Differential effects of periopathogens on host protease inhibitors SLPI, elafin, SCCA1, and SCCA2

Abstract

ObjectiveSecretory leukocyte peptidase inhibitors (SLPI), elafin, squamous cell carcinoma antigen 1 and 2 (SCCA1 and SCCA2) are specific endogenous serine protease inhibitors expressed by epithelial cells that prevent tissue damage from excessive proteolytic enzyme activity due to inflammation. To determine the effects of various periopathogens on these protease inhibitors, we utilized human gingival epithelial cells (GECs) challenged with cell-free bacteria supernatants of various periopathogens Porphyromonas gingivalis, Tannerella forsythia, Aggregatibacter actinomycetemcomitans, and Fusobacterium nucleatum. DesignThe gene expression and secretion of SLPI, elafin, SCCA1, and SCCA2 were determined using real-time PCR and ELISA, respectively. The direct effects of periopathogens and P. gingivalis gingipain mutants on these inhibitors were examined in vitro by Western Blot. The effect on the innate immune response of GECs was measured by expression of antimicrobial peptides: human beta-defenisin-2 (hBD2) and chemokine (C-C motif) ligand 20 (CCL20).ResultsWe found that SLPI, SCCA2, elafin, hBD2, and CCL20 gene expression levels were significantly induced (p<0.001) in response to P. gingivalis, whose virulence factors include cysteine proteases, but not in response to stimulation by other bacteria. P. gingivalis reduced the secretion of SLPI and elafin significantly in GECs, and degraded recombinant SLPI, elafin, SCCA1, and SCCA2. Differential degradation patterns of SLPI, elafin, SCCA1, and SCCA2 were observed with different bacteria as well as P. gingivalis mutants associated with the loss of specific gingipains secreted by P. gingivalis. In addition, pretreatment of GECs with SLPI, SCCA1, or SCCA2 partially blocked hBD2 and CCL20 mRNA expression in response to P. gingivalis, suggesting a protective effect.ConclusionOur results suggest that different periopathogens affect the host protease inhibitors in a different manner, suggesting host susceptibility may differ in the presence of these pathogens. The balance between cellular protease inhibitors and their degradation may be an important factor in susceptibility to periodontal infection.

Periodontal Disease and Gingival Innate Immunity – Who Has the Upper Hand?

“…A protective effect of these protease inhibitors in gingival health is shown by our study that demonstrated pre-treatment of GECs with SLPI, SCCA1 or SCCA2 partially attenuated antimicrobial proteins hBD-2 and CCL20 mRNA expression in response to P. gingivalis (Yin, Swanson et al 2010). However, the same study showed the presence of P. gingivalis disrupted the function of these serine protease inhibitors, suggesting that the presence of an organism colonizing oral plaque prior to the establishment by pathogens enhances expression of protease inhibitors that protect GECs, while P. gingivalis secretes proteases that degrade cellular protease inhibitors (Yin, Swanson et al 2010). It is of interest to note that various periodontal pathogens which secrete proteases (P. gingivalis, T. forsythia, A. actinomycetemcomitans) were tested, but P. gingivalis was most effective at degrading protease inhibitors (Figure 2) (Yin, Swanson et al 2010).…”

“…P. gingivalis gingipains have shown to degrade extracellular matrix components such as laminin, fibronectin, and collagen type III, IV, and V in vitro (Potempa, Banbula et al 2000), and are thought to account for at least 85% of the general proteolytic activity displayed by P. gingivalis (Imamura 2003). Our previous in vitro study found that the secretion of SLPI and ELAFIN was significantly reduced in response to P. gingivalis and that P. gingivalis supernatants digested recombinant SLPI, ELAFIN, SCCA1 and 2 (Figure 2) (Yin, Swanson et al 2010). These data suggest degradation of protease inhibitors by P. gingivalis may result in decreased host protective capacity and higher susceptibility to P. gingivalis infection (Yin, Swanson et al 2010).…”

“…However, the same study showed the presence of P. gingivalis disrupted the function of these serine protease inhibitors, suggesting that the presence of an organism colonizing oral plaque prior to the establishment by pathogens enhances expression of protease inhibitors that protect GECs, while P. gingivalis secretes proteases that degrade cellular protease inhibitors (Yin, Swanson et al 2010). It is of interest to note that various periodontal pathogens which secrete proteases (P. gingivalis, T. forsythia, A. actinomycetemcomitans) were tested, but P. gingivalis was most effective at degrading protease inhibitors (Figure 2) (Yin, Swanson et al 2010). The degradation of protease inhibitors by P. gingivalis may result in decreased host protective capacity, and the balance between cellular protease inhibitors and their degradation by P. gingivalis and/or other periodontal pathogens may be an important factor in susceptibility to P. gingivalis infection.…”

“…Our data suggest that F. nucleatum, a bridging organism between non-pathogenic commensal and pathogenic bacteria, enhances expression of protease inhibitors that protect GECs in anticipation of virulent proteases secreted by pathogenic bacteria. Both host cell-derived proteases, such as neutrophil elastase, and pathogen-derived proteases, such as the gingipains, are targeted by these protease inhibitors, and therefore, the protease inhibitors may play an important role in maintaining the extent of inflammatory tissue damage (Into, Inomata et al 2006;Williams, Brown et al 2006;Yin, Swanson et al 2010 (Yin and Dale 2007).…”

“…Control: recombinant protein only. The controls shown with P. gingivalis also apply to the recombinant proteins treated with T. forsythia and F. nucleatum (Yin, Swanson et al 2010). Permission from Co-Action Publishing.…”