), a senescence marker (p16 ink4a ) and of the TLR-2 receptor itself. Moreover, we con rmed that protein expression of SASP factors (NGF, IL-1b, TNF-a and IL-8) was higher in the TLR-2 activated cells. These proteins were chosen since they have been associated to be IVD degeneration and TLR-2 induction and have been reported to be highly expressed in degenerate human and mice IVDs (30,37,38). Taken together our results validate that IVD cells from patients with back pain and IVD degeneration at both gene and protein level respond to TLR-2 activation.
The use of synthetic antagonists aimed towards TLR-2 and TLR-4 has been evaluated in a variety of in ammatory diseases (39). Antagonists such as TAK-242, a TLR-4 antagonist, has been shown to diminish LPS-induced TLR-4 signaling and in ammation in peritoneal macrophages (39). Furthermore, our own previous study demonstrated that TAK-242 reduced pain but did not provide tissue regeneration in a mouse model of back pain (18). Similar to our study, anti-in ammatory properties of o-Vanillin was reported previously in NP cells from patients undergoing surgery for disc herniation or spinal stenosis following induction by high mobility group box-1 (40). Furthermore, the capability of o-Vanillin to reduce SASP factors has been previously depicted in IVD cell pellet cultures (18,41). o-Vanillin has also been shown to reduce cytokines, chemokines and proteases in vitro by in human HEK-TLR2 and THP-1 cells and to reduce a tumor-promoting phenotype of microglia in vivo (34,42). It has also previously been shown that o-Vanillin incorporated to Poly (Lactic-co-Glycolic Acid) scaffolds elicited more proteoglycan production and decreased in ammatory response of annulus brous cells compared to cells in unsupplemented scaffolds (43). As well, o-Vanillin has been shown to signi cantly decrease the production of pro-in ammatory cytokines and signi cantly attenuated UVB irradiation-induced cytotoxicity in human keratinocyte stem cells (44).
Senolytic drugs target selective signaling pathways involved in cell survival and apoptosis (25). These drugs could potentially be used therapeutically to treat disc degeneration, recover loss of disc height in already degenerate discs, or prophylactically to prevent future degeneration either in individuals at risk or following fusion for adjacent disc disease (45,46). Our previous study demonstrated that o-Vanillin, reduced senescenct cells and enhanced matrix production in cell pellet cultures generated from organ donor IVDs without known history of backpain (18). Here we show that o-Vanillin was able to reduce in ammation, remove senescent cells and enhance proteoglycan production in cell pellets from surgically removed symptomatic IVDs of patients with low back pain.
We further demonstrated that by targeting TLRs and senescent cells with o-Vanillin we can decrease in ammatory processes found in IVD cells from patients with back pain and IVD degeneration. Interestingly, our study demonstrates that both gene and protein expression of SASP factors (CCL2,5,7,8, GM-CSF, BDNF, NGF, TNF-a, CLCX1, CLCX8 and CLCX10, IL-1b, IL-8) were signi cantly reduced following TLR activation and o-Vanillin treatment.
The higher expression of TLR-2 in IVD cells from patients with back pain and IVD degeneration leads us to evaluate its expression level in senescent cells and investigate its role in disc cell senescence and associated SASP factors release. We found TLR-2 activation increased the expression of TLR-2 in the senescent cells. Also, treatment with o-Vanillin signi cantly reduced the number of senescent cells expressing TLR-2. One limitation of our study is that the degenerate cell population is a mix of NP and AF cells from patients suffering from chronic lower back pain. This is because the di culty to accurately distinguish and separate NP and AF tissue from surgically removed IVD tissue.This limitation does not allow us to know whether the TLR-2/p-16 co-localization is in both cell types or in AF or NP cells speci cally. To our knowledge this is the rst study to show a potential link between TLR-2 and cellular senescence in IVD cells. Further studies using genetically modi ed TLR-2 knock-out human IVD cell lines are needed to better decipher which mechanisitic pathways are shared between o-Vanillin's senolytic activity and TLR-2's antagonistic effect.

Conclusions
We showed that TLR-2/6 activation increased TLR-2 expression and senescent cells in IVD cells from both organ donors without degeneration and backpain and patients with disc degeneration and backpain. Tissues were collected during discectomies to allievate pain or during organ harvesting. Participants provided written informed consent to participate in the study and to allow their biological samples to be tested. All procedures performed were approved by the ethical review board at RI-MUHC (IRB # Tissue Biobank 2019-4896, Extracellular Matrix 2020-564 and A08-M22-17B).

Consent for publication: Not Applicable
Availbility of data and materials: The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.