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1.
Front Immunol ; 13: 806825, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35250977

RESUMO

Porphyromonas gingivalis is a Gram-negative pathogenic bacterium associated with chronic periodontitis. The development of a chimeric peptide-based vaccine targeting this pathogen could be highly beneficial in preventing oral bone loss as well as other severe gum diseases. We applied a computational framework to design a multi-epitope-based vaccine candidate against P. gingivalis. The vaccine comprises epitopes from subunit proteins prioritized from the P. gingivalis reference strain (P. gingivalis ATCC 33277) using several reported vaccine properties. Protein-based subunit vaccines were prioritized through genomics techniques. Epitope prediction was performed using immunoinformatic servers and tools. Molecular modeling approaches were used to build a putative three-dimensional structure of the vaccine to understand its interactions with host immune cells through biophysical techniques such as molecular docking simulation studies and binding free energy methods. Genome subtraction identified 18 vaccine targets: six outer-membrane, nine cytoplasmic membrane-, one periplasmic, and two extracellular proteins. These proteins passed different vaccine checks required for the successful development of a vaccine candidate. The shortlisted proteins were subjected to immunoinformatic analysis to map B-cell derived T-cell epitopes, and antigenic, water-soluble, non-toxic, and good binders of DRB1*0101 were selected. The epitopes were then modeled into a multi-epitope peptide vaccine construct (linked epitopes plus adjuvant) to enhance immunogenicity and effectively engage both innate and adaptive immunity. Further, the molecular docking approach was used to determine the binding conformation of the vaccine to TLR2 innate immune receptor. Molecular dynamics simulations and binding free energy calculations of the vaccine-TLR2 complex were performed to highlight key intermolecular binding energies. Findings of this study will be useful for vaccine developers to design an effective vaccine for chronic periodontitis pathogens, specifically P. gingivalis.


Assuntos
Vacinas Bacterianas , Infecções por Bacteroidaceae , Porphyromonas gingivalis , Infecções por Bacteroidaceae/prevenção & controle , Periodontite Crônica/prevenção & controle , Biologia Computacional , Epitopos de Linfócito T , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Porphyromonas gingivalis/imunologia , Receptor 2 Toll-Like , Vacinas de Subunidades
2.
J Stroke Cerebrovasc Dis ; 31(3): 106301, 2022 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-35032756

RESUMO

OBJECTIVES: There is accumulating evidence that periodontal disease is associated with atrial fibrillation (AF) or stroke, but it is unclear which causative species of periodontal disease are present in stroke patients with AF. We aimed to investigate the associations between AF and specific periodontal pathogens using serum titers of IgG antibodies of bacteria in acute stroke patients. MATERIALS AND METHODS: Acute stroke patients were registered at two hospitals. Serum samples were evaluated for titers of antibodies against 9 periodontal pathogens (16 genotypes) using ELISAs. We identified AF in patients according to the following criteria: (1) a history of sustained or paroxysmal AF or (2) AF detection upon arrival or during admission. We carried out propensity score matching to categorize the patients as those with AF and those without. RESULTS: Of the 664 acute stroke patients, 123 (18.5%) had AF. After propensity score matching, 234 patients were selected. Patients with AF had a higher prevalence of positive serum titers of antibodies against Porphyromonas gingivalis (FimA type III) and Porphyromonas gingivalis (FimA type V) than those without AF (59.0% vs. 39.3%, p=0.004 and 58.2% vs. 40.2%, p=0.009, respectively). CONCLUSIONS: Porphyromonas gingivalis, especially FimA type III and type V, might be associated with AF in stroke patients.


Assuntos
Fibrilação Atrial , Imunoglobulina G , Doenças Periodontais , Porphyromonas gingivalis , Acidente Vascular Cerebral , Fibrilação Atrial/sangue , Fibrilação Atrial/epidemiologia , Humanos , Imunoglobulina G/sangue , Doenças Periodontais/epidemiologia , Doenças Periodontais/microbiologia , Porphyromonas gingivalis/imunologia , Acidente Vascular Cerebral/sangue , Acidente Vascular Cerebral/epidemiologia
3.
Int J Mol Sci ; 22(24)2021 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-34948405

RESUMO

Periodontitis is characterized by bacterially induced inflammatory destruction of periodontal tissue. This also affects fibroblasts of the human periodontal ligaments (HPdLF), which play a coordinating role in force-induced tissue and alveolar bone remodeling. Excessive inflammation in the oral tissues has been observed with simultaneous stimulation by pathogens and mechanical forces. Recently, elevated levels of growth differentiation factor 15 (GDF15), an immuno-modulatory member of the transforming growth factor (TGFB) superfamily, were detected under periodontitis-like conditions and in force-stressed PdL cells. In view of the pleiotropic effects of GDF15 in various tissues, this study aims to investigate the role of GDF15 in P. gingivalis-related inflammation of HPdLF and its effect on the excessive inflammatory response to concurrent compressive stress. To this end, the expression and secretion of cytokines (IL6, IL8, COX2/PGE2, TNFα) and the activation of THP1 monocytic cells were analyzed in GDF15 siRNA-treated HPdLF stimulated with P. gingivalis lipopolysaccharides alone and in combination with compressive force. GDF15 knockdown significantly reduced cytokine levels and THP1 activation in LPS-stimulated HPdLF, which was less pronounced with additional compressive stress. Overall, our data suggest a pro-inflammatory role for GDF15 in periodontal disease and demonstrate that GDF15 partially modulates the force-induced excessive inflammatory response of PdLF under these conditions.


Assuntos
Infecções por Bacteroidaceae/imunologia , Fibroblastos/imunologia , Fator 15 de Diferenciação de Crescimento/imunologia , Inflamação/imunologia , Lipopolissacarídeos/imunologia , Porphyromonas gingivalis/imunologia , Células Cultivadas , Humanos , Ligamento Periodontal/citologia , Ligamento Periodontal/imunologia , Periodontite/imunologia
4.
J Immunol Res ; 2021: 9577695, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34734092

RESUMO

Periodontitis involves chronic inflammation of the tissues around the teeth caused by plaque and the corresponding immune response. Growth arrest-specific protein 6 (GAS6) and AXL receptor tyrosine kinase (AXL) are known to be involved in inflammatory diseases, while signal transducer and activator of transcription-1 (STAT1) and suppressor of cytokine signaling (SOCS) are related to inflammatory processes. Moreover, miRNA34a directly targets AXL to regulate the AXL expression. However, the specific roles of GAS6 and AXL in periodontitis remain unclear. This study was designed to explore the effect and mechanism of AXL on the expression of inflammatory cytokines induced by Porphyromonas gingivalis lipopolysaccharide (P. gingivalis LPS) in human periodontal ligament cells (hPDLCs). The effects of different concentrations of P. gingivalis LPS on the expression of GAS6/AXL in hPDLCs were observed. Additionally, the effect of LPS on AXL was investigated by transfection of the miRNA34a inhibitor. AXL was knocked down or overexpressed to observe the release of inflammatory cytokines interleukin- (IL-) 8 and IL-6. The results showed that the expression levels of GAS6 and AXL decreased after P. gingivalis LPS infection. Transfection of a miR-34a inhibitor to hPDLCs demonstrated a role of miR-34a in the downregulation of AXL expression induced by LPS. Moreover, AXL knockdown or overexpression influencing the expression of IL-8 and IL-6 was investigated under LPS stimulation. AXL knockdown decreased the expression of STAT1 and SOCS1/3. Overall, these results demonstrate that AXL inhibits the expression of LPS-induced inflammatory cytokines in hPDLCs and that STAT1 and SOCS1/3 are involved in the regulation of inflammation by GAS6/AXL.


Assuntos
Periodontite/imunologia , Porphyromonas gingivalis/imunologia , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Linhagem Celular , Técnicas de Silenciamento de Genes , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Lipopolissacarídeos/imunologia , Ligamento Periodontal/citologia , Ligamento Periodontal/imunologia , Ligamento Periodontal/microbiologia , Ligamento Periodontal/patologia , Periodontite/microbiologia , Periodontite/patologia , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/genética , Fator de Transcrição STAT1/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Proteína 1 Supressora da Sinalização de Citocina/metabolismo , Proteína 3 Supressora da Sinalização de Citocinas/metabolismo
5.
Front Immunol ; 12: 695227, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34484192

RESUMO

Aims: Periodontitis is an independent risk factor for cardiovascular disease, but the mechanistic link is not fully understood. In atherosclerotic cardiovascular disease, monocytes can adopt a persistent hyperresponsive phenotype, termed trained immunity. We hypothesized that periodontitis-associated bacteria can induce trained immunity in monocytes, which subsequently accelerate atherosclerosis development. Materials and Methods: We combined in vitro experiments on human primary monocytes and in vivo techniques in patients with periodontitis to test this hypothesis. Adherent peripheral blood mononuclear cells (PBMCs) were transiently exposed in vitro to Porphyromonas gingivalis for 24 hours, and restimulated with lipopolysaccharide (LPS) or Pam3CysK4 (P3C) six days later, to measure interleukin-6 (IL-6) and tumor necrosis factor α (TNFα) production. In an exploratory observational study, patients with severe periodontitis (63 ± 6 years, n=14) and control subjects with no-to-mild periodontitis (54 ± 10 years, n=14) underwent venipuncture and 2'-deoxy-2'-[18F]fluoro-D-glucose positron-emission-tomography ([18F]FDG PET/CT) scanning. Results: When adherent peripheral blood mononuclear cells (PBMCs) were transiently exposed in vitro to Porphyromonas gingivalis for 24 hours, and restimulated with LPS or P3C six days later, IL-6 and TNFα production was significantly increased (TNFα/P3C, p<0.01). Circulating leukocytes, IL-6 and interleukin-1 receptor antagonist (IL-1Ra) concentrations were generally higher in patients compared to controls (leukocytes: p<0.01; IL-6: p=0.08; IL-1Ra: p=0.10). Cytokine production capacity in PBMCs after 24h stimulation revealed no differences between groups. [18F]FDG PET/CT imaging showed a trend for increased [18F]FDG-uptake in the periodontium [mean standard uptake value (SUVmean), p=0.11] and in femur bone marrow (SUVmean, p=0.06), but no differences were observed for vascular inflammation. Positive correlations between severity of periodontitis, measured by The Dutch Periodontal Screening Index and pocket depth, with circulating inflammatory markers and tissue inflammation were found. Conclusions: P. gingivalis induces long-term activation of human monocytes in vitro (trained immunity). Patients with severe periodontitis did have signs of increased systemic inflammation and hematopoietic tissue activation. However, their circulating monocytes did not show a hyperresponsive phenotype. Together we suggest that trained immunity might contribute to local periodontal inflammation which warrants further investigation.


Assuntos
Aterosclerose/imunologia , Citocinas/metabolismo , Mediadores da Inflamação/metabolismo , Monócitos/imunologia , Periodontite/imunologia , Porphyromonas gingivalis/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Aterosclerose/diagnóstico por imagem , Aterosclerose/metabolismo , Aterosclerose/microbiologia , Estudos de Casos e Controles , Células Cultivadas , Feminino , Interações Hospedeiro-Patógeno , Humanos , Lipopeptídeos/farmacologia , Lipopolissacarídeos/farmacologia , Masculino , Pessoa de Meia-Idade , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Monócitos/microbiologia , Periodontite/diagnóstico por imagem , Periodontite/metabolismo , Periodontite/microbiologia , Fenótipo , Porphyromonas gingivalis/patogenicidade , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Medição de Risco , Fatores de Risco , Índice de Gravidade de Doença
6.
Biochim Biophys Acta Mol Basis Dis ; 1867(11): 166236, 2021 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-34389473

RESUMO

Periodontal diseases are common inflammatory diseases that are induced by infection with periodontal bacteria such as Porphyromonas gingivalis (Pg). The association between periodontal diseases and many types of systemic diseases has been demonstrated; the term "periodontal medicine" is used to describe how periodontal infection/inflammation may impact extraoral health. However, the molecular mechanisms by which the factors produced in the oral cavity reach multiple distant organs and impact general health have not been elucidated. Extracellular vesicles (EVs) are nano-sized spherical structures secreted by various types of cells into the tissue microenvironment, and influence pathophysiological conditions by delivering their cargo. However, a detailed understanding of the effect of EVs on periodontal medicine is lacking. In this study, we investigated whether EVs derived from Pg-infected macrophages reach distant organs in mice and influence the pathophysiological status. EVs were isolated from human macrophages, THP-1 cells, infected with Pg. We observed that EVs from Pg-infected THP-1 cells (Pg-inf EVs) contained abundant core histone proteins such as histone H3 and translocated to the lungs, liver, and kidneys of mice. Pg-inf EVs also induced pulmonary injury, including edema, vascular congestion, inflammation, and collagen deposition causing alveoli destruction. The Pg-inf EVs or the recombinant histone H3 activated the NF-κB pathway, leading to increase in the levels of pro-inflammatory cytokines in human lung epithelial A549 cells. Our results suggest a possible mechanism by which EVs produced in periodontal diseases contribute to the progression of periodontal medicine.


Assuntos
Vesículas Extracelulares/imunologia , Lesão Pulmonar/imunologia , Macrófagos/imunologia , Periodontite/complicações , Porphyromonas gingivalis/imunologia , Células A549 , Animais , Infecções por Bacteroidaceae , Modelos Animais de Doenças , Vesículas Extracelulares/metabolismo , Feminino , Humanos , Lesão Pulmonar/patologia , Macrófagos/citologia , Macrófagos/metabolismo , Camundongos , Periodontite/imunologia , Periodontite/microbiologia , Porphyromonas gingivalis/patogenicidade , Células THP-1
7.
Int J Mol Sci ; 22(15)2021 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-34360848

RESUMO

Titanium is often used in the medical field and in dental implants due to its biocompatibility, but it has a high rate of leading to peri-implantitis, which progresses faster than periodontitis. Therefore, in the present study, the expression of cytokines from gingival epithelial cells by nanotitania was investigated, which is derived from titanium in the oral cavity, and the additional effect of Porphyromonasgingivalis (periodontopathic bacteria) lipopolysaccharide (PgLPS) was investigated. Ca9-22 cells were used as a gingival epithelial cell model and were cultured with nanotitania alone or with PgLPS. Cytokine expression was examined by reverse transcription-quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. In addition, cellular uptake of nanotitania was observed in scanning electron microscopy images. The expression of interleukin (IL)-6 and IL-8 significantly increased in Ca9-22 cells by nanotitania treatment alone, and the expression was further increased by the presence of PgLPS. Nanotitania was observed to phagocytose Ca9-22 cells in a dose- and time-dependent manner. Furthermore, when the expression of IL-11, related to bone resorption, was investigated, a significant increase was confirmed by stimulation with nanotitania alone. Therefore, nanotitania could be associated with the onset and exacerbation of peri-implantitis, and the presence of periodontal pathogens may worsen the condition. Further clinical reports are needed to confirm these preliminary results.


Assuntos
Infecções por Bacteroidaceae/imunologia , Células Epiteliais/imunologia , Gengiva/imunologia , Nanocompostos/efeitos adversos , Peri-Implantite/imunologia , Titânio/efeitos adversos , Linhagem Celular , Citocinas/imunologia , Células Epiteliais/citologia , Gengiva/citologia , Humanos , Lipopolissacarídeos/imunologia , Peri-Implantite/patologia , Porphyromonas gingivalis/imunologia
8.
mBio ; 12(3): e0050221, 2021 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-34182783

RESUMO

Periodontal disease (PD) is an inflammatory disease of the supporting tissues of the teeth that develops in response to formation of a dysbiotic biofilm on the subgingival tooth surface. Although exacerbated inflammation leads to alveolar bone destruction and may cause tooth loss, the molecular basis of PD initiation and progression remains elusive. Control over the inflammatory reaction and return to homeostasis can be efficiently restored by negative regulators of Toll-like receptor (TLR) signaling pathways such as monocyte chemoattractant protein-induced protein 1 (MCPIP-1), which is constitutively expressed in gingival keratinocytes and prevents hyperresponsiveness in the gingiva. Here, we found that inflammophilic periodontal species influence the stability of MCPIP-1, leading to an aggravated response of the epithelium to proinflammatory stimulation. Among enzymes secreted by periodontal species, gingipains-cysteine proteases from Porphyromonas gingivalis-are considered major contributors to the pathogenic potential of bacteria, strongly influencing the components of the innate and adaptive immune system. Gingipain proteolytic activity leads to a rapid degradation of MCPIP-1, exacerbating the inflammatory response induced by endotoxin. Collectively, these results establish a novel mechanism of corruption of inflammatory signaling by periodontal pathogens, indicating new possibilities for treatment of this chronic disease. IMPORTANCE Periodontitis is a highly prevalent disease caused by accumulation of a bacterial biofilm. Periodontal pathogens use a number of virulence strategies that are under intensive study to find optimal therapeutic approaches against bone loss. In our work, we present a novel mechanism utilized by the key periodontal pathogen Porphyromonas gingivalis, based on the selective degradation of the negative regulator of inflammation, MCPIP-1. We found that the diminished levels of MCPIP-1 in gingival keratinocytes-cells at the forefront of the fight against bacteria-cause sensitization to endotoxins produced by other oral species. This results in an enhanced inflammatory response, which promotes the growth of inflammophilic pathobionts and damage of tooth-supporting tissues. Our observation is relevant to understanding the molecular basis of periodontitis and the development of new methods for treatment.


Assuntos
Gengiva/citologia , Inflamação , Queratinócitos/imunologia , Lipopolissacarídeos/metabolismo , Porphyromonas gingivalis/imunologia , Porphyromonas gingivalis/metabolismo , Ribonucleases/metabolismo , Transdução de Sinais , Animais , Biofilmes/crescimento & desenvolvimento , Células Cultivadas , Feminino , Cisteína Endopeptidases Gingipaínas , Queratinócitos/metabolismo , Queratinócitos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Periodontite/microbiologia , Porphyromonas gingivalis/fisiologia , Ribonucleases/genética , Ribonucleases/imunologia , Organismos Livres de Patógenos Específicos
9.
J Leukoc Biol ; 110(3): 461-473, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34057740

RESUMO

Periodontitis induced by bacteria especially Porphyromonas gingivalis (P. gingivalis) is the most prevalent microbial disease worldwide and is a significant risk factor for systemic diseases such as rheumatoid arthritis (RA). RA and periodontitis share similar clinical and pathologic features. Moreover, the prevalence of RA is much higher in patients with periodontitis than in those without periodontitis. To explore the immunologic mechanism of periodontitis involved in RA, we established a mouse model of periodontitis and then induced RA. According to the results of paw thickness, arthritis clinical score, arthritis incidence, microscopic lesion using H&E staining, and micro-CT analysis, periodontitis induced by P. gingivalis promoted the occurrence and development of collagen-induced arthritis (CIA) in mice. Furthermore, periodontitis enhanced the frequency of CD19+ B cells, Th17, Treg, gMDSCs, and mMDSCs, whereas down-regulated IL-10 producing regulatory B cells (B10) in CIA mice preinduced for periodontitis with P. gingivalis. In vitro stimulation with splenic cells revealed that P. gingivalis directly enhanced differentiation of Th17, Treg, and mMDSCs but inhibited the process of B cell differentiation into B10 cells. Considering that adoptive transfer of B10 cells prevent RA development, our study, although preliminary, suggests that down-regulation of B10 cells may be the key mechanism that periodontitis promotes RA as the other main immune suppressive cells such as Treg and MDSCs are up-regulated other than down-regulated in group of P. gingivalis plus CIA.


Assuntos
Artrite Reumatoide/imunologia , Artrite Reumatoide/microbiologia , Periodontite/microbiologia , Porphyromonas gingivalis/patogenicidade , Animais , Antígenos CD19/metabolismo , Artrite Experimental/imunologia , Artrite Experimental/microbiologia , Artrite Reumatoide/diagnóstico por imagem , Modelos Animais de Doenças , Regulação para Baixo , Inflamação/patologia , Camundongos , Células Supressoras Mieloides/metabolismo , Periodontite/diagnóstico por imagem , Periodontite/imunologia , Porphyromonas gingivalis/imunologia , Linfócitos T Reguladores/imunologia , Células Th17/imunologia
10.
J Microbiol Biotechnol ; 31(5): 705-709, 2021 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-33820889

RESUMO

Porphyromonas gingivalis (P. gingivalis) is a major bacterial pathogen that causes periodontitis, a chronic inflammatory disease of tissues around the teeth. Periodontitis is known to be related to other diseases, such as oral cancer, Alzheimer's disease, and rheumatism. Thus, a precise and sensitive test to detect P. gingivalis is necessary for the early diagnosis of periodontitis. The objective of this study was to optimize a rapid visual detection system for P. gingivalis. First, we performed a visual membrane immunoassay using 3,3',5,5'-tetramethylbenzidine (TMB; blue) and coating and detection antibodies that could bind to the host laboratory strain, ATCC 33277. Antibodies against the P. gingivalis surface adhesion molecules RgpB (arginine proteinase) and Kgp (lysine proteinase) were determined to be the most specific coating and detection antibodies, respectively. Using these two selected antibodies, the streptavidin-horseradish peroxidase (HRP) reaction was performed using a nitrocellulose membrane and visualized with a detection range of 103-105 bacterial cells/ml following incubation for 15 min. These selected conditions were applied to test other oral bacteria, and the results showed that P. gingivalis could be detected without crossreactivity to other bacteria, including Streptococcus mutans and Escherichia fergusonii. Furthermore, three clinical strains of P. gingivalis, KCOM 2880, KCOM 2803, and KCOM 3190, were also recognized using this optimized enzyme immunoassay (EIA) system. To conclude, we established optimized conditions for P. gingivalis detection with specificity, accuracy, and sensitivity. These results could be utilized to manufacture economical and rapid detection kits for P. gingivalis.


Assuntos
Técnicas Bacteriológicas/métodos , Porphyromonas gingivalis/isolamento & purificação , Colorimetria , Cisteína Endopeptidases Gingipaínas/imunologia , Humanos , Técnicas Imunoenzimáticas , Limite de Detecção , Periodontite/diagnóstico , Periodontite/microbiologia , Porphyromonas gingivalis/imunologia
11.
PLoS One ; 16(4): e0250177, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33857232

RESUMO

Our previous studies have shown that inoculation of the oral cavity of "humanized" B6.DR1/4 mice with the periodontal pathogen Porphyromonas gingivalis results in an increase in the percentage of circulating Th17 cells, loss of bone and an exacerbation of experimental autoimmune arthritis. The aim of this study was to assess the role played by the human HLA-DRß molecule containing the shared epitope supplied as a transgene to I-A˚ (murine class II null) C57BL/6 (B6) mice in driving these findings. We compared various immune response parameters as well as alveolar and peri-articular bone loss between humanized B6.DR1 (or B6.DR4) mice and their WT (B6) counterparts. We found that the presence of the shared epitope in the context of inoculation with P. gingivalis enhanced the percentage of Th17 cells generated, dramatically enhanced bone loss and importantly allowed for the generation of CCP2⁺ ACPAs that are not found in C57BL/6 or DBA/1 arthritic mouse serum. Due to the exceedingly complex nature of environmental factors impacting on genetic elements, it has been difficult to unravel mechanisms that drive autoimmune arthritis in susceptible individuals. The findings in this study may provide one small piece of this puzzle that can help us to better understand part of this complexity.


Assuntos
Artrite Experimental/imunologia , Artrite Reumatoide/imunologia , Epitopos , Perda do Osso Alveolar/imunologia , Animais , Artrite Experimental/sangue , Artrite Reumatoide/sangue , Biomarcadores/sangue , Humanos , Camundongos , Porphyromonas gingivalis/imunologia
12.
Front Immunol ; 12: 634923, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33717178

RESUMO

Our previous studies showed that a combination of a DNA plasmid encoding Flt3 ligand (pFL) and CpG oligodeoxynucleotides 1826 (CpG ODN) (FL/CpG) as a nasal adjuvant provoked antigen-specific immune responses. In this study, we investigated the efficacy of a nasal vaccine consisting of FimA as the structural subunit of Porphyromonas gingivalis (P. gingivalis) fimbriae and FL/CpG for the induction of FimA-specific antibody (Ab) responses and their protective roles against nasal and lung infection by P. gingivalis, a keystone pathogen in the etiology of periodontal disease. C57BL/6 mice were nasally immunized with recombinant FimA (rFimA) plus FL/CpG three times at weekly intervals. As a control, mice were given nasal rFimA alone. Nasal washes (NWs) and bronchoalveolar lavage fluid (BALF) of mice given nasal rFimA plus FL/CpG resulted in increased levels of rFimA-specific secretory IgA (SIgA) and IgG Ab responses when compared with those in controls. Significantly increased numbers of CD8- or CD11b-expressing mature-type dendritic cells (DCs) were detected in the respiratory inductive and effector tissues of mice given rFimA plus FL/CpG. Additionally, significantly upregulated Th1/Th2-type cytokine responses by rFimA-stimulated CD4+ T cells were noted in the respiratory effector tissues. When mice were challenged with live P. gingivalis via the nasal route, mice immunized nasally with rFimA plus FL/CpG inhibited P. gingivalis colonization in the nasal cavities and lungs. In contrast, controls failed to show protection. Of interest, when IgA-deficient mice given nasal rFimA plus FL/CpG were challenged with nasal P. gingivalis, the inhibition of bacterial colonization in the respiratory tracts was not seen. Taken together, these results show that nasal FL/CpG effectively enhanced DCs and provided balanced Th1- and Th2-type cytokine response-mediated rFimA-specific IgA protective immunity in the respiratory tract against P. gingivalis. A nasal administration with rFimA and FL/CpG could be a candidate for potent mucosal vaccines for the elimination of inhaled P. gingivalis in periodontal patients.


Assuntos
Adjuvantes Imunológicos/administração & dosagem , Anticorpos Antibacterianos/metabolismo , Vacinas Bacterianas/administração & dosagem , Infecções por Bacteroidaceae/prevenção & controle , Proteínas de Fímbrias/administração & dosagem , Imunogenicidade da Vacina , Imunoglobulina A Secretora/metabolismo , Porphyromonas gingivalis/imunologia , Sistema Respiratório/efeitos dos fármacos , Administração Intranasal , Animais , Vacinas Bacterianas/genética , Vacinas Bacterianas/imunologia , Infecções por Bacteroidaceae/imunologia , Infecções por Bacteroidaceae/microbiologia , Modelos Animais de Doenças , Feminino , Proteínas de Fímbrias/genética , Proteínas de Fímbrias/imunologia , Imunidade nas Mucosas/efeitos dos fármacos , Esquemas de Imunização , Proteínas de Membrana/administração & dosagem , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Camundongos Endogâmicos C57BL , Oligodesoxirribonucleotídeos/administração & dosagem , Oligodesoxirribonucleotídeos/imunologia , Porphyromonas gingivalis/patogenicidade , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Sistema Respiratório/imunologia , Sistema Respiratório/metabolismo , Sistema Respiratório/microbiologia , Fatores de Tempo , Vacinas de DNA/administração & dosagem , Vacinas de DNA/imunologia
13.
Infect Immun ; 89(4)2021 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-33361202

RESUMO

Sphingolipids (SLs) are essential structural components of mammalian cell membranes. Our group recently determined that the oral anaerobe Porphyromonas gingivalis delivers its SLs to host cells and that the ability of P. gingivalis to synthesize SLs limits the elicited host inflammatory response during cellular infection. As P. gingivalis robustly produces outer membrane vesicles (OMVs), we hypothesized that OMVs serve as a delivery vehicle for SLs, that the SL status of the OMVs may impact cargo loading to OMVs, and that SL-containing OMVs limit elicited host inflammation similar to that observed by direct bacterial challenge. Transwell cell culture experiments determined that in comparison to the parent strain W83, the SL-null mutant elicited a hyperinflammatory immune response from THP-1 macrophage-like cells with elevated tumor necrosis factor alpha (TNF-α), interleukin 1ß (IL-1ß), and IL-6. Targeted assessment of Toll-like receptors (TLRs) identified elevated expression of TLR2, unchanged TLR4, and elevated expression of the adaptor molecules MyD88 and TRIF (Toll/IL-1 receptor domain-containing adaptor-inducing beta interferon) by SL-null P. gingivalis No significant differences in gingipain activity were observed in our infection models, and both strains produced OMVs of similar sizes. Using comparative two-dimensional gel electrophoresis, we identified differences in the protein cargo of the OMVs between parent and SL-null strain. Importantly, use of purified OMVs recapitulated the cellular inflammatory response observed in the transwell system with whole bacteria. These findings provide new insights into the role of SLs in P. gingivalis OMV cargo assembly and expand our understanding of SL-OMVs as bacterial structures that modulate the host inflammatory response.


Assuntos
Infecções por Bacteroidaceae/imunologia , Infecções por Bacteroidaceae/microbiologia , Macrófagos/imunologia , Porphyromonas gingivalis/imunologia , Porphyromonas gingivalis/metabolismo , Esfingolipídeos/imunologia , Vesículas Transportadoras/imunologia , Infecções por Bacteroidaceae/patologia , Transporte Biológico , Interações Hospedeiro-Patógeno , Imunomodulação , Mutação , Porphyromonas gingivalis/genética , Proteômica/métodos , Esfingolipídeos/metabolismo , Vesículas Transportadoras/metabolismo
14.
Biochim Biophys Acta Mol Basis Dis ; 1867(1): 165991, 2021 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-33080346

RESUMO

Our previous study demonstrated that IL-10 secreting B (B10) cells alleviate inflammation and bone loss in experimental periodontitis. The purpose of this study is to determine whether antigen-specificity is required for the local infiltration of B10 cells. Experimental periodontitis was induced in the recipient mice by placement of silk ligature with or without the presence of live Porphyromonas gingivalis (P. gingivalis). Donor mice were pre-immunized by intraperitoneal (IP) injection of formalin-fixed P. gingivalis, or PBS as non-immunized control. Spleen B cells were purified and treated with LPS and CpG for 48 h to expand the B10 population in vitro. Fluorescence-labelled B10 cells were transferred into the recipient mice by tail vein injection and were tracked on day 0, 3, 5 and 10 using IVIS Spectrum in vivo imaging system. The number of B10 cells and P. gingivalis-binding B cells were significantly increased after in vitro treatment of LPS and CpG. On day 5, the fluorescence intensity in gingival tissues was the highest in mice transferred with B10 cells from pre-immunized donor mice. Gingival expression of IL-6, TNF-α, RANKL/OPG ratio and periodontal bone loss in recipient mice were significantly reduced, and the expression of IL-10 and the number of CD19+ B cells were significantly increased after pre-immunized B10 cell transfer in the presence of antigen, compared to those with non-immunized B10 cell transfer or no antigen presence. This study suggests that antigen specificity dictate the local infiltration of B10 cells into periodontal tissue and these antigen-specific B10 cells promote anti-inflammatory responses.


Assuntos
Antígenos de Bactérias/imunologia , Linfócitos B Reguladores/imunologia , Infecções por Bacteroidaceae , Gengiva , Periodontite , Porphyromonas gingivalis/imunologia , Animais , Infecções por Bacteroidaceae/diagnóstico por imagem , Infecções por Bacteroidaceae/imunologia , Infecções por Bacteroidaceae/microbiologia , Citocinas/imunologia , Diagnóstico por Imagem , Gengiva/diagnóstico por imagem , Gengiva/imunologia , Gengiva/microbiologia , Camundongos , Periodontite/diagnóstico por imagem , Periodontite/imunologia , Periodontite/microbiologia
15.
FEBS Open Bio ; 11(2): 446-455, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33332733

RESUMO

Porphyromonas gingivalis (Pg) is a periodontopathic pathogen that may affect MUC5AC-related mucus hypersecretion along airway epithelial cells. Here, we attempted to establish whether Pg virulence factors (lipopolysaccharide, FimA fimbriae, gingipains) affect MUC5AC in immortalized and primary bronchial cells. We report that MUC5AC gene expression and protein levels are affected by Pg culture supernatant, but not by lipopolysaccharide or FimA fimbriae. Cells treated with either Pg single (Kgp or Rgp) or double (Kgp/Rgp) mutants had altered levels of MUC5AC gene expression and protein levels, and MUC5AC staining of double mutant-treated mouse lung cells showed that MUC5AC protein levels were unaffected. Taken together, we propose that Pg gingipains may be the primary virulence factor that influences both MUC5AC gene expression and protein levels.


Assuntos
Mucina-5AC/metabolismo , Doenças Periodontais/complicações , Porphyromonas gingivalis/imunologia , Infecções Respiratórias/imunologia , Animais , Brônquios/imunologia , Brônquios/metabolismo , Brônquios/patologia , Modelos Animais de Doenças , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Proteínas de Fímbrias/metabolismo , Fímbrias Bacterianas/metabolismo , Cisteína Endopeptidases Gingipaínas/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Masculino , Camundongos , Mucina-5AC/análise , Doenças Periodontais/imunologia , Doenças Periodontais/microbiologia , Porphyromonas gingivalis/metabolismo , Cultura Primária de Células , Mucosa Respiratória/imunologia , Mucosa Respiratória/metabolismo , Infecções Respiratórias/microbiologia , Infecções Respiratórias/patologia , Organismos Livres de Patógenos Específicos , Fatores de Virulência/metabolismo
16.
Ann Rheum Dis ; 80(2): 162-168, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33004333

RESUMO

OBJECTIVES: An increased prevalence of periodontitis and perturbation of the oral microbiome has been identified in patients with rheumatoid arthritis (RA). The periodontal pathogen Porphyromonas gingivalis may cause local citrullination of proteins, potentially triggering anti-citrullinated protein antibody production. However, it is not known if oral dysbiosis precedes the onset of clinical arthritis. This study comprehensively characterised the oral microbiome in anti-cyclic citrullinated peptide (anti-CCP) positive at-risk individuals without clinical synovitis (CCP+at risk). METHODS: Subgingival plaque was collected from periodontally healthy and diseased sites in 48 CCP+at risk, 26 early RA and 32 asymptomatic healthy control (HC) individuals. DNA libraries were sequenced on the Illumina HiSeq 3000 platform. Taxonomic profile and functional capability of the subgingival microbiome were compared between groups. RESULTS: At periodontally healthy sites, CCP+at risk individuals had significantly lower microbial richness compared with HC and early RA groups (p=0.004 and 0.021). Microbial community alterations were found at phylum, genus and species levels. A large proportion of the community differed significantly in membership (523 species; 35.6%) and structure (575 species; 39.1%) comparing CCP+at risk and HC groups. Certain core species, including P. gingivalis, had higher relative abundance in the CCP+at risk group. Seventeen clusters of orthologous gene functional units were significantly over-represented in the CCP+at risk group compared with HC (adjusted p value <0.05). CONCLUSION: Anti-CCP positive at-risk individuals have dysbiotic subgingival microbiomes and increased abundance of P. gingivalis compared with controls. This supports the hypothesis that the oral microbiome and specifically P. gingivalis are important in RA initiation.


Assuntos
Artrite Reumatoide/microbiologia , Disbiose/imunologia , Microbiota/imunologia , Periodontite/microbiologia , Porphyromonas gingivalis/imunologia , Adulto , Anticorpos Anti-Proteína Citrulinada/sangue , Anticorpos Anti-Proteína Citrulinada/imunologia , Artrite Reumatoide/imunologia , Autoanticorpos/sangue , Autoanticorpos/imunologia , Disbiose/microbiologia , Feminino , Gengiva/imunologia , Gengiva/microbiologia , Humanos , Masculino , Pessoa de Meia-Idade , Periodontite/imunologia , Fatores de Risco
17.
Methods Mol Biol ; 2210: 157-166, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32815136

RESUMO

Bacteria release spherical nanobodies, known as membrane vesicles (MVs), during various growth phases. MVs have been gaining recognition as structurally stable vehicles in the last two decades because they deliver a wide range of antigens, virulence factors, and immunomodulators to the host. These functions suggest not only the possible contribution of MVs to pathogenicity but also the potential applicability of low-dose MVs for use as vaccines. Here, we describe a series of methods for isolating MVs of Porphyromonas gingivalis, which is an important species among periodontopathic bacteria. The present chapter also introduces a mouse model of intranasal immunization using MVs from P. gingivalis.


Assuntos
Membrana Externa Bacteriana/imunologia , Vacinas Bacterianas/uso terapêutico , Infecções por Bacteroidaceae/prevenção & controle , Porphyromonas gingivalis/imunologia , Administração Intranasal , Animais , Vacinas Bacterianas/administração & dosagem , Vacinas Bacterianas/imunologia , Infecções por Bacteroidaceae/imunologia , Centrifugação com Gradiente de Concentração/métodos , Modelos Animais de Doenças , Feminino , Imunização , Camundongos , Camundongos Endogâmicos BALB C , Ultracentrifugação/métodos
18.
Arthritis Rheumatol ; 73(4): 568-575, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33205531

RESUMO

OBJECTIVE: Both periodontal disease and cardiovascular disease (CVD) are overrepresented in rheumatoid arthritis (RA). This study was undertaken to investigate the contribution of periodontal pathogens to CVD in RA. METHODS: RA patients underwent assessments of coronary artery calcification (CAC), carotid intima-media thickness and plaque, and ankle-brachial index via computed tomography, ultrasound, and Doppler ultrasound, respectively. Sera were assayed for antibodies targeting Porphyromonas gingivalis (Pg), Aggregatibacter actinomycetemcomitans serotype B (Aa), and Aa-derived leukotoxin A (LtxA). Associations of antibodies against these periodontal pathogens with measures of atherosclerosis were explored using generalized linear models. RESULTS: Among 197 RA patients, anti-Pg was detected in 72 patients (37%), anti-Aa in 41 patients (21%), and anti-LtxA in 84 patients (43%). Adjusting for relevant confounders and reported tooth loss, the mean CAC score was 90% higher in those with anti-Aa and/or anti-LtxA compared with those without either antibody (19 units versus 10 units; P = 0.033). The adjusted odds of CAC ≥100 units were 2.23-fold higher in those with anti-Aa and/or anti-LtxA compared with those without either antibody (P = 0.040). Anti-Aa and/or anti-LtxA seropositivity was significantly associated with all other assessed measures of atherosclerosis except carotid plaque. Anti-Pg was not associated with any measure of atherosclerosis. Higher swollen joint count was associated with CAC exclusively in the group with anti-Aa and/or anti-LtxA. CONCLUSION: Immunoreactivity against Aa and/or its major virulence factor LtxA was associated with atherosclerosis in multiple vascular beds of RA patients and amplified the effect of swollen joints on coronary atherosclerosis, suggesting a role for treatment/prevention of periodontal disease in the prevention of CVD in RA.


Assuntos
Aggregatibacter actinomycetemcomitans/imunologia , Anticorpos Antibacterianos/imunologia , Artrite Reumatoide/imunologia , Aterosclerose/imunologia , Doença da Artéria Coronariana/imunologia , Porphyromonas gingivalis/imunologia , Calcificação Vascular/imunologia , Idoso , Artrite Reumatoide/complicações , Artrite Reumatoide/diagnóstico por imagem , Aterosclerose/complicações , Aterosclerose/diagnóstico por imagem , Espessura Intima-Media Carotídea , Estudos de Casos e Controles , Doença da Artéria Coronariana/complicações , Doença da Artéria Coronariana/diagnóstico por imagem , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fatores de Risco , Índice de Gravidade de Doença , Tomografia Computadorizada por Raios X , Ultrassonografia Doppler , Calcificação Vascular/complicações , Calcificação Vascular/diagnóstico por imagem
19.
Infect Immun ; 89(2)2021 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-33257533

RESUMO

The majority of Gram-negative bacteria elicit a potent immune response via recognition of lipid A expressed on the outer bacterial membrane by the host immune receptor Toll-like receptor 4 (TLR4). However, some Gram-negative bacteria evade detection by TLR4 or alter the outcome of TLR4 signaling by modification of lipid A species. Although the role of lipid A modifications on host innate immunity has been examined in some detail, it is currently unclear how lipid A remodeling influences host adaptive immunity. One prototypic Gram-negative bacterium that modifies its lipid A structure is Porphyromonas gingivalis, an anaerobic pathobiont that colonizes the human periodontium and induces chronic low-grade inflammation that is associated with periodontal disease as well as a number of systemic inflammatory disorders. P. gingivalis produces dephosphorylated and deacylated lipid A structures displaying altered activities at TLR4. Here, we explored the functional role of P. gingivalis lipid A modifications on TLR4-dependent innate and adaptive immune responses in mouse bone marrow-derived dendritic cells (BMDCs). We discovered that lipid A 4'-phosphate removal is required for P. gingivalis to evade BMDC-dependent proinflammatory cytokine responses and markedly limits the bacterium's capacity to induce beta interferon (IFN-ß) production. In addition, lipid A 4'-phosphatase activity prevents canonical bacterium-induced delay in antigen degradation, which leads to inefficient antigen cross-presentation and a failure to cross-prime CD8 T cells specific for a P. gingivalis-associated antigen. We propose that lipid A modifications produced by this bacterium alter host TLR4-dependent adaptive immunity to establish chronic infections associated with a number of systemic inflammatory disorders.


Assuntos
Linfócitos T CD8-Positivos/metabolismo , Apresentação Cruzada/fisiologia , Células Dendríticas/metabolismo , Imunidade Inata/fisiologia , Lipopolissacarídeos/metabolismo , Porphyromonas gingivalis/metabolismo , Porphyromonas gingivalis/patogenicidade , Variação Genética , Genótipo , Interações Hospedeiro-Patógeno , Humanos , Periodonto/microbiologia , Porphyromonas gingivalis/genética , Porphyromonas gingivalis/imunologia
20.
FASEB J ; 35(2): e21171, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33197074

RESUMO

Skeletal muscles have a high metabolic capacity, which play key roles in glucose metabolism. Although periodontal disease increases the risk of metabolic syndrome, the relationship between periodontal bacterial infection and skeletal muscle metabolic dysfunction is unclear. We found that anti-Porphyromonas gingivalis (Pg) antibody titers positively correlated with intramuscular adipose tissue content (IMAC), fasting blood glucose, and HOMA-IR in metabolic syndrome patients. In C57BL/6J mice fed a high-fat diet, recipients of oral Pg (HFPg) had impaired glucose tolerance, insulin resistance, and higher IMAC compared to recipients of saline (HFco). The soleus muscle in HFPg mice exhibited fat infiltration and lower glucose uptake with higher Tnfa expression and lower insulin signaling than in HFco mice. Gene set enrichment analysis showed that TNFα signaling via NFκB gene set was enriched in the soleus muscle of HFPg mice. Moreover, TNF-α also decreased glucose uptake in C2C12 myoblast cells in vitro. Based on 16S rRNA sequencing, Pg administration altered the gut microbiome, particularly by decreasing the abundance of genus Turicibacter. Microbial network of the gut microbiome was dramatically changed by Pg administration. Our findings suggest that infection with Pg is a risk factor for metabolic syndrome and skeletal muscle metabolic dysfunction via gut microbiome alteration.


Assuntos
Infecções por Bacteroidaceae/metabolismo , Glicemia/metabolismo , Microbioma Gastrointestinal/genética , Síndrome Metabólica/sangue , Músculo Esquelético/metabolismo , Doenças Periodontais/sangue , Porphyromonas gingivalis/metabolismo , Tecido Adiposo/metabolismo , Adulto , Idoso , Animais , Anticorpos Antibacterianos/sangue , Anticorpos Antibacterianos/imunologia , Infecções por Bacteroidaceae/microbiologia , Linhagem Celular Transformada , Dieta Hiperlipídica , Fezes/microbiologia , Feminino , Intolerância à Glucose/metabolismo , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Resistência à Insulina , Japão/epidemiologia , Masculino , Síndrome Metabólica/complicações , Síndrome Metabólica/epidemiologia , Síndrome Metabólica/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Mioblastos/metabolismo , Doenças Periodontais/complicações , Doenças Periodontais/epidemiologia , Doenças Periodontais/microbiologia , Porphyromonas gingivalis/genética , Porphyromonas gingivalis/imunologia , RNA Ribossômico 16S/genética
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