Aggregatibacter actinomycetemcomitans Cytolethal Distending Toxin Induces Cellugyrin-(Synaptogyrin 2) Dependent Cellular Senescence in Oral Keratinocytes
<p>Cdt induces durable cell cycle arrest in OE. TIGK cells were treated with Cdt for varying periods of time as indicated and then monitored for cell cycle arrest. Panels (<b>A</b>) (control cells) and (<b>B</b>) (Cdt-treated cells) show the effect of Cdt (20 pg/mL) on TIGK proliferation after 72 h. Cell cycle progression was assessed using dual parameter flow cytometry; DNA content was assessed by monitoring propidium iodide fluorescence (PI-A) and incorporation of BrdU-FITC. Boxes indicate gates for cell cycle analysis: G1/G0 (black), G2/M (blue) and S (red); numbers indicate the percentage of cells within each gated box. Panel (<b>C</b>) shows the analysis of cell cycle progression in control (blue line) and toxin-treated (red line) cells stained with Viafluor 488 and incubated for 72 h. The results for panels (<b>A</b>–<b>C</b>) are each representative of three experiments. Panel (<b>D</b>) shows cell cycle analysis of TIGK cells treated with medium (blue) and Cdt [20 pg/mL; (red)] for 3–7 days using propidium iodide. Cells were analyzed for cell phase as described in <a href="#sec2-pathogens-13-00155" class="html-sec">Section 2</a>; the percentage of G2/M cells is plotted versus time. Results are compiled from three experiments and represent the mean ± SEM; all data points for Cdt-treated cells are significantly different from those observed in control cells (<span class="html-italic">p</span> < 0.01). Individual cell cycle histograms are shown in <a href="#app1-pathogens-13-00155" class="html-app">Figure S1</a>.</p> "> Figure 2
<p>Cdt induces a cellular senescent phenotype in OEs characterized by increases in both SA-β-gal activity and lipofuscin content. TIGK cells were treated with 0–25 pg/mL Cdt for 72 h and then analyzed for SA-β-gal activity as described in <a href="#sec2-pathogens-13-00155" class="html-sec">Section 2</a>. Data are plotted as fluorescence (MCF) vs. Cdt concentration. The inset (panel (<b>A</b>)) shows results for exposure to Cdt for 7 days. Panel (<b>B</b>) shows the effect of Cdt on SA-β-gal activity in toxin-treated PGK cells following 3 days of exposure to Cdt. Panel (<b>C</b>) shows the effect of Cdt (20 pg/mL) on lipofuscin levels in TIGK cells following 3–7 days of exposure to toxin. Lipofuscin was detected using biotinylated SenTraGor, followed by staining with an anti-biotin antibody conjugated to AF488. Results are plotted as fluorescence (MCF) versus time of exposure to Cdt (days). Panel (<b>D</b>) shows the effect of Cdt on lipofuscin levels in PGK cells after 4 days of treatment with toxin. Results are plotted as the MCF (mean ± SEM) of three experiments; * indicates statistical significance (<span class="html-italic">p</span> < 0.05).</p> "> Figure 3
<p>Cdt induces SASP in TIGK cells. TIGK cells were treated with Cdt (0–100 pg/mL) for 72 h; cell supernatants were harvested and analyzed by ELISA for release of IL-8 (panel (<b>A</b>)), IL-6 (panel (<b>B</b>)) and RANKL (panel (<b>C</b>)). In a second series of experiments, TIGK cells were pre-treated with the GSDMD inhibitor NSA (0–1 μM) for one h, followed by the addition of Cdt (100 pg/mL). Supernatants were harvested 72 h later and analyzed for release of IL-8 (panel (<b>D</b>)), IL-6 (panel (<b>E</b>)) and RANKL (panel (<b>F</b>)). Results are the mean ± SEM of three experiments; * indicates statistical significance (<span class="html-italic">p</span> < 0.01).</p> "> Figure 4
<p>Cdt-induced OE senescent cells exhibit a breakdown in epithelial barrier function. PGKs were grown to confluence until a stable TEER was established. Medium or Cdt was then added and the cells assessed daily for changes in TEER as described in <a href="#sec2-pathogens-13-00155" class="html-sec">Section 2</a>. Results of three experiments were plotted as mean resistance (Ω × cm<sup>2</sup>) at 24 h (solid bars), 48 h (hatched bars) and 72 h (cross hatched bars); * indicates statistical significance (<span class="html-italic">p</span> < 0.01).</p> "> Figure 5
<p>Cdt treatment alters cell–cell contacts in PGKs. (<b>A</b>) Confocal images showing control (untreated) and Cdt (10 pg/mL, 48 h)-treated PGKs immunostained with ß-catenin (green). Nuclei stained with Hoechst are pseudo-colored in cyan. (<b>B</b>) Boxed regions in the panel (<b>A</b>) were enlarged and shown. (<b>C</b>) The boxed regions in panel (<b>B</b>) were further enlarged, highlighting the appearance of distinct gaps between cells in the Cdt-treated set (right) relative to the control cells. (<b>D</b>) Line intensity profiles for ß-catenin (green) and Hoechst nuclear stain (cyan) across the white dotted line in panel (<b>C</b>). The gaps between the adjacent cells identified by the ß-catenin staining pattern are depicted by dotted black lines.</p> "> Figure 6
<p>Cdt-induced cellular senescence is dependent on the host cell protein cellugyrin. Cellugyrin-deficient TIGK cells (TIGK<sup>Cg−</sup>) were prepared using CRISPR/Cas9 gene editing (inset panel (<b>A</b>)). In panel A, TIGK<sup>Cg−</sup> (cross-hatched bars) were compared with TIGK<sup>WT</sup> cells (solid bars) for susceptibility to Cdt-induced cell cycle arrest. The percentage of G2/M cells was determined using propidium iodide fluorescence and flow cytometry; the results are plotted as the percentage of G2/M cells (mean ± SEM) versus Cdt concentration. Panel (<b>B</b>) compares the effect of Cdt (10 pg/mL) on TIGK<sup>WT</sup> and TIGK<sup>Cg−</sup> cell SA-β-gal activity after 72 h; the data are plotted as SA-β-gal fluorescence [MCF; (mean ± SEM)]. Panel (<b>C</b>) shows the effect of Cdt on lipofuscin content in TIGK<sup>WT</sup> and TIGK<sup>Cg−</sup> cells following 96 h exposure to the toxin; results are plotted as lipofuscin content [MCF; (mean ± SEM)]. * indicates statistical significance (<span class="html-italic">p</span> < 0.05) when compared to similarly treated TIGK<sup>WT</sup> cells.</p> "> Figure 7
<p>Model depicting the role of <span class="html-italic">Aa</span>Cdt-induced senescence in the pathogenesis of MIPP. The left panel shows healthy tissue at risk for MIPP due to the presence of supragingival <span class="html-italic">A. actinomycetemcomitans</span> (<span class="html-italic">Aa</span>). Initial exposure to <span class="html-italic">Aa</span>Cdt occurs while the bacteria are at the gingival margin, leading to cell cycle arrest and senescence within the epithelium and concomitant loss of barrier function indicated as distinct gaps between epithelial cells (middle panel). Continued exposure to Cdt along with OE-derived SASP-associated proinflammatory mediators further contribute to increased OE senescence and translocation of <span class="html-italic">A. actinomycetemcomitans</span> into the subgingival tissue (right panel); collectively, the mediators contribute to an altered gingival microenvironment conducive to supporting infection by inflammophilic organisms. Noteworthy, continued exposure to <span class="html-italic">Aa</span>Cdt and/or SASP perpetuates the induction of OE senescence (and possibly fibroblasts) in the face of constant epithelial turnover. Ultimately, these events lead to the recruitment of both innate and acquired immune cells, chronic inflammation and bone destruction.</p> ">
Abstract
:1. Introduction
2. Materials and Methods
2.1. Oral Keratinocyte Culture and Gene Editing
2.2. Preparation of Cdt
2.3. Assessment of Cell Proliferation
2.4. Assessment of SA-β-Gal Activity
2.5. Assessment of Lipofuscin Content
2.6. Analysis of Cytokine Release from TIGK Cells
2.7. Analysis of Epithelial Barrier Integrity
3. Results
3.1. Cdt Induces a Senescent Cell Phenotype in Oral Epithelial Cells
3.2. Cdt Induces Oral Epithelial Cells to Exhibit the Senescent Associated Secretory Phenotype (SASP)
3.3. Cdt-Induced Senescent PGKs Exhibit Loss of Barrier Function
3.4. Cdt-Induced Cellular Senescence Is Dependent on the Host Cell Protein Cellugyrin
4. Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Shenker, B.J.; Korostoff, J.; Walker, L.P.; Zekavat, A.; Dhingra, A.; Kim, T.J.; Boesze-Battaglia, K. Aggregatibacter actinomycetemcomitans Cytolethal Distending Toxin Induces Cellugyrin-(Synaptogyrin 2) Dependent Cellular Senescence in Oral Keratinocytes. Pathogens 2024, 13, 155. https://doi.org/10.3390/pathogens13020155
Shenker BJ, Korostoff J, Walker LP, Zekavat A, Dhingra A, Kim TJ, Boesze-Battaglia K. Aggregatibacter actinomycetemcomitans Cytolethal Distending Toxin Induces Cellugyrin-(Synaptogyrin 2) Dependent Cellular Senescence in Oral Keratinocytes. Pathogens. 2024; 13(2):155. https://doi.org/10.3390/pathogens13020155
Chicago/Turabian StyleShenker, Bruce J., Jonathan Korostoff, Lisa P. Walker, Ali Zekavat, Anuradha Dhingra, Taewan J. Kim, and Kathleen Boesze-Battaglia. 2024. "Aggregatibacter actinomycetemcomitans Cytolethal Distending Toxin Induces Cellugyrin-(Synaptogyrin 2) Dependent Cellular Senescence in Oral Keratinocytes" Pathogens 13, no. 2: 155. https://doi.org/10.3390/pathogens13020155