IADR Abstract Archives
Autophagy induction by the periodontal pathogen Aggregatibacter actinomycetemcomitans
Objectives: There is evidence indicating that, in periodontitis, autophagy is stimulated by adverse environmental conditions generated by the immunoinflammatory process. Herein, we investigate if the periodontal pathogen A. actinomycetemcomitans is able to induce host-cell autophagy, and evaluate the role of this homeostatic process during the onset of the periodontitis pathogenesis.
Methods: We have developed an in vitro infection model using A. actinomycetemcomitans serotype b, its purified LPS and junctional epithelium keratinocytes (OKF6/TERT-2 human cell line). Expression of host-cell autophagy markers was assessed by immunoblotting, qRT-PCR and high-resolution confocal microscopy. The effect of A. actinomycetemcomitans on host-cell viability was analyzed by flow cytometry.
Results: Immunofluorescence confocal analysis of OKF6/TERT2 cells infected with A. actinomycetemcomitans serotype b or treated with LPS revealed an extensive Transcription Factor EB (TFEB) nuclear translocation as compared with uninfected cells. Accordingly, proteins regulated by this transcription factor (LC3) and FOXO (Atg5, Atg12 and Becli1) exhibited an increased expression as determined by western blot. Anti-LC3B antibody detected an increase in autophagosome formation in OKF6/TERT2 infected cells as observed by confocal immunofluorescence analysis. 3D reconstruction of confocal images displayed a close association between intracellular bacteria and LC3-positives vesicles. Similarly, OKF6/TERT2 cells treated with LPS, showed an increase amount of LC3-reactive vesicles after 2 hours of incubation. In addition, palatal periodontal tissue from mice infected with A. actinomycetemcomitans serotype b showed Atg5 increased expression, 30 days after infection. In the same context, we evaluate the role of autophagy in cell survival. Treatment of OKF6/TERT2 cells with 3-methyladenine, a recognized autophagy inhibitor, induced an increase in necrotic cell death as determined by flow cytometry.
Conclusions: A. actinomycetemcomitans and its purified LPS induce autophagy in the host-cell and this process has a protective effect on host-cell in early stages of infection.
Methods: We have developed an in vitro infection model using A. actinomycetemcomitans serotype b, its purified LPS and junctional epithelium keratinocytes (OKF6/TERT-2 human cell line). Expression of host-cell autophagy markers was assessed by immunoblotting, qRT-PCR and high-resolution confocal microscopy. The effect of A. actinomycetemcomitans on host-cell viability was analyzed by flow cytometry.
Results: Immunofluorescence confocal analysis of OKF6/TERT2 cells infected with A. actinomycetemcomitans serotype b or treated with LPS revealed an extensive Transcription Factor EB (TFEB) nuclear translocation as compared with uninfected cells. Accordingly, proteins regulated by this transcription factor (LC3) and FOXO (Atg5, Atg12 and Becli1) exhibited an increased expression as determined by western blot. Anti-LC3B antibody detected an increase in autophagosome formation in OKF6/TERT2 infected cells as observed by confocal immunofluorescence analysis. 3D reconstruction of confocal images displayed a close association between intracellular bacteria and LC3-positives vesicles. Similarly, OKF6/TERT2 cells treated with LPS, showed an increase amount of LC3-reactive vesicles after 2 hours of incubation. In addition, palatal periodontal tissue from mice infected with A. actinomycetemcomitans serotype b showed Atg5 increased expression, 30 days after infection. In the same context, we evaluate the role of autophagy in cell survival. Treatment of OKF6/TERT2 cells with 3-methyladenine, a recognized autophagy inhibitor, induced an increase in necrotic cell death as determined by flow cytometry.
Conclusions: A. actinomycetemcomitans and its purified LPS induce autophagy in the host-cell and this process has a protective effect on host-cell in early stages of infection.