Chlamydia pneumoniae is present in the dental plaque of periodontitis patients and stimulates an inflammatory response in gingival epithelial cells

Authors

Cassio Almeida-da-Silva, University of the Pacific, School of DentistryFollow
Tamer Alpagot, University of the Pacific, School of DentistryFollow
Sonho Sierra Lee, University of the Pacific, School of Dentistry; University of California, BerkeleyFollow
Brian Roberts, University of the Pacific, School of Dentistry; University of California, Los AngelesFollow
Norina Tang, University of the Pacific, School of DentistryFollow
David Ojcius, University of the Pacific, School of DentistryFollow

Lead Author Affiliation

Biomedical Sciences

Lead Author Program & Year

Faculty/Staff/Researcher

Presentation Category

Research

Introduction/Context/Diagnosis

Periodontitis is the most common oral inflammatory disease. It involves inflammation and destruction of the attachment apparatus of the teeth. Bacterial invasion of the gingiva elicits a host immune response which begins with inflammation of the gingiva, progressing to destruction of deep periodontal tissues and loss of alveolar bone. Even though over 600 species of bacteria are present in the human oral microbiome, only about ten have been identified as putative pathogens causing periodontal disease. Chlamydiae are obligate, intracellular bacteria that have a biphasic developmental cycle. C. pneumoniae has also been linked to asthma, arthritis, atherosclerosis, stroke, multiple sclerosis and Alzheimer’s disease. Currently, it is still not known whether C. pneumoniaei nfects gingival epithelium and subsequently promotes a host-mediated immune response that leads to periodontitis.

Methods/Treatment Plan

Clinical subjects, samples and PCR analysis:

Eighty total subgingival dental plaque samples were obtained from 20 human subjects: 10 with periodontitis and 10 without periodontitis. For each patient, four subgingival plaque samples were collected from different locations of the mouth. All study participants signed consent forms approved by the Institutional Review Board of the University of the Pacific. Exclusion criteria for this study were: a) individuals < 25 years old, b) smokers, c) persons having fewer than 20 teeth, d) individuals requiring premedication with antibiotics prior to periodontal exam, e) persons who took antibiotics, anti-inflammatory drugs, or immunosuppressive drugs within the past 6 months, and f) those who had periodontal therapy within the past 6 months.

Clinical subjects, samples and PCR analysis:

- PCR was performed to amplify the 16S ribosomal RNA gene of C. pneumoniae, P. gingivalis and A. actinomycetemcomitans. Amplicon size was examined by electrophoresis in a 2% agarose gel stained with ethidium bromide and visualized under UV light.

Cell culture and infection with C.pneumoniae

- Human GECs were cultured in supplemented Keratinocyte Serum-Free Medium, human cervical epithelial cell (Hela cells) were obtained from ATCC® (CCL-2™) and cultured in supplemented Minimum Essential Medium. Human lung epithelial cells, A549, were obtained from ATCC® (CCL-185™) and cultured in supplemented F-K12 medium. All cell lines were grown and maintained at 37ºC, 5% CO2. Cells were grown to 70 - 80% confluence and infected with C.pneumoniae at different multiplicity of infection and incubated for different time lengths (as indicated).

C.pneumoniae/ NF-KBimmunofluorescence:

- GECs were infected with C. pneumoniae at a MOI of 5. To visualize the C.pneumoniae in GECs, infected cells were incubated with a fluorescein isothiocyanate (FITC)-conjugated monoclonal antibody from Invitrogen following manufacturer’s instructions.

- GECs were infected with C. pneumoniae and after 3 days samples were collected. A rabbit monoclonal antibody against NF-KB/p65 was used to analyzed NF-кB activation. Fluorescent images were captured using a DMI4000B confocal microscope.

qPCR:

- GECs, HeLa cells, and A549 cells were grow in 6-well plates and infected with C.pneumoniae at indicated MOI. Total RNA was extracted from the infected cells and the concentration was determined using a Nano Drop instrument. cDNAs were synthesized from 2ug RNA using random hexamers and reagents from the Taqman ReverseTranscription Kit.

ELISA

- GECs were infected with C.pneumoniae at MOI=5. Supernatants were collected 0-4 days post infection and ELISA was carried out using a human IL-1B ELISA kit following manufacturer’s instructions.

Western Blot

Cells were seeded into 6-well plates and infected with C. pneumoniae. Supernatants were collected and then subjected to trichloroacetic acid precipitation. Samples were loaded onto 12% SDS-polyacrylamide gels, transferred to PVDF membranes and incubated with rabbit anti-human caspase-1 antibody. Chemiluminescent images were acquired using ChemiDocXRS+ system and analyzed using ImageJ software.

Results/Outcome

The demographic characteristics of the studied population (healthy and periodontitis patients) are presented in Table 1; no statistically significant differences were observed among groups. Table 2 shows the clinical characteristics for our study population.In the periodontitis group, diseased sites exhibited generalized moderate chronic periodontitis.

In our sample population, we found that C. pneumoniae, as well as the well-known periodontal pathogens P. gingivalis and A. actinomycetemcomitans, was significantly more frequent in the diseased group compared with the healthy group (Fig. 1).

Figure 2 shows C. pneumoniae is able to infect GECs in the same extend as it infects cervical and lung epithelial cells. Also, we show that infection increases on day 2, stabilize until day 4 when it starts to decrease.

Figure 3 shows that C. pneumoniae is able to induce activation of the immune transcription factor NF-kB.

Figure 4 shows C. pneumoniae induces IL-1b secretion and caspase-1 activation over time of infection with a peak of secretion at day 4 post infection.

Figure 5 shows that less expression of NLRP3 by siRNA (part of the NLRP3 inflammasome) led to a decreased activation of caspase-1.

Significance/Conclusions

Our study showed for the first time the presence of C. pneumoniae in dental plaques more often in periodontitis samples and in healthy sites, comparing healthy and diseased groups (80 sites from 20 patients). We found that C. pneumoniae can infect human GECs, with maximal growth and replication within inclusions inside the host which was able to induce immune responses. Thus, we hypothesize that, upon C. pneumoniae infection, epithelial cells, macrophages and fibroblasts in the periodontium may secrete pro-inflammatory cytokines involved in the pathogenesis of periodontal disease.

Format

Event