1887

Abstract

activates protease-activated receptors (PARs) on oral keratinocytes, resulting in downstream signalling for an innate immune response. Activation depends on gingipains, but could be confounded by lipopolysaccharide signalling through Toll-like receptors. We therefore hypothesized that cleaves oral keratinocyte PARs in an Arg- (Rgp) or Lys- (Kgp) gingipain-specific manner to upregulate pro-inflammatory cytokines. Immortalized human oral keratinocytes (TERT-2) were incubated with wild-type (ATCC 33277) or strains from a panel of isogenic gingipain deletion mutants: Kgp-deficient (KDP 129); Rgp-deficient (KDP 133); or Kgp- and Rgp-deficient (KDP 136). After incubation with , keratinocytes were probed with specific antibodies against the N-terminus of PAR-1 and PAR-2. Using flow cytometry and immunofluorescence, receptor cleavage was marked by loss of specific antibody binding to the respective PARs. TERT-2 cells constitutively expressed high levels of PAR-1 and PAR-2, and lower levels of PAR-3. ATCC 33277 cleaved PAR-1 and PAR-2 in a dose-dependent manner, while the receptors were unaffected by the protease-negative double mutant (KDP 136) at all m.o.i. tested. The single Kgp-negative mutant preferentially cleaved PAR-1, whereas the Rgp-negative mutant cleaved PAR-2. Wild-type or Kgp-negative mutant cleavage of PAR-1 upregulated expression of IL-1, IL-1, IL-6 and TNF-; the Rgp-negative mutant did not modulate these cytokines. Selective cleavage of PAR-1 on oral epithelial cells by Rgp therefore upregulates expression of pro-inflammatory cytokines.

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2009-10-01
2020-01-27
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