Subgingival biofilm microbiome in individuals with asthma and periodontitis: Metagenomic analysis.

OBJECTIVE: This observational study aimed to explore the metagenomics of subgingival biofilms in individuals with varying degrees of asthma, from severe to none, to elucidate the association between the subgingival microbiome and asthma. MATERIALS AND METHODS: Subgingival biofilm samples were collected from thirty participants at the Asthma Control Program Outpatient Clinic in Bahia (ProAR). These samples were categorized into six groups based on the severity of asthma and the presence or absence of periodontitis. We employed next-generation sequencing (Illumina MiSeq), targeting the 16S rRNA gene, to characterize the microbial communities present. Our analysis included descriptive statistics and sequencing data, evaluated using multivariate statistical methods such as the Shannon index, principal coordinate analysis, and the Bray-Curtis dissimilarity. RESULTS: Our findings indicate a higher prevalence of periodontally detrimental bacterial genera in individuals with severe asthma and periodontitis. Additionally, individuals with asthma, but without periodontitis, exhibited a tendency toward dysbiosis, particularly in cases of severe asthma. CONCLUSION: This research provides new insights into the composition of the subgingival microbiome in individuals with varying severities of asthma and periodontitis. The genera identified in this study underscore the need for further investigations to build upon these findings.

Single nucleotide variants in the IL33 and IL1RL1 (ST2) genes are associated with periodontitis and with Aggregatibacter actinomycetemcomitans in the dental plaque biofilm: A putative role in understanding the host immune response in periodontitis.

The Interleukin (IL)-33 is important in several inflammatory diseases and its cellular receptor is the Interleukin 1 receptor-like 1 (IL1RL1), also called suppression of tumorigenicity 2 ligand (ST2L). This study investigated associations between single nucleotide variants (SNVs) in the IL33 gene and in the IL1RL1 (ST2) gene with periodontitis. Additionally, aimed to determine the role of Aggregatibacter actinomycetemcomitans (Aa) relative amount in the subgingival biofilm in these associations. A cross-sectional study was carried out with 506 individuals that answered a structured questionnaire used to collect their health status, socioeconomic-demographic, and behavioral characteristics. Periodontal examination was performed to determine the presence and severity of periodontitis, and subgingival biofilm samples were collected to quantify the relative amount of Aa by real time polymerase chain reaction. Human genomic DNA was extracted from whole blood cells and SNV genotyping was performed. Logistic regression estimated the association measurements, odds ratio (OR), and 95% confidence interval (95%CI), between the IL33 and ST2 genes with periodontitis, and subgroup analyses assessed the relative amount of Aa in these associations. 23% of individuals had periodontitis. Adjusted measurements showed a statistically significant inverse association between two SNVs of the ST2; rs148548829 (C allele) and rs10206753 (G allele). These two alleles together with a third SNV, the rs11693204 (A allele), were inversely associated with moderate periodontitis. One SNV of the IL33 gene also showed a statistically significant inverse association with moderate periodontitis. Nine SNVs of the ST2 gene were inversely associated with the relative amount of Aa. In the high Aa subgroup, there was a direct association between 11 SNVs of the ST2 gene and moderate periodontitis and two SNVs of the ST2 gene and severe periodontitis, and eight SNVs of the ST2 gene and periodontitis. These exploratory findings of genetic variants in IL-33/ST2 axis support the concept that the different tissue responses among individuals with periodontitis may be modulated by the host's genetics, influencing the physiopathology of the disease.

Apoptosis Transcriptional Profile Induced by Porphyromonas gingivalis HmuY.

This study aimed at evaluating the transcriptional profile of apoptosis-related genes after in vitro stimulation of peripheral blood mononuclear cells (PBMCs) derived from individuals with periodontitis (P) and healthy nonperiodontitis (NP) control subjects with P. gingivalis HmuY protein. PBMCs from the P and NP groups were stimulated with HmuY P. gingivalis protein, and the expression of genes related to apoptosis was assessed by custom real-time polymerase chain reaction array (Custom RT2 PCR Array). Compared with the NP group, the P group showed low relative levels of apoptosis-related gene expression, downregulated for FAS, FAS ligand, TNFSF10 (TRAIL), BAK1, CASP9, and APAF1 after P. gingivalis HmuY protein stimulation. Furthermore, the P group exhibited low levels of relative gene expression, downregulated for CASP7 when the cells were not stimulated. Our data suggest that P. gingivalis HmuY protein might participate differently in the modulation of the intrinsic and extrinsic apoptosis pathways.

Production of interferon-gamma, interleukin-6, and interleukin-1ß by human peripheral blood mononuclear cells stimulated with novel lys-gingipain synthetic peptides.

BACKGROUND: Periodontitis is a progressive inflammatory process, and its pathogenesis is related to the presence of a dysbiotic subgingival biofilm that elicits the immune response. Porphyromonas gingivalis is a keystone pathogen, and its Lys-gingipain (Kgp) virulence factor is involved in the pathogen-host interaction through the production of cytokines by host cells, but the specific mechanisms of this interaction have not been elucidated. The present study evaluated the in vitro production of interferon-gamma (IFN-γ), interleukin (IL)-6, and IL-1ß cytokines in response to antigenic stimulation of peripheral blood mononuclear cells (PBMCs) with novel Kgp synthetic peptides. METHODS: Our previous in silico study predicted 16 immunogenic peptides from Kgp protein. Nine peptides derived from different regions of the protein were chemically synthesized. The synthetic peptides Kgp12, 17, and 18 were selected based on the immunoglobulin G immunoreactivity in the serum of patients with periodontitis (P) and individuals without periodontitis (WP), and they were used in in vitro stimulation of PBMC derived from groups P and WP. Enzyme-linked immunosorbent assay and microsphere-based flow cytometric assay were used to verify the levels of the cytokines produced in PBMC cultures after 48 hours. RESULTS: Kgp12, 17, and 18 peptides induced lower production of IFN-γ. Kgp12 induced higher levels of IFN-γ in WP than in P individuals. Kgp12 induced higher production of IL-6 and IL-1ß compared with the other stimuli. CONCLUSION: The novel Kgp synthetic peptides tested herein are immunogenic peptides (epitopes) since they induced the production of cytokines by PBMC and therefore may be useful tools in evaluating the pathogen-host interaction.

Porphyromonas gingivalis HmuY stimulates expression of Bcl-2 and Fas by human CD3+ T cells.

BACKGROUND: Apoptosis is a highly controlled process of cell death that can be induced by periodontopathogens. The present study aimed to investigate the expression of Fas and Bcl-2 proteins by CD3+ T cells in vitro under stimulation by total Porphyromonas gingivalis antigens and purified recombinant P. gingivalis HmuY protein. RESULTS: CD3+ T cells derived from CP patients and stimulated with HmuY expressed higher levels of Bcl-2 compared to identical cells stimulated with P. gingivalis crude extract or cells derived from NP control subjects (p = 0.043). CONCLUSION: The authors hypothesize that P. gingivalis HmuY plays a role in the pathogenesis of chronic periodontitis, possibly by reducing or delaying apoptosis in T cells through a pathway involving the Bcl-2 protein.

Porphyromonas gingivalis antigens differently participate in the proliferation and cell death of human PBMC.

OBJECTIVE: Modulation of cell-mediated immunity by microorganisms in periodontal diseases has been widely studied; however, the proliferative activity and/or programmed death of mononuclear cells under periodontopathogenic stimuli are not yet well understood. The aim of this study was to investigate in vitro proliferation and death of peripheral blood mononuclear cells (PBMC) upon stimulation with Porphyromonas gingivalis (Pg) antigens. DESIGN: In 19 patients with chronic periodontitis (CP) and 16 controls without periodontitis (NP) the following clinical parameters were evaluated: bleeding on probing, probing depth, and clinical attachment level. PBMC were cultured under Pg stimuli and apoptosis/necrosis and proliferation assays were carried out for 18 and 48 h, respectively. Fluorescence of labelled cells was determined using flow cytometry. RESULTS: PBMC of CP and NP subjects exhibited a lower proliferative response to Pg LPS (p<0.05) and HmuY protein (p<0.001) compared with non-stimulated cells. Early apoptosis was induced by Pg LPS (p<0.01) and Pg extract (p<0.05), whilst all antigens induced late apoptosis (Pg LPS: p<0.001; Pg extract: p<0.001; HmuY: p<0.01) and necrosis (Pg LPS: p<0.01; Pg extract: p<0.001; HmuY: p<0.001). Pg LPS induced higher late apoptosis than HmuY (p<0.05). Only Pg LPS-induced necrosis tended to be higher in CP compared with NP. CONCLUSIONS: The inhibitory effect of cell proliferation caused by Pg LPS and HmuY protein is not observed when these antigens comprise Pg extract. Despite induced apoptosis, some still unknown mechanism determines the inflammatory outcome in cell death stimulated by HmuY.