Cell activation with a TLR-2/6 agonist, caused an increase in the number of senescent cells and SASP factor release in cells from non-degenrate human IVDs
We have previously reported that TLR expression and the number of senescent cells are positively correlated with level of IVD degeneration (20, 23). Moreover, we have seen that treating cells from degenerating human IVDs with TLR agonists PAM2csk4 (TLR-2/6), PAM3csk4 (TLR- 1/2) and LPS (TLR-4) increased expression of pain mediators and pro-inflammatory cytokines when compared to vehicle control (23). Here we aimed to determine the effect and the relation of TLR activation and cell senescence in human IVD cells from non-degenrate IVDs. Monolayer cultures were treated with TLR agonists activating TLR-2/6, 1/2 and 4 for 6, 12, 24 and 48 hours. p16ink4a was used to identify senescent cells and the number of senescent cells were significantly increased following TLR-2/6 activation with a 11% ± 1.732 increase at 24 hours and a 22.67% ± 4.163 increase at 48 hours (Fig. 1A-B). Furthermore, using RT-qPCR the gene expression levels of TLRs, senescense markers and SASP factors were evaluated in the treated cell pellets. The expression of the TLR-1,2,4,6 in pellets treated with TLR agonists for 48 hours was investigated. A significant increase (7.24-fold ± 3.458) in expression of the TLR-2 in the cells exposed to the TLR-2/6 agonist was observed when normalized to the untreated control. Significance was not reached for TLR-1, 4 and 6 (Fig. 1C). Moreover, a significant increase (2.56-fold ± 0.288) of p16ink4a gene expression only in cells exposed to the TLR-2/6 agonist when compared to control was observed. Of note, no significant difference was found in the expression of p21 gene expression following exposure to any of the TLR agonists (Fig. 1D). When evaluating SASP factors after 48 hours of exposure to the TLR agonists, the most significant increase was observed following TLR2/6 exposure (Fig. 1E). Comparing to the control, an increase in CCL2 (12.70 -fold ± 2.541), CCL5 (55.11- fold ± 2.696), CCL7 (13.78-fold ± 2.440), CCL8 (12.68-fold ± 1.534), IL-6 (1079.12-fold ±43.135), IL-8 (1890.28-fold ± 85.617), TNF-α (5.30-fold ± 0.214), NGF (3.49-fold ± 0.250) and BDNF (3.31-fold ± 0.051) was seen following TLR2/6 activation (Fig. 1E). Altogether, these results validate that activation of TLR-2/6 increases both the number of senescent cells and SASP factors produced in cells from non-degenerate IVDs.
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 (100µM) 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 p16ink4a (Fig. 2A). The percentage of p16ink4a positive cells decreased significantly from 14.66% ± 2.758 in the untreated control to 6.38% ± 0.4973 in the o-Vanillin treated pellets (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, DMMB assay was performed to assesses sGAG content in the culture media (32). Pooled media from all post-treatment time points in the o-Vanillin treated cell pellets (1.62 µg/ml ± 0.4134) had significantly higher sGAG content then the untreated control pellets (0.33µ g/ml ± 0.2876) (Fig. 2D). We then evaluated o-Vanillin’s ability to reduce SASP factors (IL-1β, IL-8, IL-6 and TNF-α) that are commonly produced by senescent IVD cells (20). Using ELISA immunoassay, we compared the percent difference of the pooled post-treated media over the pre-treated media. A significant 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-1β (13.75% ± 3.473 vs 30.63% ± 3.279), IL-8 (38.38% ± 12.903 vs 61.5% ± 18.821), IL-6 (13.38% ± 5.867 vs 25.75% ± 1.652) and TNF-α (19.38% ± 0.408 vs 46% ± 0.750) (Fig. 2E).
o-Vanillin reduced gene expression of p16, TLR-2 and SASP factors following TLR-2 activation in IVD cells from patients with back pain and IVD degeneration
IVD cells from patients with back pain and IVD degeneration were exposed the TLR-2/6 agonist for 48 hours in the presence or absence of o-Vanillin (100µM) during the last 6 hours of the treatment. We first assessed gene expression of senescece markers p16 and p21. Similar to the effect observed in cells from non-degenerate IVDs, there was a significant increase in p16ink4a expression (3.83-fold ± 1.055) and no significant difference in the expression of p21 following TLR-2/6 exposure when compared to the untreated control (Fig. 3A). Interestingly, treatment with o-Vanillin significantly decreased p16ink4a expression (1.07-fold ± 0.308) while no significant change was found for p21 expression (Fig. 3A). When assessing TLR expression in the cells from patients with back pain and IVD degeneration, a significant increase in TLR-2 gene expression (9.17-fold ± 1.594) following TLR-2/6 exopsure, compared to the control was seen. However, there was no significant increase in TLR-1,4 or 6 (Fig. 3B). These samples, when treated with o-Vanillin showed a significant decrease (1.67-fold ± 0.565) in TLR-2 expression (Fig. 3B). Exposure of IVD cells from patients with back pain and IVD degeneration to TLR-2/6 agonist significantly increased the expression of SASP factors CCL2 (42.32-fold ± 11.337), CCL5 (49.03-fold ± 11.487), CCL7 (9.30-fold ± 1.430), CCL8 (28.40-fold ± 4.936), GM-CSF (118.55-fold ± 10.067), BDNF (1.77-fold ± 0.126), NGF (2.75-fold ± 0.586), TNF-α (7.36-fold ± 2.361), CLCX1 (594.16-fold ± 44.718), CLCX8 (594.5-fold ± 98.644) and CLCX10 (745.23-fold ± 107.787) when compared to the untreated control (Fig. 3C). o-Vanillin significantly reduced this increase, CCL2 (5.89-fold ± 2.075), CCL5 (6.21-fold ± 2.156), CCL7 (3.51-fold ± 1.521), CCL8 (2.8-fold ± 2.281), GM-CSF (6.33-fold ± 2.39), BDNF (0.85-fold ± 0.368), NGF (0.62-fold ± 0.135), TNF-α (1.09-fold ± 0.656), CLCX1 (24.67-fold ± 5.132), CLCX8 (75.49-fold ± 18.608) and CLCX10 (10.8-fold ± 3.087) (Fig. 3C).
o-Vanillin reduced the protein expression of SASP factors (IL-1β, NGF, IL-8 and TNF-α) following TLR-2/6 activation of IVD cells from patients with back pain and IVD degeneration
Protein expression of (TNF-α, IL-1β, IL-8 and NGF) was evaluated by immunohistochemistry following a 48 hrs exposure to TLR-2/6 agonist. (Fig. 4A-D). Quantification of the percentage of positive cells for each SASP factor was compared to untreated controls. A significant increase of TNF-α (56.6% ± 2.881 vs 95% ± 1.155), IL-1β (56.4% ± 3.050 vs 94.25% ± 0.957), IL-8 (44.2% ± 1.924 vs 84% ± 2.236) and NGF (63.6% ± 2.408 vs 92.15% ± 1.388) positive cells was observed. When evaluating the effect of o-Vanillin, a significant decrease was observed in protein expression in the treated samples for TNF-α (74.5% ± 3.109), IL-1β (75% ± 0.816), IL-8 (48.4% ± 5.550) and NGF (70.46% ± 2.416) (Fig. 4E). Additionally, to measure the concentrations of SASP factors affected by TLR-2/6 activation and o-Vanillin treatment we performed ELISAs immunoassay of the culture media for TNF-α, IL-1β, IL-8 and on the cell lysate for NGF. A significant increasewas found in the SASP factors in the culture media following TLR-2/6 activation, TNF-α (72.03 pg/ml ± 9.044 vs 284.25 pg/ml ± 30.972), IL-1β (10.97 pg/ml ± 1.09 vs 42.11 pg/ml ± 3.022), IL-8 (92.95 pg/ml ± 8.385 vs 406.25 pg/ml ± 39.891) and in the cell lysate for NGF (186 pg/ml ± 5.957 vs 355.03 pg/ml ± 27.086) when compared to the control and that this induction was significantly decreased for all evaluated SASP factors following treatment with o-Vanillin (TNF-α: 104.5 pg/ml ± 28.831, IL-1β: 21.15 pg/ml ± 2.123, IL-8: 117.8 pg/ml ± 36.944 and NGF: 262.83 pg/ml ± 5.208) (Fig. 4F).
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 findings that exposure to TLR-2/6 agonist caused a significant increase in p16ink4a 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 p16ink4a 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). Quantification of TLR-2 and p16ink4a was done by measuring the percent of cells positive for the two markers. Following TLR-2/6 activation it was found that there was a significant increase in the expression of TLR-2 (53.17% ± 8.684) and p16ink4a (47.19% ± 7.951) when compared to untreated controls; TLR-2 (29.92% ± 9.448) and p16ink4a (25.95% ± 6.071) (Fig. 5B-C). Furthermore, treatment with o-Vanillin for the final 6 hrs significantly reduced this increase for TLR-2 (36.3% ± 8.057) and p16ink4a (31.07% ± 3.854) (Fig. 5B-C). Finally, to verify this link between TLR-2 and cell senescence in IVD cells, we assessed the percentage of cells co-expressing p16ink4a and 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 significantly to 61.05% ± 6.946 (Fig. 5D). The most noteworthy finding was that o-Vanillin significantly reduced the number of senescent cells expressing TLR-2 27.57% ± 2.509 when exposed to TLR-2/6 agonist. (Fig. 5D). These findings 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-inflammatory effects of o-Vanillin.