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: rcasarin@unicamp.br (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