Effect of Periodontal Treatment on Reducing Chronic Inflammation in Systemically Healthy Patients With Periodontal Disease.

BACKGROUND: We determined the effects and an accurate marker of periodontal treatment on serum interleukin (IL)-6 and high-sensitivity C-reactive protein (HsCRP) levels in systemically healthy individuals with periodontal disease. METHODS: This multicenter study included systemically healthy individuals with periodontal disease who received initial periodontal treatment and had no periodontal treatment history. Periodontal parameters, including periodontal inflamed surface area, masticatory efficiency, and periodontal disease classification; serum IL-6 and HsCRP levels; and serum immunoglobulin (Ig)G titers against periodontal pathogens were evaluated at baseline and after treatment. Subjects were classified as low or high responders (group) based on periodontal inflamed surface area changes. RESULTS: There were 153 participants. Only periodontal inflamed surface area changes were markedly different between low and high responders. Periodontal treatment (time point) decreased both serum IL-6 and HsCRP levels. The interaction between group and time point was remarkable only for serum IL-6 levels. Changes in serum immunoglobulin (Ig)G titers against periodontal pathogens were not associated with IL-6 changes in high responders. We analyzed the indirect effect of serum anti-Porphyromonas gingivalis type 2 IgG titer changes using mediation analysis and found no significance. However, the direct effect of group (low or high responder) on IL-6 changes was considerable. CONCLUSIONS: Periodontal treatment effectively decreased serum IL-6 levels, independent of periodontal pathogen infection, in systemically healthy individuals with periodontal disease.

Ingenuity pathway analysis of gingival epithelial cells stimulated with estradiol and progesterone.

OBJECTIVE: Periodontal disease is a risk factor for preterm delivery, and elevated female hormone levels during pregnancy promote hormone-dependent periodontopathogenic bacterial growth and gingivitis. Although the saliva of pregnant women contains female hormones at elevated levels, their effects on the gingiva are poorly understood. Therefore, in this study, we investigated the effects of estradiol and progesterone stimulation on gingival epithelial cells via ingenuity pathway analysis. METHODS: Human gingival epithelial progenitors were cultured in a CnT-Prime medium; 17ß-estradiol (E2) and progesterone (P4) were used as the reagents. Cells treated with dimethyl sulfoxide alone were used as the control group. Cells in the control and experimental groups were incubated for 12 h. RNA was extracted from the cultured cells, RNA-Seq was performed, and pathway analysis was conducted. RESULTS: Differentially expressed genes were detected for 699 (over 2-fold increase) and 348 (decrease) genes in group E2 and for 1448 (increase) and 924 (decrease) genes in group P4 compared with those in the control group (FDR <0.05, n = 4). The z-scores of the pathways suggest that E2 and P4 increased the activity of the wound healing signaling pathway. The activation of this pathway was higher in the E2 and P4 groups than that in the control group. CONCLUSIONS: The results of this study suggest that estradiol and progesterone may affect gingival homeostasis and wound healing.

Increase in Bifidobacterium is a characteristic of the difference in the salivary microbiota of pregnant and non-pregnant women.

BACKGROUND: The establishment of symbiotic microbiota in pregnant women is important for both the mother and her offspring. Little is known about the salivary symbiotic bacteria in pregnancy, and analysis of composition of microbiome (ANCOM) is useful to detect small differences in the number of bacteria. The aim of this study was to investigate the differences in the salivary bacteria between healthy pregnant and non-pregnant women using ANCOM. METHODS: Unstimulated saliva samples were collected from 35 healthy pregnant women at 35 weeks gestation and 30 healthy non-pregnant women during menstruation. All participants underwent a periodontal examination. Estradiol and progesterone levels were examined by enzyme-linked immunosorbent assay. DNA extracted from the saliva was assessed by 16S ribosomal RNA amplicon sequencing and real-time PCR. RESULTS: Salivary estradiol and progesterone levels were significantly increased in pregnant women. The alpha and beta diversities were higher in pregnant women than in non-pregnant women. The largest effect size difference noted when the microbiota of the pregnant and non-pregnant women were analyzed was that for Bifidobacteriales. Levels of Bifidobacterium dentium, but not of Bifidobacterium adolescentis, were significantly increased in pregnant women, and the levels were significantly correlated with progesterone concentration. CONCLUSION: The results suggest that Bifidobacterium and progesterone levels are elevated in the saliva of healthy pregnant women compared with non-pregnant women.

Gut flora alterations due to lipopolysaccharide derived from Porphyromonas gingivalis.

Gut dysbiosis induces 'leaky gut,' a condition associated with diabetes, NASH, and various auto-immune diseases. Porphyromonas gingivalis is a periodontopathic bacterium which causes periodontal tissue breakdown, and often enters the systemic blood flow. Oral administration of P. gingivalis induced gut dysbiosis in mice model, but no systemic administration of P. gingivalis has been reported thus far. In the present study, we investigated the effect of P. gingivalis-derived lipopolysaccharide (Pg-LPS) on the intestinal flora of our established mouse model. Eight-week-old C57BL/6J mice were intraperitoneally administered Pg-LPS. Three months later, DNA was extracted from stool, and RNA from the small and large intestines. After euthanizing the mice, pathological sections of the intestinal tract were prepared and stained with hematoxylin and eosin (H&E). Tumor necrosis factor alpha (TNF-α), interleukin (IL)-1ß, and IL-6 expression levels were evaluated using quantitative PCR. 16S rRNA gene PCR amplicon analysis data were acquired using NGS. Microbial diversity and composition were analyzed using Quantitative Insights into Microbial Ecology 2. Furthermore, alterations in microbial function were performed by PICRUSt2. No significant inflammatory changes were observed in the H&E. No significant differences in the mRNA levels of IL-1ß, IL-6, and TNF-α were observed between the groups. Pg-LPS administration decreased the abundance of Allobacterium in the gut. A predictive metagenomic analysis by PICRUSt2 and STAMP showed that 47 pathways increased and 17 pathways decreased after Pg-LPS administration. Systemic application of periodontal pathogens may cause changes in the intestinal flora which may affect the physiological functions of the intestinal tract.

Draft Genome Sequence of Neisseria mucosa Strain HSUH001, Isolated from an Aggressive Periodontal Lesion.

We report the draft genome sequence (143 contigs, with a total length of 2,424,805 bp and an N 50 value of 36,066 bp) of a bacterium isolated from an aggressive periodontal lesion in a patient. We assigned strain HSUH001 to Neisseria mucosa through a multilocus sequence analysis.

Lamin B receptor-mediated chromatin tethering to the nuclear envelope is detrimental to the Xenopus blastula.

In the nucleus of eukaryotic cells, chromatin is tethered to the nuclear envelope (NE), wherein inner nuclear membrane proteins (INMPs) play major roles. However, in Xenopus blastula, chromatin tethering to the NE depends on nuclear filamentous actin that develops in a blastula-specific manner. To investigate whether chromatin tethering operates in the blastula through INMPs, we experimentally introduced INMPs into Xenopus egg extracts that recapitulate nuclear formation in fertilized eggs. When expressed in extracts in which polymerization of actin is inhibited, only lamin B receptor (LBR), among the five INMPs tested, tethered chromatin to the NE, depending on its N2 and N3 domains responsible for chromatin-protein binding. N2-3-deleted LBR did not tether chromatin, although it was localized in the nuclei. We subsequently found that the LBR level was very low in the Xenopus blastula but was elevated after the blastula stage. When the LBR level was precociously elevated in the blastula by injecting LBR mRNA, it induced alterations in nuclear lamina architecture and nuclear morphology and caused DNA damage and abnormal mitotic spindles, depending on the N2-3 domains. These results suggest that LBR-mediated chromatin tethering is circumvented in the Xenopus blastula, as it is detrimental to embryonic development.

Evaluation of the Virulence of Aggregatibacter actinomycetemcomitans Through the Analysis of Leukotoxin.

Aggregatibacter actinomycetemcomitans is frequently isolated from localized aggressive periodontitis and periodontitis associated with systemic diseases. A. actinomycetemcomitans produces a leukotoxin, which induces apoptosis in human leukocytes. The leukotoxin expression is dependent on the upstream sequence, likely including the promoter, of the gene encoding leukotoxin; strains with the truncated/short upstream sequence express more leukotoxin than strains with the general/long upstream. This chapter addresses the determination of the type of the leukotoxin promoter by PCR analysis, and detection of the apoptosis in the coculture of human monocyte cell line (THP-1) with A. actinomycetemcomitans by the DNA ladder formation, membrane perturbation, and lactate dehydrogenase release.

Involvement of Toll-like receptor 2 in apoptosis of Aggregatibacter actinomycetemcomitans-infected THP-1 cells.

BACKGROUND/PURPOSE: Aggregatibacter (Actinobacillus) actinomycetemcomitans is a gram-negative bacterium that has been associated with aggressive periodontitis. A actinomycetemcomitans infection induces apoptosis in the human monocytic cell line THP-1, and p38 mitogen-activated protein kinase (p38) activity and tumor necrosis factor-α (TNF-α) production are enhanced by A actinomycetemcomitans infection. However, mechanisms governing the recognition of A actinomycetemcomitans by monocytes during apoptosis have not been elucidated. A actinomycetemcomitans cell wall components stimulate Toll-like receptor (TLR)2 and/or TLR4. The authors examined whether TLR2 and/or TLR4 were involved in the apoptosis of A actinomycetemcomitans-infected THP-1 cells. METHODS: A actinomycetemcomitans-infected THP-1 cells were transferred to six-well culture plates and incubated for 0 to 6 hours. In some experiments, THP-1 cells were incubated with anti-TLR2, anti-TLR4, or isotype control antibody (10 µg/mL) for 30 minutes prior to A actinomycetemcomitans infection. Expression of messenger RNA (mRNA) was examined by reverse transcription-polymerase chain reaction. Intracellular bacteria recovered from A actinomycetemcomitans-infected cells and apoptotic cells were detected by APOPercentage dye (Biocolor Ltd, Northern Ireland, UK) staining. Cellular p38 activity and TNF-α production were assessed with enzyme-linked immunosorbent assay. RESULTS: The expression of TLR2 mRNA was increased by A actinomycetemcomitans infection. Phagocytosis and apoptosis in A actinomycetemcomitans-infected THP-1 cells were inhibited by the addition of anti-TLR2 antibody. Also, anti-TLR2 antibody suppressed the activation of p38 and production of TNF-α in A actinomycetemcomitans-infected THP-1 cells. CONCLUSION: These results suggest that A actinomycetemcomitans induces increased expression of TLR2, leading to phagocytosis and apoptosis of THP-1 cells via p38 activation and TNF-α production.

Actinobacillus actinomycetemcomitans induces apoptosis in human monocytic THP-1 cells.

It has previously been reported that the murine macrophage cell line J774.1 and the human oral epithelial cell line KB undergo apoptosis as a result of Actinobacillus actinomycetemcomitans infection. Recent studies have demonstrated that apoptosis regulation is modulated by multiple phosphorylation of several different protein kinases, including the major subtypes of the mitogen-activated protein kinase (MAPK) family. The MAPK family promotes cell survival and/or proliferation in response to growth factor stimulation, or apoptosis in response to various stress stimuli. The primary objective of the present investigation was to clarify whether human immune cells undergo apoptosis following A. actinomycetemcomitans infection and, if so, to establish the involvement of the MAPK family. Human monocytic THP-1 cells were infected with A. actinomycetemcomitans in microtubes. Lactate dehydrogenase release into the culture supernatant and DNA fragmentation in the cells were monitored. DNA fragmentation was also identified by agarose gel electrophoresis. Cell death following A. actinomycetemcomitans infection occurred by apoptosis, shown by an increase in the proportion of fragmented DNA and the typical ladder pattern of DNA fragmentation indicative of apoptosis. Furthermore, p38 MAPK activity and tumour necrosis factor alpha (TNF-alpha) levels increased following A. actinomycetemcomitans infection. In contrast, cell death and TNF-alpha levels in infected cells decreased upon addition of a p38 inhibitor or an anti-TNF-alpha antibody. However, exogenous TNF-alpha could not induce apoptosis in uninfected THP-1 cells. Interestingly, p38 MAPK activity diminished in the presence of anti-TNF-alpha antibody. These findings indicated that A. actinomycetemcomitans infection induces apoptosis in THP-1 cells and that p38 MAPK activity is directly involved in apoptosis. TNF-alpha may play an indirect role in apoptosis via enhanced p38 MAPK activity. A. actinomycetemcomitans-induced apoptosis of human immune cells may be important in terms of initiation and progression of periodontal diseases.

Interaction of Actinobacillus actinomycetemcomitans outer membrane vesicles with HL60 cells does not require leukotoxin.

Outer membrane derived vesicles (MVs) secreted by Actinobacillus actinomycetemcomitans JP2 contain a membranolytic leukotoxin and are toxic to human HL60 cells. To determine how MVs interact with human target cells, HL60 cells were incubated with vesicles, reacted with anti-vesicle antibodies and a FITC-labelled reporter, and visualized by confocal scanning laser microscopy. Target cells rapidly became reactive with anti-vesicle antibodies upon exposure to vesicles. Confocal microscopy showed that labelling occurred primarily in the cytoplasmic membrane and that very little internal fluorescence was observed. The cytoplasmic membrane of HL60 cells was also strongly labelled after exposure to MVs that contained the fluorescent phospholipid, SP-DiOC18. In contrast, incubation of cells with free SP-DiOC18 resulted primarily in the labelling of internal structures of HL60 cells. These results suggest that A. actinomycetemcomitans MVs associate with, or are incorporated into the cytoplasmic membrane of HL60 cells. The leukotoxin is a membranolytic cytotoxin and cells exposed to MVs were lysed by vesicle-associated toxin in a time and dose-dependent manner. However, cells became reactive with anti-vesicle antibodies when MVs were added in the presence of inhibitors of leukotoxin-mediated lysis or when sublytic doses of MVs were analysed. In addition, MVs produced by an isogenic leukotoxin-deficient strain of A. actinomycetemcomitans JP2 were non-toxic but rapidly interacted with HL60 cells. These results suggest that A. actinomycetemcomitans MVs can deliver leukotoxin to HL60 cells but that the association of vesicles with the cytoplasmic membrane occurs independently of the leukotoxin polypeptide.

Nitric oxide-mediated protection of A. actinomycetemcomitans-infected murine macrophages against apoptosis.

We investigated apoptotic cell death in murine macrophage cell line J774.1 following Actinobacillus actinomycetemcomitans infection. Infected macrophages generally kill bacteria within phagosomes with nitric oxide (NO). Our previous study demonstrated that DNA fragmentation in infected cells increased significantly on addition of S-Methylisothiourea (SMT), a selective inhibitor of inducible NO synthetase (iNOS). The purpose of the present study was to determine the mechanism via which NO affects apoptosis of infected macrophages. J774.1 cells were infected with A. actinomycetemcomitans Y4 at a bacterium/cell ratio of 500:1. The infected cells were then cultured in the presence or absence of SMT (400 microM). Culture supernatant was removed 21 h after the infection to measure LDH activity. Additionally, cellular proteins were extracted from the infected cells and measured for histone-associated DNA fragmentation and caspase-1, -3, -5, -6, -8, -9 activities. LDH activity and DNA fragmentation were significantly elevated by the infection; moreover, levels increased further on addition of SMT. Caspase activity of infected cells, particularly caspase-3, was significantly higher than that of uninfected cells. Furthermore, caspase activity increased on addition of SMT. These findings indicate that NO protects infected J774.1 cells, at least in part, against apoptotic cell death via a decrease in caspase activity.

Outer membrane-like vesicles secreted by Actinobacillus actinomycetemcomitans are enriched in leukotoxin.

Actinobacillus actinomycetemcomitans is associated with early onset periodontal diseases and secretes membranous vesicles that appear to contain several virulence-associated proteins. However, the composition of these vesicles and the process leading to their secretion are not well defined. Electron micrographs of thin sectioned bacterial cells and purified vesicle preparations showed that vesicles are spherical lipid bilayers, 50-100 nm in diameter, that appear to form by budding from the outer membrane of the bacterium. Thin layer chromatography identified the predominant lipid components of vesicles as lipopolysaccharide, phosphatidylethanolamine and cardiolipin, similar to the main lipid constituents of the outer membrane. However, vesicles also contained minor lipids that were not detected in outer membrane samples. The major protein constituents of vesicles co-migrated with proteins in outer membrane extracts of A. actinomycetemcomitans, but the outer membrane preparations possessed polypeptides that were not detected in vesicles. Three vesicle proteins were identified; the heat-modifiable OmpA homologue of A. actinomycetemcomitans, a 28 kDa lipoprotein related to the major outer membrane lipoprotein of Mannheimia haemolytica and leukotoxin. Incubation of leukotoxin-sensitive human HL60 cells with vesicles from A. actinomycetemcomitans strains JP2 and 652 resulted in cell lysis, indicating that vesicle-associated leukotoxin is biologically active. Vesicles from the highly leukotoxic strain JP2 were five- to 10-fold more toxic than vesicles from the minimally leukotoxic 652 strain. Furthermore, the specific leukotoxic activity of JP2 vesicles was approximately four- to five-fold higher than isolated outer membrane preparations from JP2, suggesting that vesicles are enriched in leukotoxin. Together, these results suggest that the formation of A. actinomycetemcomitans vesicles occurs by a process that results in the enrichment of leukotoxin.

Human epithelial cell death caused by Actinobacillus actinomycetemcomitans infection.

The gingival sulcus is the shallow crevice around the tooth, and its epithelium is a gateway for initial bacterial infection in periodontal disease. Recent studies have shown that Actinobacillus actinomycetemcomitans invades an epithelial cell line, KB cells, in vitro. The aim of the present study was to clarify the changes in KB cells after A. actinomycetemcomitans infection. The cytotoxic effects of A. actinomycetemcomitans on KB cells were determined at 72, 96 and 120 h after infection by an MTT assay. Nuclear morphological changes were observed by staining with Hoechst 33258. Cytoplasmic histone-associated DNA fragmentation in the infected KB cells was determined by ELISA. A. actinomycetemcomitans was cytotoxic on KB cells, and condensation and degradation of the nuclei were observed. DNA fragmentation was increased after the infection. In addition, A. actinomycetemcomitans showed similar cytotoxic effects on human gingival epithelial cells. The present study demonstrated that A. actinomycetemcomitans induces apoptotic cell death of oral epithelial cells in an in-vitro culture system. This induced apoptosis might be involved in the initiation and progression of periodontitis.