*2.4. Functional Analysis of TRPA1 and TRPV1 in IVD Cells*

Allyl isothiocyanate (AITC, 3 and 10 μM), which is an agonist for TRPA1 and TRPV1, was used to test the involvement of these channels in (1) inflammation responses and (2) matrix homeostasis within the IVD compartment. The activity of AITC was tested by Calcium flux assay (*n* = 3). AITC was applied in untreated IVD cells (expressing TRPV1) as well as in cells that were treated with pro-inflammatory cytokines (expressing TRPA1 and TRPV1). AITC did not induce calcium flux in the untreated IVD cells. AITC significantly induced calcium flux in both IL-1β and TNF-α-treated cells, suggesting the involvement of TRPA1 (Figure 3). Calcium flux in the TNF-α treated cells was significantly higher than in IL-1β-treated cells, likely due to the overall higher induction of TRPA1 expression by TNF-α. Therefore, it is possible that TRPA1 (not TRPV1) can be functionally involved in the calcium-induced responses of inflamed IVD cells. To verify the function of TRPA1/TRPV1, AITC was used to study gene and protein expression of inflammation and catabolic mediators in untreated as well as cytokine-treated IVD cells.

**Figure 3.** Calcium flux in IVD cells untreated (control) and treated with 100 μM Allyl isothiocyanate (AITC) with and without 10 ng/mL IL-1β or 10 ng/mL TNF-α. Graph shows calcium flux as 340/380 signal ratio (mean ± SD, *n* = 3). Asterisks indicate statistical significance (\* *p* < 0.05, Kruskal–Wallis test and Dunn's multiple comparison test). The arrow indicates AITC application.

Cytokine untreated IVD cells (expressing TRPV1) were used to test the downstream effects of TRPV1 activation (*n* = 3–4). In unstimulated IVD cells, the gene expression of inflammation/pain mediators (IL-6, IL-8, NGF, COX-2) (Figure 4A–D) and most of the ECM remodeling enzymes (ADAMTS4, ADAMTS5, MMP3, TIMP1, TIMP2) was unchanged by AITC (Figure 4E–J). IL-6 and IL-8 concentration in culture media was close to the lower detection limit (~zero) (not shown). MMP1 was significantly induced by 10μM AITC (Figure 4H). The gene expression of COL1A1, COL2A1, and aggrecan in AITC-treated cells was not significantly different from the control (Figure 4K–M). Due to the fact that cytokine-untreated cells did not express TRPA1, the observed changes in MMP1 expression were not TRPA1-dependent. MMP1 upregulation by AITC may be an unrelated non-specific effect (as AITC did not induce calcium flux in untreated IVD cells).

IVD cells treated with pro-inflammatory cytokines (expressing TRPA1 and TRPV1) were used to test the downstream effects of TRPA1 activation (*n* = 3–4). In IL-1β-treated cells, AITC did not influence gene expression of inflammation mediators IL-6 and IL-8 (Figure 5A,D) and pain mediators NGF and COX-2 (Figure 5C,F). Interestingly, the protein release of IL-8 was significantly reduced by 3 μM AITC (Figure 5E). 10 μM AITC significantly reduced the gene expression of ADAMTS5 (Figure 5H), while MMP1 was induced (Figure 5J). Gene expression of other ECM remodeling enzymes (ADAMTS4, MMP3, TIMP1, TIMP2) and ECM genes (Figure 5M–O) in AITC + IL-1β-treated cells was not different from the IL-1β-only controls.

10 μM AITC significantly induced the gene and protein expression of IL-8 (Figure 6D,E) in TNF-α-treated cells (expressing TRPA1, reduced TRPV1). AITC did not influence the expression of IL-6 (Figure 6A,B), NGF, and COX-2 (Figure 6C,F). The gene expression of ADAMTS5 was significantly reduced (Figure 6H), while MMP1 was induced by 10 μM AITC in TNF-α-treated cells (Figure 6J). Gene expression of other ECM remodeling enzymes (MMP3, ADAMTS4, TIMP1, and TIMP2) was not different from TNF-α-treated control. Gene expression of COL1A1 was significantly reduced (Figure 6M), while the other tested ECM proteins (COL2A1 and aggrecan) were not influenced by AITC in TNF-α-treated cells. AITC did not affect the gene expression of TRPA1 itself (Supplementary Figure S3).

**Figure 4.** The effects of TRPA1 agonist allyl isothiocyanate (AITC) on the gene expression of inflammation markers and extracellular matrix (ECM) molecules in IVD cells without cytokine pre-treatment. Gene expression of (**A**) interleukin-6 (IL-6), (**B**) nerve growth factor (NGF), (**C**) interleukin 8 (IL-8), (**D**) cyclooxygenase-2 (COX-2), (**E**) ADAMTS4, (**F**) ADAMTS5, (**G**) tissue inhibitor of matrix metalloproteinase 1 (TIMP1), (**H**) matrix metalloproteinase 1 (MMP1), (**I**) matrix metalloproteinase 3 (MMP3), (**J**) tissue inhibitor of matrix metalloproteinase 2 (TIMP2), (**K**) COL1A1, (**L**) COL2A1, and (**M**) Aggrecan in IVD cells treated with 3 and 10 μM AITC. Graphs show gene expression and protein release calculated relative control (2−dd*C*<sup>t</sup> , mean ± SD, *n* = 3). Asterisks indicate statistical significance (\* *p* < 0.05, Kruskal–Wallis test and Dunn's multiple comparison test).

**Figure 5.** The effects of TRPA1 agonist allyl isothiocyanate (AITC) on the expression of inflammation markers and ECM molecules in IL-1β-treated cells. Gene expression of (**A**) IL-6 and (**D**) IL-8 in IVD cells treated with 10 ng/mL IL-1β ± 3 and 10 μM AITC. Protein release of (**B**) IL-6 and (**E**) IL-8 in IVD cells that were treated with 10 ng/mL IL-1β ± 3 and 10 μM AITC. Gene expression of (**C**) NGF, (**F**) COX-2, (**G**) ADAMTS4, (**H**) ADAMTS5, (**J**) MMP1, (**K**) MMP3, (**I**) TIMP1 and (**L**) TIMP2, (**M**) COL1A1, (**N**) COL2A1, and (**O**) Aggrecan in IVD cells that were treated with 10 ng/mL IL-1β ± 3 or 10 μM AITC. Graphs show gene expression and protein release calculated relative to IL-1β treatment (mean ± SD, *n* = 3–4). Asterisks indicate statistical significance (\* *p* < 0.05, Kruskal–Wallis test and Dunn's multiple comparison test).

**Figure 6.** The effects of TRPA1 agonist allyl isothiocyanate (AITC) on gene expression of inflammation markers and ECM molecules in TNF-α-treated cells. Gene expression of (**A**) IL-6 and (**D**) IL-8 in IVD cells treated with 10 ng/mL TNF-α ± 3 or 10 μM AITC. Protein release of (**B**) IL-6 and (**E**) IL-8 in IVD cells treated with 10 ng/mL TNF-α ± 3 or 10 μM AITC. Gene expression of (**C**) NGF, (**F**) COX-2, (**G**) ADAMTS4, (**H**) ADAMTS5, (**I**) TIMP1, (**J**) MMP1, (**K**) MMP3, and (**L**) TIMP2, (**M**) COL1A1, (**N**) COL2A1, and (**O**) Aggrecan in IVD cells treated with 10 ng/mL TNF-α ± 3 and 10 μM AITC. Graphs show gene expression and protein release calculated relative to TNF-α treatment (mean ± SD, *n* = 3-4). Asterisks indicate statistical significance (\* *p* < 0.05, Kruskal–Wallis test and Dunn's multiple comparison test).
