Written by: John Perrier, Masters Student, Pryzdial Lab

Edited by: Alex Witt, PhD Candidate, Pryzdial Lab

Have you ever wondered how our immune system is intertwined with our oral health? More specifically, how proteins secreted by cytotoxic T-cells and natural killer cells can affect the progression of advanced gum disease, also known as periodontitis?

Researchers in the Kim lab at the Centre for Blood Research have discovered a new role for serine protease Granzyme B (GzmB) in signaling within the connective tissue cells of our periodontium, such as fibroblasts.¹The periodontium refers to the tissues which surround and support the tooth. It has previously been established that gingival fibroblasts contribute to periodontitis by secreting matrix metalloproteinases (MMP) in response to biofilm buildup.² Because GzmB degrades extracellular matrix proteins, activates pro-inflammatory cytokines, and interacts with a receptor present on gingival fibroblasts, protease-activated receptor-1 (PAR-1), this study hoped to uncover a role for GzmB in the degradation of periodontium connective tissue.^3-5

Cover of the Journal of Periodontal Research, 2023.

To assess whether GzmB can contribute to MMP-1 release, the team made use of in vitro and in vivo assays, as well as gingival crevicular fluid (GCF) samples collected from a cohort of over 100 individuals. Looking at healthy individuals, people with gingivitis, and people with periodontitis, the researchers used a GzmB-specific enzyme-linked immunosorbent assay (ELISAs) to establish that the concentration of GzmB is elevated when damage to the periodontium has occurred. GCF samples showed that those with periodontitis or gingivitis had nearly 5 times the concentration of GzmB present, relative to healthy samples.

Using cultured human gingival fibroblasts, the way that GzmB can trigger MMP-1 release from gingival fibroblasts was revealed. MMP1-specific ELISAs of GzmB treated cells showed a significant increase in MMP-1 release over a 24h period, and even more interestingly showed a 2-3-fold higher MMP activity. Building on previous reports showing a link between pro-inflammatory stimuli and Erk1/2 phosphorylation⁶, the authors of this study went further, showing that MMP-1 upregulation by GzmB is dependent on Erk1/2 MAPK signaling. This was confirmed through immunoblot analysis of gingival fibroblast lysates, showing an increase of phosphorylated Erk1/2 following addition of GzmB.

But how does GzmB initiate this signaling pathway? That would be through the aforementioned receptor, PAR-1. GzmB can proteolytically activate this transmembrane receptor, which in turn leads to the downstream activation of Erk1/2 MAPK. An antibody which inhibits PAR-1 function, ATAP-2, was used to dissect the role of PAR1 signaling in GzmB-mediated MMP-1 release. ATAP-2 blocking of PAR-1 greatly reduced MMP-1 secretion in gingival fibroblasts treated with GzmB.

This study establishes a novel signaling pathway, implicating GzmB in the pathogenesis of periodontal disease for the first time. Using human clinical samples, the Kim lab showed that the local concentration of GzmB is higher in those impacted by both gingivitis and periodontitis. This finding is put into context as the authors define the Erk1/2- and PAR-1-dependant signaling pathway for GzmB which results in MMP-1 release from gingival fibroblasts. The current study highlights the potential for GzmB to be used as a therapeutic target to mitigate tissue damage caused by periodontitis.

Link to paper: https://pubmed.ncbi.nlm.nih.gov/37873693/

References

1. Ben-Eltriki M, Ahmadi AR, Nakao Y, et al. Granzyme B promotes matrix metalloproteinase-1 (MMP-1) release from gingival fibroblasts in a PAR1- and Erk1/2-dependent manner: A novel role in periodontal inflammation. Journal of Periodontal Research. 2023;n/a(n/a).
2. Theilade E, Wright WH, Jensen SB, Löe H. Experimental gingivitis in man. Journal of Periodontal Research. 1966;1(1):1-13.
3. Buzza MS, Zamurs L, Sun J, et al. Extracellular matrix remodeling by human granzyme B via cleavage of vitronectin, fibronectin, and laminin. J Biol Chem. 2005;280(25):23549-23558.
4. Afonina IS, Tynan GA, Logue SE, et al. Granzyme B-dependent proteolysis acts as a switch to enhance the proinflammatory activity of IL-1α. Mol Cell. 2011;44(2):265-278.
5. Lee PR, Johnson TP, Gnanapavan S, et al. Protease-activated receptor-1 activation by granzyme B causes neurotoxicity that is augmented by interleukin-1β. Journal of Neuroinflammation. 2017;14(1):131.
6. Omori K, Naruishi K, Nishimura F, Yamada-Naruishi H, Takashiba S. High glucose enhances interleukin-6-induced vascular endothelial growth factor 165 expression via activation of gp130-mediated p44/42 MAPK-CCAAT/enhancer binding protein signaling in gingival fibroblasts. J Biol Chem. 2004;279(8):6643-6649.