<i>Porphyromonas gingivalis</i> Gingipain-Dependently Enhances IL-33 Production in Human Gingival Epithelial Cells

<div><p>The cytokine IL-33 is constitutively expressed in epithelial cells and it augments Th2 cytokine-mediated inflammatory responses by regulating innate immune cells. We aimed to determine the role of the periodontal pathogen, <i>Porphyromonas gingivalis</i>, in the enhanced expression of IL-33 in human gingival epithelial cells. We detected IL-33 in inflamed gingival epithelium from patients with chronic periodontitis, and found that <i>P</i>. <i>gingivalis</i> increased IL-33 expression in the cytoplasm of human gingival epithelial cells <i>in vitro</i>. In contrast, lipopolysaccharide, lipopeptide, and fimbriae derived from <i>P</i>. <i>gingivalis</i> did not increase IL-33 expression. Specific inhibitors of <i>P</i>. <i>gingivalis</i> proteases (gingipains) suppressed IL-33 mRNA induction by <i>P</i>. <i>gingivalis</i> and the <i>P</i>. <i>gingivalis</i> gingipain-null mutant KDP136 did not induce IL-33 expression. A small interfering RNA for protease-activated receptor-2 (PAR-2) as well as inhibitors of phospholipase C, p38 and NF-κB inhibited the expression of IL-33 induced by <i>P</i>. <i>gingivalis</i>. These results indicate that the PAR-2/IL-33 axis is promoted by <i>P</i>. <i>gingivalis</i> infection in human gingival epithelial cells through a gingipain-dependent mechanism.</p></div

Induction of IL-33 mRNA expression by <i>P</i>. <i>gingivalis</i> requires activation of the NF-κB pathway.

<p>(A) Ca9-22 cells stimulated with 50 μg/ml of whole <i>P</i>. <i>gingivalis</i> W83 cells for indicated periods. (B) Whole <i>P</i>. <i>gingivalis</i> W83 cells (50 μg/ml) were incubated with gingipain inhibitors (0.3 μM FPR-cmk plus 0.3 μM KYT-36) for 15 min at 37°C, and then used to stimulate Ca9-22 cells for 9 h. (C) Ca9-22 cells stimulated with 50 μg/ml of whole <i>P</i>. <i>gingivalis</i> ATCC 33277 wild-type or KDP136 cells for 9 h. (A-C) Cells were transiently transfected with pNFκB-<i>Metridia</i> luciferase reporter or control p<i>Metridia</i> luciferase reporter plasmids. Amount of secreted luciferase in culture supernatants were analyzed using a luminometer. (D) Ca9-22 cells incubated with indicated concentrations of PDTC (NF-κB inhibitor) for 1 h and then stimulated with 50 μg/ml of whole <i>P</i>. <i>gingivalis</i> W83 cells for 48 h. Total cellular RNA was extracted and transcripts were analyzed by RT-qPCR. Data are representative of three independent experiments and are shown as means ± SD of triplicate assays. Statistical significant differences are indicated (*, <i>P</i><0.05 compared with respective unstimulated control; §, <i>P</i><0.05 compared with <i>P</i>. <i>gingivalis</i> alone). (E) Ca9-22 cells were incubated with 25 μM PCTC for 1 h and then stimulated for 18 h with 50 μg/ml of whole <i>P</i>. <i>gingivalis</i> W83 cells in medium containing 1% FBS. Cell lysates were analyzed by Western blotting against anti-phospho-p38 and anti-p38 antibodies. Data are representative of three independent experiments. Relative expression levels of phosphorylated p38 were normalized to p38 and quantified by densitometry. Control media was adjusted to contain 0.1% (v/v) DMSO during incubation with inhibitor.</p

Production of IL-33 is increased in chronic periodontitis.

<p>Cryosections of normal periodontal tissues from healthy donors (A and B) and inflamed periodontal tissues from patients with chronic periodontitis (C and D) were stained with rabbit anti-human IL-33 mAb (B and D) or rabbit anti-human IgG isotype control (A and C) and HRP-conjugated anti-rabbit IgG and then visualized with DAB. Sections were counterstained with Mayer’s hematoxylin. GE, gingival epithelium; LP, lamina propria. Bar = 3 μm. Data are representative of five experiments.</p

Induction of IL-33 mRNA expression by <i>P</i>. <i>gingivalis</i> requires activation of the p38 pathway.

<p>(A) Ca9-22 cells stimulated for indicated periods with 50 μg/ml of whole <i>P</i>. <i>gingivalis</i> W83 cells in medium containing 1% FBS. Phosphorylation of p38 was detected in cell lysates by Western blotting against anti-phospho-p38 antibody (p-p38). Controls comprised antibody against total p38. Data are representative of three independent experiments. Relative expression of phosphorylated p38 was quantified using densitometry. Relative expression of phosphorylated p38 was normalized to that of p38. (B) Ca9-22 cells incubated with 10 μM PD98059, SP600125, or SB203580 for 30 min and then stimulated with 50 μg/ml of whole <i>P</i>. <i>gingivalis</i> W83 cells for 48 h in medium containing 5% FBS. Total cellular RNA was extracted and transcripts were analyzed by RT-qPCR. Data are representative of three independent experiments, and are shown as means ± SD of triplicate assays. Statistical significant differences are indicated (*, <i>P</i><0.05 compared with untreated control; §, <i>P</i><0.05 compared with <i>P</i>. <i>gingivalis</i> alone). (C) Whole <i>P</i>. <i>gingivalis</i> W83 cells (50 μg/ml) were incubated with 0.3 μM FPR-cmk plus 0.3 μM KYT-36 for 15 min at 37°C and then used to stimulate Ca9-22 cells for 18 h in medium containing 1% FBS. (D) Ca9-22 cells were stimulated with 50 μg/ml of <i>P</i>. <i>gingivalis</i> ATCC 33277 wild-type or KDP 136 whole cells for 18 h. Cell lysates were analyzed by Western blotting using anti-phospho-p38 and anti-p38 antibodies. Data are representative of three independent experiments. Relative expression levels of phosphorylated p38 were normalized to levels of p38 and quantified by densitometry. Controls were adjusted to contain 0.1% (v/v) DMSO in media during incubation with inhibitors.</p

Participation of gingipains in <i>P</i>. <i>gingivalis</i>-induced IL-33 mRNA expression in gingival/oral epithelial cells.

<p>Whole <i>P</i>. <i>gingivalis</i> W83 cells (50 μg/ml) were incubated with 0.3 μM FPR-cmk (Rgp inhibitor) or 0.3 μM KYT-36 (Kgp inhibitor) for 15 min at 37°C and then used to stimulate Ca9-22 (A) or primary oral epithelial (B) cells for 48 h. (C) Ca9-22 cells stimulated with 50 μg/ml of whole cells of <i>P</i>. <i>gingivalis</i> ATCC 33277 wild-type, <i>P</i>. <i>gingivalis</i> KDP131 (Δ<i>rgpA</i>), KDP132 (Δ<i>rgpB</i>), KDP129 (Δ<i>kgp</i>), KDP133 (Δ<i>rgpA</i> Δ<i>rgpB</i>), or KDP136 (Δ<i>kgp</i> Δ<i>rgpA</i> Δ<i>rgpB</i>) gingipain-null mutant for 48 h. (D) Primary oral epithelial cells stimulated with 50 μg/ml of <i>P</i>. <i>gingivalis</i> ATCC 33277 wild-type or KDP136 for 48 h. (E) Ca9-22 cells cultured for 48 h with 50 μg/ml of whole <i>P</i>. <i>gingivalis</i> W83 cells after incubation with or without at 70°C for 1 h. Total cellular RNA was extracted and transcripts were analyzed by RT-qPCR. Data are representative of three independent experiments, and are shown as means ± SD of triplicate assays. Statistical significant differences are indicated (*, <i>P</i><0.05 compared with respective unstimulated control; §, <i>P</i><0.05 compared with <i>P</i>. <i>gingivalis</i> alone).</p

<i>P</i>. <i>gingivalis</i> increases IL-33 mRNA expression in gingival epithelial cells.

<p>Ca9-22 cells were infected with fresh <i>P</i>. <i>gingivalis</i> W83 cultures at MOI of 0.5 for the indicated periods (A) or indicated MOI for 48 h (B). Ca9-22 cells were stimulated with 50 μg/ml (MOI of 0.1) of whole <i>P</i>. <i>gingivalis</i> W83 cells for the indicated periods (C) and indicated amounts of whole <i>P</i>. <i>gingivalis</i> W83 cells, fimbriae, PGTP2-RL, and LPS derived from <i>P</i>. <i>gingivalis</i> for 48 h (D). Cells were incubated with 10 μg/ml cycloheximide for 45 min (E), 1 μg/ml nocodazole for 1 h (F) or 0.5 μM cytochalasin D for 30 min (G) and then stimulated for 48 h with 50 μg/ml of <i>P</i>. <i>gingivalis</i> W83 whole cells with the respective inhibitors. Total cellular RNA was then extracted at the indicated times, and IL-33 transcripts were analyzed by RT-qPCR. Data are representative of three independent experiments, and are shown as means ± SD of triplicate assays. Statistically significant differences are indicated. *, <i>P</i><0.05 compared with untreated control; §, <i>P</i><0.05 compared with <i>P</i>. <i>gingivalis</i> alone.</p

<i>P</i>. <i>gingivalis</i> increases IL-33 protein expression in gingival epithelial cells.

<p>(A) Ca9-22 cells were stimulated with 50 μg/ml of whole <i>P</i>. <i>gingivalis</i> W83 cells for the indicated periods. Cell lysates were analyzed by Western blotting with an anti-human IL-33 mAb. Expression levels of IL-33 were quantified by densitometry using ImageJ software and normalized to medium alone. (B) Ca9-22 cells were stimulated with 50 μg/ml of whole <i>P</i>. <i>gingivalis</i> W83 cells for 4 d, and intracellular IL-33 protein was stained with PE-conjugated anti-human IL-33 mAb. Nuclei were stained with DAPI. Bar = 25 μm. Data are representative of three independent experiments.</p

Induction of apoptosis in cultured DU145 and PC3 cells in response to PL treatment.

<p>The cells were treated with 1 mg/ml or 2 mg/ml for 48 h, and subsequently stained with Annexin V. Error bars represent the standard deviation from 3 independent experiments.</p

Analysis of side-effects of PL.

<p><b>A</b> and <b>B</b>. After the tumor nodules were formed, the body weight (<b>A</b>) and water consumption (<b>B</b>) of each mouse with or without treated with PL were measured. Error bars represent the standard deviation from 3–6 mice.</p

Histopathology or immunohistochemistry staining of PL-treated or control tumors isolated from the mice.

<p><b>A</b>. The tumors were prepared for hematoxylin and eosin staining. <b>B</b> and <b>C</b>. The slides mounted with the tumor tissues were stained with TUNEL agent (<b>B</b>) or anti-caspase 3 antibody (<b>C</b>).</p