Abstract
BackgroundPorphyromonas gingivalis is an important bacterial etiological agent involved in periodontitis. The bacterium expresses two kinds of cysteine proteases called gingipains: arginine gingipains (RgpA/B) and lysine gingipain (Kgp). This study evaluated the interaction between P. gingivalis and THP-1 cells, a widely used monocytic cell line, in vitro with a focus on CXCL8 at the gene and protein levels and its fate thereafter in cell culture supernatants. THP-1 cells were stimulated with viable and heat-killed wild-type strains ATCC 33277 or W50 or viable isogenic gingipain mutants of W50, E8 (Rgp mutant) or K1A (Kgp mutant), for 24 hours.ResultsELISA and qPCR results show an elevated CXCL8 expression and secretion in THP-1 cells in response to P. gingivalis, where the heat-killed ATCC33277 and W50 induced higher levels of CXCL8 in comparison to their viable counterparts. Furthermore, the Kgp-deficient mutant K1A caused a higher CXCL8 response compared to the Rgp-deficient E8. Chromogenic quantification of lipopolysaccharide (LPS) in supernatant showed no significant differences between viable and heat killed bacteria except that W50 shed highest levels of LPS. The wild-type strains secreted relatively more Rgp during the co-culture with THP-1 cells. The CXCL8 degradation assay of filter-sterilized supernatant from heat-killed W50 treated cells showed that Rgp was most efficient at CXCL8 hydrolysis. Of all tested P. gingivalis strains, adhesion and internalization in THP-1 cells was least conspicuous by Rgp-deficient P. gingivalis (E8), as demonstrated by confocal imaging.ConclusionsW50 and its Kgp mutant K1A exhibit a higher immunogenic and proteolytic function in comparison to the Rgp mutant E8. Since K1A differs from E8 in the expression of Rgp, it is rational to conclude that Rgp contributes to immunomodulation in a more dynamic manner in comparison to Kgp. Also, W50 is a more virulent strain when compared to the laboratory strain ATCC33277.
Highlights
Porphyromonas gingivalis is an important bacterial etiological agent involved in periodontitis
The results show a marked increase in the levels of CXCL8 (2000–70,000 pg/ml) in P. gingivalis stimulated cells in comparison to unstimulated cells that serve as control
The highest amounts of CXCL8 were observed in the group treated with heat-killed W50 (72,000 pg/ml) and ATCC 33277 (65,000 pg/ml) respectively and this was 30 times higher compared to the levels in cells treated with viable ATCC 33277 and W50 strains
Summary
Porphyromonas gingivalis is an important bacterial etiological agent involved in periodontitis. THP-1 cells were stimulated with viable and heat-killed wild-type strains ATCC 33277 or W50 or viable isogenic gingipain mutants of W50, E8 (Rgp mutant) or K1A (Kgp mutant), for 24 hours. Monocytes and neutrophils are sentinel cells of innate immunity and are found in abundance during periodontal infection [8]. Toll-like recptors (TLRs) are germline encoded pattern recognition receptors (PRRs) present on various cells and they have been evolved to recognize conserved products unique to microbial metabolism and alert the immune system along signaling cascades. The PARs are a unique category of transmembrane receptors that are activated on the cleavage of the receptor N-terminal part to expose a new, previously cryptic sequence. The exposed sequence remains tethered to the receptor and acts further as a receptor-activating ligand which results in Ca2+ increase and production of CXCL8 [13]. It has been shown that platelet activation with gingipains is associated with PARs and PAR1 and 4 are involved in response to Rgp [14,15]
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