Differential modulation of the EC death related gene expression with <i>Pg</i> and its LPS in Ox-LDL and TNF-α pre-treated cells.

<p><b>(A)</b> Gene expression of Caspase-1,-3 and -9 in HUVECs infected with <i>Pg</i> at a MOI of 100 or with heat inactivated <i>Pg</i> and stimulated by <i>Pg</i>-LPS (1μg/ml) or <i>E</i>.<i>Coli</i>-LPS (1μg/ml) at 24h. <b>(B)</b> Gene expression for the same described genes in Ox-LDL pre-treated HUVECs at 24h. <b>(C)</b> Gene Expression for the same described genes in TNF- α pre-treated HUVECs at 24h. Data were expressed as mean ± SD. *: difference between non-stimulated/infected and stimulated/infected cells, <i>p</i> < 0.05, ┼: difference between non pre-treated/stimulated/infected and treated cells, <i>p</i> < 0.05.</p

<i>Pg</i> and its LPS modulate the expression of EC death related gene expression.

<p><b>(A)</b> Gene expression of Bcl-2, Bax-1, and Apaf-1 in HUVECs infected with <i>Pg</i> at a MOI of 100 or with heat inactivated <i>Pg</i> and stimulated by <i>Pg</i>-LPS (1μg/ml) or <i>E</i>.<i>Coli</i>-LPS (1μg/ml) at 24h. <b>(B)</b> Gene expression for the same described genes in Ox-LDL (50μg/ml) pre-treated HUVECs infected with <i>Pg</i> at a MOI of 100 or with heat inactivated <i>Pg</i> and stimulated by <i>Pg</i>-LPS (1μg/ml) or <i>E</i>.<i>Coli</i>-LPS (1μg/ml) at 24h. <b>(A)</b> Gene expression for the same described genes in TNF- α (10ng/ml) pre-treated HUVECs infected with <i>Pg</i> at a MOI of 100 or with heat inactivated <i>Pg</i> and stimulated by <i>Pg</i>-LPS (1μg/ml) or <i>E</i>.<i>Coli</i>-LPS (1μg/ml) at 24h. Data were expressed as mean ± SD. *: difference between non-stimulated/infected and stimulated/infected cells, <i>p</i> < 0.05, ┼: difference between non pre-treated/stimulated/infected and treated cells, <i>p</i> < 0.05.</p

Differential modulation of the EC death related caspase activity after infection with <i>Pg</i> and its LPS in Ox-LDL and TNF-α pre-treated cells.

<p><b>(A)</b> Enzymatic activity of Caspase-1, -3 and -9 in HUVECs infected with <i>Pg</i> at a MOI of 100 or with heat inactivated <i>Pg</i> and stimulated by <i>Pg</i>-LPS (1μg/ml) or <i>E</i>.<i>Coli</i>-LPS (1μg/ml) at 24h. <b>(B)</b> Enzymatic activity of Caspase-1, -3 and -9 in Ox-LDL pre-treated HUVECs at 24h. <b>(C)</b> Enzymatic activity of Caspase-1, -3 and -9 in in TNF- α pre-treated HUVECs at 24h. Data were expressed as mean ± SD. *: difference between non-stimulated/infected and stimulated/infected cells, <i>p</i> < 0.05.</p

Pre-treatment of EC leads to different types of cell death induced by <i>Pg</i>.

<p><b>(A)</b> Percentage of cell death of Ox-LDL (50μg/ml) pre-treated ECs infected with <i>Pg</i> at a MOI of 100 or Heat-inactivated <i>Pg</i> (H<i>Pg</i>) and stimulated by <i>Pg</i>-LPS (1μg/ml) or <i>E</i>.<i>Coli</i>-LPS (1μg/ml) for 24h. <b>(B)</b> The apoptosis/necrosis ratio of Ox-LDL (50μg/ml) pre-treated ECs with <i>Pg</i> at a MOI of 100 or Heat-inactivated <i>Pg</i> (H<i>Pg</i>) and stimulated by <i>Pg</i>-LPS (1μg/ml) or <i>E</i>.<i>Coli</i>-LPS (1μg/ml) for 24h. <b>(C)</b> Percentage of cell death of TNF-α (10ng/ml) pre-treated ECs infected with <i>Pg</i> at a MOI of 100 or Heat-inactivated <i>Pg</i> (H<i>Pg</i>) and stimulated by <i>Pg</i>-LPS (1μg/ml) or <i>E</i>.<i>Coli</i>-LPS (1μg/ml) for 24h. <b>(D)</b> The apoptosis/necrosis ratio of TNF- α (10ng/ml) pre-treated ECs infected with <i>Pg</i> at a MOI of 100 or Heat-inactivated <i>Pg</i> (H<i>Pg</i>) and stimulated by <i>Pg</i>-LPS (1μg/ml) or <i>E</i>.<i>Coli</i>-LPS (1μg/ml) for 24h. Data were expressed as mean ± SD. ✷: difference between non pre-treated/stimulated/infected and infected/stimulated cells, <i>p</i> < 0.05; <b>(E)</b> Infected and stimulated Ox-LDL (50μg/ml) and TNF- α (10ng/ml) pre-treated HUVECs cell death was evaluated for each condition qualitatively using Annexin V-IP staining at 24h and 48h (in green: Annexin V positive staining; in red: Iodure propidium positive staining; in blue: DAPI nuclear staining). Images were acquired under fluorescence microscopy (10x) after Annexin V-IP and DAPI staining for all previously described condition. All scale bars indicate 100 μm.</p

Infection of ECs leads to cell death mediated by apoptosis.

<p><b>(A)</b> Percentage of cell death of ECs infected with <i>Pg</i> or Heat inactivated <i>Pg</i> (H<i>Pg</i>) at a MOI of 100 and stimulated by <i>Pg</i>-LPS (1μg/ml) or <i>E</i>.<i>Coli</i>-LPS (1μg/ml). Each percentage was calculated on account of total cells counted in triplicate for each experiment. <b>(B)</b> The apoptosis/necrosis ratio of ECs infected with <i>Pg</i> or Heat inactivated <i>Pg</i> (H<i>Pg</i>) at a MOI of 100 and stimulated by <i>Pg</i>-LPS (1μg/ml) or <i>E</i>.<i>Coli</i>-LPS (1μg/ml). Each value was calculated from the ratio between the total number of apoptotic cells and necrotic cells and for each count nine images were used of each experimentation. Data were expressed as mean ± SD. ✷: difference between non pre-treated/stimulated/infected and infected/stimulated cells, <i>p</i> < 0.05; <b>(C)</b> Infected and stimulated HUVECs cell death was evaluated for each condition qualitatively using Annexin V-IP staining at 24h (in green: Annexin V positive staining; in red: Iodure propidium positive staining; in blue: DAPI nuclear staining) Images were acquired under fluorescence microscopy (10x) after Annexin V-IP and DAPI staining for all previously described condition. All scale bars indicate 100 μm.</p

Qualitative evaluation of the EC death.

<p><b>(A)</b> Viability of HUVECs infected with <i>Pg</i> at a MOI of 100 or Heat-inactivated <i>Pg</i> (H<i>Pg</i>) and stimulated by <i>Pg</i>-LPS (1μg/ml) or <i>E</i>.<i>Coli</i>-LPS (1μg/ml) at 24h. All different conditions have been evaluated quantitatively and qualitatively by Live-Dead staining assays. <b>(B)</b> Viability of Ox-LDL (50μg/ml) pre-treated HUVECs on cell cultures infected with <i>Pg</i> or H<i>Pg</i> at a MOI of 100 and stimulated by <i>Pg</i>-LPS (1μg/ml) or <i>E</i>.<i>Coli</i>-LPS (1μg/ml) at 24h. <b>(C)</b> Viability of TNF- α (10ng/ml) pre-treated HUVECs on cell cultures infected with <i>Pg</i> or H<i>Pg</i> at a MOI of 100 and stimulated by <i>Pg</i>-LPS (1μg/ml) or <i>E</i>.<i>Coli</i>-LPS (1μg/ml) at 24h. <b>(D)</b> Percentage of dead cells infected with <i>Pg</i> or H<i>Pg</i> and stimulated by <i>Pg</i>-LPS (1μg/ml) or <i>E</i>.<i>Coli</i>-LPS (1μg/ml) at 24h. <b>(E)</b> Percentage of dead cells in OxLDL qnd TNF- α pre-treated HUVECs. All images were acquired under fluorescence microscopy (in green: viable cells; in red: dead cells). All scale bars indicate 100 μm.</p

<i>Pg</i> infection increased the expression of RANK-L and IL-33 mRNAs in human oral epithelial cells.

<p>Human oral epithelial cells (OKF6/TERT2) were cultured with <i>Pg</i> at 10:1 or 100:1 MOI for 6, 12 or 24 hours. mRNAs encoding for IL-33 (A) and RANK-L ((B) were quantified by RT-qPCR. Three separate sets of experiment were performed. Data are shown as mean ± SEM. *p<0.05; **p<0.01.</p

Time-course of alveolar bone loss in the ligature-induced murine model of experimental periodontitis.

<p>CD1 Swiss mice (n = 90) were subjected to experimental periodontitis for 4, 14 and 28 days. At each time point, animals were sacrificed and maxillary samples were harvested. A. After 4, 14 and 28 days, μCT analysis was performed. Longitudinal sections through the middle of the palatal root of the first maxillary molar (left images) and transversal sections from the apices of the three roots of the first maxillary molar to the summit of the alveolar bone crest (right images) are presented for each time points. B. Alveolar bone loss was assessed using 2D μCT. At each time point, data of ligatured groups (Lig and <i>Pg</i> L) were compared to their respective Sham groups. Data are shown as means ± SEM. * p<0.05.</p

Characterization of human gingival samples in healthy and patients affected by chronic periodontitis.

<p>A. Samples were stained for the T lymphocytes marker CD3 (arrows). Sections were counterstained with Harris Hematoxylin staining. EP: Epithelium; CT: Connective Tissue. B. TNF-α and IL-6 expression in healthy and CP patients were measured by RT-qPCR. Data are shown as mean ± SD. Healthy samples n = 9; chronic periodontitis samples n = 13. Bar = 250μm. *p<0.05, **p<0.01.</p

Characteristics of the patients.

<p>Characteristics of the patients.</p