| |

Received: 27 February 2019    Revised: 14 November 2019    Accepted: 25 November 2019

DOI: 10.1111/jre.12721

ORIGINAL ARTICLE

FOXP3+ and CD25+ cells are reduced in patients with stage IV,
grade C periodontitis: A comparative clinical study

Raphael J. G. da Motta1  | Luciana Yamamoto Almeida2  | Kelly R. V. Villafuerte3  |

Alfredo Ribeiro-Silva4  | Jorge E. León5  | Camila Tirapelli1

1
Integrated Dental Clinic, Department of
Dental Materials and Prosthodontics, School Abstract
of Dentistry of Ribeirão Preto (FORP/USP), Background and Objective: Some studies suggest that regulatory T cells (Tregs) have
University of São Paulo, Ribeirão Preto,
Brazil suppressive effects on inflammatory osteolysis. The aim of this study was to evaluate
2
Haematology Division, Department of Treg immunomarkers in periodontitis-affected tissues from patients with periodonti-
Clinical Medicine, Ribeirão Preto Medical
tis and clinically healthy gingiva (control).
School (FMRP/USP), University of São Paulo,
Ribeirão Preto, Brazil Material and Methods: The presence and distribution of positive cells for CD4, CD25
3
Department of Oral and Maxillofacial and FOXP3 (Treg immunomarkers) in periodontitis-affected tissues (epithelium and
Surgery and Periodontology, School of
Dentistry of Ribeirão Preto (FORP/USP),
lamina propria) of 30 patients (ten per group) with a diagnosis of stage IV, grade C
University of São Paulo, Ribeirão Preto, periodontitis (IV-C), stage III, grade B periodontitis (III-B) and the control were evalu-
Brazil
4
ated. A two-way ANOVA followed by Fisher's LSD test was used to demonstrate
Department of Pathology, Ribeirão Preto
Medical School (FMRP/USP), University of differences between the groups and immunomarkers; Student's t test was used to
São Paulo, Ribeirão Preto, Brazil demonstrate differences between the epithelium and the lamina propria.
5
Oral Pathology, Department of
Results: Both IV-C and III-B periodontitis presented a significantly high proportion
Stomatology, Public Oral Health, and
Forensic Dentistry, School of Dentistry of of immune-stained cells for all immunomarkers when compared to the control group.
Ribeirão Preto (FORP/USP), University of
Notably, CD25+ and FOXP3+ cells were detected in a significantly higher number in
São Paulo, Ribeirão Preto, Brazil
III-B than IV-C periodontitis (P < .05).
Correspondence
Conclusion: Our results suggest the participation of Tregs on the osteoimmunologi-
Jorge E. León, Oral Pathology, Department
of Stomatology, Public Oral Health, and cal mechanisms in IV-C and III-B periodontitis patients, notably contributing to strat-
Forensic Dentistry, School of Dentistry of
egies for alveolar bone regeneration in clinical treatment decisions.
Ribeirão Preto (FORP/USP), University of
São Paulo, Ribeirão Preto, SP, Brazil.
Email: jleon@forp.usp.br KEYWORDS

immunohistochemistry, immunology, periodontitis, Tregs

Funding information
Fundação de Amparo à Pesquisa do Estado
de São Paulo-FAPESP, Grant/Award
Number: 2013/08589-3, 2016/02713-2 and
2016/11419-0

1 |  I NTRO D U C TI O N
Periodontitis is a chronic inflammatory disease of periodontal tis-
sues that occurs when complex immune/inflammatory mechanisms
1
are initiated in response to bacterial aggression. The current prev-
alence of periodontitis in the world population is 11%, and it is con-
sidered as the main cause of tooth loss. 2 In this context, a better
understanding of the relationship between the immune system, in-
cluding cell types and their activation profiles, and periodontitis is
fundamental because it will help in understanding the pathogenesis
of this disease.
According to the new classification of periodontal disease,3,4
periodontitis can be classified according to the stage and grade. The
stages (I-IV) will depend on the severity, complexity and extent of

J Periodont Res. 2019;00:1–7. wileyonlinelibrary.com/journal/jre © 2019 John Wiley & Sons A/S.     1 |
Published by John Wiley & Sons Ltd
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2       da
the disease, where stages I and II are the early stages and stages III
and IV are the most severe stages of periodontitis. The grades (A-C)
reflect the biological characteristics, including the risk of rapid pro-
gression, the treatment response and systemic modifying factors.
Several studies indicate that the host immune response plays a
5,6
central role in the pathogenesis of the periodontal disease.
worth noting that some patients demonstrate rapid bone destruc-
tion and elevated levels of pro-inflammatory cytokines, regardless
of the presence or absence of pathogenic bacteria. Therefore, the
genetic variation in the immunological responses and/or the regu-
lation of inflammatory factors may explain the pathogenesis of the
periodontal disease, as well as the unusual aggressive clinicopatho-
logical presentation of the disease.7
In periodontitis, active biological substances that are present in
dental biofilm lead to local inflammatory responses.8 The resulting in-
flux of inflammatory cells and cytokines (notably interferon-gamma
[IFN-γ], prostaglandin E2 [PGE2], interleukin [IL]-1, IL-12, IL-17 and
the receptor activator of NF-kappaB ligand [RANKL]) promotes
bone resorption by osteoclasts interfering with the host protection
against periodontal destruction.9 These interesting cell mechanisms,
involving different immune cell types on bone metabolism, can be
better explained by the “osteoimmunology" which assesses interac-
tions between bone homeostasis and the immune system.10,11
Regarding immune cells, while T cells predominate in the early
and stable stages of periodontal disease, B cells are often observed
in more advanced lesions with established bone destruction.12
Among T cells, several T helper subsets (Th1, Th2, Th17 and regula-
tory T cells [Tregs]) have been described in periodontal disease.13-16
Different from Th2 cytokines (IL-4 and IL-10), Th1 cytokines (IFN-γ
and IL-12) have been associated with bone destruction. Similarly,
with the Th1 pathway, interestingly, Th17 cells (through the secre-
tion of IL-6, IL-17, IL-23 and RANKL, cytokines with osteoclastogenic
properties) have been associated with osteolytic processes, includ-
ing periodontitis,17 as well as with pro-inflammatory properties
in infectious and autoimmune diseases. On the other hand, Tregs
and Th1 cells present suppressive effects on inflammatory osteol-
ysis, mediated by transforming growth factor-beta (TGF-β), cyto-
14,18,19
toxic T lymphocyte-associated antigen 4 (CTLA-4) and IL-10.
Although some studies suggest that Tregs play a protective role
against bone resorption in periodontal disease,14,18 it is interesting
that a detailed immunohistochemical (IHC) characterization of Tregs
in periodontitis-affected tissue samples (especially considering the
recent classification) is unknown to date.
Several studies have been developed to study the presence and
distribution of immune cell subsets in periodontal disease tissues in
an attempt to elucidate the mechanisms involved in its pathogene-
sis, aiming to find more efficient treatments in the future.5,20 Thus,
considering only lymphocyte subsets in periodontal disease, they
were assessed by immunohistochemistry through CD45, 21 CD3, 21,22
21,23 21,22 22
CD4, CD8, TIA-1, granzyme B, CD16, CD28 and CD56
for T cells, as well as CD2022 and CD1921 for B cells. The Tregs were
evaluated through FOXP313,16,18,24,25 and CD4.13,16,26 In a recent
study, which assessed healthy/gingivitis and chronic periodontitis
MOTTA et al.
specimens, a similar distribution of FOXP3+ cells was observed. 25
However, none of the previous studies has evaluated the presence
and distribution of Treg immunomarkers (CD4, CD25 and FOXP3)
in periodontitis-affected tissue samples considering its different
stages and degrees.
It is Thus, considering that Tregs exert suppressive effects on inflam-
matory osteolysis, the present study assessed Treg immunomarkers
to better understand their osteoimmunological mechanisms on the
pathogenesis of periodontitis, aiming to establish immunotherapeu-
tic protocols that can improve the quality of life of these patients.
The authors hypothesized that patients with stage IV, grade C peri-
odontitis (IV-C) had reduced Treg levels, which would be associated
with a greater progression of the disease.
2 | M ATE R I A L A N D M E TH O DS
2.1 | Subject selection
This clinical study was approved by the Research Ethics Committee
of the School of Dentistry of Ribeirão Preto, the University of
São Paulo, which follows the Declaration of Helsinki (Protocol
12945713.1.0000.5419).
Considering the last World Workshop on the classification of
periodontal and peri-implants diseases and conditions,3,4 periodon-
titis was classified in stages (I, II, III and IV), according to the sever-
ity and complexity of the treatment. I and II are the initial stages,
while III and IV are the moderate-to-severe stages of periodontitis,
wherein stage IV there is a combination of periodontium destruction
with the loss of teeth, including pathological migration, poor tooth
positions, dental hypermobility and masticatory dysfunction.
The degree of periodontitis is estimated with the direct or indi-
rect evidence of the progression rate in three categories: slow (grade
A), moderate (grade B) and rapid (grade C) progression. Direct evalu-
ation is based on longitudinal observation, and indirect evaluation is
based on the evaluation of bone loss of the most affected tooth as a
function of age (percentage of root length divided by subject's age).
Extraoral and intraoral examinations, considering periodontal
and radiographic complete examinations (periapical and panoramic
radiographs), to define a periodontal diagnosis were performed by
a trained periodontist (KRVV) in patients from the Integrated Clinic
Area, seeking the selection of 30 patients (ten per group).
1. Patients with stage III, grade B periodontitis (III-B): Clinical
attachment loss (CAL) ≥5  mm, probing depth (PD) ≥6  mm,
≥20 teeth and radiographic bone loss extending to the middle
third of the root. Grade B was evaluated indirectly consider-
ing radiographic bone loss in the most affected tooth in the
dentition as a function of age (0.25-1.0), and the presence of
21
supragingival and subgingival calculus is consistent with the
severity of periodontal breakage.
2. Patients with stage IV, grade C periodontitis (IV-C): CAL ≥ 5 mm,
PD  ≥  6  mm, <20 teeth (10 opposing pairs) with the loss of
da MOTTA et al.
masticatory function and the presence of mobility due to occlusal
trauma, radiographic bone loss extending to more than a third of
the root. Grade C was assessed considering the radiographic bone
loss in the most affected tooth in the dentition as a function of age
(>1.0), and supragingival and subgingival calculus is inconsistent
with the severity of periodontal breakage.
3. Control patients (clinically healthy gingiva with intact periodon-
tium): Patients without any clinical signs of inflammation with-
out a history of periodontitis; PD  ≤  3  mm; <10% of sites with
bleeding on probing (BOP) and the presence of a dental biofilm;
CAL < 1 mm detected by periodontal probing and the absence
of bone loss detected by radiographic examination; without ex-
tensive caries or restorations and at least 28 permanent teeth. In
addition, all the control patients showed the absence of any local
or systemic pathology.
The exclusion criteria were as follows: patients under 18 and over
65 years of age; periodontal treatment or antibiotic therapy in progress
or in the last six months; long-term administration of anti-inflammatory
medication; use of mouthwashes in the previous two months; patients
with orthodontic treatment; pregnancy or lactation; chronic diseases
(such as autoimmune diseases, diabetes mellitus and other endocrine
disorders); smokers and ex-smokers; drug-induced gingival hyperplasia
and the use of antibiotics, corticosteroids or non-steroidal anti-inflam-
matory drugs in the previous six months.
The variables evaluated in this study were immune-positive cells,
counted from the fields obtained in each slide, in the epithelium (Ep)
and lamina propria (LP) of the periodontitis-affected tissue samples.
2.2 | Clinical measurements
Oral hygiene was assessed by the plaque index (PI) of the patients,
which determined the presence or absence of plaque at the gingival
margin (GM), and it was expressed as a percentage of biofilm on the
total of the tooth surfaces. BOP was recorded based on the pres-
ence or absence of bleeding up to 30 seconds after probing with a
manual probe, and it was expressed as the percentage of bleeding
faces of the total examined.
The PD and CAL were measured with a manual probe at six sites
per tooth (mesiobuccal, buccal, distobuccal, distolingual, lingual and
mesiolingual). The PD was measured from the free GM to the bot-
tom of the periodontal pocket, whereas CAL from the cementoe-
namel junction to the bottom of the periodontal pocket.
All probing measurements were performed using a manual
University of North Carolina—UNC Periodontal probe (Hu-Friedy).
2.3 | Collection of periodontitis-affected
tissue samples
Periodontitis-affected tissue samples were chosen strictly fol-
lowing the inclusion criteria above commented and based on the
|
      3
clinical parameters recorded per patient, as well as previously de-
tailed methodologies.12,17,18,27 Accordingly, gingival biopsies (one
biopsy per patient) were obtained from the site with greater PD
(≥6 mm), and in the case of more than one site with the same PD, the
followed criteria were radiographic bone loss in the most affected
tooth, BOP and PI, in order to assess the most diseased site. The
samples were collected with adequate depth from either buccal or
lingual sites (medium size of 5 mm) including epithelium and lamina
propria, which are fundamental to understanding the anatomy of
blood supply to gingival tissue, and they were obtained during the
first procedure for the treatment of periodontal disease (periodontal
surgery). Specific sites corresponded to the followed teeth in III-B
27, 26, 16, 26, 26, 41, 27, 41, 16, 16 and IV-C 26, 27, 16, 26, 17, 36,
46, 16, 16, 17.
In the control patients, gingival tissue was removed during sur-
gical procedures inherent to their original treatment plan (eg third
molar extraction, clinical crown increase or gingivectomy). In both
periodontitis-affected and healthy sites, under local anaesthesia
(mepivacaine 2% with adrenaline [1:100 000]), and following bio-
safety standards, gingival tissue samples were obtained by surgical
excision performed with a 15C scalpel blade. The removed gingival
tissue (about 5 × 2 × 2 mm) was fixed in 4% buffered formaldehyde
for 24 hours.
2.4 | Analysis of tissue samples
Following the previous methodology, 28 serial sections of 3 µm
in thickness were obtained. For each block, four-section tissues
were prepared: one section was stained with haematoxylin and
eosin (H&E), and the other three were submitted to IHC reactions.
For the antigen retrieval of the CD4, CD25 and FOXP3 markers,
the tissue sections received a pre-treatment on a pressure cooker
containing 10 mmol/L of sodium citrate buffer (pH 6.0). Sections
were successively incubated with the primary monoclonal anti-
bodies against CD4 (clone 1F6, 1:200 dilution, Leica Biosystems),
CD25 (clone 4C9, 1:400 dilution; Leica Biosystems) and FOXP3
(clone SP97, 1:500 dilution, Spring Bioscience). After the primary
antibodies were incubated, the conjugated secondary antibodies
were incubated with the method of streptavidin-biotin-peroxi-
dase (K0690, Universal Dako LSAB+ Kit, Peroxidase), followed by
diaminobenzidine chromogen and counterstained with Carazzi's
haematoxylin.
2.5 | Analysis of immunohistochemical reactions
The cell analysis for CD4, CD25 and FOXP3 markers was per-
formed using a light microscope (Leica DM500) connected to
a high-resolution camera (Leica ICC500) and interconnected
to a monitor and computer, such as described by Motta et al. 28
Positive cells (brownish colouration) for each antibody were eval-
uated at a magnification of x100, and areas of a higher density of
 |
4       da MOTTA et al.

positive cells were recorded at a magnification of x400 (area of for age or gender were done. Student's t test was applied to evaluate
2
0.785 mm ). CD4 and CD25 showed a cell membrane staining pat- the differences between the Ep and LP. Prism software, version 6.0
tern, whereas FOXP3 exhibited a nuclear staining pattern. These (GraphPad) was used to run the tests (confidence level of 95%).
antibodies were evaluated in consecutive serial histological sec-
tions for successful comparative analysis of the immunostained
areas. 3 | R E S U LT S

The demographic characteristics of the sample are shown in

2.6 | Statistical analysis
The measurement variable was the immunostained cell count. The fac-
tors of variation were the three periodontal conditions (control, III-B
periodontitis and IV-C periodontitis) and the three immunomarkers
(CD4, CD25 and FOXP3). For immunostained cell count, each tissue
specimen was divided into two areas: (a) Ep and (b) LP. For each area, the
cell count was performed in ten fields (at a magnification of x400). For
each immunomarker and periodontal condition, the mean and stand-
ard deviation of each patient were recorded. The Shapiro-Wilk test
was used to observe normality and homoscedasticity, and a two-way
ANOVA, followed by Fisher's LSD test, was used to observe the effect
of the two factors of variation on the discrete variable. No corrections
Table 1, and the values found for the immunopositive cells are shown
in Table 2. Figure 1 shows the IHC findings, morphologically illus-
trating these values. In general, CD4+ cells were more frequently
observed, followed by similar amounts of CD25+ and FOXP3+ cells
(IV-C, P  <  .05; III-B, P  >  .05; control group, P  <  .05). Assessing the
distribution of immunopositive cells in Ep and LP, all immunomarkers
showed significant LP localization in all the groups. When compared
to the control group, IV-C and III-B periodontitis patients showed
a significantly higher number of immunostained cells for all immu-
nomarkers. Notably, both CD25+ and FOXP3+ cells were detected in
a significantly higher number in III-B than IV-C periodontitis patients
(P < .05). No interaction between the immunomarkers and periodon-
tal disease was observed.

TA B L E 1   Patient's clinical and demographic characteristics

Patients group Age range/

(Stage-grade) n Gender % mean MTPPa ATPPb PD CAL BOP PI

Periodontitis 10 6 (60%) female 35 to 65/ 51 22.08 ± 1.50 5 7.29 ± 0.94 6.6 ± 1.15 55 ± 28.03 69.8 ± 33.7

(III-B) 4 (40%) male
Periodontitis 10 7 (70%) female 18 to 42/ 36 17.5 ± 1.93 6 7.5 ± 0.92 7.5 ± 1.16 37.65 ± 6.05 35.15 ± 4.40
(IV-C) 3 (30%) male
Control 10 7 ( 70% female 18 to 35/ 25 27.33 ± 1.55 0 1.7 ± 0.48 1.7 ± 0.48 10.45 ± 6.04 6 ± 3.81
3 (30%) male

Note: Mean PD and CAL were obtained by taking the sum of all PDs and CAL of affected teeth divided by the number of affected teeth per patient.
Mean of BOP and PI was obtained the mean of all BOP and PI, and expressed as a percentage on the total of tooth surfaces.
Abbreviations: ATPP, affected teeth per patient; BOP, Bleeding on probing (%); CAL, clinical attachment loss (mean/mm); MTPP, mean of teeth per
patient; n, number; PD, probing depth (mean/mm); PI, Plaque index (%); SD, standard deviation.
a
Mean of number of teeth per patient.
b
Mean of number of teeth affected per patient.

TA B L E 2   Distribution (average and standard deviation) of CD4−, CD25− and FOXP3-positive cells in all groups

Periodontal diagnoses

Stage IV, grade C periodontitis (n = 10) Stage III, grade B periodontitis (n = 10) Control (n = 10)

Markers Ep LP Ep LP Ep LP

CD4 3.6 (±1.7)*aA 12.0 (±5.6) aA 3.4 (±1.1)*aA 12.0 (±3.9) aA 2.5 (±1.5)*aA 5.5 (±2.9) aA
CD25 3.3 (±2.0) aA 7.8 (±4.4) bA 5.5 (±4.1) bB 8.9 (±3.8) aA 1.5 (±0.9) aA 3.1 (±5.4) bA
FOXP3 2.6 (±1.7) aA 7.6 (±3.9) bA 3.3 (±1.7)*aA 11.0 (±2.9) aA 1.9 (±1.2) aA 2.8 (±0.7) bB

Note: In accordance with 2-way analyses of variance and Fisher's LSD test (P < .05). Different uppercase letters on the lines indicate statistically
different means (P < .05) of immunostained cells considering the periodontal diagnosis. Different lowercase letters on the columns indicate
statistically different means (P < .05) of immunostained cells considering the CD4, CD25 and FOXP3 immunomarkers. Asteristics indicate statistical
differences between Ep and LP according to Student's t test (P < .05).
Abbreviations: Ep, epithelium; LP, lamina propria.
da MOTTA et al. |
      5

F I G U R E 1   Illustrative image of the

immunohistochemical findings of Tregs
in periodontitis-affected tissue samples
(epithelium [Ep] and lamina propria [LP])
from patients with IV-C periodontitis,
III-B periodontitis and clinically healthy
gingiva (Control). Note that CD4+ cells are
observed in higher number than CD25+
and FOXP3+ cells, in all groups. Different
from III-B periodontitis, patients with IV-C
periodontitis presented significant lower
amount of CD25+ and FOXP3+ cells in
the periodontitis-affected tissues. The
arrows indicate some immunopositive
cells for each immunomarker (original
magnification, x400; scale bar = 50 µm)

4 | D I S CU S S I O N Treg immunomarkers (CD4/CD25/FOXP3) regarding the staging

Several studies have been developed to analyse the presence and
distribution of cells of the immune system following the old clas-
27
sification of periodontal disease from 1999, in order to clarify its
immunological mechanisms that could help in the treatment plan, as
well as to point out new therapies.13,18,22,29,30 Although some stud-
ies indicate that Tregs play a protective role against bone resorption
in periodontal disease, probably by the downregulation of RANKL
expression mediated by Th1 and Th17 cells in periodontitis,14,18 to
date, notably, no study has focused on the comparative analysis of
and grading of periodontitis, in an attempt to better understand the
complex link between the immune system and periodontal bone tis-
sue homeostasis.13,15,16,18,29
Our results, evaluating human periodontitis-affected tissue
samples through immunohistochemistry, show for the first time
that CD25+ and FOXP3+ cells are present in a significantly higher
number in III-B than IV-C periodontitis patients. These findings may
attempt to explain the clinicopathological differences observed
in these patient subgroups, especially considering the relation-
ships between the immune system and bone resorption processes
 |
6       da
(osteoimmunology), and perhaps the model that may help to eluci-
date the complex mechanisms involved in the pathogenesis of the
periodontal disease.
In fact, some studies have shown that several subgroups of T cells
can be observed in periodontal disease, including Th1, Th2, Th17 and
13,14,17,18,25,26,31
Tregs. However, this is the first study emphasizing
the presence and distribution of Tregs in periodontitis-affected tis-
sue samples based on the staging and grading of periodontitis.3,4 The
importance of this comparative analysis is supported by the profile
of the osteoimmunological mechanisms of these lymphocyte pop-
ulations. Thus, Th1 and Th17 are associated with bone destruction
and osteoclastogenic properties, whereas Th2 and Tregs present sup-
pressive effects on inflammatory osteolysis.18,19 Interestingly, when
comparing patients affected by gingivitis and periodontitis, Treg im-
munomarkers were expressed more highly in periodontitis than gingi-
vitis.13 Taken together, our results suggest that the significantly higher
number of both CD25+ and FOXP3+ cells in patients with III-B than
IV-C periodontitis may explain, at least in part, the clinicopathological
differences observed in these patient subgroups. This is corroborated
by previous studies that show that Tregs attenuate the progression of
experimental periodontitis in mice6 and play a protective role against
bone resorption in human periodontal disease.14,18 In these stud-
ies, Tregs were associated with the expression of IL-10, CTLA-4 and
TGF-β cytokines. Interestingly, treatment with anti-glucocorticoid-in-
duced tumour necrosis factor (TNF)-related protein (GITR) monoclo-
nal antibody, an inhibitor of Treg function, showed increased alveolar
bone loss, as well as the migration of inflammatory cells.6
In this study, as mentioned above, the differential expression of
Treg immunomarkers in III-B and IV-C periodontitis patients may be
influencing its clinical course. In fact, patients with IV-C periodonti-
tis appear to have a dysregulated immune system response leading
to an inflammatory process and the release of inflammatory media-
tors that mediate accelerated bone loss. 28 In this context, we have
recently shown that the predominance of immature dendritic cells in
patients with high rates of periodontal bone resorption seems to be
implicated in its pathogenesis, probably due to its inability to induce
the activation of immune cells. 28 These observations are relevant if
we also consider other studies which show that Tregs are important
in the immunoregulation of the periodontal disease13,14,18,19,31 and,
in view of our results, Tregs probably mediate the inhibition or the
impairment of osteoclast activity.
Unlike CD4, FOXP3 and CD25 appear to be Treg-specific mark-
ers. This may explain our results, which show the CD4 marker being
prevalent in all groups when compared with CD25 and FOXP3 im-
munomarkers. For a better interpretation of our results, consec-
utive serial histological sections were evaluated, allowing us to
efficiently compare immunostained areas, being relevant for CD25
and FOXP3, which identified similar an immunostaining pattern.
In the last decade, the field of “osteoimmunology” has emerged
with great interest indicating a link between immune system cells
and bone tissue homeostasis, including studies evaluating the patho-
genesis of the periodontal disease.10,11 The understanding of the bi-
ological mechanisms that control the bone remodelling processes
MOTTA et al.
will clarify not only the events involved in the regulation of bone tis-
sue homeostasis but also the pathophysiology of accelerated bone
loss, as observed in stage IV periodontitis and in other diseases such
as osteoporosis and rheumatoid arthritis.32
In conclusion, our results show that IV-C, when compared with
III-B periodontitis patients, present a significantly lower number of
CD25+ and FOXP3+ cells, when evaluating periodontitis-affected
tissue samples comparatively by immunohistochemistry. These
findings may explain some of the clinicopathological differences ob-
served in IV-C and III-B periodontitis patients.
AC K N OW L E D G E M E N T S
Financial support came from the State of São Paulo Research
Foundation (FAPESP) grants 2016/11419-0 (Jorge Esquiche León)
2013/08589-3 (Camila Tirapelli and Raphael Jurca G da Motta) and
2016/02713-2 (Luciana Yamamoto de Almeida).
C O N FL I C T O F I N T E R E S T
The authors declare that there are no conflicts of interest in this
study.
ORCID
Raphael J. G. da Motta  https://orcid.org/0000-0002-9149-1831
Luciana Yamamoto Almeida  https://orcid.
org/0000-0001-5403-4052
Kelly R. V. Villafuerte  https://orcid.org/0000-0002-0231-5482
Alfredo Ribeiro-Silva  https://orcid.org/0000-0002-3579-5048
Jorge E. León  https://orcid.org/0000-0002-9668-5870
Camila Tirapelli  https://orcid.org/0000-0001-5020-6515
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How to cite this article: da Motta RJG, Almeida LY,
Villafuerte KRV, Ribeiro-Silva A, León JE, Tirapelli C. FOXP3+
and CD25+ cells are reduced in patients with stage IV, grade
C periodontitis: A comparative clinical study. J Periodont Res.
2019;00:1–7. https​://doi.org/10.1111/jre.12721​