MMP-9/TIMP-1 imbalance induced in human dendritic cells by Porphyromonas gingivalis

Abstract

Matrix metalloproteinase-9 (MMP-9) cleaves collagen, allowing leukocytes to traffic toward the vasculature and the lymphatics. When MMP-9 is unregulated by tissue inhibitor of metalloproteinase-1 (TIMP-1), this can lead to tissue destruction. Dendritic cells (DCs) infiltrate the oral mucosa increasingly in chronic periodontitis, characterized by infection with several pathogens including Porphyromonas gingivalis. In this study, human monocyte-derived DCs were pulsed with different doses of lipopolysaccharide of P. gingivalis 381 and of Escherichia coli type strain 25922, as well as whole live isogenic fimbriae-deficient mutant strains of P. gingivalis 381. Levels of induction of MMP-9 and TIMP-1, as well as interleukin-10 (IL-10), which reportedly inhibits MMP-9 induction, were measured by several approaches. Our results reveal that lipopolysaccharide of P. gingivalis, compared with lipopolysaccharide from E. coli type strain 25922, is a relatively potent inducer of MMP-9, but a weak inducer of TIMP-1, contributing to a high MMP-9/TIMP-1 ratio.Whole live P. gingivalis strain 381, major fimbriae mutant DPG-3 and double mutant MFB were potent inducers of MMP-9, but minor fimbriae mutant MFI was not. MMP-9 induction was inversely proportional to IL-10 induction. These results suggest that lipopolysaccharide and the minor and the major fimbriae of P. gingivalis may play distinct roles in induction by DCs of MMP-9, a potent mediator of local tissue destruction and leukocyte trafficking.

Fig. 1. Semi-quantitative analysis of MMP-9 production by MoDCs in response to P. gingivalis LPS and E. coli LPS

(A) Western blotting analysis of supernatants from MoDCs stimulated with 10, 100 and 1000 ng/ml of P. gingivalis (Pg) LPS and E.coli LPS or no LPS (control [ctl]) for 24 hrs. Equal amounts of protein were loaded onto SDS-PAGE and separated by electrophoresis and transferred to nitrocellulose membranes as described in the Materials and Methods. Representative bands corresponding to 92 kDa (MMP-9). (B) Western blot bands corresponding to 92 kDa (MMP-9) were quantified by optical densitometry (GelPro Analyzer, Media Cybernetics, Silver Spring, MD) and results expressed as densitometric units (DU). Results represent means ± S.D. of densitometric units (DU). * Significant difference between P. gingivalis LPS and E.coli LPS at the same dose (p < 0.01, Kruskal–Wallis test).

Fig. 2. Enzymatic and quantitative analysis of MMP-9 and of the MMP-9/TIMP-1 ratio in MoDCs exposed to P. gingivalis LPS

(A) MoDCs were stimulated with 100, 200, 400 and 800 ng/ml of P. gingivalis LPS and E.coli LPS for 24 hrs. MMP-9 in cell supernatants was analyzed by gelatin zymography. Equal amounts of protein were loaded and separated by electrophoresis as described in the Materials and Methods. (B) Gelatin zymogram bands corresponding to 92 kDa (MMP-9) were quantified by optical densitometry (GelPro Analyzer, Media Cybernetics, Silver Spring, MD). Results represent means ± S.D. of densitometric units (DU). (C) ELISA analysis of MMP-9 production in pg/ml by MoDCs in response to 100 ng/ml of either LPS or no LPS (control). Shown are the means ± S.E. of assay performed in triplicate. * Significant increase in MMP-9 induction by P. gingivalis LPS compared to E. coli LPS and DC control (p<0.05, Student’s t-test (D) ELISA analysis of TIMP-1 production in pg/ml by MoDCs in response to 100 ng/ml of either LPS or no LPS (control). Shown are the means ± S.E. of assay performed in triplicate.

Fig. 3. Induction of MMP-9, TIMP-1 and IL-10 by P.gingivalis 381, its fimbriae deficient mutants

MoDCs were pulsed with whole cells of wild type Pg 381, its minor fimbriae deficient strain (MFI), major fimbriae deficient strain (DPG-3) and double-fimbriae deficient mutant strain (MFB) for 18 h at a MOI of 1:25. Secretion of MMP-9, TIMP-1 and IL-10 in pg/ml were assessed by ELISA, as described in Materials and Methods. The data are the mean ± S.D. of triplicate assays. (A) MMP-9 production in response to different fimbriae deficient strains. (B) Ratio of MMP-9/TIMP-1. (C) Comparison of MMP-9 and IL-10 produced by MoDCs in response to different fimbriae deficient strains.