Salivary metabolomic profile in adolescents with juvenile systemic lupus erythematosus

Abstract The aim of this study was to characterize the salivary metabolomic profile in adolescents with juvenile systemic lupus erythematosus (jSLE). A total of 24 adolescents with jSLE (15.92 ± 2.06 years) and 12 systemically healthy controls (15.25 ± 2.7 years) were included in the study. Participants underwent rheumatologic testing and periodontal examination, with the recording of plaque index (PI), probing depth (PD), clinical attachment level (CAL), and bleeding on probing index (BPI). Unstimulated whole saliva was collected from both groups and stored at -80 ºC. The salivary proton nuclear magnetic resonance (1H-NMR) spectra were acquired in a spectrometer operating at 500 MHz. Partial least squared discriminant analysis (PLS-DA) and orthogonal PLS-DA (O-PLS-DA) were used for statistical analysis. Mean CAL and PI were significantly increased in the group with jSLE (p < 0.01). Patients with jSLE presented a significantly different salivary metabolic profile (accuracy = 0.54; R2 = 0.86; Q2 = -0.293), significantly higher salivary levels of N-acetyl sugars, and significantly reduced levels of phenylalanine, glycine, taurine, hydroxybutyrate, and valerate compared with healthy controls (p < 0.05). It is suggested that the salivary metabolomic profile analyzed by 1H NMR in patients with jSLE presents a different fingerprint that the systemically healthy subjects. Integrating the variation of metabolites with the identification of the metabolic pathways involved seems to provide a better understanding of the influence of systemic disease on salivary metabolites.

O impacto do lúpus eritematoso sistêmico juvenil na inflamação gengival: achados clínicos, imunológicos e microbiológicos / The impact of juvenile systemic lupus erythematosus on gingival inflammation: clinical, immunological and microbiological findings

Os objetivos desse estudo foram avaliar a expressão de citocinas no soro e fluido gengival, a produção de arginina-peptidil-deiminase (anti-PPAD) e o perfil microbiológico de pacientes com lúpus eritematoso sistêmico juvenil (LESj) e comparar com indivíduos saudáveis sistemicamente. Como objetivo secundário, avaliamos o impacto do tratamento da inflamação gengival sobre a expressão das citocinas e dos níveis de anti-PPAD. Participaram do estudo 30 pacientes com LESj (idade média: 16,2 ± 1,5 anos) e 29 sem doença sistêmica (idade média 15,5 ± 2,3 anos), ambos com gengivite. Foram coletados dados reumatológicos, periodontais, sangue, fluido gengival e biofilme intrassulcular. As citocinas foram analisadas pelo multiensaio multiplex; anti-PPAD pelo ensaio de imunoabsorção enzimática (ELISA) e níveis bacterianos pelo checkerboard DNA-DNA hybridization. Para avaliar variáveis categóricas foi utilizado o teste Qui-quadrado de Pearson; para as numéricas, o teste U de Mann-Whitney e para as correlações a estatística tau-b de Kendall. No estudo longitudinal, foi utilizado o teste de McNemar para dados qualitativos, e o de Wilcoxon para dados numéricos. No estudo transversal, o grupo teste apresentou maiores níveis de profundidade de bolsa à sondagem (PBS), nível de inserção clínica (NIC), % de placa e sangramento do que o controle. Na análise do soro, G-CSF foi significativamente maior e TNF-α significativamente menor no grupo teste. Na análise do fluido gengival, IL-1ß, IL-7, IL-8, IL-13, G-CSF, IFN-γ e MCP-1 foram significativamente maiores e IL-4, IL-12(p70) e GM-CSF significativamente menores no grupo teste. Não houve diferença significativa nos níveis de anti-PPAD entre os grupos. S. constellatus, A. actinomycetencomytans, E. saburreum, V. parvula, S. intermedius, C. showae e F. nucleatum foram significantemente mais numerosas no grupo teste e A. gerencseriae e T. denticola no grupo controle. Após o tratamento da inflamação gengival, o SLEDAI, %NIC 1-2 e NIC reduziram significantemente. Já os valores de PBS e %NIC 0 aumentaram. No soro, houve diminuição significativa da IL-4 e IL-5 e aumento significativo dos níveis de anti-PPAD após o tratamento. Já no fluido gengival, houve diminuição significativa da IL-1ß, IL-10 e MCP-1 e aumento significativo da IL-4, IL-12(p70), IL-17, GM-CSF e INF-α. Sendo assim, podemos concluir que pacientes com LESj apresentaram piores condições periodontais, PBS, NIC, % de placa e sangramento do que pacientes saudáveis sistemicamente. A análise de citocinas mostrou um aumento do G-CSF e TNF-α no soro e de IL-1ß, IL-7, IL-8, IL-13, G-CSF, IFN-γ e MCP-1 no fluido gengival dos pacientes com LESj. Foram identificados anticorpos anti-PPAD nos pacientes com LESj, o que pode servir como gatilho para a quebra da tolerância imunológica. O estudo longitudinal intervencionista demonstrou que o tratamento da inflamação gengival melhorou significantemente os parâmetros %NIC 1-2 e NIC. Houve uma pequena, porém significante, piora na PBS, a qual acreditamos não ter relevância clínica. Observamos também uma melhora significante no SLEDAI e dos níveis de IL-4 e IL-5 no soro e um aumento das citocinas IL-12, IL-17 e GM-CSF no fluido gengival. Já em relação ao anticorpo anti-PPAD, observamos um aumento significativo após o tratamento da inflamação gengival.

The objectives of this study were to evaluate the expression of cytokines in serum and gingival fluid, the production of arginine-peptidyl-deiminase (anti-PPAD) and the microbiological profile of patients with juvenile systemic lupus erythematosus (jSLE) and compare with systemically healthy individuals. As a secondary objective, we evaluated the impact of treatment of gingival inflammation on cytokine expression and anti-PPAD levels. Thirty patients with jSLE (mean age: 16.2 ± 1.5 years) and 29 without systemic disease (mean age 15.5 ± 2.3 years), both with gingivitis, participated in the study. Rheumatological and periodontal data, blood, gingival fluid and intrassulcular biofilm were collected. Cytokines were analyzed by multiplex multi-assay; anti-PPAD by enzyme-linked immunosorbent assay (ELISA) and bacterial levels by checkerboard DNA-DNA hybridization. Pearson's Chi-square test was used to evaluate categorical variables; Mann-Whitney U test for numerical variables and Kendall's tau-b statistic for correlations. In longitudinal study, McNemar test was used for qualitative data, and Wilcoxon test for numerical data. In cross-sectional study, test group presented higher levels of probing depth (PD), clinical attachment level (CAL), % of plaque and bleeding than control group. In serum analysis, G-CSF were significantly higher and TNF-α significantly lower in test group. In analysis of gingival fluid, IL-1ß, IL-7, IL-8, IL-13, G-CSF, IFN-γ and MCP-1 were significantly higher and IL-4, IL-12(p70) and GM-CSF were significantly lower in test group. There was no significant difference in anti-PPAD levels between groups. S. constellatus, A. actinomycetencomytans, E. saburreum, V. parvula, S. intermedius, C. showae and F. nucleatum were significantly more numerous in test group and A. gerencseriae and T. denticola in control group. After treatment of gingival inflammation, SLEDAI, % CAL 1-2 and CAL decreased significantly. Already the values of PD and % CAL 0 increased. In serum, there was a significant decrease in IL-4 and IL-5 and a significant increase in anti-PPAD levels after treatment. In gingival fluid, there was a significant decrease in IL-1ß, IL-10 and MCP-1 and significant increase in IL-4, IL-12 (p70), IL-17, GM-CSF and INF-α. Thus, we can conclude that patients with jSLE presented worse periodontal conditions, PBS, NIC, % plaque and bleeding than systemically healthy patients. Cytokine analysis showed an increase in serum G-CSF and TNF-α and IL-1ß, IL-7, IL-8, IL-13, G-CSF, IFN-γ and MCP-1 in gingival fluid of patients with jSLE. Anti-PPAD antibodies have been identified in patients with jSLE, which may serve as a trigger for impaired immune tolerance. Longitudinal interventional study demonstrated that treatment of gingival inflammation significantly improved % CAL 1-2 and CAL parameters. There was a small, but significant worsening in PBS, which we believe has no clinical relevance. We also observed a significant improvement in SLEDAI and levels of IL-4 and IL-5 in serum and an increase in cytokines IL-12, IL-17 and GM-CSF in gingival fluid. Regarding the anti-PPAD antibody, we observed a significant increase after the treatment of gingival inflammation.

Infecção periodontal por porphyromonas gingivalis: de uma inflamação oral a um possível "gatilho" para a autoimunidade via citrulinação de proteínas / Periodontal infection by Porphyromonas gingivalis: from oral inflammation to a possible "trigger" for autoimmunity by protein citrulination

A infecção periodontal é causada por uma disbiose polimicrobiana sinérgica onde o Porphyromonas gingivalis pode ser considerado um microrganismo-chave. Recentemente, este microrganismo tem sido associado à produção de autoanticorpos comuns à autoimunidade, sendo assim, o objetivo deste estudo foi revisar a literatura sobre como a infecção periodontal por Porphyromonas gingivalis pode iniciar uma resposta autoimune. A citrulinação fisiológica pode não ser suficiente para gerar doenças autoimunes, porém fontes externas de citrulinação como traumas e infecções podem modificar quantitativa e qualitativamente os peptídeos citrulinados. Diversos estudos têm fornecido valiosas informações a respeito dos fatores de virulência do Porphyromonas gingivalis, e seus efeitos no sistema imunológico do indivíduo. A Porphyromonas gingivalis-peptidilarginina-deiminase (PPAD), expressa pelo Porphyromonas gingivalis, gera peptídeos citrulinados que podem levar a produção de autoanticorpos, e assim, induzir a iniciação de uma resposta autoimune, amplificada e perpetuada pela citrulinação fisiológica (AU)

Periodontal disease is caused by a synergistic polymicrobial dysbiosis where Porphyromonas gingivalis can be considered a keystone pathogen. Recently,this pathogen has been associated with production of autoantibodies common in autoimmunity, therefore the purpose of this study was to review the literature about how periodontal infection with Porphyromonasgin givaliscan initiate an immune response. Physiological citrulination can be not sufficient to induce autoimmune diseases, however external sources of citrullination like trauma and infections can modify the quantity and quality of citrullinated peptides. Several studies has provided valuable information regarding virulence factors of Porphyromonas gingivalis and its effects on the individual's immune system. Porphyromonas gingivalis-peptidylargininedeiminase (PPAD), expressed by Porphyromonas gingivalis, generates citrullinated peptides that can lead to production of autoantibodies and than induce the initiation of autoimmune response, amplified and perpetuated by physiological citrulination.(AU)

Cell Derived Microparticles in Gingival Crevicular Fluid from Periodontitis Patients with Type 2 Diabetes

Abstract Cell-derived microparticles (MPs) have been described as vital contributors to the inflammatory process. However, its role in the periodontal disease pathogenesis remains unclear. Therefore, we aimed to detect the presence neutrophil (CD66b+) and platelet (CD41b+) derived microparticles in gingival crevicular fluid from individuals having periodontitis aggravated by type 2 diabetes. Twelve patients (56.2 ±7.2 yrs) with severe form of chronic periodontitis aggravated by type 2 diabetes were included. Clinical and metabolic data were gathered. Gingival crevicular fluid was collected using filter strips from deep and shallow sites. MPs were detected by flow cytometry according to their size (< 1 µm) and the expression of surface markers (CD66b for neutrophil-derived MPs and CD41b for platelet-derived MPs). All samples were positive for the antibodies. Median levels of CD66b+ MPs and CD41b+ MPs were, respectively, 3,677.0 (2,553.2 - 9,059.8) MP/µL and 520.7 (432.9 - 766.1) MP/µL in deep sites. In shallow sites, the corresponding values were 2,644.9 (1,451.5 - 3,858.9) MP/µL and 371.2 (287.2 - 692.7) MP/µL. There was no significant difference between deep and shallow sites (p>0.05). In conclusion, this study reported the presence of neutrophil and platelet derived microparticles in gingival crevicular fluid from individuals having severe periodontitis and type 2 diabetes.

Resumo As micropartículas derivadas de células (MPs) têm sido descritas como contribuintes vitais para o processo inflamatório. No entanto, seu papel na patogênese da doença periodontal permanece obscuro. Por isso, nosso objetivo foi detectar a presença de micropartículas derivadas de neutrófilos (CD66b +) e plaquetas (CD41b +) no fluido gengival de indivíduos com periodontite e diabetes tipo 2. Doze pacientes (56,2 ± 7,2 anos) com periodontite crônica severa e diabetes tipo 2 foram incluídos no estudo. Foram coletados dados clínicos e metabólicos. O fluido gengival foi coletado usando tiras de filtro de papel em sítios rasos e profundos. As MPs foram detectadas por citometria de fluxo de acordo com o seu tamanho (<1 μm) e pela expressão de marcadores de superfície (CD66b para MPs derivadas de neutrófilos e CD41b para MPs derivadas de plaquetas). Todas as amostras foram positivas para os anticorpos. Os níveis médios de CD66b + MPs e CD41b + MPs foram, respectivamente, 3.677.0 (2,553.2 - 9,059.8) MP/μL e 520.7 (432.9 - 766.1) MP/μL nos sítios profundos. Nos sítios rasos, os valores correspondentes foram 2,644.9 (1,451.5 - 3,858.9) MP/μL e 371.2 (287.2 - 692.7) MP/μL. Não houve diferença significativa entre os sítios rasos e profundos (p>0.05). Concluindo, o presente estudo reportou a presença de micropartículas derivadas de neutrófilos e plaquetas no fluido gengival de pacientes com periodontite e com diabetes tipo 2 .

Periodontitis and systemic lupus erythematosus / Doença periodontal e lúpus eritematoso sistêmico

ABSTRACT A large number of studies have shown a potential association between periodontal and autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus (SLE). Similar mechanisms of tissue destruction concerning periodontitis and other autoimmune diseases have stimulated the study of a possible relationship between these conditions. This study aims to review the literature about this potential association and their different pathogenic mechanisms. Considering that periodontal disease is a disease characterized by inflammation influenced by infectious factors, such as SLE, it is plausible to suggest that SLE would influence periodontal disease and vice-versa. However, this issue is not yet fully elucidated and several mechanisms have been proposed to explain this association, as deregulation mainly in innate immune system, with action of phagocytic cells and proinflammatory cytokines such as IL-1β and IL-18 in both conditions’ pathogenesis, leading to tissue destruction. However, studies assessing the relationship between these diseases are scarce, and more studies focused on common immunological mechanisms should be conducted to further understanding.

RESUMO Um grande número de estudos tem mostrado uma potencial associação entre doenças periodontais e doenças autoimunes, como artrite reumatoide e lúpus eritematoso sistêmico (LES). Os mecanismos de destruição tecidual semelhantes entre a periodontite e as demais doenças autoimunes têm estimulado o estudo de possíveis relações entre essas condições. O presente estudo tem como objetivo revisar a literatura acerca dessa potencial associação e dos seus diferentes mecanismos patogênicos. Considerando-se a doença periodontal uma doença de caráter inflamatório que sofre influência de fatores infecciosos, assim como o LES, é plausível sugerir que o LES influenciaria sua progressão, assim como a periodontite influenciaria a progressão do LES. Entretanto, essa questão ainda não é totalmente elucidada e vários mecanismos têm sido propostos para explicar tal associação, como desregulações, principalmente no sistema imune inato, com ações de células fagocíticas e de citocinas pró-inflamatórias, como IL-1β e IL-18, na patogênese de ambas as condições, o que contribui para a destruição tecidual. Existem, contudo, poucos estudos na literatura que avaliam a relação entre essas doenças e mais trabalhos focados nos mecanismos imunológicos comuns a ambas as condições devem ser feitos para um maior entendimento.

Serum Adipokine Levels and their Relationship with Fatty Acids in Patients with Chronic Periodontitis

Adipokines are present in inflammatory processes and may be directly related to periodontal disease. Moreover, their activities may be regulated by fatty acids. The goal of this study was to quantify the concentrations of the main adipokines, leptin, adiponectin and resistin, and the docosahexaenoic (DHA), docosapentaenoic (DPA), eicosapentaenoic (EPA) and arachidonic (AA) fatty acids, in patients with generalized chronic periodontitis. As a secondary objective, the ratios of these substances in the blood of these patients were evaluated. The study included 15 systemically healthy patients with generalized chronic periodontitis (test group) and 15 patients with gingivitis (control group). Medical and periodontal parameters and blood samples were collected. Serum concentrations of fatty acids were analyzed by gas chromatography and adipokines by multiplex bead immunoassay. There was no significant difference in adipokines between groups. However, there was a tendency for lower values of adiponectin in periodontitis patients. Regarding the fatty acids, they were significantly higher in the test group compared with controls. The res/DHA, res/AA, adipon/DHA, adipon/AA and adipon/DPA ratios were significantly lower in the test group. There was no significant correlation between adipokines and clinical parameters and between adipokines and fatty acids levels. It was concluded that generalized chronic periodontitis patients showed significantly higher levels of fatty acids in comparison to gingivitis; adiponectin revealed a trend to lower values in the periodontitis group, even after Ancova correction. The ratios suggest a minor proportion of adiponectin and resistin in relation to the fatty acids in patients with generalized chronic periodontitis.

As adipocinas estão presentes em processos inflamatórios e podem estar diretamente relacionadas à doença periodontal. Além disso, suas atividades podem ser reguladas pelos ácidos graxos. Este estudo teve como objetivo quantificar as concentrações das principais adipocinas, leptina, adiponectina e resistina, e dos ácidos graxos: ácido docosahexaenóico (DHA), ácido docosapentaenóico (DPA), ácido eicosapentaenóico (EPA) e ácido araquidônico (AA), em pacientes com periodontite crônica generalizada. Como objetivo secundário, avaliar as proporções destas substâncias no sangue desses pacientes. O estudo incluiu 15 pacientes sistemicamente saudáveis com periodontite crônica generalizada (grupo teste) e 15 com gengivite (grupo controle). Foram coletados parâmetros médicos e periodontais e amostras de sangue. As concentrações séricas dos ácidos graxos foram analisadas por cromatografia gasosa e as das adipocinas foram analisadas pelo método multiensaio multiplex. Não houve diferença significativa entre os níveis de adipocinas entre os grupos. No entanto, houve uma tendência para menores valores nos níveis da adiponectina nos pacientes com periodontite. Com relação aos ácidos graxos, os valores foram significativamente maiores no grupo teste em comparação com os controles. As razões entre res/DHA, res/AA, adipon/DHA, adipon/AA e adipon/DPA foram significativamente menores no grupo teste. Não houve correlação significativa entre as adipocinas e os parâmetros clínicos e entre os níveis de adipocinas e ácidos graxos. Conclui-se que pacientes com periodontite crônica generalizada apresentaram níveis significativamente maiores de ácidos graxos em relação à gengivite, adiponectina apresentou uma tendência a valores menores no grupo periodontite, mesmo após a correção de Ancova. Os resultados das razões sugerem uma menor proporção de adiponectina e resistina em relação aos ácidos graxos em pacientes com periodontite crônica generalizada.

Use of minimally invasive gingival biopsies in the study of inflammatory mediators expression and their correlation with gingival fluid in patients with chronic periodontitis.

Background: There are no studies comparing the gingival crevicular fluid (GCF) cytokines expression with its corresponding values from the same tissue’s sites. Such comparison might be of great value since most of the cytokine function is related to cell and/or tissue receptors. Aims: Our aim was to use minimally invasive biopsies to evaluate the expression of interferon‑gamma, interleukin 1 (IL‑1) β, IL‑6, IL‑17A, IL‑17F, and their correlation with the expression in gingival fluid in patients with chronic periodontitis. Materials and Methods: The collection of gingival fluid comprised 22 samples from 11 patients (mean age 46.73 ± 10.16 standard deviation years) with chronic periodontitis. The collection of biopsies comprised 22 samples from the same patients. Gingival fluid and biopsy were taken from the same site in one shallow and one deep site per patient. Gingival fluid samples were collected with periopaper® and analyzed using Luminex®. Biopsies were taken with a 2 mm diameter punch and analyzed for the same mediators using immunohistochemistry. Results: The gingival fluid showed higher amounts for IL‑1‑β in deep sites. Immunohistochemical markers were observed in the analyzed cells groups, both in deep and shallow sites, without significant differences between them. In the comparative analysis between immunohistochemical markers and GCF, IL‑1‑β showed high concordance in shallow and deep sites. Conclusions: The use of a standardized punch of 2 mm diameter for periodontal tissue biopsies seems to be suitable for immunohistochemistry analysis and showed that the GCF may not express all the markers in the same proportion at the corresponding tissue.