Fig 2: (A) Macroscopic IVD schematic with zoomed in proposed mechanistic models of (B) MC invasion into the intradiscal environment in acute onset and chronic state as visualized through (1) Acute injury or matrix perturbation provides physical access of MC progenitors into usually immune-privileged intra-discal microenvironment acutely and (2) in the chronic state access can occur through vascularization, (3) facilitated by the MC chemoattractant SCF (KITLG). (C) Biochemical pathways of immune modulation in DBP as follows; (4) MCs in the IVD respond to mechanical load and nutrient deprivation in the harsh microenvironment by releasing tryptase (Wiet et al., 20171; Zhang et al., 20122; Fowlkes et al., 20133). Tryptase can activate (5) PAR2 directly (Molino et al., 19974) as well as (5’) matrix enzymes, matrix metalloproteinases 3 and 13 (MMPs) (Magarinos et al., 20135). (6) PAR2 activation by tryptase, abated by PAR2A (Chen et al., 20116) in the present study, leads to downstream factors related to catabolism, (6’) such as MMPs (Lee et al., 20108), which can degrade the proteoglycan aggrecan (Magarinos et al., 20135) creating an increasingly permissive environment for inflammation and stimulating increased activation of endogenous pro-MMP (Vo et al., 20139). (7) PAR2’s activation also leads to downstream factors related to inflammation, neo-vascularization/neo-innervation, and MC chemotaxis contributing to a synergistic positive feedback loop, whereas PAR2’s activation upregulates its own expression through IL-1ß, ad general inflammation, perpetuating a chronic degenerate state (Ritchie et al., 20077). Black Arrows represent labeling or directionality, Blue arrows represent forward mechanism steps, Green arrows represent feedback interactions. (C) Blunt arrow ends represent effects coming from, pointed ends represent effects going to, and bidirectional pointed ends represent co-regulatory interactions.

Fig 3: Expression of PAR2 in HFL-1 cells. RTqPCR was used to confirm the expression of the serine protease receptor PAR2 on HFL-1. The relative gene expression was related to the housekeeping gene 18 s (n = 2, 1 experiment). Human lung fibroblasts, HFL-1, showed a high gene expression for PAR2, while LAD2 cells did not show any PAR2 gene expression (a). Expression of PAR2 examined in monolayer cultures of HFL-1, LAD2, and in co-culture of HFL-1 and LAD2. Representative images are shown with PAR2 staining in yellow, mast cell specific tryptase-staining in red and nuclei stained with DAPI in blue (b-m). LAD2 cells stained by PAR2 antibodies showed no positive receptor staining (d), while HFL-1 fibroblasts showed a positive expression (h). HFL-1 showed no positive staining for tryptase (g), while mast cells were immunopositive (c). Representative scale bars are shown at the beginning of each figure (b, f, j) and indicated as 20 µm

Fig 4: (A) Representative images of immunohistochemical (IHC) PAR2 staining in IVD tissue from all regions; NP, AF and CEP including relevant positive and negative lung tissue controls. (B) PAR2 expression presented as % of cells expressing PAR2 as assessed by IHC and confirmed with western blot using the same PAR2 primary antibodies. Red scale bar = 100 µm, Black scale bar = 50 µm. Arrows point to same cells at both magnifications.