Universidade Estadual de Campinas
Faculdade de Odontologia de Piracicaba
Thiago Perez Rangel
Relação entre o perfil de citocinas e níveis de lipopolissacarídeos e
ácido lipoteicóico no ambiente subgengival de indivíduos
diabéticos e não diabéticos.
Relation between cytokines profiles and lipopolysaccharide and Lipoteichoic Acid on
subgingival microbiota in diabetics and non-diabetics individuals.
Piracicaba
2019
Thiago Perez Rangel
Relação entre o perfil de citocinas e níveis de lipopolissacarídeos e ácido
lipoteicóico no ambiente subgengival de indivíduos diabéticos e não
diabéticos.
Relation between cytokines profiles and lipopolysaccharide and Lipoteichoic
Acid on subgengival microbiota in diabetics and non-diabetics individuals.
Dissertação apresentada à Faculdade de
Odontologia de Piracicaba da Universidade
Estadual de Campinas como parte dos
requisitos para obtenção do título de mestre
em Clínica Odontológica, na Área de
Periodontia.
Dissertation presented to the Piracicaba Dental
School of the University of Campinas in partial
fulfillment of the requirements for the degree of
Master in Clinical Dentistry, in Periodontics
area.
Orientador: Prof. Dr. Renato Corrêa Viana Casarin
Este exemplar corresponde à versão final da dissertação defendida pelo aluno
Thiago Perez Rangel e orientada pelo Prof. Dr. Renato Corrêa Viana Casarin.
Piracicaba
2019
Ficha de Aprovação
Dedicatória
Dedico esse trabalho a todos que me ajudaram de alguma forma durante essa
caminhada.
E a meu avô que, de forma celestial, vem mostrando meu futuro.
“A morte não é nada.
Eu somente passei para o outro lado do Caminho.
Eu sou eu, vocês são vocês.
O que eu era para vocês, eu continuarei sendo.
Me deem o nome que vocês sempre me deram, falem comigo como vocês sempre fizeram.
Vocês continuam vivendo no mundo das criaturas, eu estou vivendo no mundo do Criador.
Não utilizem um tom solene ou triste, continuem a rir daquilo que nos fazia rir juntos.
Rezem, sorriam, pensem em mim. Rezem por mim.
Que meu nome seja pronunciado como sempre foi, sem ênfase de nenhum tipo.
Sem nenhum traço de sombra ou tristeza.
A vida significa tudo o que ela sempre significou, o fio não foi cortado.
Porque eu estaria fora de seus pensamentos, agora que estou apenas fora de suas vistas?
Eu não estou longe, apenas estou do outro lado do Caminho…
Você que aí ficou, siga em frente, a vida continua, linda e bela como sempre foi.”
Oração Santo Agostinho
Agradecimentos
À Divina Trindade que me permite levantar todas as manhãs para vencer meus
desafios.
Aos meu avô e avó, Prof. Dr. Humberto de Araújo Rangel e Celene de Oliveira Rangel,
por me suportarem durante minha infância, durante meus momentos aventureiros e
mostrarem como a vida deve ser vivida.
Aos meus Pais, Alexandre de Oliveira Rangel e Maria Angélica Perez Rangel, por
serem pais e me ensinarem da melhor forma que puderam tudo que sei hoje, por todo
o companheirismo e genes que me fizeram ser o quem sou.
À minha irmã, Carolina Perez Rangel, pelos incontáveis resumos que me fizeram
passar da graduação e horas e horas de conversas durante nossas viagens.
À Linda, Caroline Simplicio, que me acompanha desde a graduação e compartilha
comigo os piores e melhores momentos, sem ela com toda certeza a caminhada seria
muito penosa.
Aos meus futuros sogros, Adilson e Sueli, por todo carinho e apoio que me delegam
de todo coração, me tratando como se fosse um filho.
Ao meu orientador, Prof. Dr. Renato Corrêa Viana Casarin, que me mostra e orienta
para que meu sonhe se realize.
A todos os Professores da área de Periodontia, Prof. Dr. Antonio Wilson Sallum, Prof.
Dr. Enilson Antonio Sallum, Prof. Dr. Francisco Humberto Nociti Júnior, Profa. Dra.
Karina Gonzalez Silvério Ruiz, Prof. Dr. Márcio Zaffalon Casati, Prof. Dr. Renato
Corrêa Viana por sempre compartilharem seus conhecimentos e aprendizados
buscando nos tornar grandes profissionais.
À Aurélio Amorim Reis e Rocharles Fontenele, meus amigos de apartamento, por
todas as risadas compartilhadas nesse último ano.
Aos meus colegas de Pós-graduação por todos os momentos no “caipiras” que
fizeram esses 2 anos serem ainda mais saborosos. Em especial, à Aurélio Amorim
Reis, Thiago Bueno, Mabelle Monteiro, Ana Livia Mazzonetto, Amanda Bandeira,
Rafaela Videira pela inestimável amizade.
À Periodontia da FOP, todos os professores e funcionários, por se transformarem na
minha família odontológica.
Aos colegas que levo desde a graduação, Maria Claúdia Cuzzulin e Rafaela Chapola
que além de muitas risadas me ajudaram nos períodos de dúvidas e desesperos.
O presente trabalho foi realizado com apoio da Coordenação de Aperfeiçoamento de
Pessoal de Nível Superior – Brasil (CAPES) – Código de Financiamento 001,
agradeço o apoio financeiro para a realização dessa pesquisa.
E a todos que de alguma forma, que mesmo não mencionados, me fizeram chegar
onde cheguei, meus sinceros agradecimentos e minhas orações para que Deus
retribua sua bondade.
Resumo
A doença periodontal e o diabetes mellitus são doenças altamente prevalentes e inter-
relacionadas, resultando em alterações no sistema imunológico, resposta do
hospedeiro e microbiota. Em relação a microbiota, a liberação de fatores de virulência
se mostrou capaz de regular o perfil inflamatório do hospedeiro. Assim, o objetivo
deste estudo de caso-controle foi avaliar os níveis de ácido lipoteicóico (LTA) e
lipopolissacarídeos (LPS) presentes nas bolsas periodontais e sua relação com
citocinas e metaloproteínas (MMPs) em pacientes diabéticos em comparação com
pacientes normoglicêmicos. Trinta pacientes foram selecionados para este estudo
caso-controle, diabético (DM) (n = 15) e normoglicêmico (n = 15). De 4 bolsas
profundas (PS> 7mm) foram inseridas tiras de papel filtro para coletar FGC. LPS e
LTA foram analisados por ensaio imunoenzimático (ELISA), enquanto IFN-γ, IL-10, IL-
17, IL-1β, IL-4, MMP-2 e MMP-9 foram medidos por LUMINEX/MAGpix. No grupo
diabético, a concentração de LTA foi maior, 3899,8±3643,5 ng/mL, do que em
normoglicêmicos, 767,9 ± 410,4 (p <0,001). LPS também foi encontrado em maior
nível de concentração no grupo DM (p<0,001). Níveis mais elevados de IL-10 e MMP-
2 (p<0,05) foram encontrados em indivíduos diabéticos. Em pacientes diabéticos, LTA
e LPS modularam a liberação de citocinas no GCF de forma diferente, alterando o
perfil inflamatório do hospedeiro em relação aos pacientes normoglicêmicos. LTA em
pacientes normoglicêmicos e com DM foi positivamente correlacionada com IL-17 e
MMP-2 e negativamente com IL-10. Em relação ao LPS, apenas no DM foi
correlacionado com IFN-γ, IL-17, MMP-2 e negativamente com IL-10. No DM o LTA e
LPS expressos alteram o perfil pró-inflamatório através da modulação de citocinas e
MMPs.
Palavras-chaves: Diabetes Mellitus, Citocinas, Periodontite Crônica, Fatores de Virulência.
Abstract
Periodontal disease and diabetes mellitus are highly prevalent and interrelated
diseases, resulting in changes in the immune system, host response and microbiota.
In relation to microbiota, the release of virulence factors proved to be able to regulate
the inflammatory profile of the host. Thus, the objective of this case-control study was
to evaluate the levels of lipoteicóico acid (LTA) and lipopolysaccharides (LPS) present
in the periodontal pockets and their relation with cytokines and metalloproteins (MMPs)
in diabetic patients in comparison to normoglycemic patients. Thirty patients were
selected for this case-control study, diabetic (DM) (n = 15) and normoglycemic (n =
15). From 4 deep pockets (PS> 7mm) filter paper strips were inserted to collect FGC.
LPS and LTA were analyzed by enzyme-linked immunosorbent assay (ELISA), while
IFN-γ, IL-10, IL-17, IL-1β, IL-4, MMP-2 and MMP-9 were measured by LUMINEX /
MAGpix. In the diabetic group, the concentration of LTA was higher, 3899.8 ± 3643.5
ng / mL, than in normoglycemics, 767.9 ± 410.4 (p <0.001). LPS was also found in a
high level of concentration in the DM group (p <0.001). Higher levels of IL-10 and MMP-
2 (p <0.05) were found in diabetic subjects. In diabetic patients, LTA and LPS modified
the release of cytokines in the GCF differently, altering the inflammatory profile of the
host in relation to normoglycemic patients. LTA in normoglycemic and DM patients was
positively correlated with IL-17 and MMP-2 and negatively with IL-10. Regarding LPS,
only in DM was correlated with IFN-γ, IL-17, MMP-2 and negatively with IL-10. In DM
the LTA and LPS expressed alter the pro-inflammatory profile through the modulation
of cytokines and MMPs.
Keywords: Diabetes Mellitus, Cytokines, Chronic Periodontitis, Virulence Factors.
Sumário
1. Introdução 11
2. Artigo: Diabetes mellitus alters Lipopolysaccharide- and Lipoteichoic
Acid-mediated Pro-inflammatory Cytokine Production in periodontal
tissues. 13
3. Conclusão 37
Referências
Anexos 41
Anexo 1 - Parecer Consubstanciado CEP 41
Anexo 2 - Relatório Similaridade 42
Anexo 3 – Comprovante de Submissão 43
11
1. Introdução
A periodontite é uma doença inflamatória de etiologia multifatorial,
desencadeada pela resposta imune-inflamatória do hospedeiro aos periodonto
patógenos presentes no biofilme subgengival, caracterizada clinicamente pela
destruição óssea e perda de inserção conjuntiva, que se não tratada pode levar
a perda dental (Papapanou et al., 2018). A instalação e progressão dessa doença
está relacionada a um perfil pró-inflamatório, com um desequilíbrio entre a
liberação de citocinas pró e anti-inflamatórias, após a ativação de células
inflamatórias por periodonto patógenos (Bartold & Van Dyke, 2013). Diversos
estudos in vitro tem demonstrado que o componente da membrana externa de
bactérias gram-negativas e gram-positivas chamados Lipopolissacarídeos (LPS)
e ácido lipoteicóico (LTA) podem modular a liberação de citocinas pelas células
(Hessle et al., 2005; Kang et al., 2016; Koch et al., 2014; Tsigou et al., 2014).
Lipopolissacarídeos (LPS), são componentes da membrana externa de
bactérias gram-negativas, frequentemente encontrados em lesões de gengivite e
periodontite. Estes são reconhecidos por receptores do próprio hospedeiro, como
receptores Toll-like (TLRs). O TLR2 e o TLR4 podem reconhecer uma variedade
de componentes bacterianos e suas interações com o LPS desencadeiam uma
resposta inflamatória que, quando desequilibrada ou excessiva, pode resultar na
destruição do tecido periodontal (Yoshioka et al., 2008). Estudos prévios
mostraram que os patógenos reconhecidamente associados às doenças
periodontais apresentam LPS que induzem a resposta celular. Porphyromonas
gingivalis (Pg), Aggregatibacter actinomycetemcomitans (Aa), Prevotella
intermedia (Pi), Fusobacterium nucleatum (Fn) e Tannerella forsythia (Tf)
apresentam LPS em sua constituição e mostraram, por meio da ligação com
TLR2 e TLR4, a indução a produção de citocinas pró-inflamatórias como
interleucina 1β (IL-1β) e Fator de necrose tumoral (TNF-α), bem como induziram
a reabsorção óssea e progressão da destruição dos tecidos de suporte
periodontal em ratos (Gölz et al., 2014; Kang et al., 2016; Loos, et al., 2000; Zhu
et al., 2016). Essas conclusões foram obtidas em estudos em animais e/ou cultura
de células e, até o presente momento, não há estudos que avaliaram os níveis de
LPS no ambiente subgengival e sua relação com as citocinas liberadas
localmente.
12
Em bactérias Gram-positivas o principal fator de virulência é o ácido
lipoteicóico (LTA), uma endotoxina bacteriana (Klukowska et al., 2017) que é
liberado após lise (Ginsburg, 2002). Estes se ligam a receptores como toll-like 2
(TLR2), que são expressos nas células hospedeiras do periodonto. A ativação de
TLR2 dependente de LTA em células dendríticas imaturas, segundo Keller et al.,
2010, leva à produção significativa de TNF-alfa e IL-1beta, exacerbando a
resposta inflamatória. Também foi sugerido o papel do LTA, associado à
persistência de lesões, ativando macrófagos e o sistema complemento do
hospedeiro, levando a autólise tecidual (Endo et al., 2013).
Indivíduos com diabetes tipo 2 (DM2) apresentam uma relação bi -
direcional com a doença periodontal. Apresentando maior severidade da
periodontite por conta da hiperglicemia e um pior controle glicêmico por conta da
periodontite (Hsu et al., 2018; Preshaw et al., 2012; Zhou et al., 2013). Essa inter-
relação é ocasionada, segundo estudos prévios, por alterações no funcionamento
celular de monócitos/macrófagos, o que resulta na superprodução de citocinas
pró-inflamatórias em resposta a patógenos periodontais (Jiang et al., 2018; Salvi
et al., 1997).
Em relação ao perfil microbiano de indivíduos diabéticos, parece haver
diferenças na composição que poderiam laterar vias de inflamação. Exemplo
disso é o trablaho de Casarin et al., 2013 que demostraram uma mudança na
microbiota subgengival de pacientes diabéticos, com um maior número de
bactérias gram-positivas em relação a pacientes normoglicêmicos. Essa presença
elevada de gram-positivos é confirmada por outros pesquisadores, e sugere que
essas bactérias possam ter um papel modulador na doença periodontal (Ganesan
et al., 2017; Longo et al., 2018).
Crê-se que, uma vez que o ambiente subgengival em pacientes
diabéticos possui essa alteração em sua microbiota e a presença de uma maior
quantidade de bactérias gram-positivas possa estar relacionada a progressão
acelerada da doença periodontal, gerando perda precoce dos dentes e suas
consequências sistêmicas e sociais o conhecimento da relação entre
microrganismos presentes nas bolsas periodontais e seus fatores de virulência,
LPS e LTA e seu padrão de modulação em citocinas e proteases (MMP), torna-
se de ainda maior importância.
13
2. Artigo
Diabetes mellitus alters Lipopolysaccharide- and Lipoteichoic Acid-mediated Pro-inflammatory
Cytokine Production in periodontal tissues.
Thiago Perez Rangel*, Aurelio Amorim Reis*, Lara Caponi*, Larissa Pena Spirito*, Karina Gonzales Silvério
Ruiz*, Mauro Pedrine Santamaria†, Ingrid Fernandes Mathias†, Marcio Zaffalon Casati*, Renato Corrêa
Viana Casarin*
*Campinas State University, Dental School of Piracicaba. Department of Prosthodontic and Periodontics
†São Paulo State University - FOSJC, College of Dentistry. Division of Periodontology.
Corresponding author:
Renato Corrêa Viana Casarin
Department of Prosthodontic and Periodontics
Avenida Limeira, 901 – Areião, Piracicaba, ZC 13414-903
email: [email protected] (can be published)
phone: (19) 2106-5301
Number of Tables: 2
Number of Figure: 2
Number of Words: 3258
Diabetes mellitus alters cytokine/protease levels in gingival crevicular fluid, what could be associated to
increased LTA/LPS levels.
Abstract
14
Background: Periodontal disease and diabetes mellitus (DM) are highly prevalent and
interrelated diseases, resulting in altered host response microbiota. Thus, the aim of this study
was to evaluate the impact of DM on local levels of Lipopolysaccharide (LPS) and Lipoteichoic
Acid (LTA) and the impact on cytokines and matrix metalo-proteinases (MMPs).
Methods: Thirty patients were included in this case-control study, diabetic (n=15) and
normoglycemic (n=15). Gingival crevicular fluid from deep pockets (PPD> 7mm) was collected
and LPS and LTA levels analyzed by enzyme-linked immunosorbent assay (ELISA), while IFN-γ,
IL-10, IL-17, IL-1β, IL-4, MMP-2 and MMP-9 were measured by LUMINEX/MAGpix.
Results: Higher levels of LTA, LPS, IL-10, IL-1β and MMP-2 (p<0.05) and lower level of IL-17 were
found in DM group (p<0.05). Local levels of LTA were positively correlated with IL-17 and MMP-
2 and negatively with IL-10 in DM and Normoglycemic (p<0.05). However, in relation to LPS,
only in DM there was a positive correlation IFN-γ, IL-17, MMP-2 and negatively with IL-10.
Conclusion: LTA and LPS alters the inflammatory profile through the modulation of cytokines
and MMPs in different manner in DM and normoglycemic subjects.
Keywords: Diabetes Mellitus, Cytokines, Chronic Periodontitis, Virulence Factors.
15
Introduction
Diabetes Mellitus (DM) is defined as a group of diseases of metabolic origin resulting from
failure of secretion or action of insulin. It is divided into two main types - type I - related to the
autoimmune destruction of pancreatic cells; and type II - related to alteration in production
and cellular resistance to insulin1. Regardless of the degree of development of the country, DM
is an important and growing global health problem2. Only in Brazil, the most recent data point
to over sixteen million diagnoses, which has led to an increase in incidence of more than sixty
percent in the last ten years2. Among the systemic effects of DM, medicine includes
periodontitis as an important occurrence of this disease.
Periodontal disease and diabetes mellitus are highly prevalent and interrelated chronic
diseases, having a bidirectional relationship3. Hyperglycemia results in changes in the immune
system that may exacerbate periodontal disease induced by bacteria; on the other hand,
periodontal infection can jeopardize glycemic metabolic control3–5. This two-sided pathway
suggests that the control of periodontal infection is essential for the management of diabetes
mellitus, as glycemic control is important for the prevention and control of periodontal
disease3.This pathological cycle is associated not only to the host-response alteration as well as
to an altered microbiota.6–8
Casarin et al. (2013) 7 showed that a change of the subgingival microbiota occurs in
periodontal patients with DM, generating specific local factors in the periodontal pocket,
creating a differentiated microbial constitution. This finding is corroborated by different
authors that showed higher levels of well-recognized pathogen as T. forsythia, P. gingivalis, F.
nucleatum 9–13. Interestingly, although the most common diseased-associated species has been
described as anaerobic gram negatives, several studies shown the diabetic-associated
microbiome has a higher number of gram-positive bacteria, suggesting a greater role of these
16
bacteria in disease progression and treatment failure 7,9. Selenomonas spp, Gemella spp and
Capnocytophaga spp are examples of gram positive detected in higher prevalence or levels in
subgingival environment of DM subjects7. Although out of focus, these bacteria also could lead
to an imbalanced host-response, once some virulence factors can be detected in them.
The main virulence factor of gram-positive bacteria is lipoteichoic acid (LTA), a bacterial
endotoxin that is released after lysis14,15. These bind to receptors such as toll-like 2 (TLR2),
which are expressed in the host cells of the periodontium. Activation of LTA-dependent TLR2
in immature dendritic cells, according to Keller et al. (2010)16, leads to significant IL-1beta
production, exacerbating the inflammatory response. It has also been suggested the role of
LTA, associated with the persistence of lesions, activating macrophages and the complement
system of the host, leading to tissue autolysis17. In gram-negative bacteria, this virulence factor
is played by lipopolysaccharides (LPS), an external membrane component. These are also
recognized by host receptors, such as TLR2 and TLR4 and can recognize a variety of bacterial
components and their interactions, triggering an inflammatory response that, when
unbalanced or excessive, can result in the destruction of periodontal tissue 18. Not surprisingly,
a robust evidence shown a different cytokine pattern in DM19.
In this vein, since the subgingival environment in the periodontal disease condition has a
large concentration of gram-positive and gram-negative bacteria, the presence of these
virulence factors can directly influence the production of cytokines and proteases altering the
progression of the disease periodontal. This pathological pattern may still be worsened in
individuals with systemic conditions that alter the pattern of cellular response, such as Diabetes
mellitus. However, the LPS and also LTA levels in subgingival environment and its impact on
locally released cytokines, still remains unknown.
17
Thus, the objective of the study was to evaluate the levels of LTA and LPS present in the
periodontal pockets in diabetic patients and their relationship with the cytokine and MMP
profile in the subgingival environment of diabetic individuals compared to normoglycemic
subjects.
18
Material and Methods
Study Design
This study was a case-control trial, comparing DM and Normoglycemic subjects and were
approved by the Institutional Review Board (IRB) of Piracicaba Dental School (CAAE
89303418.8.0000.5418). After acceptance in participate on study, written consent was taken
and data/ samples were collected.
Subjects were designed to each group according to inclusion criteria:
DM group (n=15) - Diagnosis of type 2 diabetes mellitus for at least 2 years; Have at least
20 teeth; Presence of generalized severe chronic periodontal disease (≥10 periodontal pockets
with probing pocket depths of >4 mm and marginal alveolar bone loss of >30%), age superior
than 35 years.
Normoglycemic group (n=15) - Systemic health; Have at least 20 teeth; Presence of
generalized severe chronic periodontal disease (≥10 periodontal pockets with probing pocket
depths of >4 mm and marginal alveolar bone loss of >30%); age superior than 35 years.
The exclusion criteria were: 1. Presence of other type of periodontal disease; 2. Are on
diet or nutritional monitoring; 3. Pregnant and lactating women; 4. Smokers; 5. Be in
orthodontic treatment; 6. Have completed periodontal treatment in the last year; 7. Use
antimicrobial mouthwashes in the last 30 days. 8. Use of medications that alter the course of
periodontal disease.
19
Clinical evaluation
After inclusion, all patients were instructed on the causes and consequences of
periodontal disease, as well as on preventive techniques, including the technique of brushing
and flossing. The usual clinical parameters were collected, plague index, gingival index, Pocket
Depth, Bleeding on Probing and Clinical Attachment Level. All parameters were evaluated by a
calibrated examiner (intra-class correlation of 87% for CAL). After sample collection, patients
were treated with Full-Mouth Ultra-sonic Disinfection protocol and included in a supportive
therapy.20
Cytokine/Protease profile
From each individual, crevicular gingival fluid (GCF) was collected from 4 deep pockets
(PPD>7mm), one per quadrant. After removal of the supragingival biofilm, the teeth were
washed and the area were isolated with the help of cotton rollers and gently dried with air jets.
The GFC was collected through the insertion of filter paper strips‡ into the periodontal pocket
for a period of 15 seconds. Two paper strips were used per site to obtain an adequate volume
of GCF. For analysis of the local cytokine profile, GCF samples were analyzed for the detection
of IL-10, IL-1beta, IL-17, IL-4 IFN-γ, MMP-2 and MMP-9 by Luminex/MAGpix technology§, using
commercially available kits‖ following manufacturer instructions. For analysis, besides
individuals’ values, cytokine was grouped according biological function (Pro-inflammatory ones
(PRO) = sum of IL-1beta, IL-17 and IFN-γ; Anti-inflammatory ones (ANTI) = sum of IL-10 and IL-
4) and ratios between them were also done.
20
LPS Analysis
At the same sites and after GCF collection, 2 apyrogenic paper point** were inserted in
periodontal pockets for 30 seconds. These samples were used for LPS and LTA analysis. LPS
levels was analyzed using the General Endotoxin ELISA Kit††, according to instructions from the
manufacturer. Briefly, after plate and reagents preparation, standard solution, supplied by the
manufacturer, as well as blanks and 50uL of samples were added to wells. Then, primary
antibody was inserted and the plate incubated for 1 hour at 37° C. After incubation, plate was
washed, and then a secondary antibody added. Plate was incubated for 45 min at 37° C, washed
and the substrate and stop solution added to each well. LPS levels will be analyzed through an
ELISA reader‡‡ at 450 nm.
LTA Analysis
From the same samples, LTA levels were analyzed using the human LTA ELISA Kit††
according to manufacturer’s instruction. The ELISA plate was conditioned with the LTA
monoclonal antibody supplied by the kit manufacturer, and the standard, control and 50uL
sample solutions inserted. Incubate for 60 min at 37° C, after which it will be washed and a
substrate were added thus allowing the plate to read. LTA levels were analyzed through an
ELISA reader‡‡ at 450 nm.
From all samples, total protein was determined using a Bradford reaction¶. The total
protein of each sample was used for correction during cytokines, LPS and LTA concentration, in
order to adjust for different volume collection.
21
Statistical analysis
This transversal study, presenting as primary variable the levels of LTA, set a sample size
based on previous study21, once this is the first one to analyze this virulence factor in
periodontal pockets. For all analysis, subject was considered the experimental unit and all
comparisons were done by a blinded statistician (RCVC). Firstly, the value of each
cytokine/ratios, LPS or LTA of each group was compared by Student's t or Mann-Whitney test,
depending on the normality evidenced by the Shapiro-Wilk test. Moreover, a
Spearman/Pearson correlation tests were used to assess the relationship between LPS and LTA
levels and locally released cytokines/ratios. In all analyzes, a significance level of 5% was
considered.
Results
Demographic and clinical data
From June 2017 to September 2018, a total of 1134 subjects referred to Graduated Clinic
of Piracicaba Dental School were examined and from this total, 15 type-2 diabetics and 15
normoglycemic were included in the study. At table 1 are displayed demographic and clinical
data of subject from both groups. Only on fasting plasma glucoses a statistical difference
(p>0.05) was seen, confirming the hyperglycemic status of DM group.
22
Table 1. Demographical and Clinical parameters of subjects included in the study.
DM Group
(n=15)
Normoglycemic Group
(n=15)
Age (years±SD) 45.1±4.32 42.2±3.81
Gender (n female) 11 12
Fasting Plasma Glucoses (dg/mL±SD) 157.5 ±64.0* 93.5±7.4
Plaque Index (%±SD) 34.2±14.1 38.5±16.9
Gingival Index (%±SD) 8.9±2.0 10.5±2.6
Full Mouth Probing Depth (mm±SD) 2.7±0.06 2.2±0.06
Full Mouth Clinical Attachment Level (mm±SD) 3.36±0.80 2.3±0.3
Bleeding on Probing (%±SD) 35.9±11.7 25.5±12.3
*Indicate Statistical difference between groups (Student’s t test, p>0.05). dg – Decigram; SD – Standard Deviation.
LTA, LPS and cytokines/protease levels
Diabetic subjects presented higher levels of LPS as well as LTA in subgingival environment
than normoglycemic ones (p<0.05) (Figure 1). Regarding cytokines levels, DM subjects
presented higher levels of IL-10, IL-1ß and MMP-2 (p<0.05). Moreover, the ratio IL-1ß/IL-10 and
PRO/ANTI were higher in DM subjects than normoglycemics (p<0.05). No difference between
groups was noted regarding other cytokines/ratios (Table 2).
23
Figure 1. Concentration of LPS (EU/mL) and LTA (ng/mL) in subgingival samples of DM and
Normoglycemic subjects.
*indicate statistical difference between groups (Mann-Whitney test, p<0.05)
L P S
EU
/mL
DM
No
rmo
gly
cem
ic
0 .0 1
0 .1
1
1 0
1 0 0
1 0 0 0
1 0 0 0 0 *L T A
ng
/mL
DM
No
rmo
gly
cem
ic
1 0
1 0 0 0
1 0 0 0 0 0 *
24
Table 2. GFC levels (mean (standard error)) of cytokines and MMP’s (pg/mL) in DM and
normoglycemic subjects.
IFN-γ IL-10 IL-17 IL-1β IL-4 MMP-2 MMP-9
DM 12,28 (3,87)
0,94 (0,16) *
2,22 (0,47) *
551,29 (157,59) *
1,52 (0,76)
71152,01 (20181,21) *
171415,12 (53053,87)
Normoglycemic 19,69 (4,73)
0,43 (0,08)
7,08 (1,94)
106,23 (38,30)
1,80 (0,78)
19002,96 (9098,23)
172679,69 (33131,18)
*indicate statistical difference between groups (Student’s t and Mann-Whitney tests, p<0.05)
Correlation between virulence factors and GFC profile
At table 2 it could be noted that LPS was not significantly correlated to any
cytokine/protease in normoglycemic subjects (p>0.05), while LTA directly and significantly
correlated with IL-17, as well as IL-17/IL-10 ratio and MMP-2 levels, while negatively correlated
with IL-10 levels (p<0.05). On the other hand, in DM subjects, LPS was directly correlated to
IFN-γ, IL-17, IL-1β/IL-10 ratio, PRO/ANTI ratio and MMP-2, while negatively correlated with IL-10
(p<0.05). Regarding LTA, directly and significantly correlated with IL-17 and MMP-2 levels, while
negatively correlated with IL-10 levels (p<0.05).
Table 2. Correlation (r(p)) between cytokines/proteases and LPS/LTA levels in diabetic and
normoglycemic subjects.
Diabetes Group (n=15) Normoglycemic Group (n=15)
LPS LTA LPS LTA
IFN-γ (pg/mL) 0.538(0.04) 0.459(0.09) 0.042(0.88) 0.397(0.138)
IL-10 (pg/mL) -0.798(<0.001) -0.731(0.003) -0.323(0.25) -0.615(0.01)
IL-17 (pg/mL) 0.820(<0.0001) 0.740(0.003) 0.231(0.42) 0.572(0.025)
25
IL-1β (pg/mL) -0.112 (0.69) -0.340(0.22) 0.169(0.55) 0.125(0.65)
IL-4 (pg/mL) 0.288 (0.30) 0.430(0.12) -0.094(0.738) 0.087(0.74)
MMP-2 (pg/mL) 0.880(<0.0001) 0.644(0.01) 0.202(0.47) 0.736(0.001)
MMP-9 (pg/mL) -0.020 (0.92) -0.156(0.583) -0.059(0.83) 0.408(0.127)
IL-1β/IL-10 (pg/mL) 0.539 (0.046) -0.042 (0.886) 0.423 (0.132) 0.397 (0.149)
IL-17/IL-10 (pg/mL) 0.360 (0.206) -0.062 (0.834) -0.003 (0.992) 0.666 (0.007)
PRO/ANTI (pg/mL) 0.551 (0.041) -0.015 (0.958) 0.385 (0.174) 0.360 (0.187)
Discussion
Diabetes mellitus is a metabolic disease, considered as a risk factor of periodontitis.
Several studies tried to clarify how this systemic condition affect periodontal homeostasis,
leading to higher degree of destruction and inflammation. Within several pathogenic aspects
cited by literature, changes in microbiota has been described as an important aspect and some
authors suggested not only an increase in gram negative pathogens colonization but also a
higher level of gram positives. Since both type of bacteria presents a membrane virulence factor
(LPS and LTA, respectively), the present study evaluated the LPS and LTA levels in subgingival
environment of DM and normoglycemics and the impact on cytokine profile. Results showed
that although LPS and LTA was augmented in DM, the local release of cytokines and MMPs was
differently affect in normoglycemic and DM subjects, indicating different role of each virulence
factor in systemically-affected subjects.
Hyperglycemia has been well described as one of the major negative factors affecting
periodontal tissues. Changes in cytokine release, collagen production, bone metabolism and
clinical response has been described22. Considering the bidirectional relationship (periodontitis
affecting DM control), understand how it this interaction became essential. In 2013, our group
26
suggest a possible role of gram positive in this condition, once Gemella, Eikenella, Selenomonas,
Actinomyces, and Streptococcus genera – all of them gram positive - were highly detected in
deep pockets of DM than in non-DM7. Recently, Longo et al, 20188, also identified an
enrichment in facultative gram positives in DM, corroborating other studies 7,10–13. This higher
number of gram positives could explain the higher level of LTA in DM than in normoglycemic
subjects observed in the present study. To the best of our knowledge, this is the first study
assessing LTA levels in periodontal pockets.
LTA is a potent cell-stimulator, promoting the release of different molecules 23. Its binding
on TLR2 and induce the release of IL-1β and TNF-alpha by human monocytes24, IL-18 by
macrophages25, induced β‑catenin pathway activation (and NF‑κB activity) and
proinflammatory cytokine expression by epithelial cells26, IL-6 and TNF-alpha by blood cells 27.
Interestingly, this is the first in vivo study trying to correlate LTA levels and its potential as local
host-response modulator. The results showed that both populations, DM and normoglycemics,
presented a similar correlation between LTA and the cytokine profile in periodontal pockets -
the higher LTA level, the higher MMP-2 and IL-17 and lower IL-10 release in subgingival
environment.
Ahn et al, 201828 in a mice model, showed that LTA isolated from Lactobacillus plantarum
reduced the IL-10 production. On the same way, Volz et al, 201829, identified a reduction in IL-
10 release by dendritic cells after LTA stimulation. However, the authors highlighted that this
reduction only occurs when cells were stimulated by S. aureus-LTA and not S. epidermidis-LTA,
suggesting a possible specie-dependent way on IL-10 regulation. Considered as an anti-
inflammatory cytokine, IL-10 can down-regulate the synthesis of proinflammatory cytokines
and chemokines such as IL-1, IL-6, TNF-𝛼, nitric oxide30,31, regulating the host homeostasis.
Therefore, IL-10 has also been regarded as an important regulator of bone homeostasis in
27
homeostatic and inflammatory conditions32,33. Another interesting finding is the fact that IL-10
down-regulate gelatinase (MMP2) and type IV collagenase (MMP-9) production 31. This
information colaborate to understand an higher production of MMP-2 and the positive and
strong association between LTA.
Matrix metalloproteinase-2 (MMP-2), also named gelatinase A, has played a major role
on matrix destruction during periodontal disease 34. In the present study, beside LTA, also LPS
was correlated to higher MMP2 release, and DM subjects showed higher levels than
normoglycemics. MMPs have been proposed as master regulators of inflammation, through
proteolysis of chemokines, growth factors, receptors and their binding proteins 34. MMP also
acts as an intracellular multifunctional protein, resulting in pro- or anti-inflammatory functions,
leading to either tissue homeostasis or pathology 34,35. In DM subjects, higher levels of MMP-2
has been described in DM population, also associated to systemic adverse effects, as
retinopathy, nephropathy and cardiovascular disease 36–39. In an esophageal cancer cell culture,
MMP-2 was induced by IL-17A, through ROS/NF-κB/MMP-2/9 signaling pathway activation40.
In the present study, IL-17 also was modulated by LTA in DM and Normoglycemic subjects.
Regarding IL-17, the present study showed a positive correlation with LTA in DM and
normoglycemic ones. Although no difference between groups has been observed, IL-17 has
been suggested as an important marker in periodontal destruction in DM subjects 41–43,
regulating other inflammatory features 43. This relationship between LTA and IL-17 also could
be affected by another way. Recently, in an animal model, diabetes increased the pathogenicity
of the oral microbiome, as shown by increased inflammation, osteoclastogenesis, and
periodontal bone loss when transferred to normal germ-free hosts, playing IL-17 an essential
role41. The authors also discuss that hyperglycemia could modify bacterial behavior and
virulence – for example, stimulation of LTA production by them, what create a vicious cycle.
28
This possible pathway should be better explored in future studies. Moreover, there are other
virulence factors that also could be founded in gram positive, as lipoproteins, what has been
proved to be capable of induce inflammation and could interfere in disease progression44. In
summary, it is clear that LTA is an important feature on periodontitis destruction, although it is
quite forgiven by literature. The present study is, to the best of our knowledge, the first to
evaluate it levels in periodontal pocket and future studies should consider it on pathogenesis.
Meanwhile one of most striking result of the present study is the dissimilarity between
LPS-cytokines relationship observed in DM and normoglycemic. While in normoglycemic LPS
did not significantly correlated to anyone cytokine/protease, in diabetics, LPS was positively
correlated to IL-17, MMP-2 (as also observed in LTA), IFN-γ, IL-1ß/IL-10 and PRO/ANTI ratios.
Moreover, it was negatively associated to IL-10 only in DM. Interestingly, diabetic subjects
presented a higher level of LPS in subgingival content than normoglycemic. LPS is a well-known
and studied virulence factor from gram negatives that has been historically associated to the
release of pro-inflammatory cytokines and reduction in anti-inflammatory ones 45,46. However,
in local release of cytokines, there was a different result in DM and non-DM subjects. This
differential modulation could be attributed to changes in cells behavior after a long period of
hyperglycemia. An interesting and recent study in monkeys evaluated the different pathways
activated during periodontal disease development in hyperglycemia condition. The authors
showed an increase in AGES (advanced glycation products), beta-defensin and also in IL-17 in
DM monkeys compared to non-DM ones. They suggest that a hyperglycemic environment
might lead to the destruction of periodontal tissues by accelerating inflammatory response and
weakening the defense system in periodontal tissues46. This conclusion corroborates with
Acharya et al47 who suggest different cytokines ratios in diabetic patients. These altered cell
function could be the explanation why several studies, including the present one, detected a
29
local dissimilarity in cytokine production in diabetic condition, in special in high level of blood
glycemia 48–50.
The imbalance in DM host response became clear when analyzing the axis IL-10-IL-1ß. An
apparently contradictory result is the negative correlation of LPS (and also LTA) with IL-10,
although in DM a higher level of IL-10 and LPS/LTA was seen. When looking for results, although
as increased IL-10 could bring the idea of a more anti-inflammatory environment, the ratio IL-
1ß/IL-10 and also PRO/ANTI ratio were higher in DM, showing the hyper-inflamed local
condition, what corroborate another study 47. So, in this vein, the higher presence of virulence
factors and altered cell behavior promoted an increase not only in pro-inflammatory markers
but also in anti-inflammatory ones, trying to “equilibrate” the host defense. However, based
on the fact that LPS/LTA negatively correlated to IL-1028,29 - summed to their higher levels in
DM, this expected equilibrium became a weaken response and induce an exacerbated
destruction. This consortium could bring a new light on periodontal disease in DM subjects and
might be considered in future.
Conclusion
Diabetic patients had a higher local level of LTA and LPS than normoglycemics, resulting
in a disequilibrium on host response. In addition to these elevated levels, in diabetic patients,
LTA modulated increasing levels of the IL-17 and MMP-2 cytokines while decreasing those of
IL-10 while LPS also demonstrated the modulation of different cytokines, demonstration a
change of modulation due hyperglycemic environment, positively modulating IFN-gamma, IL-
17 and MMP-2 while negatively modulating IL-10.
Footnotes
30
‡ Periopaper, Oraflow
§ MAGpix
‖ HCYTOMAG-60K and HMMPMAG, Merck, Darmstadt, Germany
** Tanari, Manaus, AM
† †My BioSource, San Diego, CA
‡‡ ELISA
¶ BioRad, Hercules, CA
ACNOWLEDGEMENTS
This study was financed by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
– Brasil (CAPES) – Finance Code 001.
31
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37
3. Conclusão
Pacientes diabéticos tiveram um nível mais alto de LTA e LPS do que
normoglicêmicos, alterando o perfil inflamatório do hospedeiro. Além desses níveis
elevados, em pacientes diabéticos, o LTA modulou aumentando a concentração das
citocinas IL-17 e MMP-2 e diminuiu os da IL-10. Em pacientes diabéticos o LPS
também demonstrou a modulação de diferentes citocinas do que em relação aos
pacientes normoglicêmicos, demonstrando uma mudança de modulação devido ao
ambiente hiperglicêmico, modulando positivamente o IFN-γ, a IL-17 e a MMP-2,
enquanto modulava negativamente a IL-10.
38
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Anexo 1. Parecer Consubstanciado CEP.
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Anexo 2. Relatório Similaridade.
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Anexo 3. Comprovante de Submissão