This study investigates the role of Interleukin 17 (IL-17) in exacerbating periapical lesions caused by Porphyromonas gingivalis (Pg) lipopolysaccharides (LPS) in the context of metabolic disease and its potential impact on glucose tolerance. Researchers developed a unique mouse model where mice were monocolonized with Pg to induce periapical lesions. After 1 month, they were fed a high-fat diet (HFD) for 2 months to simulate metabolic disease and oral microbiota dysbiosis. To explore the role of LPS from Pg, wild-type (WT) mice were challenged with purified LPS from Porphyromonas gingivalis, as well as with LPS-depleted and non-depleted Pg bacteria; IL-17 knockout (KO) mice were also included to assess the role of IL-17 signaling. The impact on bone lysis, periapical injury, glucose intolerance, and immune response was assessed. Results showed that in WT mice, the presence of LPS significantly worsened bone lysis, Th17 cell recruitment, and periapical injury. IL-17 KO mice exhibited reduced bone loss, glucose intolerance, and immune cell infiltration. Additionally, inflammatory markers in adipose tissue were lower in IL-17 KO mice, despite increased dysbiosis. The findings suggest that IL-17 plays a critical role in amplifying Pg-induced periapical lesions and systemic metabolic disturbances. Targeting IL-17 recruitment could offer a novel approach to improving glycemic control and reducing type 2 diabetes (T2D) risk in individuals with periapical disease.
Animals ; Porphyromonas gingivalis/immunology* ; Th17 Cells/immunology* ; Lipopolysaccharides/immunology* ; Mice ; Glucose Intolerance/microbiology* ; Interleukin-17/metabolism* ; Mice, Knockout ; Mice, Inbred C57BL ; Disease Models, Animal ; Diet, High-Fat ; Periapical Diseases/microbiology* ; Male ; Dysbiosis

Tight junctions (TJs) are the most apical intercellular junctions of epithelial cells formed by occludin, claudins, junctional adhesion molecules (JAMs), and zonula occludens (ZO). Tight junction proteins can sense the presence of bacteria and regulate the transcription of target genes that encode effectors and regulators of the immune response. The aim of this study was to determine the impact of TJ proteins in response to Porphyromonas gingivalis (P. gingivalis), P. gingivalis lipopolysaccharide (P. gingivalis LPS), and extracellular adenosine triphosphate (ATP) in the oral epithelial cell culture model. Quantified real time-polymerase chain reaction (RT-PCR), immunoblots, and immunostaining were performed to assess the gene and protein expression in TJs. It was found that P. gingivalis infection led to transient upregulation of the genes encoding occludin, claudin-1, and claudin-4 but not JAM-A, claudin-15, or ZO-1, while P. gingivalis LPS increased claudin-1, claudin-15, and ZO-1 and decreased occludin, JAM-A, and claudin-4. Tight junction proteins showed significant upregulation in the above two groups when cells were pretreated with ATP for 3 h. The findings indicated that P. gingivalis induced the host defence responses at an early stage. P. gingivalis LPS exerted a more powerful stimulatory effect on the disruption of the epithelial barrier than P. gingivalis. ATP stimulation enhanced the reaction of TJ proteins to P. gingivalis invasion and LPS destruction of the epithelium.International Journal of Oral Science (2018) 10, e8; doi:10.1038/ijos.2017.51; published online 10 January 2018.
Adenosine Triphosphate ; pharmacology ; Cells, Cultured ; Epithelial Cells ; cytology ; Gene Expression ; Humans ; Immunoblotting ; Lipopolysaccharides ; pharmacology ; Mouth Mucosa ; cytology ; Porphyromonas gingivalis ; immunology ; Real-Time Polymerase Chain Reaction ; Tight Junction Proteins ; metabolism ; Up-Regulation

OBJECTIVEA study was conducted to investigate the effects of Porphyromonas gingivalis lipopolysaccharide (Pg-LPS) on the expression of regulated upon activation normal T-cell expressed and secreted (RANTES) and fractalkine in human umbilical vein endothelial cells (HUVECs).

METHODSHUVECs were incubated with different concentrations of Pg-LPS (200, 500, and 1000 ng x mL(-1)) for 1, 6, 12, and 24 h, respectively. Then real time quantitative polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent method (ELISA) were adopted to detect the protein levels and mRNA levels of RANTES and fractalkine.

RESULTSThe RANTES protein levels and mRNA levels, as well as fractalkine mRNA levels, were significantly higher in all experimental groups of 1, 6, and 12 h than in the control group (P<0.05), except the expression of RANTES mRNA in 200 ng x mL(-1) group of 12 h and RANTES protein in 200 ng x mL(-1) group of 1 h. The expression levels of RANTES mRNA and fractalkine mRNA were highest in 1000 ng x mL(-1) group of 6 h and were 4.88- and 6.20-fold higher, respectively, than those in the control group. The expression levels of RANTES protein, mRNA, and fractalkine mRNA decreased 6 h after stimulation, and were significantly higher than those in the control group (P<0.05) in the RANTES and fractalkine in HUVEC, and such expression is important in the development of atherosclerosis 500 ng x mL(-1) group of 24 h. There was a significant difference between the expression of fractalkine mRNA in 1000 ng x mL(-1) group of 6 and 12 h than in the control group (P<0.05).

CONCLUSIONPg-LPS infection might up-regulate the expression of RANTES and fractalkine in HUVEC, and such expression is important in the development of atherosclerosis.

Atherosclerosis ; Cells, Cultured ; Chemokine CCL5 ; genetics ; metabolism ; Chemokine CX3CL1 ; analysis ; genetics ; metabolism ; Enzyme-Linked Immunosorbent Assay ; Human Umbilical Vein Endothelial Cells ; metabolism ; Humans ; Lipopolysaccharides ; pharmacology ; Porphyromonas gingivalis ; immunology ; isolation & purification ; RNA, Messenger ; analysis ; Reverse Transcriptase Polymerase Chain Reaction ; Up-Regulation

Oral mucosal and salivary lipids exhibit potent antimicrobial activity for a variety of Gram-positive and Gram-negative bacteria; however, little is known about their spectrum of antimicrobial activity or mechanisms of action against oral bacteria. In this study, we examine the activity of two fatty acids and three sphingoid bases against Porphyromonas gingivalis, an important colonizer of the oral cavity implicated in periodontitis. Minimal inhibitory concentrations, minimal bactericidal concentrations, and kill kinetics revealed variable, but potent, activity of oral mucosal and salivary lipids against P. gingivalis, indicating that lipid structure may be an important determinant in lipid mechanisms of activity against bacteria, although specific components of bacterial membranes are also likely important. Electron micrographs showed ultrastructural damage induced by sapienic acid and phytosphingosine and confirmed disruption of the bacterial plasma membrane. This information, coupled with the association of treatment lipids with P. gingivalis lipids revealed via thin layer chromatography, suggests that the plasma membrane is a likely target of lipid antibacterial activity. Utilizing a combination of two-dimensional in-gel electrophoresis and Western blot followed by mass spectroscopy and N-terminus degradation sequencing we also show that treatment with sapienic acid induces upregulation of a set of proteins comprising a unique P. gingivalis stress response, including proteins important in fatty acid biosynthesis, metabolism and energy production, protein processing, cell adhesion and virulence. Prophylactic or therapeutic lipid treatments may be beneficial for intervention of infection by supplementing the natural immune function of endogenous lipids on mucosal surfaces.
Anti-Bacterial Agents ; pharmacology ; Bacterial Proteins ; drug effects ; Colony Count, Microbial ; Fatty Acids ; pharmacology ; Humans ; Lipids ; pharmacology ; Microscopy, Electron ; Mouth Mucosa ; chemistry ; immunology ; microbiology ; Porphyromonas gingivalis ; chemistry ; drug effects ; ultrastructure ; Saliva ; chemistry ; microbiology ; Sphingolipids ; pharmacology ; Virulence ; drug effects

China ; Chronic Periodontitis ; microbiology ; Dental Restoration, Permanent ; Genotype ; Humans ; Periodontal Diseases ; surgery ; Periodontics ; trends ; Periodontitis ; genetics ; immunology ; surgery ; Porphyromonas gingivalis ; isolation & purification

AIMTo elucidate the genetic basis for the pronounced resistance that the oral pathogen, Porphyromonas gingivalis (P. gingivalis), exhibits towards the cationic antimicrobial peptide, polymyxin B.

METHODOLOGYA genetic screen of P. gingivalis clones generated by a Tn4400'-based random insertion mutagenesis strategy was performed to identify bacteria harboring novel genetic mutations that render P. gingivalis susceptible to killing by the cationic antimicrobial peptide, polymyxin B (PMB, 50 microg x mL(-1)).

RESULTSP. gingivalis (ATCC 33277) is unusually resistant to the cationic antimicrobial peptide, PMB at relatively high concentrations (200 microg x mL(-1)). Approximately 2,700 independent Tn4400'-derived mutants of P. gingivalis were examined for increased sensitivity to PMB killing at a relatively low dose (50 microg x mL(-1)). A single PMB-sensitive mutant was obtained in this phenotypic screen. We determined that the Tn4400' transposon was integrated into the gene encoding the lipid A 4'-phosphatase, PGN_0524, demonstrating that this insertion event was responsible for its increased susceptibility of this clone to PMB-dependent killing. The resulting mutant strain, designated 0524-Tn4400', was highly sensitive to PMB killing relative to wild-type P. gingivalis, and exhibited the same sensitivity as the previously characterized strain, 0524KO, which bears a genetically engineered deletion in the PGN_0524 locus. Positive ion mass spectrometric structural (MALDI-TOF MS) analyses revealed that lipid A isolates from 0524-Tn4400' and 0524KO strains displayed strikingly similar MALDI-TOF MS spectra that were substantially different from the wildtype P. gingivalis lipid A spectrum. Finally, intact 0524-Tn4400' and 0524KO mutant bacteria, as well as their corresponding LPS isolates, were significantly more potent in stimulating Toll-like receptor 4 (TLR4)-dependent E-selectin expression in human endothelial cells relative to intact wild-type P. gingivalis or its corresponding LPS isolate.

CONCLUSIONThe combined molecular evidence provided in this report suggests that PGN_0524, a lipid A 4'-phosphatase, is the sole genetic element conferring the ability of the periodontopathogen, P. gingivalis, to evade the killing activity of cationic antimicrobial peptides, such as PMB. These data strongly implicate PGN_0524 as a critical virulence factor for the ability of P. gingivalis to evade front-line host innate defenses that are dependent upon cationic antimicrobial peptide activity and TLR 4 sensing.

Anti-Bacterial Agents ; pharmacology ; Chromosome Mapping ; DNA Transposable Elements ; genetics ; Drug Resistance, Bacterial ; genetics ; E-Selectin ; analysis ; immunology ; Endothelial Cells ; immunology ; microbiology ; Gene Deletion ; Humans ; Lipid A ; analysis ; immunology ; Lipopolysaccharides ; analysis ; immunology ; Mutagenesis, Insertional ; genetics ; Open Reading Frames ; genetics ; Phosphoric Monoester Hydrolases ; genetics ; physiology ; Polymyxin B ; pharmacology ; Porphyromonas gingivalis ; enzymology ; genetics ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ; Toll-Like Receptor 4 ; analysis ; immunology ; Virulence Factors ; physiology

OBJECTIVETo establish the model of cellular inflammatory responses of gingival epithelial cells in vitro induced by Porphyromonas gingivalis vesicle and to probe into the pathogenesis of Porphyromonas gingivalis in periodontitis.

METHODSThe effect of Porphyromonas gingivalis vesicle on prostaglandin E(2) (PGE(2)) production of gingival epithelial cells was detected by ELISA and the effects of Porphyromonas gingivalis vesicle on cyclooxygenase-2 (COX-2), interleukin (IL)-6 and IL-8 mRNA expression in gingival epithelial cells were determined by Real-time reverse transcription-polymerase chain reaction (RT-PCR).

RESULTSPorphyromonas gingivalis vesicle dose-dependently induced PGE(2) production and up-regulated COX-2, IL-6 and IL-8 mRNA expression in gingival epithelial cells significantly.

CONCLUSIONSCellular inflammatory responses of gingival epithelial cells induced by Porphyromonas gingivalis vesicle may contribute to the initiation and progression of periodontitis.

Bacterial Adhesion ; Cells, Cultured ; Cyclooxygenase 2 ; immunology ; metabolism ; Dinoprostone ; immunology ; metabolism ; Epithelial Cells ; immunology ; metabolism ; microbiology ; Gingiva ; immunology ; metabolism ; microbiology ; Humans ; Interleukin-6 ; immunology ; metabolism ; Interleukin-8 ; immunology ; metabolism ; Porphyromonas gingivalis ; immunology ; pathogenicity

OBJECTIVETo evaluate the effect of mechanical periodontal treatment combined with tetracycline on periodontal attachment, distances of cementum-enamel junction (CEJ) to the bone defect bottom and to the alveolar crest, and avidity of serum IgG against Porphyromonas gingivalis (Pg) lipopolysaccharide (LPS) in patients with aggressive periodontitis.

METHODSTwenty-six patients with aggressive periodontitis were divided equally into two groups to receive mechanical periodontal treatment combined with tetracycline or exclusive mechanical periodontal treatment, with another 20 periodontally healthy subjects serving as the control group. In the two patient groups, routine clinical examination of the subjects was performed to record periodontal attachment level and distances of CEJ to the bottom of the bone defects and to the alveolar crest before and 3 and 6 months after the treatment. The avidity of serum IgG against Pg LPS was measured by ELISA with thiocyanate elution before and after periodontal treatment.

RESULTSCompared with patients with exclusive mechanical periodontal treatment, patients with combined treatments showed significant improvement in periodontal attachment level and the distance of CEJ to the bone defect bottom after the treatment (P<0.01 and P<0.05, respectively) with also significantly reduced avidity of serum IgG against Pg LPS (P<0.01).

CONCLUSIONMechanical periodontal treatment combined with tetracycline produces favorable effects in patients with aggressive periodontitis.

Adult ; Anti-Bacterial Agents ; therapeutic use ; Antibodies, Bacterial ; blood ; Combined Modality Therapy ; Female ; Humans ; Immunoglobulin G ; blood ; Lipopolysaccharides ; immunology ; Male ; Periodontitis ; immunology ; therapy ; Porphyromonas gingivalis ; immunology ; Tetracycline ; therapeutic use

OBJECTIVETo observe the protection against periodontal bone loss in the Sprague-Dawley (SD) rats periodontitis model, with the recombined plasmid pcDNA3.1+/kgpcd as DNA gene vaccine.

METHODSPcDNA3.1+/kgpcd was delivered into rats by submandibular gland-targeted injection. The anti-KGPcd sIgA in saliva was measured by indirect ELISA method. Immunohistochemistry staining was used to assess the protection in the animal model.

RESULTSThe level of specific anti-KGPcd sIgA in saliva of the experimental group was significantly higher than that of control group. HE staining showed that immunization with recombined plasmid pcDNA3.1+/kgpcd could protect or minimize tissue destruction caused by subsequent P. gingivalis challenge in the rat model.

CONCLUSIONSThe results indicate that pcDNA3.1+/kgpcd was a good candidate for anti-periodontitis gene vaccine and could provide protection against Porphyromonas gingivalis-caused periodontitis in rat lesion model.

Animals ; Bacterial Vaccines ; immunology ; therapeutic use ; Immunoglobulin A, Secretory ; analysis ; Periodontitis ; immunology ; microbiology ; prevention & control ; Porphyromonas gingivalis ; genetics ; immunology ; Rats ; Rats, Sprague-Dawley ; Vaccines, DNA ; immunology ; therapeutic use

OBJECTIVEThe aim of this study was to investigate effects of immunodeficiency on the periodontal infection characters of the specific pathogens of juvenile periodontitis.

METHODSA total of 36 immunodeficient guinea pigs produced by twice whole-body irradiation with 60Co were divided randomly into four groups, in which Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, and A. actinomycetemcomitans with P. gingivalis were inoculated into the gingival sulcus of two mandibular incisors respectively. The pigs in the control group did not receive any inoculation. At 2, 3 and 6 weeks after inoculation, three animals in each group were sacrificed successively. Clinical and histological examinations were used to examine the changes in the periodontal tissues. The other 36 normal guinea pigs were divided into four groups and treated in a similar way described above.

RESULTSSignificant periodontal damages were noted in immunodeficient pigs inoculated with A. actinomycetemcomitans, P. gingivalis or A. actinomycetemcomitans and P. gingivalis in 2 and 3 weeks after bacterial inoculation. The damages were more severe than in the normal groups. The immunodeficient groups demonstrated larger numbers of osteoclasts than the normal groups (P < 0.05).

CONCLUSIONThe loss of periodontal tissue in immunodeficient hosts is much serious than those with normal defence system, after they are infected with A. actinomycetemcomitans and P. gingivalis. Abnormal defence system in hosts may play an important role in onset and development of juvenile periodontitis.

Actinobacillus Infections ; immunology ; Aggregatibacter actinomycetemcomitans ; Aggressive Periodontitis ; immunology ; microbiology ; Animals ; Bacteroidaceae Infections ; immunology ; Female ; Immunocompromised Host ; immunology ; Male ; Porphyromonas gingivalis ; Random Allocation