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Published in final edited form as: Int Immunopharmacol. 2019 Feb 10;69:299–306. doi: 10.1016/j.intimp.2019.01.031

Down-regulated Treg Cells in Exacerbated Periodontal Disease during Pregnancy

Aislinn Hays a, Xingyu Duan a, Jianxin Zhu a, Wei Zhou b, Satya Upadhyayula a, Juili Shivde a, Li Song c, Huizhi Wang a, Li Su d, Xuyu Zhou e, Shuang Liang a,*
PMCID: PMC6411422  NIHMSID: NIHMS1521256  PMID: 30753969

Abstract

Pregnancy is a special period marked with complicated changes in various immune responses. Although pregnant women are prone to developing gingival inflammation, its immunological mechanism remains to be clarified. In a modified ligature-induced periodontal disease murine model, pregnant mice developed more severe alveolar bone loss. Using this model, we investigated the Treg responses during exacerbated periodontal disease in pregnant mice. We tested Treg-associated molecules in gingival tissues by quantitative real-time PCR and found decreased gingival expression of Foxp3, TGFβ, CTLA-4, and CD28 in pregnant mice after periodontal disease induction. We further confirmed that lower number of Treg cells were present in the cervical lymph nodes of pregnant periodontitis mice. Treg cells from the cervical lymph nodes of ligated pregnant mice and non-pregnant mice were tested for their suppressive function in vitro. We manifested that Treg suppressive function was also down-regulated in the pregnant mice. Additionally, we demonstrated that more inflammatory Th17 cells were present in the cervical lymph nodes of ligated pregnant mice. Therefore, impaired Treg development and function, together with upregulated Th17 response, may contribute to the exacerbated periodontal disease during pregnancy.

Keywords: Treg, Th17, alveolar bone loss, periodontal disease, pregnancy, Porphyromonas gingivalis

1. Introduction

Pregnant women are more prone to gingival inflammation and periodontal disease [1-14], which can be detrimental to pregnant women’s health and dangerous to their fetuses [15-20]. It is critical to understand the immunological mechanisms of more severe periodontal disease during pregnancy. Various immune cells are responsible for the aggravated periodontal conditions during pregnancy [21]. As one of the most important components of host immunity, T cells are involved in almost all immune interactions. Regulatory T cells (Treg) are a subset of T cells that emerge as critical immune regulators and maintain immune homeostasis. Transcription factor Foxp3 has been found to program Treg cell development as well as function [22, 23]. Treg cells suppress T cell proliferation and cytokine production [24-26] as well as the innate immune responses [27]. Treg cells are enriched in the periodontal lesions and play critical roles in periodontal diseases [28-30]. In certain infectious diseases, Treg cells can be exploited by pathogens to down-regulate host immune responses and facilitate the survival of the pathogens [31]. On the other hand, Treg cells can down-regulate excessive host immune responses and control the diseases. The balance between Treg and other immune cells satisfies the survival strategy of both pathogens and the hosts [31, 32].

Treg is believed to play a protective role in periodontal disease by suppressing excessive inflammation and collateral tissue damages [33, 34]. During pregnancy, Treg cells in uterus-draining lymph nodes and systemic Treg are up-regulated [35, 36]. The upregulation of Treg cells seems contradictory to the symptom of elevated periodontal inflammation during pregnancy. To this date there is no research on the Treg change responding to oral infection and its role in periodontal disease during pregnancy. It is possible that the Treg response at the fetal-maternal interface is different from that in the oral cavity during pregnancy.

Periodontal pathogens are reported to be critical in exacerbated periodontal diseases during pregnancy [10, 37]. Porphyromonas gingivalis, one of the most widely investigated oral pathogens, has been found in pregnant women with aggravated gingival inflammation [1, 5, 38, 39]. P. gingivalis is well known for its function to regulate host immunity and induce oral microbiota dysbiosis, thus causing inflammation and collateral tissue damage. Interestingly, P. gingivalis infection induced Treg change in vitro and in vivo [40]. P. gingivalis can also promote Th17 development [41]. Th17 is one of the newly discovered inflammatory T cell subsets, which is involved in various inflammatory diseases including periodontitis [41-43]. RORγt has been regarded as specific for Th17 and responsible for the production of pro-inflammatory cytokines in Th17 cells [44]. Th17 development is closely related to Treg development in that TGFβ is critical for the development of both cell subsets [45, 46]. It is also intriguing to assess the Th17 response in periodontal disease during pregnancy.

In our report, by the placement of silk ligature around 2nd molar and P. gingivalis infection, we induced more severe inflammatory bone loss in the pregnant mice, accompanied by the down-regulated gingival expression of Treg-related molecules. We illustrated that there were fewer Treg cells in the cervical lymph nodes from the pregnant mice with periodontal disease, accompanied by an increased presence of Th17 cells. Additionally, the Treg cells from the pregnant mice exhibited compromised suppressive function. These finding demonstrated that the mechanism of more severe periodontal disease might be attributed to fewer Treg cells and impaired anti-inflammatory regulation by Treg cells.

2. Materials and Methods:

2.1. Mice

C57BL/6 mice (8–10 wk of age) were purchased from Jackson Lab and kept in animal facilities at University of Louisville, in appliance with the established Federal and State policies. All handling and processing were approved by Institutional Animal Care and Use Committee.

2.2. Ligature-induced periodontal disease model

Female C57BL/6 mice were paired with male mice and checked for vaginal plugs, which indicates successful copulation. As soon as vaginal plugs were found, females were separated from males and counted as day 0 of gestation, upon confirmation of pregnancy at a later date. The pregnant mice were ligated around the maxillary 2nd molar with 6-0 silk suture at day 8 of gestation; the suture resided within the gingival sulcus for the remainder of the experiment. During the experiments, the mice were infected with 109 CFU P. gingivalis ATCC 33277 in 2% carboxymethylcellulose every other day. Ten days after ligature placement, the experiments were terminated and jawbones were harvested. The distance from the cementoenamel junction to the alveolar bone crest (CEJ-ABC) was measured on the ligated second molar (three sites corresponding to mesial cusp, palatal groove, and distal cusp) and the affected adjacent regions (sites corresponding to distal cusp and distal groove of the first molar, and palatal cusp of the third molar). To calculate pro-inflammatory periodontal bone loss, the mean CEJ-ABC distance from the group of sham-ligated mice was subtracted from the CEJ-ABC distance for each mouse. The results were presented in mm; the values indicated bone loss relative to controls. Gingival tissues or cervical lymph nodes were harvested for the later assays, depending on the purpose of the experiments.

2.3. Gingival mRNA expression

Gingival tissue was excised from around the maxillary molars for mRNA harvest. The expression of the interested molecules was determined by quantitative real-time PCR. Briefly, mRNA was extracted from gingival tissue, using the PerfectPure RNA kit (5 Prime; Fisher, Waltham, MA, USA). High-Capacity cDNA Archive kit (Applied Biosystems, Foster City, CA, USA) was used to reverse-transcribe mRNA, and quantitative real-time PCR (qPCR) was performed using the ABI 7500 System, according to the manufacturer’s protocol (Applied Biosystems). TaqMan probes, sense primers, and antisense primers for genes of interest and a housekeeping gene (glyceraldehyde 3-phosphate dehydrogenase, GAPDH) were purchased from Applied Biosystems.

2.4. Lymphocyte isolation and detection

Cervical lymph nodes were removed from mice and homogenized through a nylon mash (70 μM). The cells were then stained with fluorescence-conjugated anti-CD3, anti-CD4, anti-RORγt, and anti-Foxp3 (eBioscience). Transcription factor staining buffer set (eBioscience) was used for RORγt and Foxp3 intracellular staining. Stained cells were then acquired and analyzed on FACSCalibur using CellQuest (BD Biosciences) on FACSCelesta using Flowjo.

2.5. T cell in vitro proliferation

Foxp3-EGFP transgenic mice (B6.Cg-Foxp3tm2(EGFP)Tch /J) [47], in which EGFP and the Treg-specific transcription factor Foxp3 are co-expressed, were induced for gingival inflammation during pregnancy as mentioned before. Treg cells were sorted by fluorescence flow cytometry based on EGFP expression from gingival tissue of B6.Cg-Foxp3tm2(EGFP)Tch/J mice. As previously reported as a standard method to test Treg suppressive function, we determined the ability of these Tregs to suppress effector T cell proliferation [48, 49]. Briefly, CD4+CD25- naïve T cells were harvested from spleens and lymph nodes of naive WT C57BL/6 mice using magnetic column and anti-CD4 and anti-CD25 beads (Miltenyi Biotec, Auburn, CA). Irradiated spleen cells were used as antigen presenting cells. Treg cells (GFP+ cells) were sorted from infected pregnant or non-pregnant B6.Cg-Foxp3tm2(EGFP)Tch/J mice. Treg cells were then co-cultured with 25,000 CD4+ CD25− effector cells at different ratios in the presence of antigen presenting cells (100,000 cells) and 10 μg/ml anti-CD3 antibody. Cells were cultured in complete media (RPMI 1640, 10% heat-inactivated FCS, 2 mM glutamine, 10 mM Hepes, 100 U/ml penicillin G sodium, 100 μg/ml streptomycin sulfate, and 10 −5 M 2-mercaptoethanol) at 37°C and 5% CO2 for 3 days, after which the CCK-8 solution was added to each well to a final concentration of 100 μl/ml. The cells were incubated for an additional 2 hours, and the absorbance was then measured at 450nm with a microplate reader.

2.6. Statistics

Data were evaluated by two-way ANOVA (GraphPad Prism7). Two-tailed t tests were also performed where appropriate (comparison of two groups only). Differences were considered statistically significant at thep < 0.05 level.

3. Results

3.1. Exacerbated periodontal conditions in pregnant mice

A ligature induced periodontal disease animal model, in which periodontal disease was induced by molar ligation and P. gingivalis infection, was modified as described in our previous publication [50]. Our results validated the model by showing that ligation and P. gingivalis infection caused significantly higher alveolar bone loss in pregnant mice (Fig. 1).

Fig. 1.

Fig. 1

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Periodontal bone loss in ligated pregnant and non-pregnant mice. (A) Representative images from the maxillae of ligated (lower panels) or non-ligated (upper panels) non-pregnant (Non-preg; left panels) and pregnant (Preg; right panels) mice. (B) The mm distance from the cementoenamel junction (CEJ) to the alveolar bone crest (ABC) was measured at 6 most affected maxillary palatal sites and the readings were totaled for each mouse. The CEJ-ABC reading of each mouse was represented by each dot. The data from CEJ-ABC readings were transformed to indicate bone loss, as outlined in Materials and Methods. Asterisks indicate statistically significant (***, p < 0.001) differences between different groups.

3.2. Reduced gingival expression of Treg-related molecules in pregnant mice

As one of the most important cell subsets in regulating host immune responses, Treg can curb both innate immunity and adaptive immunity. Foxp3 is a transcription factor that is specific to Treg cells and is critical in Treg development and function [22, 23]. TGFβ [48, 51, 52], CTLA-4 [53, 54], and CD28 [55-58] are important for Treg cell development and function. We tested the gingival expression of these Treg-related molecules. In non-pregnant mice, periodontal disease induction led to higher Foxp3 expression. While in pregnant mice, Foxp3 expression is significantly lower than that in ligated non-pregnant mice (Fig. 2A). After ligation, the expression of TGFβ (Fig. 2B) in pregnant mice was also significantly lower than non-pregnant mice. Similarly, both CTLA-4 and CD28 were expressed at lower level in the pregnant mice with periodontal disease (Fig. 2C and D).

Fig. 2.

Fig. 2

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Relative expression of Treg-related cytokines and molecules in the gingivae of ligated pregnant and non-pregnant mice. Quantitative real-time PCR (qPCR) was used to determine gingival mRNA expression levels for the indicated molecules (normalized against GAPDH mRNA levels). The gingivae used were excised from pregnant (Preg) and non-pregnant (Non-Preg) C57/BL6 mice. Results are shown as fold change relative to non-pregnant sham-ligated mice. Each group represents the mean ± SD of at least 5 separate expression values, corresponding to qPCR analysis of individual mice. Asterisks indicate statistically significant (*, p < 0.05; **, p < 0.01) differences between different groups.

3.3. Lower number of Treg cells in cervical lymph nodes from pregnant periodontal disease mice

In order to confirm the Treg change during exacerbated periodontal disease in pregnant mice, the lymphocytes from cervical lymph nodes were tested by flow cytometry. The percentage of CD3+CD4+Foxp3+ cells (Treg) in CD3+CD4+ cells (Th) was determined. Consistent with the expression of Treg related molecules in gingival tissues, the frequency of Treg cells in ligated pregnant mice was significantly lower than ligated non-pregnant mice (Fig. 3).

Fig. 3.

Fig. 3

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Treg cells in the cervical lymph nodes of ligated pregnant and non-pregnant mice. (A) Representative flow cytometry images of the gated CD3+CD4+ cells from non-ligated (upper panels) or ligated (lower panels), non-pregnant (left panels) and pregnant (right panels) mice; (B) the frequency of Treg cells were shown as percentage of CD3+CD4+Foxp3+ cells in CD3+CD4+ cells. Each mouse was represented by each dot. Asterisks indicate statistically significant (*, p < 0.05;) differences between different groups.

3.4. Impaired suppressive function of Treg cells from pregnant mice

Besides the ratio/number of Tregs, the regulatory/suppressive function of these cells is also critical in affecting the immune response and disease progression. To determine the suppressive function of Treg cells, GFP+ Treg cells were sorted by fluorescence flow cytometry from cervical lymph nodes and co-cultured with effector T cells at different dosages. Treg cell itself did not proliferate in this experiment [59] (Fig. 4). At lower dosages (1:16 and 1:8), Treg cells from ligated pregnant mice were less potent than the ones from non-pregnant mice in suppressing effector cell proliferation (Fig. 4), indicating that the Treg cells from ligated pregnant mice exhibited an impaired regulatory function.

Fig. 4.

Fig. 4

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Treg cells from ligated pregnant mice exhibited less potent regulatory function in vitro than those from ligated non-pregnant mice. After periodontal disease induction in pregnant mice and non-pregnant mice, GFP+ cells (Treg cells) were sorted from cervical lymphocytes by fluorescence-activated cell sorter and tested for regulatory function in vitro. For this assay, 25,000 freshly harvested CD4+ CD25− cells (effector cells) were purified and cocultured at varying (Treg/effector cells) ratios with GFP+ cells from pregnant mice (Preg) or non-pregnant mice (Non-preg) in the presence of irradiated spleen cells (APC) and anti-CD3 for 3 days. Asterisks indicate statistically significant (*, p < 0.05;) differences between different groups. #, indicate statistically significant difference from the group without Treg (group 0).

3.5. Higher number of Th17 cells in cervical lymph nodes from pregnant periodontal disease mice

We further tested the presence of Th17 cells in cervical lymph nodes. The lymphocytes from cervical lymph nodes were stained with fluorescence-conjugated anti-CD3, CD4, and Th17 specific transcription factor RORγt before tested by flow cytometry. The percentage of CD3+CD4+ RORγt+ cells (Th17) in CD3+CD4+ cells (Th) was determined. We found that the frequency of Th17 cells in ligated pregnant mice was significantly higher than ligated non-pregnant mice (Fig. 5).

Fig. 5.

Fig. 5

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Th17 cells in the cervical lymph nodes of ligated pregnant and non-pregnant mice. (A) Representative flow cytometry images of the gated CD3+CD4+ cells from non-ligated (upper panels) or ligated (lower panels), non-pregnant (left panels) and pregnant (right panels) mice; (B) the frequency of Th17 cells were shown as percentage of CD3+CD4+RORγt+ cells in CD3+CD4+ cells. Each mouse was represented by each dot. Asterisks indicate statistically significant (*, p < 0.05; **, p < 0.01) differences between different groups.

4. Discussion:

While the inflammation during pregnancy can be detrimental to pregnant women’s health, negatively impact their quality of life, and may even cause adverse pregnancy outcomes, the mechanism of the more severe oral inflammation is illusive. We modified a widely applied ligature model and utilized it to understand the pathogenesis of the disease in pregnant mice, as described in our previous publication [50]. This model induced inflammation in a shorter time period, which enabled us to fit the experiment into the time frame of pregnancy. We ligated the maxillary 2nd molar of the pregnant mice with sutures and infected the mice with P. gingivalis which induced significantly more severe alveolar bone loss in ligated pregnant mice than ligated non-pregnant mice (Fig. 1), confirming that periodontal deterioration could be accelerated by pregnancy [10, 14].

P. gingivalis is one of the most important periodontal pathogens and readily found in the pregnant women with gingival inflammation [38]. A series of recent research of periodontal disease pathogenesis indicated that P. gingivalis infection caused disruption of immune homeostasis and normal oral microflora, subsequently leading to periodontitis [60, 61]. P. gingivalis might exploit similar pathogenic mechanisms to induce more severe periodontal disease during pregnancy. In this paper, our research investigated the complicated yet seldom investigated T cell responses to periodontal pathogens in the oral cavity during pregnancy; whereas previous research focused on the immune responses at the fetal-maternal interface. Treg cells have manifested their critical roles in down-regulating the host immunity and inflammation [24-27]. Indeed, we found that after periodontal disease induction, IL-10 expression was lower in the pregnant mice than non-pregnant mice [50], which led us to further investigate Treg cell responses in the exacerbated periodontal disease during pregnancy. We tested the gingival mRNA expression of Treg-related molecules, including Foxp3, TGFβ, CTLA4, and CD28. We found that after ligation and P. gingivalis infection, the gingival expressions of the Treg-specific transcription factor Foxp3 was significantly lower in pregnant mice than non-pregnant mice (Fig. 2A). TGFβ, which is famous for its anti-inflammatory function, has been well manifested to be critical in Treg development and function [48, 51, 52]. Gingival expression of TGFβ was also lower in the pregnant mice with periodontal diseases (Fig. 2B). CTLA-4, which is highly expressed in Treg cells and important for Treg development [51, 53, 54], is expressed in T cells from patients with periodontitis [62]. CD28, which shares ligands CD80/CD86 on the antigen presenting cells with CTLA-4, is also critical for Treg development and maintenance [55-58]. Lower gingival expression of these molecules in pregnant mice indicated that Treg cell development was impaired in the pathogenic environment (Fig. 2C and D).

To confirm that the periodontal pathogen-induced Treg cell development is down-regulated during pregnancy, we harvested lymphocytes from cervical lymph nodes and tested the frequency of Treg cells through flow cytometry. Our results in Fig. 2A and Fig. 3 showed that Treg cells were up-regulated in non-pregnant mice after periodontal disease induction. This observation is consistent with previous reports, which have shown that Treg cells were enriched in periodontal lesions [28, 29], and fulfilled protective function in inflammation-induced bone loss [30]. This protective mechanism from Treg cells might be impaired in the pregnant mice. Indeed, in the pregnant mice that had been induced for periodontal disease, we found lower frequency of Treg cells in helper T cells than the non-pregnant mice with periodontal disease (Fig. 3). Pregnancy status reversed the periodontal disease-induced Treg development. No significant difference in Foxp3 expression (Fig. 2A) or Treg cell (Fig. 3) was found between non-pregnant and non-ligated mice versus pregnant and ligated mice. These results imply that the periodontal pathogenesis during pregnancy could be attributed, at least partially, to the compromised Treg development.

Besides their roles in Treg development and maintenance, Foxp3, TGFβ, and IL-10 are also important for Treg suppressive function [51, 54, 63-65]. While CTLA-4 also plays a critical role in Treg development and maintenance, its role in Treg function is controversial [66, 67]. Since Treg-related molecules were down-regulated in the pregnant mice with periodontal disease, it is possible that Treg cells from these mice exhibit impaired regulatory function and are less effective in suppressing effector T cell proliferation. Therefore, we co-cultured Treg cells with effector T cells and tested the ability of Treg cells to suppress effector T cell proliferation in a commonly accepted method [48, 49]. Indeed, we showed that after periodontal disease induction, the suppressive function of Treg cells from pregnant mice were not as potent as the ones from non-pregnant ones (Fig. 4). Our results showed that both Treg number and Treg function were down-regulated in the pregnant mice with periodontal disease.

Th17 cells are a subset of inflammatory T cells and are involved in various inflammatory diseases [41-43]. Th17 development is closely related to Treg development. While TGFβ promotes Treg development, it directs Th17 differentiation with the presence of IL-6 [45]. Th17 can be induced in periodontal disease or in response to periodontal pathogen stimulation; however, its effect on periodontal disease progression is still unclear. It is intriguing to reveal whether the Th17 response is up-regulated in periodontal disease during pregnancy. Transcription factor RORγt is specifically expressed in Th17 and was labeled to determine the presence of Th17 by flow cytometry. Indeed, we detected higher number of Th17 cells in the cervical lymph nodes from pregnant periodontal disease mice (Fig. 5). Recent publication showed that Treg cells converted into inflammatory Th17 cell in periphery in the inflammatory environment [68, 69], which may be the reason that fewer Treg cells and more Th17 cells were detected in pregnant mice. Treg/Th17 plasticity will be our future research interest.

In summary, pregnant mice showed exacerbated inflammatory alveolar bone loss after periodontal disease induction in our animal model. Lower gingival expression of Treg-related molecules implied that down-regulation of Treg is involved the pathogenesis of the disease. Pregnant mice with periodontal disease showed decreased Treg cells in their cervical lymph nodes. Besides, these Treg cells exhibited less potent regulatory function than the cells from non-pregnant periodontal disease mice. Furthermore, Th17 cell increased in the pregnant mice with periodontal disease, which implies that an imbalance in Th17/Treg might contribute to the more severe periodontal disease during pregnancy. Our finding has therefore helped us to better understand the mechanisms underlying periodontal disease susceptibility and/or progression during pregnancy.

Highlights.

  • Fewer Treg cells were present in the pregnant mice with periodontal disease.

  • Treg cells from the pregnant mice with periodontal disease exhibited impaired suppressive function.

  • More Th17 cells were present in the pregnant mice with periodontal disease.

  • Imbalance in Th17/Treg might contribute to the exacerbated periodontal disease during pregnancy.

Acknowledgement:

This work was supported by NIH DE025388 (S.L.). The authors report no conflicts of interest related to this work.

Footnotes

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References:

  • 1.Carrillo-de-Albornoz A, Figuero E, Herrera D, and Bascones-Martinez A (2010). Gingival changes during pregnancy: II. Influence of hormonal variations on the subgingival biofilm. Journal of clinical periodontology 37, 230–240. [DOI] [PubMed] [Google Scholar]
  • 2.Arafat AH (1974). Periodontal status during pregnancy. Journal of periodontology 45, 641–643. [DOI] [PubMed] [Google Scholar]
  • 3.Loe H, and Silness J (1963). Periodontal Disease in Pregnancy. I. Prevalence and Severity. Acta odontologica Scandinavica 21, 533–551. [DOI] [PubMed] [Google Scholar]
  • 4.Cohen DW, Shapiro J, Friedman L, Kyle GC, and Franklin S (1971). A longitudinal investigation of the periodontal changes during pregnancy and fifteen months post-partum. II. Journal of periodontology 42, 653–657. [DOI] [PubMed] [Google Scholar]
  • 5.Raber-Durlacher JE, van Steenbergen TJ, Van der Velden U, de Graaff J, and Abraham-Inpijn L (1994). Experimental gingivitis during pregnancy and post-partum: clinical, endocrinological, and microbiological aspects. Journal of clinical periodontology 21, 549–558. [DOI] [PubMed] [Google Scholar]
  • 6.Emmatty R, Mathew JJ, and Kuruvilla J (2013). Comparative evaluation of subgingival plaque microflora in pregnant and non-pregnant women: A clinical and microbiologic study. Journal of Indian Society of Periodontology 17, 47–51. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Cohen DW, Friedman L, Shapiro J, and Kyle GC (1969). A longitudinal investigation of the periodontal changes during pregnancy. Journal of periodontology 40, 563–570. [DOI] [PubMed] [Google Scholar]
  • 8.Gursoy M, Pajukanta R, Sorsa T, and Kononen E (2008). Clinical changes in periodontium during pregnancy and post-partum. Journal of clinical periodontology 35, 576–583. [DOI] [PubMed] [Google Scholar]
  • 9.Taani DQ, Habashneh R, Hammad MM, and Batieha A (2003). The periodontal status of pregnant women and its relationship with socio-demographic and clinical variables. Journal of oral rehabilitation 30, 440–445. [DOI] [PubMed] [Google Scholar]
  • 10.Lieff S, Boggess KA, Murtha AP, Jared H, Madianos PN, Moss K, Beck J, and Offenbacher S (2004). The oral conditions and pregnancy study: periodontal status of a cohort of pregnant women. Journal of periodontology 75, 116–126. [DOI] [PubMed] [Google Scholar]
  • 11.Moss KL, Beck JD, and Offenbacher S (2005). Clinical risk factors associated with incidence and progression of periodontal conditions in pregnant women. Journal of clinical periodontology 32, 492–498. [DOI] [PubMed] [Google Scholar]
  • 12.Machuca G, Khoshfeiz O, Lacalle JR, Machuca C, and Bullon P (1999). The influence of general health and socio-cultural variables on the periodontal condition of pregnant women. Journal of periodontology 70, 779–785. [DOI] [PubMed] [Google Scholar]
  • 13.Christensen K, Gaist D, Jeune B, and Vaupel JW (1998). A tooth per child? Lancet 352, 204. [DOI] [PubMed] [Google Scholar]
  • 14.Gonzalez-Jaranay M, Tellez L, Roa-Lopez A, Gomez-Moreno G, and Moreu G (2017). Periodontal status during pregnancy and postpartum. PloS one 12, e0178234. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Lopez NJ, Da Silva I, Ipinza J, and Gutierrez J (2005). Periodontal therapy reduces the rate of preterm low birth weight in women with pregnancy-associated gingivitis. Journal of periodontology 76, 2144–2153. [DOI] [PubMed] [Google Scholar]
  • 16.Offenbacher S, Lin D, Strauss R, McKaig R, Irving J, Barros SP, Moss K, Barrow DA, Hefti A, and Beck JD (2006). Effects of periodontal therapy during pregnancy on periodontal status, biologic parameters, and pregnancy outcomes: a pilot study. Journal of periodontology 77, 2011–2024. [DOI] [PubMed] [Google Scholar]
  • 17.Wang X, Buhimschi CS, Temoin S, Bhandari V, Han YW, and Buhimschi IA (2013). Comparative microbial analysis of paired amniotic fluid and cord blood from pregnancies complicated by preterm birth and early-onset neonatal sepsis. PloS one 8, e56131. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Sanz M, and Kornman K (2013). Periodontitis and adverse pregnancy outcomes: consensus report of the Joint EFP/AAP Workshop on Periodontitis and Systemic Diseases. Journal of clinical periodontology 40 Suppl 14, S164–169. [DOI] [PubMed] [Google Scholar]
  • 19.Horton AL, and Boggess KA (2012). Periodontal disease and preterm birth. Obstetrics and gynecology clinics of North America 39, 17–23, vii. [DOI] [PubMed] [Google Scholar]
  • 20.Michalowicz BS, Hodges JS, DiAngelis AJ, Lupo VR, Novak MJ, Ferguson JE, Buchanan W, Bofill J, Papapanou PN, Mitchell DA, et al. (2006). Treatment of periodontal disease and the risk of preterm birth. The New England journal of medicine 355, 1885–1894. [DOI] [PubMed] [Google Scholar]
  • 21.Wu M, Chen SW, and Jiang SY (2015). Relationship between gingival inflammation and pregnancy. Mediators of inflammation 2015, 623427. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Fontenot JD, Gavin MA, and Rudensky AY (2003). Foxp3 programs the development and function of CD4+CD25+ regulatory T cells. Nature immunology 4, 330–336. [DOI] [PubMed] [Google Scholar]
  • 23.Hori S, Nomura T, and Sakaguchi S (2003). Control of Regulatory T Cell Development by the Transcription Factor Foxp3. Science 299, 1057–1061. [DOI] [PubMed] [Google Scholar]
  • 24.Cottrez F, Hurst SD, Coffman RL, and Groux H (2000). T Regulatory Cells 1 Inhibit a Th2-Specific Response In Vivo. Journal of immunology 165, 4848–4853. [DOI] [PubMed] [Google Scholar]
  • 25.Gorelik L, Constant S, and Flavell RA (2002). Mechanism of Transforming Growth Factor {beta}-induced Inhibition of T Helper Type 1 Differentiation. J. Exp. Med 195, 1499–1505. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Suvas S, Kumaraguru U, Pack CD, Lee S, and Rouse BT (2003). CD4+CD25+ T Cells Regulate Virus-specific Primary and Memory CD8+ T Cell Responses. J. Exp. Med 198, 889–901. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Maloy KJ, Salaun L, Cahill R, Dougan G, Saunders NJ, and Powrie F (2003). CD4+CD25+ TR Cells Suppress Innate Immune Pathology Through Cytokine-dependent Mechanisms. J. Exp. Med 197, 111–119. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Cardoso CR, Garlet GP, Moreira AP, Junior WM, Rossi MA, and Silva JS (2008). Characterization of CD4+CD25+ natural regulatory T cells in the inflammatory infiltrate of human chronic periodontitis. Journal of leukocyte biology 84, 311–318. [DOI] [PubMed] [Google Scholar]
  • 29.Dutzan N, Gamonal J, Silva A, Sanz M, and Vernal R (2009). Over-expression of forkhead box P3 and its association with receptor activator of nuclear factor-kappa B ligand, interleukin (IL) -17, IL-10 and transforming growth factor-beta during the progression of chronic periodontitis. Journal of clinical periodontology 36, 396–403. [DOI] [PubMed] [Google Scholar]
  • 30.Glowacki AJ, Yoshizawa S, Jhunjhunwala S, Vieira AE, Garlet GP, Sfeir C, and Little SR (2013). Prevention of inflammation-mediated bone loss in murine and canine periodontal disease via recruitment of regulatory lymphocytes. Proceedings of the National Academy of Sciences of the United States of America 110, 18525–18530. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Belkaid Y, Piccirillo CA, Mendez S, Shevach EM, and Sacks DL (2002). CD4+CD25+ regulatory T cells control Leishmania major persistence and immunity. Nature 420, 502–507. [DOI] [PubMed] [Google Scholar]
  • 32.Hesse M, Piccirillo CA, Belkaid Y, Prufer J, Mentink-Kane M, Leusink M, Cheever AW, Shevach EM, and Wynn TA (2004). The Pathogenesis of Schistosomiasis Is Controlled by Cooperating IL-10-Producing Innate Effector and Regulatory T Cells. Journal of immunology 172, 3157–3166. [DOI] [PubMed] [Google Scholar]
  • 33.Garlet GP, Cardoso CR, Mariano FS, Claudino M, de Assis GF, Campanelli AP, Avila-Campos MJ, and Silva JS (2010). Regulatory T cells attenuate experimental periodontitis progression in mice. Journal of clinical periodontology 37, 591–600. [DOI] [PubMed] [Google Scholar]
  • 34.Wang L, Wang J, Jin Y, Gao H, and Lin X (2014). Oral administration of all-trans retinoic acid suppresses experimental periodontitis by modulating the Th17/Treg imbalance. Journal of periodontology 85, 740–750. [DOI] [PubMed] [Google Scholar]
  • 35.Chen T, Darrasse-Jeze G, Bergot AS, Courau T, Churlaud G, Valdivia K, Strominger JL, Ruocco MG, Chaouat G, and Klatzmann D (2013). Self-specific memory regulatory T cells protect embryos at implantation in mice. Journal of immunology 191, 2273–2281. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Kahn DA, and Baltimore D (2010). Pregnancy induces a fetal antigen-specific maternal T regulatory cell response that contributes to tolerance. Proceedings of the National Academy of Sciences of the United States of America 107, 9299–9304. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Kruger M, Casarin RP, Goncalves LB, Pappen FG, Bello-Correa FO, and Romano AR (2017). Periodontal Health Status and Associated Factors: Findings of a Prenatal Oral Health Program in South Brazil. International journal of dentistry 2017, 3534048. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Kornman KS, and Loesche WJ (1980). The subgingival microbial flora during pregnancy. Journal of periodontal research 15, 111–122. [DOI] [PubMed] [Google Scholar]
  • 39.Laine MA (2002). Effect of pregnancy on periodontal and dental health. Acta odontologica Scandinavica 60, 257–264. [DOI] [PubMed] [Google Scholar]
  • 40.Wang L, Guan N, Jin Y, Lin X, and Gao H (2015). Subcutaneous vaccination with Porphyromonas gingivalis ameliorates periodontitis by modulating Th17/Treg imbalance in a murine model. International immunopharmacology 25, 65–73. [DOI] [PubMed] [Google Scholar]
  • 41.Moutsopoulos NM, Kling HM, Angelov N, Jin W, Palmer RJ, Nares S, Osorio M, and Wahl SM (2012). Porphyromonas gingivalis promotes Th17 inducing pathways in chronic periodontitis. Journal of autoimmunity 39, 294–303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Cheng WC, Hughes FJ, and Taams LS (2014). The presence, function and regulation of IL-17 and Th17 cells in periodontitis. Journal of clinical periodontology 41, 541–549. [DOI] [PubMed] [Google Scholar]
  • 43.Eskan MA, Jotwani R, Abe T, Chmelar J, Lim JH, Liang S, Ciero PA, Krauss JL, Li F, Rauner M, et al. (2012). The leukocyte integrin antagonist Del-1 inhibits IL-17-mediated inflammatory bone loss. Nature immunology 13, 465–473. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Ruan Q, Kameswaran V, Zhang Y, Zheng S, Sun J, Wang J, DeVirgiliis J, Liou HC, Beg AA, and Chen YH (2011). The Th17 immune response is controlled by the Rel-RORgamma-RORgamma T transcriptional axis. The Journal of experimental medicine 208, 2321–2333. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Bettelli E, Carrier Y, Gao W, Korn T, Strom TB, Oukka M, Weiner HL, and Kuchroo VK (2006). Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells. Nature 441, 235–238. [DOI] [PubMed] [Google Scholar]
  • 46.Weaver CT, Harrington LE, Mangan PR, Gavrieli M, and Murphy KM (2006). Th17: An Effector CD4 T Cell Lineage with Regulatory T Cell Ties. Immunity 24, 677–688. [DOI] [PubMed] [Google Scholar]
  • 47.Lin W, Haribhai D, Relland LM, Truong N, Carlson MR, Williams CB, and Chatila TA (2007). Regulatory T cell development in the absence of functional Foxp3. Nature immunology 8, 359–368. [DOI] [PubMed] [Google Scholar]
  • 48.Liang S, Alard P, Zhao Y, Parnell S, Clark SL, and Kosiewicz MM (2005). Conversion of CD4+ CD25- cells into CD4+ CD25+ regulatory T cells in vivo requires B7 costimulation, but not the thymus. J. Exp. Med 201, 127–137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49.Liang S, Hosur KB, Nawar HF, Russell MW, Connell TD, and Hajishengallis G (2009). In vivo and in vitro adjuvant activities of the B subunit of Type IIb heat-labile enterotoxin (LT-IIb-B5) from Escherichia coli. Vaccine 27, 4302–4308. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50.Duan X, Hays A, Zhou W, Sileewa N, Upadhyayula S, Wang H, and Liang S (2018). Porphyromonas gingivalis induces exacerbated periodontal disease during pregnancy. Microbial pathogenesis 124, 145–151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 51.Tang Q, Boden EK, Henriksen KJ, Bour-Jordan H, Bi M, and Bluestone JA (2004). Distinct roles of CTLA-4 and TGF-beta in CD4+CD25+ regulatory T cell function. European journal of immunology 34, 2996–3005. [DOI] [PubMed] [Google Scholar]
  • 52.Chen W, Jin W, Hardegen N, Lei K.-j., Li L, Marinos N, McGrady G, and Wahl SM (2003). Conversion of Peripheral CD4+CD25- Naive T Cells to CD4+CD25+ Regulatory T Cells by TGF-{beta} Induction of Transcription Factor Foxp3. J. Exp. Med 198, 1875–1886. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 53.Zheng Y, Manzotti CN, Liu M, Burke F, Mead KI, and Sansom DM (2004). CD86 and CD80 differentially modulate the suppressive function of human regulatory T cells. Journal of immunology 172, 2778–2784. [DOI] [PubMed] [Google Scholar]
  • 54.Tang Q, Adams JY, Tooley AJ, Bi M, Fife BT, Serra P, Santamaria P, Locksley RM, Krummel MF, and Bluestone JA (2006). Visualizing regulatory T cell control of autoimmune responses in nonobese diabetic mice. Nature immunology 7, 83–92. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 55.Tang Q, Henriksen KJ, Boden EK, Tooley AJ, Ye J, Subudhi SK, Zheng XX, Strom TB, and Bluestone JA (2003). Cutting edge: CD28 controls peripheral homeostasis of CD4+CD25+ regulatory T cells. Journal of immunology 171, 3348–3352. [DOI] [PubMed] [Google Scholar]
  • 56.Guo F, Iclozan C, Suh WK, Anasetti C, and Yu XZ (2008). CD28 controls differentiation of regulatory T cells from naive CD4 T cells. Journal of immunology 181, 2285–2291. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 57.Zhang R, Huynh A, Whitcher G, Chang J, Maltzman JS, and Turka LA (2013). An obligate cell-intrinsic function for CD28 in Tregs. The Journal of clinical investigation 123, 580–593. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 58.He X, Smeets RL, van Rijssen E, Boots AM, Joosten I, and Koenen HJ (2017). Single CD28 stimulation induces stable and polyclonal expansion of human regulatory T cells. Scientific reports 7, 43003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 59.Tarbell KV, Yamazaki S, and Steinman RM (2006). The interactions of dendritic cells with antigen-specific, regulatory T cells that suppress autoimmunity. Seminars in immunology 18, 93–102. [DOI] [PubMed] [Google Scholar]
  • 60.Hajishengallis G, Liang S, Payne MA, Hashim A, Jotwani R, Eskan MA, McIntosh ML, Alsam A, Kirkwood KL, Lambris JD, et al. (2011). Low-abundance biofilm species orchestrates inflammatory periodontal disease through the commensal microbiota and complement. Cell host & microbe 10, 497–506. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 61.Hajishengallis G (2014). Immunomicrobial pathogenesis of periodontitis: keystones, pathobionts, and host response. Trends in immunology 35, 3–11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 62.Aoyagi T, Yamazaki K, Kabasawa-Katoh Y, Nakajima T, Yamashita N, Yoshie H, and Hara K (2000). Elevated CTLA-4 expression on CD4 T cells from periodontitis patients stimulated with Porphyromonas gingivalis outer membrane antigen. Clin Exp Immunol 119, 280–286. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 63.Presser K, Schwinge D, Wegmann M, Huber S, Schmitt S, Quaas A, Maxeiner JH, Finotto S, Lohse AW, Blessing M, et al. (2008). Coexpression of TGF-beta1 and IL-10 enables regulatory T cells to completely suppress airway hyperreactivity. Journal of immunology 181, 7751–7758. [DOI] [PubMed] [Google Scholar]
  • 64.Yagi H, Nomura T, Nakamura K, Yamazaki S, Kitawaki T, Hori S, Maeda M, Onodera M, Uchiyama T, Fujii S, et al. (2004). Crucial role of FOXP3 in the development and function of human CD25+CD4+ regulatory T cells. International immunology 16, 1643–1656. [DOI] [PubMed] [Google Scholar]
  • 65.Kasprowicz DJ, Smallwood PS, Tyznik AJ, and Ziegler SF (2003). Scurfin (FoxP3) controls T-dependent immune responses in vivo through regulation of CD4+ T cell effector function. Journal of immunology 171, 1216–1223. [DOI] [PubMed] [Google Scholar]
  • 66.Wing K, Onishi Y, Prieto-Martin P, Yamaguchi T, Miyara M, Fehervari Z, Nomura T, and Sakaguchi S (2008). CTLA-4 control over Foxp3+ regulatory T cell function. Science 322, 271–275. [DOI] [PubMed] [Google Scholar]
  • 67.Paterson AM, Lovitch SB, Sage PT, Juneja VR, Lee Y, Trombley JD, Arancibia-Carcamo CV, Sobel RA, Rudensky AY, Kuchroo VK, et al. (2015). Deletion of CTLA-4 on regulatory T cells during adulthood leads to resistance to autoimmunity. The Journal of experimental medicine 212, 1603–1621. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 68.Zhou X, Bailey-Bucktrout SL, Jeker LT, Penaranda C, Martinez-Llordella M, Ashby M, Nakayama M, Rosenthal W, and Bluestone JA (2009). Instability of the transcription factor Foxp3 leads to the generation of pathogenic memory T cells in vivo. Nature immunology 10, 1000–1007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 69.Massoud AH, Charbonnier LM, Lopez D, Pellegrini M, Phipatanakul W, and Chatila TA (2016). An asthma-associated IL4R variant exacerbates airway inflammation by promoting conversion of regulatory T cells to TH17-like cells. Nature medicine 22, 1013–1022. [DOI] [PMC free article] [PubMed] [Google Scholar]

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