RESUMO
Aggregatibacter actinomycetemcomitans is a Gram-negative oral bacterium with high immunostimulatory and pathogenic potential involved in the onset and progression of periodontitis, a chronic disease characterized by aberrant immune responses followed by tooth-supporting bone resorption, which eventually leads to tooth loss. While several studies have provided evidence related to the virulence factors of A. actinomycetemcomitans involved in the host cell death and immune evasion, such as its most studied primate-specific virulence factor, leukotoxin, the role of specific lipopolysaccharide (LPS) domains remain poorly understood. Here, we analyzed the role of the immunodominant domain of the LPS of A. actinomycetemcomitans termed O-polysaccharide (O-PS), which differentiates the distinct bacterial serotypes based on its antigenicity. To determine the role of the O-PS in the immunogenicity and virulence of A. actinomycetemcomitans during periodontitis, we analyzed the in vivo and in vitro effect of an O-PS-defective transposon mutant serotype b strain, characterized by the deletion of the rmlC gene encoding the α-L-rhamnose sugar biosynthetic enzyme. Induction of experimental periodontitis using the O-PS-defective rmlC mutant strain resulted in lower tooth-supporting bone resorption, infiltration of Th1, Th17, and Th22 lymphocytes, and expression of Ahr, Il1b, Il17, Il23, Tlr4, and RANKL (Tnfsf11) in the periodontal lesions as compared with the wild-type A. actinomycetemcomitans strain. In addition, the O-PS-defective rmlC mutant strain led to impaired activation of antigen-presenting cells, with less expression of the co-stimulatory molecules CD40 and CD80 in B lymphocytes and dendritic cells, and downregulated expression of Tnfa and Il1b in splenocytes. In conclusion, these data demonstrate that the O-PS from the serotype b of A. actinomycetemcomitans plays a key role in the capacity of the bacterium to prime oral innate and adaptive immune responses, by triggering the Th1 and Th17-driven tooth-supporting bone resorption during periodontitis.
Assuntos
Aggregatibacter actinomycetemcomitans/imunologia , Aggregatibacter actinomycetemcomitans/patogenicidade , Periodontite/imunologia , Periodontite/microbiologia , Polissacarídeos Bacterianos/imunologia , Virulência/imunologia , Aggregatibacter actinomycetemcomitans/genética , Perda do Osso Alveolar/diagnóstico por imagem , Perda do Osso Alveolar/etiologia , Perda do Osso Alveolar/patologia , Animais , Biologia Computacional/métodos , Modelos Animais de Doenças , Feminino , Interações Hospedeiro-Patógeno/imunologia , Lipopolissacarídeos/imunologia , Camundongos , Mutação , Periodontite/complicações , Periodontite/diagnóstico , Sorogrupo , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Fatores de Virulência , Microtomografia por Raio-XRESUMO
OBJECTIVE: To evaluate the effect of the periodontal treatment on Aggregatibacter actinomycetemcomitans JP2 clone, and the IgG serum levels against its outer membrane protein (Omp29) and A. actinomycetemcomitans serotypes in aggressive periodontitis (AgP). SUBJECTS AND METHODS: Seventeen patients with generalized (GAgP), 10 with localized (LAgP), and 10 healthy controls were included. AgP participants were submitted to periodontal treatment-scaling and root planing plus antibiotics (SRP+A). Periodontal parameters, for example, probing depth (PD) and clinical attachment loss (CAL), were evaluated at baseline and at 1-year. Serum IgG against Omp29 and serotypes a, b, and c were determined by ELISA. The levels of A. actinomycetemcomitans JP2 clone were determined in subgingival biofilm samples by qPCR. RESULTS: Periodontal treatment resulted in significant reductions of PD, CAL, and IgG levels against Omp29, serotypes b, and c. After therapy, IgG levels against A. actinomycetemcomitans serotypes, as well as the levels of the JP2 clone in AgP, became similar to controls. The reduction in JP2 clone count was correlated with a reduction of PD and IgG response against Omp29. CONCLUSION: Scaling and root planing plus antibiotics decreased IgG levels response against Omp29 and A. actinomycetemcomitans serotypes involved in the disease (b and c), while the serum response increased against tne commensal serotype (a), similar to what occurs in periodontally healthy individuals.
Assuntos
Aggregatibacter actinomycetemcomitans/crescimento & desenvolvimento , Aggregatibacter actinomycetemcomitans/imunologia , Periodontite Agressiva/microbiologia , Periodontite Agressiva/terapia , Proteínas da Membrana Bacteriana Externa/imunologia , Imunoglobulina G/sangue , Adolescente , Adulto , Periodontite Agressiva/sangue , Periodontite Agressiva/complicações , Amoxicilina/uso terapêutico , Antibacterianos/uso terapêutico , Contagem de Colônia Microbiana , Feminino , Humanos , Masculino , Metronidazol/uso terapêutico , Perda da Inserção Periodontal/microbiologia , Índice Periodontal , Estudos Prospectivos , Aplainamento Radicular , Sorogrupo , Adulto JovemRESUMO
Individuals carrying the ATC/TTC haplotype (Hap-1) in the interleukin 8 (IL8) gene were reported as more susceptible to chronic periodontitis (CP), an infectious disease associated with Gram-negative bacteria, in comparison to patients with the ATT/TTC haplotype (Hap-2). This study investigated the functionality of the IL8 haplotypes in lymphocytes and monocytes of individuals carrying the Hap-1 or Hap-2 IL8 haplotypes in the response to CP-associated Gram-negative bacteria (periodontopathogens). Peripheral blood was collected from 6 subjects carrying each haplotype, and their immune cells were challenged with periodontopathogens or phorbol 12-myristate 13-acetate (PMA) plus Ionomycin. Depending on the immune cell type (lymphocytes or monocyte-derived macrophages) the assessed outcomes were: phenotypical polarization, gene expression, phagocytic activity, chemotaxis and production of reactive oxygen species (ROS). Subjects carrying the Hap-1 haplotype showed increased expression of IL8 and TNFA and significantly skewing towards pro-inflammatory Th1/M1/Th17 phenotypes. There was increased percentage of ROS-producing monocyte-derived macrophages from individuals carrying the Hap-1 haplotype. Cells from individuals presenting the Hap-2 haplotype had an overall attenuated response to periodontopathogens, with a significant shift towards the Treg phenotype. In conclusion, the IL8 haplotypes showed to be functional both in monocyte-derived macrophages and lymphocytes. The Hap-1 haplotype previously associated with increased susceptibility to CP demonstrated greater skewing to pro-inflammatory Th1/M1/Th17 phenotypes and production of ROS.
Assuntos
Periodontite Crônica , Bactérias Gram-Negativas/imunologia , Bactérias Gram-Negativas/patogenicidade , Interleucina-8/genética , Linfócitos/metabolismo , Macrófagos/metabolismo , Aggregatibacter actinomycetemcomitans/imunologia , Aggregatibacter actinomycetemcomitans/patogenicidade , Periodontite Crônica/genética , Periodontite Crônica/imunologia , Periodontite Crônica/microbiologia , Feminino , Predisposição Genética para Doença , Infecções por Bactérias Gram-Negativas/genética , Infecções por Bactérias Gram-Negativas/imunologia , Haplótipos , Humanos , Interleucina-8/metabolismo , Linfócitos/imunologia , Macrófagos/imunologia , Masculino , Pessoa de Meia-Idade , Monócitos/imunologia , Monócitos/metabolismo , Fenótipo , Porphyromonas gingivalis/imunologia , Porphyromonas gingivalis/patogenicidadeRESUMO
OBJECTIVE: In Aggregatibacter actinomycetemcomitans, different serotypes have been described based on LPS antigenicity. Mixed infection with the different A. actinomycetemcomitans serotypes is frequent in periodontitis patients; accordingly, the role of this bacterial species in the pathogenesis of periodontitis may differ depending whether patients or periodontal lesions harbour one or more of the A. actinomycetemcomitans serotypes. We hypothesized that different combinations of these serotypes could be associated with distinct host responses and hence different inflammatory patterns. This investigation was aimed to assess whether the increased immuno-stimulatory potential attributed to the serotype b of A. actinomycetemcomitans on immune cells is able to be modified during co-infection with other A. actinomycetemcomitans serotypes. METHODS: Dendritic cells (DCs) were obtained from healthy subjects and stimulated with the different A. actinomycetemcomitans serotypes or their purified LPS using the following stimulatory conditions: serotype a, b, or c, and the combinations a+b, a+c, b+c, or a+b+c. The cytokine, CCR, and CCL levels were quantified by qPCR and ELISA. RESULTS: Higher levels of cytokines, CCRs, and CCLs were induced when DCs were stimulated with the serotype b of A. actinomycetemcomitans compared with the same cells stimulated with the other serotypes. When DCs were co-infected, these levels decreased in comparison with the serotype b-stimulation alone, in particular when the serotype a was present in the mixed infection. CONCLUSIONS: The increased immuno-stimulatory potential attributed to the serotype b was modified when DCs were co-infected with other A. actinomycetemcomitans serotypes, in particular, when the serotype a was present, the DC response diminished.
Assuntos
Aggregatibacter actinomycetemcomitans/imunologia , Citocinas/imunologia , Células Dendríticas/imunologia , Adulto , Quimiocinas/imunologia , Regulação para Baixo , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Lipopolissacarídeos , Masculino , Reação em Cadeia da Polimerase , SorogrupoRESUMO
OBJECTIVE: In Aggregatibacter actinomycetemcomitans, different serotypes have been described based on lipopolysaccharide (LPS) antigenicity. When T lymphocytes were stimulated with these serotypes, different patterns of T-helper (Th)1 and Th17-type of immune responses were reported. Recently, two new Th phenotypes have been described and named Th9 and Th22 lymphocytes; however, their role in the pathogenesis of periodontitis remains unclear. This study aimed to investigate the potential Th9 and/or Th22 lymphocyte responses when stimulated with autologous dendritic cells infected with different A. actinomycetemcomitans serotypes. METHODS: Monocyte-derived dendritic cells and naïve CD4+ T lymphocytes were obtained from healthy donors and stimulated with different serotypes of A. actinomycetemcomitans at a multiplicity of infection MOI=102 or their purified LPS (10-50ng/ml). The levels for the Th9 and Th22-associated cytokines, as well as the transcription factor master-switch genes implied in their differentiation Spi-B and AhR, were quantified by qPCR and ELISA. RESULTS: When stimulated with the serotype b of A. actinomycetemcomitans, higher levels of interleukin (IL)-6 and tumor necrosis factor (TNF)-α were detected in dendritic cells, as well as higher levels of IL-22 and AhR were detected in T lymphocytes, when compared with stimulation with the other serotypes. CONCLUSIONS: The serotype b of A. actinomycetemcomitans has a higher capacity of trigger Th22-type of immune response in both dendritic cells and T lymphocytes. These data allow us to suggest that, when the serotype b of A. actinomycetemcomitans is a significant part of the subgingival biofilm, the Th22 polarization might be triggered within the periodontal lesion.
Assuntos
Aggregatibacter actinomycetemcomitans/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Aggregatibacter actinomycetemcomitans/patogenicidade , Biofilmes , Diferenciação Celular , Citocinas/imunologia , Citocinas/metabolismo , Humanos , Fenótipo , Sorogrupo , Células Th17 , Fatores de VirulênciaRESUMO
Short-chain fatty acids (SCFAs), predominantly acetic, propionic, and butyric acids, are bacterial metabolites with an important role in the maintenance of homeostasis due to their metabolic and immunomodulatory actions. Some evidence suggests that they may also be relevant during infections. Therefore, we aimed to investigate the effects of SCFAs in the effector functions of neutrophils to an opportunistic pathogenic bacterium, Aggregatibacter actinomycetemcomitans. Using a subcutaneous model to generate a mono, isolated infection of A. actinomycetemcomitans, we demonstrated that the presence of the SCFAs in situ did not affect leukocyte accumulation but altered the effector mechanisms of migrating neutrophils by downregulating the production of cytokines, their phagocytic capacity, and killing the bacteria, thus impairing the containment of A. actinomycetemcomitans. Similar effects were observed with bacteria-stimulated neutrophils incubated with SCFAs in vitro. These effects were independent of free-fatty acid receptor 2 (FFAR2) activation, the main SCFA receptor expressed on neutrophils, occurring possibly through inhibition of histone deacetylases because similar effects were obtained by using histone deacetylase inhibitors, such as SAHA, MS-275, and RGFP 966. Considering the findings of this study, we hypothesized that in an infectious condition, SCFAs may exert a detrimental effect on the host by inhibiting neutrophil's effector functions.
Assuntos
Ácido Acético/farmacologia , Aggregatibacter actinomycetemcomitans/imunologia , Butiratos/farmacologia , Neutrófilos/imunologia , Infecções por Pasteurellaceae/imunologia , Propionatos/farmacologia , Ácido Acético/metabolismo , Acrilamidas/farmacologia , Animais , Butiratos/metabolismo , Citocinas/biossíntese , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Inflamação/imunologia , Contagem de Leucócitos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Nylons/farmacologia , Infecções por Pasteurellaceae/microbiologia , Fagocitose/efeitos dos fármacos , Fenilenodiaminas/farmacologia , Propionatos/metabolismo , Pirróis/farmacologiaRESUMO
Periodontitis is a chronic inflammatory condition characterized by destruction of non-mineralized and mineralized connective tissues. It is initiated and maintained by a dysbiosis of the bacterial biofilm adjacent to teeth with increased prevalence of Gram-negative microorganisms. Nucleotide-binding oligomerization domain containing 1 (NOD1) is a member of the Nod-like receptors (NLRs) family of proteins that participate in the activation of the innate immune system, in response to invading bacteria or to bacterial antigens present in the cytoplasm. The specific activating ligand for NOD1 is a bacterial peptidoglycan derived primarily from Gram-negative bacteria. This study assessed the role of NOD1 in inflammation-mediated tissue destruction in the context of host-microbe interactions. We used mice with whole-genome deletion of the NOD1 gene in a microbe-induced periodontitis model using direct injections of heat-killed Gram-negative or Gram-negative/Gram-positive bacteria on the gingival tissues. In vitro experiments using primary bone-marrow-derived macrophages from wild-type and NOD1 knockout mice provide insight into the role of NOD1 on the macrophage response to Gram-negative and Gram-negative/Gram-positive bacteria. Microcomputed tomography analysis indicated that deletion of NOD1 significantly aggravated bone resorption induced by Gram-negative bacteria, accompanied by an increase in the numbers of osteoclasts. This effect was significantly attenuated by the association with Gram-positive bacteria. In vitro, quantitative PCR arrays indicated that stimulation of macrophages with heat-killed Gram-negative bacteria induced the same biological processes in wild-type and NOD1-deficient cells; however, expression of pro-inflammatory mediators was increased in NOD1-deficient cells. These results suggest a bone-sparing role for NOD1 in this model.
Assuntos
Aggregatibacter actinomycetemcomitans/imunologia , Reabsorção Óssea/imunologia , Gengiva/imunologia , Limosilactobacillus fermentum/imunologia , Macrófagos/fisiologia , Proteína Adaptadora de Sinalização NOD1/metabolismo , Doenças Periodontais/imunologia , Animais , Antígenos de Bactérias/imunologia , Reabsorção Óssea/microbiologia , Células Cultivadas , Modelos Animais de Doenças , Gengiva/microbiologia , Interações Hospedeiro-Patógeno , Humanos , Mediadores da Inflamação/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Adaptadora de Sinalização NOD1/genética , Osteoclastos/patologia , Doenças Periodontais/microbiologiaRESUMO
During periodontitis, alveolar bone resorption is associated with activation of T helper type 17 (Th17) lymphocytes and receptor activator of nuclear factor-κB ligand (RANKL) -induced osteoclasts. We previously reported that serotype b of Aggregatibacter actinomycetemcomitans has a higher capacity to trigger Th17-type differentiation and function in activated T lymphocytes and its lipopolysaccharide is a more potent immunogen compared with the other serotypes. This study aimed to investigate whether serotype b of A. actinomycetemcomitans induces higher Th17-associated RANKL production, RANKL-induced osteoclast activation, and antigen-specific memory T lymphocyte proliferation. On naive CD4(+) T lymphocytes stimulated with autologous dendritic cells primed with different A. actinomycetemcomitans serotypes, RANKL production, T-bet, GATA-3, RORC2 and Foxp3 expression, RORC2/RANKL intracellular double-expression, TRAP(+) osteoclast activation, and bone resorption were quantified. The frequency of proliferating memory T lymphocytes in response to A. actinomycetemcomitans serotypes was determined in periodontitis and healthy subjects. Naive CD4(+) T lymphocytes stimulated by serotype b-primed dendritic cells elicited higher levels of RANKL, RORC2, TRAP(+) osteoclasts, and bone resorption than the same cells stimulated with the other serotypes. RANKL positively correlated and co-expressed with RORC2. Memory T lymphocytes responding to serotype b were more frequently detected in periodontitis patients than healthy subjects. These results indicate that serotype b of A. actinomycetemcomitans is associated with higher production of RANKL and these increased levels are associated with Th17 lymphocyte induction, osteoclast activation, and bone resorption.
Assuntos
Aggregatibacter actinomycetemcomitans/imunologia , Osteoclastos/imunologia , Linfócitos T/imunologia , Antígenos de Bactérias/imunologia , Antígenos de Bactérias/farmacologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/microbiologia , Diferenciação Celular/imunologia , Periodontite Crônica/imunologia , Periodontite Crônica/microbiologia , Células Dendríticas/imunologia , Fatores de Transcrição Forkhead/biossíntese , Fator de Transcrição GATA3/biossíntese , Humanos , Memória Imunológica/imunologia , Lipopolissacarídeos/farmacologia , Ativação Linfocitária , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/biossíntese , Ligante RANK/imunologia , Sorogrupo , Linfócitos T/microbiologiaRESUMO
UNLABELLED: In Aggregatibacter actinomycetemcomitans, different serotypes have been described based on LPS antigenicity. Recently, our research group has reported a differential immunogenicity when T lymphocytes were stimulated with these different serotypes. In particular, it was demonstrated that the serotype b of A. actinomycetemcomitans has a stronger capacity to trigger Th1- and Th17-type cytokine production. OBJECTIVE: This study aimed to quantify the expression of different CC chemokines (CCLs) and receptors (CCRs) in T lymphocytes stimulated with the differentA. actinomycetemcomitans serotypes. In addition, the expression of the transcription factors T-bet, GATA-3, RORC2, and Foxp3, master-switch genes implied in the Th1, Th2, Th17, and T-regulatory differentiation, respectively, was analysed in order to determine T-cell phenotype-specific patterns of CCL and CCR expression upon A. actinomycetemcomitans stimulation. MATERIAL AND METHODS: Human naïve CD4+ T lymphocytes were obtained from healthy subjects and stimulated with autologous dendritic cells primed with the differentA. actinomycetemcomitans serotypes. The expression levels for the chemokines CCL1, CCL2, CCL3, CCL5, CCL11, CCL17, CCL20, CCL21, CCL25, and CCL28, as well as the chemokine receptors CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, and CCR10 were quantified by qPCR. Similarly, the expression levels for the transcription factors T-bet, GATA-3, RORC2, and Foxp3 were quantified and correlated with the CCL and CCR expression levels. RESULTS: Higher expression levels of CCL2, CCL3, CCL5, CCL20, CCL21, CCL28, CCR1, CCR2, CCR5, CCR6, CCR7, and CCR9 were detected in T lymphocytes stimulated with the serotype b of A. actinomycetemcomitans compared with the other serotypes. In addition, these higher expression levels of CCLs and CCRs positively correlated with the increased levels of T-bet and RORC2 when T lymphocytes were stimulated with the serotype b. CONCLUSION: A T-lymphocyte response biased towards a Th1- and Th17-pattern of CCL and CCR expression was detected under stimulation with the serotype b ofA. actinomycetemcomitans.
Assuntos
Aggregatibacter actinomycetemcomitans/imunologia , Antígenos de Diferenciação de Linfócitos T/análise , Quimiocinas CC/análise , Receptores CCR/análise , Linfócitos T/imunologia , Adulto , Aggregatibacter actinomycetemcomitans/genética , Antígenos de Diferenciação de Linfócitos T/genética , Antígenos de Diferenciação de Linfócitos T/imunologia , Diferenciação Celular/imunologia , Células Cultivadas , Quimiocinas CC/genética , Quimiocinas CC/imunologia , Células Dendríticas/imunologia , Feminino , Citometria de Fluxo , Humanos , Ativação Linfocitária , Masculino , Reação em Cadeia da Polimerase , Receptores CCR/genética , Receptores CCR/imunologia , Sorogrupo , Adulto JovemRESUMO
In Aggregatibacter actinomycetemcomitans, different serotypes have been described based on LPS antigenicity. Recently, our research group has reported a differential immunogenicity when T lymphocytes were stimulated with these different serotypes. In particular, it was demonstrated that the serotype b of A. actinomycetemcomitans has a stronger capacity to trigger Th1- and Th17-type cytokine production.Objective This study aimed to quantify the expression of different CC chemokines (CCLs) and receptors (CCRs) in T lymphocytes stimulated with the differentA. actinomycetemcomitans serotypes. In addition, the expression of the transcription factors T-bet, GATA-3, RORC2, and Foxp3, master-switch genes implied in the Th1, Th2, Th17, and T-regulatory differentiation, respectively, was analysed in order to determine T-cell phenotype-specific patterns of CCL and CCR expression upon A. actinomycetemcomitans stimulation.Material and Methods Human naïve CD4+ T lymphocytes were obtained from healthy subjects and stimulated with autologous dendritic cells primed with the differentA. actinomycetemcomitans serotypes. The expression levels for the chemokines CCL1, CCL2, CCL3, CCL5, CCL11, CCL17, CCL20, CCL21, CCL25, and CCL28, as well as the chemokine receptors CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, and CCR10 were quantified by qPCR. Similarly, the expression levels for the transcription factors T-bet, GATA-3, RORC2, and Foxp3 were quantified and correlated with the CCL and CCR expression levels.Results Higher expression levels of CCL2, CCL3, CCL5, CCL20, CCL21, CCL28, CCR1, CCR2, CCR5, CCR6, CCR7, and CCR9 were detected in T lymphocytes stimulated with the serotype b of A. actinomycetemcomitans compared with the other serotypes. In addition, these higher expression levels of CCLs and CCRs positively correlated with the increased levels of T-bet and RORC2 when T lymphocytes were stimulated with the serotype b.Conclusion A T-lymphocyte response biased towards a Th1- and Th17-pattern of CCL and CCR expression was detected under stimulation with the serotype b ofA. actinomycetemcomitans.
Assuntos
Humanos , Masculino , Feminino , Adulto , Adulto Jovem , Aggregatibacter actinomycetemcomitans/imunologia , Antígenos de Diferenciação de Linfócitos T/análise , Quimiocinas CC/análise , Receptores CCR/análise , Linfócitos T/imunologia , Aggregatibacter actinomycetemcomitans/genética , Antígenos de Diferenciação de Linfócitos T/genética , Antígenos de Diferenciação de Linfócitos T/imunologia , Diferenciação Celular/imunologia , Células Cultivadas , Quimiocinas CC/genética , Quimiocinas CC/imunologia , Células Dendríticas/imunologia , Citometria de Fluxo , Ativação Linfocitária , Reação em Cadeia da Polimerase , Receptores CCR/genética , Receptores CCR/imunologia , SorogrupoRESUMO
BACKGROUND AND OBJECTIVE: Based on lipopolysaccharide (LPS) antigenicity, different Aggregatibacter actinomycetemcomitans serotypes have been described. Serotype b strains have demonstrated a stronger capacity to trigger cytokine production on dendritic cells (DCs). As DCs regulate the development of T-lymphocyte lineages, the objective of this investigation was to study the response of T lymphocytes after being stimulated with autologous DCs primed with different bacterial strains belonging to the most prevalent serotypes of A. actinomycetemcomitans in humans: a-c. MATERIAL AND METHODS: Human DCs were primed with increasing multiplicity of infection (10(-1) -10(2) ) or the purified LPS (10-50 ng/mL) of A. actinomycetemcomitans serotypes a-c and then used to stimulate autologous naïve CD4(+) T lymphocytes. The T-helper (Th) type 1, Th2, Th17 and T-regulatory transcription factors T-bet, GATA-3, RORC2 and Foxp3, which are the master-switch genes implied in their specific differentiation, as well as T-cell phenotype-specific cytokine patterns were quantified by real-time reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. In addition, the intracellular expression of T-bet/interferon-γ, GATA-3/interleukin-4, RORC2/interleukin-17A and Foxp3/transforming growth factor-ß1 was analysed by double staining and flow cytometry. RESULTS: All the A. actinomycetemcomitans serotypes led to T-lymphocyte activation; however, when T lymphocytes were stimulated with DCs primed with the A. actinomycetemcomitans serotype b strain or their purified LPS, higher levels of Th1- and Th17-associated transcription factors and cytokines were detected compared with similar experiments with the other serotypes. CONCLUSION: These results demonstrate that serotype b of A. actinomycetemcomitans has a higher capacity of trigger Th1 and Th17 phenotype and function and it was demonstrated that their LPS is a more potent immunogen compared with the other serotypes.
Assuntos
Aggregatibacter actinomycetemcomitans/imunologia , Fenótipo , Sorogrupo , Linfócitos T/imunologia , Aggregatibacter actinomycetemcomitans/classificação , Células Cultivadas , Citocinas/análise , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Perfilação da Expressão Gênica , Humanos , Imunofenotipagem , Lipopolissacarídeos/imunologia , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/análiseRESUMO
BACKGROUND: Different serotypes of Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis have been shown to induce differential dendritic cell (DC) responses. This study investigates whether cytokine and CC-chemokine receptor (CCR) production by DCs stimulated with different serotypes of A. actinomycetemcomitans or P. gingivalis is Toll-like receptor 2 (TLR2) and/or TLR4 dependent. METHODS: DCs were obtained from healthy individuals and primed at a multiplicity of infection (MOI) of 10(2) with different A. actinomycetemcomitans or P. gingivalis serotypes in the presence or absence of anti-TLR2 or anti-TLR4 blocking antibodies. TLR2 and TLR4 expression, CCR5 and CCR6 expression, and interleukin (IL)-1ß, IL-10, IL-12, and IL-23 expression and secretion were quantified by flow cytometry, real-time reverse-transcription polymerase chain reaction, and enzyme-linked immunosorbent assay. RESULTS: When DCs were stimulated with serotype b of A. actinomycetemcomitans or serotype K1 of P. gingivalis, higher levels of TLR2 or TLR4, respectively, were detected compared to DCs stimulated with the other serotypes. Similarly, higher levels of cytokines and CCRs were detected in serotype b- or serotype K1-primed DCs compared to the others, and these increased levels positively correlated with levels of TLR2 or TLR4. When TLR2 signaling was blocked using a specific anti-TLR2 monoclonal antibody, serotype b-induced cytokine and CCR expression was inhibited; when TLR4 signaling was blocked, serotype K1-induced response was inhibited. CONCLUSIONS: These results demonstrate that the variability of secretion of cytokines and expression of CCRs detected in DCs stimulated with different serotypes of A. actinomycetemcomitans or P. gingivalis is TLR2 or TLR4 dependent, respectively.
Assuntos
Aggregatibacter actinomycetemcomitans/imunologia , Células Dendríticas/microbiologia , Porphyromonas gingivalis/imunologia , Receptor 2 Toll-Like/imunologia , Receptor 4 Toll-Like/imunologia , Adulto , Aggregatibacter actinomycetemcomitans/classificação , Carga Bacteriana , Técnicas de Cultura de Células , Diferenciação Celular/fisiologia , Células Cultivadas , Células Dendríticas/imunologia , Feminino , Humanos , Interleucina-10/análise , Interleucina-12/análise , Interleucina-1beta/análise , Interleucina-23/análise , Masculino , Monócitos/fisiologia , Porphyromonas gingivalis/classificação , Receptores CCR5/análise , Receptores CCR6/análise , Sorogrupo , Receptor 2 Toll-Like/análise , Receptor 4 Toll-Like/análise , Adulto JovemRESUMO
AIMS: To evaluate the association among serum immunoglobulin G (IgG) responses to Aggregatibacter actinomycetemcomitans (Aa) serotypes a, b and c, Porphyromonas gingivalis (Pg), Tannerella forsythia (Tf) and clinical parameters in Aggressive Periodontitis (AP) subjects. Associations between periodontal pathogens and clinical and immunological parameters were also evaluated. METHODS: Thirty-eight subjects diagnosed with generalized AP (GAP) and localized AP (LAP) were included. Ten healthy controls were also evaluated. Clinical parameters were assessed and percentages of subgingival levels of Aa, Pg and Tf (beyond bacterial load), were determined by quantitative real-time polymerase chain reaction. Serum IgG antibody levels against Aa, Pg and Tf were evaluated by enzyme-linked immunosorbent assay. RESULTS: Percentages of Aa, Pg and Tf were significantly higher in AP than in controls. The response to Aa serotype c was higher in LAP subjects than in controls. There were no differences in microbial composition or antibodies responses between GAP and LAP, except for IgG response to Tf. Pg levels were correlated with probing depth (PD), BoP and CAL in GAP but not in LAP subjects. Tf levels correlated with PD and CAL in GAP subjects. In GAP, the infection levels of Aa and Pg correlated with the corresponding IgG levels to Aa serotype c and Pg. CONCLUSION: Given the evidences that IgG response in AP patients correlated with bacterial infection level in GAP, but not in LAP, and that LAP patients lack a response to Tf, despite harbouring this species, our data suggest a difference in host immune defence between these two forms of aggressive periodontitis.
Assuntos
Periodontite Agressiva/microbiologia , Anticorpos Antibacterianos/sangue , Bactérias Gram-Negativas/imunologia , Imunoglobulina G/sangue , Adulto , Aggregatibacter actinomycetemcomitans/classificação , Aggregatibacter actinomycetemcomitans/imunologia , Periodontite Agressiva/classificação , Periodontite Agressiva/imunologia , Perda do Osso Alveolar/diagnóstico por imagem , Perda do Osso Alveolar/microbiologia , Carga Bacteriana , Bacteroides/classificação , Bacteroides/imunologia , Estudos Transversais , Feminino , Interações Hospedeiro-Patógeno/imunologia , Humanos , Masculino , Perda da Inserção Periodontal/imunologia , Perda da Inserção Periodontal/microbiologia , Bolsa Periodontal/imunologia , Bolsa Periodontal/microbiologia , Porphyromonas gingivalis/classificação , Porphyromonas gingivalis/imunologia , Radiografia , Sorogrupo , Adulto JovemRESUMO
AIM: Different serotypes of Aggregatibacter actinomycetemcomitans have been described based on the lipopolysaccharide (LPS)-O-polysaccharide antigenicity. In turn, a distinct effect of A. actinomycetemcomitans serotypes has been described on cell proliferation and pro-inflammatory cytokine production in different human cells. This study was aimed to investigate the differential dendritic cell (DC) response when stimulated with different bacterial strains belonging to the most prevalent serotypes of A. actinomycetemcomitans (a-c). MATERIALS AND METHODS: Dendritic cells were obtained from healthy subjects and stimulated with increasing multiplicity of infection (MOI = 10(-1) -10(2)) of A. actinomycetemcomitans, serotypes a-c, or their lipopolysaccharide (10-50 ng/ml). The levels for interferon (IFN)-γ, tumour necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-5, IL-6, IL-10, IL-12 and IL-23 were quantified by real-time RT-PCR and ELISA. RESULTS: Variable DC responses were detected when stimulated with the different strains of A. actinomycetemcomitans. DCs stimulated with A. actinomycetemcomitans strains belonging to the serotype b or their purified LPS expressed higher levels of IL-1ß, IL-6, IL-12, IL-23, IFN-γ and TNF-α than DCs stimulated with the other serotypes. CONCLUSIONS: Aggregatibacter actinomycetemcomitans strains belonging to the serotype b demonstrated a higher capacity to trigger Th1 and Th17-type cytokine production on DCs. These increased potential is likely explained by a higher immunogenicity of their LPS.
Assuntos
Aggregatibacter actinomycetemcomitans/imunologia , Células Dendríticas/microbiologia , Aggregatibacter actinomycetemcomitans/classificação , Diferenciação Celular/imunologia , Células Dendríticas/imunologia , Humanos , Interferon gama/análise , Interleucina-10/análise , Interleucina-12/análise , Interleucina-1beta/análise , Interleucina-23/análise , Interleucina-5/análise , Interleucina-6/análise , Lipopolissacarídeos/imunologia , Monócitos/imunologia , Antígenos O/imunologia , Sorogrupo , Células Th1/imunologia , Células Th17/imunologia , Fator de Necrose Tumoral alfa/análiseRESUMO
Antibody levels to some periodontal pathogens are associated with enhanced levels of inflammatory markers. The purpose of the current study was to examine the relative contribution of serum immunoglobulin G (IgG) subclass antibody level factors and local factors on the probing pocket depth in chronic periodontitis. Serum samples were taken from 444 patients diagnosed with moderate and severe periodontitis and 223 control subjects. The IgG subclass antibody titers to Porphyromonas gingivalis (Pg), Aggregatibacter actinomycetemcomitans (Aa) and Tanerella forsythia (Tf) using indirect immunoassay (ELISA) were determined. The relative contribution of patient, tooth and site-associated parameters on the probing pocket depth were evaluated with a hierarchical multilevel model. The results indicated that periodontitis patients had detectable levels of IgG1 and IgG2. High IgG1 and IgG2 antibody levels against Aa occurred in 132 and 142 periodontitis patients, respectively. High IgG1 and IgG2 antibody levels against Pg occurred in 141 and 138, periodontitis patients, respectively, and High IgG1 and IgG2 antibody levels against Tf occurred in 121 and 136 periodontitis patients, respectively. The majority of the variance was attributed to the site level (48 percent). The multilevel analysis associated deeper probing depth with subject factors (serum IgG1 and IgG2 antibody to Pg and Aa), tooth factors (tooth type), and site factors (mesial-distal location and bleeding on probing). Elevated serum IgG1 and IgG2 antibody to Pg and Aa (subject factors) reflects destructive periodontal disease status.
Los niveles de anticuerpos en algunos patógenos periodontales están asociados con mayores niveles de marcadores inflamatorios. El propósito de este estudio fue examinar la contribución relativa de inmunoglobulina sérica G (IgG) factores de nivel de anticuerpos de subclase y factores locales en la profundidad del sondaje en periodontitis crónica. Se tomaron muestras de suero de 444 pacientes con diagnóstico de periodontitis moderada y grave y de 223 sujetos de control. Se determinaron los títulos de anticuerpos IgG subclase a Porphyromonas gingivalis (Pg), Aggregatibacter actinomycetemcomitans (Aa) y Tanerella forsythia (Tf) mediante inmunoensayo indirecto (ELISA). La contribución relativa de los pacientes, los dientes, y el sitio asociado a los parámetros en la profundidad de sondaje fueron evaluados con un modelo multinivel jerárquico. Los resultados indicaron que los pacientes con periodontitis tenían niveles detectables de IgG1 e IgG2. Altos niveles de anticuerpos IgG1 e IgG2 contra Aa fueron observados en 132 y 142 pacientes con periodontitis, respectivamente. Niveles altos de anticuerpos IgG1 e IgG2 contra Pg fueron detectados en 141 y 138 en pacientes con periodontitis respectivamente, y niveles altos de anticuerpos IgG1 e IgG2 contra Tf se produjeron en 121 y 136 pacientes con periodontitis, respectivamente. La mayor parte de la varianza se atribuyó a nivel de sitio (48 por ciento). El análisis multinivel asociados a profundidad de sondaje con factores relacionados a los sujetos, anticuerpos (suero IgG1 e IgG2 Aa y Pg), factores de los dientes (tipo) y los factores del sitio (localización mesial - distal y sangrado al sondaje). Anticuerpos elevados de suero IgG1 e IgG2 Aa y Pg (factores de los sujetos) reflejan el estado de la enfermedad periodontal destructiva.
Assuntos
Humanos , Masculino , Adulto , Feminino , Pessoa de Meia-Idade , Imunoglobulina G , Periodontite Crônica/imunologia , Periodontite Crônica/microbiologia , Anticorpos , Aggregatibacter actinomycetemcomitans/imunologia , Ensaio de Imunoadsorção Enzimática , Análise Multinível , Porphyromonas gingivalis/imunologia , Análise de Regressão , Medição de RiscoRESUMO
Aggregatibacter actinomycetemcomitans is a Gram-negative bacteria highly associated with localized aggressive periodontitis. The recognition of microbial factors, such as lipopolysaccharide from A. actinomycetemcomitans ((Aa)LPS), in the oral environment is made mainly by surface receptors known as Toll-like receptors (TLR). TLR4 is the major LPS receptor. This interaction leads to the production of inflammatory cytokines by myeloid differentiation primary-response protein 88 (MyD88) -dependent and -independent pathways, which may involve the adaptor Toll/interleukin-1 receptor-domain-containing adaptor inducing interferon-ß (TRIF). The aim of this study was to assess the involvement of MyD88 in alveolar bone loss induced by (Aa)LPS in mice. C57BL6/J wild-type (WT) mice, MyD88, TRIF or TRIF/MyD88 knockout mice received 10 injections of Aa LPS strain FDC Y4 (5 µg in 3 µl), in the palatal gingival tissue of the right first molar, every 48 h. Phosphate-buffered saline was injected in the opposite side and used as control. Animals were sacrificed 24 h after the 10th injection and the maxillae were removed for macroscopic and biochemical analyses. The injections of Aa LPS induced significant alveolar bone loss in WT mice. In the absence of MyD88 or TRIF/MyD88 no bone loss induced by (Aa)LPS was observed. In contrast, responses in TRIF(-/-) mice were similar to those in WT mice. Diminished bone loss in the absence of MyD88 was associated with fewer TRAP-positive cells and increased expression of osteoblast markers, RUNX2 and osteopontin. There was also reduced tumor necrosis factor-α production in MyD88(-/-) mice. There was less osteoclast differentiation of hematopoietic bone marrow cells from MyD88(-/-) mice after (Aa)LPS stimulation. Hence, the signaling through MyD88 is pivotal for (Aa)LPS-induced osteoclast formation and alveolar bone loss.
Assuntos
Aggregatibacter actinomycetemcomitans/imunologia , Perda do Osso Alveolar/imunologia , Lipopolissacarídeos/imunologia , Fator 88 de Diferenciação Mieloide/metabolismo , Animais , Diferenciação Celular , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Osteoclastos/fisiologia , Transdução de SinaisRESUMO
AIM(S): To explore the associations between the presence of periodontal pathogens and the expression of toll-like receptors (TLR-2 and TLR-4) in the placental tissue of patients with hypertensive disorders compared to the placentas of healthy normotensive patients. MATERIAL AND METHODS: A case-control study was performed. From a cohort composed of 126 pregnant women, 33 normotensive healthy pregnant women were randomly selected, and 25 cases of patients with hypertensive disorders of pregnancy, including gestational hypertension and pre-eclampsia, were selected. Placental biopsy was obtained after aseptic placental collection at the time of delivery. All of the samples were processed and analysed for the detection of Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, Treponema denticola and Tannerella forsythia using the polymerase chain reaction (PCR) technique. Determination of the expressions of TLR-2 and TLR-4 was performed in samples of total purified protein isolated from placental tissues and analysed by ELISA. The data were assessed using descriptive statistics. The associations among variables were estimated through multiple logistic regression models and the Mann-Whitney test to evaluate the differences between the two groups. RESULTS: A significant increase was observed in the expression of TLR-2 in the placentas of patients with hypertensive disorders (p = 0.04). Additionally, the multiple logistic regression models demonstrated an association between the presence of T. denticola and P. gingivalis in placental tissues and hypertensive disorders (OR: 9.39, p = 0.001, CI 95% 2.39-36.88 and OR: 7.59, p = 0.019, CI 95% 1.39-41.51, respectively). CONCLUSIONS: In the present study, pregnant women with periodontal disease presented an association in the placental tissue between the presence of T. denticola and P. gingivalis and hypertensive disorders. Additionally, increased expression of TLR-2 was observed. However, further studies are required to determine the specific roles of periodontal pathogens and TLRs in the placental tissue of patients with pregnancy-related hypertensive disorders.
Assuntos
Hipertensão Induzida pela Gravidez/microbiologia , Placenta/imunologia , Porphyromonas gingivalis/isolamento & purificação , Receptor 2 Toll-Like/análise , Treponema denticola/isolamento & purificação , Adulto , Aggregatibacter actinomycetemcomitans/imunologia , Aggregatibacter actinomycetemcomitans/isolamento & purificação , Bacteroides/imunologia , Bacteroides/isolamento & purificação , Estudos de Casos e Controles , Estudos de Coortes , Feminino , Fusobacterium nucleatum/imunologia , Fusobacterium nucleatum/isolamento & purificação , Gengivite/imunologia , Gengivite/microbiologia , Humanos , Hipertensão Induzida pela Gravidez/imunologia , Perda da Inserção Periodontal/imunologia , Perda da Inserção Periodontal/microbiologia , Índice Periodontal , Bolsa Periodontal/imunologia , Bolsa Periodontal/microbiologia , Periodontite/imunologia , Periodontite/microbiologia , Placenta/microbiologia , Porphyromonas gingivalis/imunologia , Pré-Eclâmpsia/imunologia , Pré-Eclâmpsia/microbiologia , Gravidez , Receptor 4 Toll-Like/análise , Treponema denticola/imunologiaRESUMO
BACKGROUND: Evidence to date shows that mast cells play a critical role in immune defenses against infectious agents, but there have been no reports about involvement of these cells in eliminating periodontopathogens. In this study, the phagocytic ability of mast cells against Aggregatibacter actinomycetemcomitans compared with macrophages is evaluated. METHODS: In vitro phagocytic assays were conducted using murine mast cells and macrophages, incubated with A. actinomycetemcomitans, either opsonized or not, with different bacterial load ratios. After 1 hour, cells were stained with acridine orange and assessed by confocal laser-scanning electron microscopy. RESULTS: Phagocytic ability of murine mast cells against A. actinomycetemcomitans was confirmed. In addition, the percentage of mast cells with internalized bacteria was higher in the absence of opsonization than in the presence of opsonization. Both cell types showed significant phagocytic activity against A. actinomycetemcomitans. However, the percentage of mast cells with non-opsonized bacteria was higher than that of macrophages with opsonized bacteria in one of the ratios (1:10). CONCLUSIONS: This is the first report about the participation of murine mast cells as phagocytes against A. actinomycetemcomitans, mainly in the absence of opsonization with human serum. Our results may indicate that mast cells act as professional phagocytes in the pathogenesis of biofilm-associated periodontal disease.
Assuntos
Aggregatibacter actinomycetemcomitans/imunologia , Mastócitos/imunologia , Doenças Periodontais/microbiologia , Fagócitos/imunologia , Laranja de Acridina , Animais , Carga Bacteriana/imunologia , Biofilmes , Células da Medula Óssea/imunologia , Técnicas de Cultura de Células , Escherichia coli/imunologia , Corantes Fluorescentes , Humanos , Macrófagos/imunologia , Masculino , Mastócitos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Doenças Periodontais/imunologia , Fagócitos/microbiologia , Fagocitose/imunologia , Fatores de TempoRESUMO
The 15-deoxy-(Δ12,14)-PG J(2) (15d-PGJ(2)) has demonstrated excellent anti-inflammatory results in different experimental models. It can be used with a polymeric nanostructure system for modified drug release, which can change the therapeutic properties of the active principle, leading to increased stability and slower/prolonged release. The aim of the current study was to test a nanotechnological formulation as a carrier for 15d-PGJ(2), and to investigate the immunomodulatory effects of this formulation in a mouse periodontitis model. Poly (D,L-lactide-coglycolide) nanocapsules (NC) were used to encapsulate 15d-PGJ(2). BALB/c mice were infected on days 0, 2, and 4 with Aggregatibacter actinomycetemcomitans and divided into groups (n = 5) that were treated daily during 15 d with 1, 3, or 10 µg/kg 15d-PGJ(2)-NC. The animals were sacrificed, the submandibular lymph nodes were removed for FACS analysis, and the jaws were analyzed for bone resorption by morphometry. Immunoinflammatory markers in the gingival tissue were analyzed by reverse transcriptase-quantitative PCR, Western blotting, or ELISA. Infected animals treated with the 15d-PGJ(2)-NC presented lower bone resorption than infected animals without treatment (p < 0.05). Furthermore, infected animals treated with 10 µg/kg 15d-PGJ(2)-NC had a reduction of CD4(+)CD25(+)FOXP3(+) cells and CD4/CD8 ratio in the submandibular lymph node (p < 0.05). Moreover, CD55 was upregulated, whereas RANKL was downregulated in the gingival tissue of the 10 µg/kg treated group (p < 0.05). Several proinflammatory cytokines were decreased in the group treated with 10 µg/kg 15d-PGJ(2)-NC, and high amounts of 15d-PGJ(2) were observed in the gingiva. In conclusion, the 15d-PGJ(2)-NC formulation presented immunomodulatory effects, decreasing bone resorption and inflammatory responses in a periodontitis mouse model.
Assuntos
Anti-Inflamatórios não Esteroides/administração & dosagem , Reabsorção Óssea/prevenção & controle , Nanocápsulas/administração & dosagem , Periodontite/tratamento farmacológico , Periodontite/imunologia , Prostaglandina D2/análogos & derivados , Infecções por Actinobacillus/imunologia , Infecções por Actinobacillus/patologia , Infecções por Actinobacillus/prevenção & controle , Aggregatibacter actinomycetemcomitans/imunologia , Animais , Anti-Inflamatórios não Esteroides/uso terapêutico , Reabsorção Óssea/imunologia , Reabsorção Óssea/microbiologia , Modelos Animais de Doenças , Gengiva/efeitos dos fármacos , Gengiva/imunologia , Gengiva/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos DBA , Nanocápsulas/uso terapêutico , Periodontite/patologia , Prostaglandina D2/administração & dosagem , Prostaglandina D2/uso terapêuticoRESUMO
AIM: To characterize the histologic and cellular response to A. actinomycetemcomitans (Aa) infection. MATERIAL & METHODS: Wistar rats infected with Aa were evaluated for antibody response, oral Aa colonization, loss of attachment, PMN recruitment, TNF-α in the junctional epithelium and connective tissue, osteoclasts and adaptive immune response in local lymph nodes at baseline and 4, 5 or 6 weeks after infection. Some groups were given antibacterial treatment at 4 weeks. RESULTS: An antibody response against Aa occurred within 4 weeks of infection, and 78% of inoculated rats had detectable Aa in the oral cavity (p < 0.05). Aa infection significantly increased loss of attachment that was reversed by antibacterial treatment (p < 0.05). TNF-α expression in the junctional epithelium followed the same pattern. Aa stimulated high osteoclast formation and TNF-α expression in the connective tissue (p < 0.05). PMN recruitment significantly increased after Aa infection (p < 0.05). Aa also increased the number of CD8(+) T cells (p < 0.05), but not CD4(+) T cells or regulatory T cells (Tregs) (p > 0.05). CONCLUSION: Aa infection stimulated a local response that increased numbers of PMNs and TNF-α expression in the junctional epithelium and loss of attachment. Both TNF-α expression in JE and loss of attachment was reversed by antibiotic treatment. Aa infection also increased TNF-α in the connective tissue, osteoclast numbers and CD8(+) T cells in lymph nodes. The results link Aa infection with important characteristics of periodontal destruction.