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J Leukoc Biol. 2013 Jan; 93(1): 21–31.
doi: 10.1189/jlb.0512220
PMCID: PMC3525832
PMID: 23077245

Role of TLR2-dependent IL-10 production in the inhibition of the initial IFN-γ T cell response to Porphyromonas gingivalis

Dalia E. Gaddis,*,1 Craig L. Maynard,† Casey T. Weaver,† Suzanne M. Michalek,* and Jannet Katz‡,2
Author information Article notes Copyright and License information PMC Disclaimer

IL-10 produced by T cells and CD11b+ cells utilizes TLR2 signaling and FimA antigen to inhibit early IFN-γ T cell responses to Porphyromonas gingivalis.

Keywords: cytokine regulation, chronic infections, programmed death 1

Abstract

P.g., a Gram-negative bacterium, is one of the main etiological agents of the chronic inflammatory disease, periodontitis. Disease progression is thought to occur as a result of an inadequate immune response, which although happens locally, can also occur distally as a result of the dissemination of P.g. into the circulation. As IL-10 and TLR2 are pivotal molecules in the immune response that P.g. elicits, we hypothesized that TLR2-mediated IL-10 production, following the initial systemic exposure to P.g., inhibits the IFN-γ T cell response. To address this hypothesis, mice were primed with P.g., and the types of cells producing IL-10 and the capacity of T cells to produce IFN-γ following blocking or neutralization of IL-10 were assessed. Our results showed that upon initial encounter with P.g., splenic T cells and CD11b+ cells produce IL-10, which when neutralized, resulted in a substantial increase in IFN-γ production by T cells. Furthermore, IL-10 production was dependent on TLR2/1 signaling, partly in response to the major surface protein, FimA of P.g. In addition, P.g. stimulation resulted in the up-regulation of PD-1 and its ligand PD-L1 on CD4 T cells and CD11b+ cells, respectively. Up-regulation of PD-1 was partially dependent on IL-10 but independent of TLR2 or FimA. These results highlight the role of IL-10 in inhibiting T cell responses to the initial systemic P.g. exposure and suggest multiple inhibitory mechanisms potentially used by P.g. to evade the host's immune response, thus allowing its persistence in the host.

Keywords: cytokine regulation, chronic infections, programmed death 1

Introduction

P.g., a Gram-negative anaerobic bacterium, is considered one of the main causative agents of adult periodontitis, a chronic inflammatory condition that occurs in the periodontal tissues surrounding the teeth and if left untreated, leads to alveolar bone resorption and tooth loss [1–4]. Various studies have shown the importance of the adaptive immune system in disease pathogenesis, as well as in the effective clearance of P.g. [5–8]. Virulence factors derived from P.g. have been shown to dampen the immune response by decreasing inflammatory cytokines and chemokines, by degrading antibodies, or by altering the host's cell signaling response [9–13]. In humans, differential involvement of T and B cells at different stages of infection has been documented [14]. Although the role of T cells in periodontal disease pathogenesis is still unclear [7, 15–18], it is interesting that a microarray analysis from mice immunized with P.g. showed down-regulation of more than 1000 genes modulating CD4 and CD8 T cell activation and function, suggesting the suppression of these cells by P.g. [19]. Moreover, in periodontal disease tissues, P.g. has been shown to colocalize with CD4 T cells [20], and furthermore, T cells from periodontal lesions have a suppressed capacity to produce IL-2 and to proliferate [7, 18]. Taken together, these reported observations point to the ability of P.g. to evade or suppress the adaptive T cell response, which likely allows for the persistence of this pathogen in periodontitis.

T cells, among other cells, produce IL-10, a critical cytokine characterized by its anti-inflammatory properties involved in the regulation of the host immune response [21]. In addition, studies have shown a role for IL-10 in impeding infection resolution [22]. IL-10 is a major cytokine produced during P.g. infection [23–26], and further, P.g.-induced IL-10 down-regulates IL-12 production by myeloid cells [27]. As IL-12 is an important cytokine in deriving IFN-γ-producing Th1 CD4 T cells, this would suggest the suppression of Th1 cells following infection with P.g. Moreover, as IFN-γ facilitates macrophage-mediated opsonophagocytosis of P.g. [28], the anti-inflammatory effect of IL-10 may, in fact, impede bacterial clearance.

Previously published reports have provided evidence that TLR2 facilitates P.g. infection, as P.g. uses TLR2 to escape phagocytosis [29], and P.g.-infected TLR2−/− mice showed rapid clearance of bacteria and less bone resorption compared with WT-infected mice [30]. TLR2 signaling has been implicated in decreasing the inflammatory state of the immune response by inducing IL-10 production or the activation of Tregs [31]. Whereas the production of IL-10 during P.g. infection has been demonstrated previously [32], the role of TLR2 signaling in P.g.-induced IL-10 has not been shown. Moreover, the exact type of cells that produce IL-10 during initial exposure to P.g. or the P.g. antigens playing a role in the regulation of IL-10 induction is not known.

Whereas most studies have focused on understanding the interaction among T cells, IL-10, and P.g. locally in the gingival tissue [32], little is known regarding the type of response induced systemically, particularly as P.g. is capable of disseminating from the local sites of infection to the circulation and to distal sites [33, 34]. In addition, as T cells isolated from gingival tissue of periodontitis patients express mostly memory phenotype [35], and periodontal antigen-specific T cells are capable of migrating from the circulation to the gingival tissue [36], exposure and priming of T cells with P.g. can probably occur systemically in the blood or in secondary lymphoid tissues. Therefore, in this study, we investigated the role of IL-10 in regulating T cell responses upon the initial systemic exposure to P.g. In addition, we determined the type of cells that produce IL-10 in response to P.g. in this setting, the influence of TLR2 signaling in this process, and the possibility that FimA, a P.g. fimbrial protein and virulence factor, is involved in the induction of IL-10 production. The results obtained from this study provide valuable information for a better understanding of the initial mechanisms that lead to the persistence of P.g. in periodontitis and the consequent establishment of a chronic infection.

MATERIALS AND METHODS

Mice

The original TLR2−/−, TLR4−/−, TLR1−/−, and TLR6−/− breeding pairs were obtained under a Materials Transfer Agreement from Dr. Shizuo Akira (Osaka University, Osaka, Japan). IL-10 reporter mice (10BiT) were generated at the University of Alabama at Birmingham (USA) [37]. C57BL/6 WT, and the above strains of mice were bred and maintained in an environmentally controlled, pathogen-free animal facility at the University of Alabama at Birmingham. Mice of both sexes, 6–12 weeks of age, were used in the studies. All experiments were done according to the guidelines of the NIH. Protocols were approved by the Institutional Animal Care and Use Committee at the University of Alabama at Birmingham.

Antibodies

Fluorescent-labeled antibodies against CD4 (clone RM4-5), CD8α (clone 53-6.7), CD11b (clone M1/70), CD25 (clone PC61.5), CD69 (clone H12F3), T-bet (clone eBio4B10), Foxp3 (clone FJK-16S), IFN-γ (clone XMG1.2), PD-1 (CD279; clone J43), PD-L1 (CD274; clone MIH5), and purified mouse α-IL-10 (clone JES2A5) were purchased from eBioscience (San Diego, CA, USA). Fluorescent-labeled antibodies against Thy1.1 (CD90.1; clone OX-7) and purified mouse α-IL-10R (CD210; clone 1B1.3a) were purchased from BD Biosciences (San Jose, CA, USA).

P.g. and priming of mice

P.g. was prepared as described previously with some modifications [38–41]. Briefly, the bacteria were cultured and maintained on enriched trypticase soy plates consisting of trypticase soy agar supplemented with 1% yeast extract, 5% defibrinated sheep blood, hemin (5 mg/l), and menadione (1 mg/l) at 37°C in an anaerobic atmosphere of 10% H2, 5% CO2, and 85% N2. Two strains of P.g. (ATCC 33277) lacking the major surface fimbrial protein FimA, designated as FimA mutant and YPF1, were a kind gift from Dr. Richard Lamont (University of Louisville, KY, USA). The generation and characterization of YPF1 have been reported previously [42]. The FimA mutant was generated by Dr. Lamont as follows. Briefly, insertional inactivation of FimA was performed by fusion PCR, as described previously [43]. Regions immediately upstream and downstream of FimA were amplified and fused to the tetQ gene from pT-COW by PCR. The final construct was introduced into P.g. by electroporation. A double-crossover recombination event was selected by plating with tetracycline selection. Insertion of the replacement allele was confirmed by PCR and Southern hybridization, and loss of mRNA was established by RT-PCR. The FimA mutant and YPF1 strains were grown on trypticase soy agar as described above and supplemented with tetracycline or erythromycin, respectively. Prior to use, bacteria were harvested from the culture plates and resuspended in PBS. The estimated number of bacteria in suspension was determined by reading the OD at 580 nm and extrapolating from a bacterial growth standard curve. For priming experiments, WT or 10BiT mice were injected i.v. through the retro-orbital plexus with 107 CFU P.g. in PBS or PBS-only as controls. Spleens were harvested 8 days following priming, and cells were analyzed as described below. For in vitro stimulation, cells were stimulated with P.g., FimA mutant, or YPF1 at a MOI of five.

CD4 T cell purification and cell stimulation

Splenocytes from naive or P.g.-primed mice were erythrocyte-depleted and used to purify total CD4 T cells using MACS CD4+ T cell isolation kit and LS or CS columns (Miltenyi Biotec, Auburn, CA, USA), according to the manufacturer's instruction. For the preparation of feeder cells, splenocyte suspensions from naive mice were depleted of CD4+ T cells using MACS CD4 beads and LS columns (Miltenyi Biotec), according to the manufacturer's instruction, and irradiated with 3000 rads. Equal numbers of purified CD4+ or CD4+CD25− T cells and feeder cells or total splenocytes were cultured in 96-well plates in RMPI-1640 media, supplemented with 10% heat-inactivated FCS, l-glutamine (2 mM), and β-ME (50 mM) at 37°C in a humidified 7.5% CO2 incubator. Cultures were stimulated (MOI=5) with freshly prepared P.g. and in some cases, the FimA mutant or YPF1 or left unstimulated as controls. For cultures where IL-10 signaling was blocked, α-IL-10 or α-IL-10R was added together with the bacteria at a final concentration of 10 μg/ml. Cultures where α-IL-10 or α-IL-10R was added without bacteria served as controls. Penicillin (50 U/ml) and streptomycin (50 mg/ml) were added to the cultures 4 h following stimulation to kill extracellular bacteria, thus allowing for only the bacteria that were internalized and processed by APCs to be presented to T cells. Unless otherwise indicated, cells were stimulated for 36 h to determine the expression levels of activation markers or transcription factors and for 5 days, for the assessment of cytokine production, as routinely done [44].

Tregs isolation and suppression assay

Splenocytes from naive WT mice were erythrocyte-depleted and used to purify Tregs (CD4+CD25+ T cells) using the MACS CD4+CD25+ T cell isolation kit and LD and MS columns (Miltenyi Biotec), according to the manufacturer's instruction. The CD4+CD25− fraction eluted from the columns was also used in the experiments. Equal numbers of CD4+CD25+ or CD4+CD25− T cells or a combination of both were cultured with feeder cells in supplemented RMPI-1640 media and stimulated as described previously.

Analysis of transcription factors, activation markers, Thy1.1, and intracellular IFN-γ expression

Purified CD4+CD25− T cells from naive WT or TLR2−/− mice were stimulated as described previously, harvested, and stained with CD4 and CD69 or CD25 antibodies in PBS supplemented with 2% BSA and 0.1% sodium azide (FACS buffer) on ice for 30 min. Cells were washed, fixed, and permeabilized with Foxp3 staining buffer (eBioscience) for 30 min on ice. Cells were washed again and stained with T-bet or Foxp3 antibodies in permeabilizing buffer for 30 min on ice, washed, and resuspended in FACS buffer before acquisition.

To determine the expression of PD-1 on CD4 or CD8 T cells ex vivo, spleens from P.g.-primed or control WT mice were harvested, and single-cell suspensions were prepared and stained with CD4, CD8, and PD-1 antibodies in FACS buffer for 30 min on ice. Cells were then washed and fixed with PBS containing 2% PFA. To determine the expression of PD-1 or PD-L1 and Thy1.1 on activated CD4 T cells or CD11b+ cells, respectively, cell cultures were harvested 2 days following in vitro stimulation and stained with CD4 and Thy1.1 and PD-1 or CD11b and Thy1.1 and PD-L1, as described previously. In instances where isolated CD4+CD25− cells were stimulated, cells were stained with CD4, CD69, and PD-1 antibodies.

For the ex vivo detection of Thy1.1 expression on cells isolated from 10BiT mice, total splenocytes from primed or control mice were stained with CD4, CD8, CD11b, and Thy1.1 antibodies. Detection of the dual expression of IFN-γ and Thy1.1 following in vitro stimulation was done with total splenocytes from primed or control mice stimulated for 3 or 5 days. Ten to 12 h prior to cell harvest, GolgiPlug (Brefeldin A, BD Biosciences) was added to cells at 1 μl/ml cell culture. Once harvested, cells were stained with CD4 or CD8 and Thy1.1 antibodies for 30 min on ice, washed, fixed, and permeabilized using IC Fixation Buffer (eBioscience) for 30 min. Cells were then washed and stained with IFN-γ antibodies in permeabilization buffer (eBioscience) for 30 min, washed again, and fixed in PFA. All samples were acquired using FACSCalibur (BD Biosciences) and analyzed using CellQuest software (BD Biosciences).

Cytokine ELISA

Culture supernatants were harvested 5 days following stimulation and were assessed for the levels of IFN-γ (eBioscience) and IL-10 (R&D Systems, Minneapolis, MN, USA) by ELISA, according to the manufacturers' instructions.

Statistical analysis

When data from two experimental groups were compared, two-tail unpaired Student's t-test was used for analysis, whereas data derived from three or more experimental groups were assessed by unpaired ANOVA, followed by post hoc analysis with the Tukey-Kramer multiple comparison test. All of the data were analyzed using the GraphPad InStat Version 3.0a (GraphPad Software, San Diego, CA, USA). Differences between groups were considered significant at the level of P < 0.05.

RESULTS

IL-10 production by T and CD11b+ cells following P.g. stimulation inhibits IFN-γ T cell response

Although several reports have shown that IL-10 is one of the main cytokines produced during P.g. infection and in periodontal lesions [26, 32, 45, 46], no studies have addressed the type of cells that produce this cytokine upon initial encounter with the bacteria. Stimulation of splenocytes from naive or P.g.-primed mice resulted in IL-10 production in the culture supernatants from naive and P.g.-primed cells stimulated with P.g. (Fig. 1A). However, the level of IL-10 was increased significantly in the culture supernatants of cells isolated from P.g.-primed mice (Fig. 1A). This suggests that P.g. induces IL-10 production upon initial encounter of naive cells, and moreover, upon secondary exposure, this bacterium promotes further up-regulation of this anti-inflammatory cytokine. As IL-10 can inhibit Th1-derived cytokines, such as IFN-γ [47], we next assessed IFN-γ production and observed minimal amounts of IFN-γ in P.g.-stimulated cell cultures from naive mice, which was increased upon priming (Fig. 1B). Neutralization of IL-10 with blocking antibodies resulted in a dramatic increase of IFN-γ levels in P.g.-stimulated splenocytes from naive mice (>15-fold) and primed mice (approximately sixfold; Fig. 1B). Similar results were seen with isolated CD4 T cells (Fig. 1) and CD8 T cells (data not shown). Upon assessing other inflammatory cytokines during neutralization of IL-10, our results showed a slight increase in TNF-α (two- to threefold) and also some increase, although inconsistent, in IL-17, yet both cytokine levels were significantly lower than IFN-γ (data not shown). These results suggest that P.g.-induced IL-10 has a substantial effect on the inhibition of IFN-γ production, especially in naive cells encountering this pathogen for the first time.

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P.g.-stimulated splenocytes and CD4 T cells produce IL-10, which inhibits IFN-γ production.

WT mice were primed with P.g. or received PBS as controls. Eight days following priming, total splenocytes or isolated CD4 T cells and irradiated CD4-depleted feeder cells from naive mice were stimulated with P.g. ± α-IL-10. Culture supernatants were harvested 5 days following stimulation and assessed for IL-10 (A) and IFN-γ (B) production by ELISA. Results are expressed as the mean ± sem of triplicate cultures from one of at least five independent experiments. Significant differences were ***P < 0.001, **P < 0.01, and *P < 0.05.

To confirm the type of cells that produce IL-10 in response to P.g., we used IL-10 reporter mice (10BiT), where a Thy1.1 gene replaced the endogenous coding segment of the first exon of the IL-10 gene [37]. These mice harbor a transgene featuring the Thy1.1 (CD90.1) allele under the control of the IL-10 promoter. Thus, surface expression of Thy1.1 enables the identification of cells programmed to express IL-10 (IL-10-competent cells). Upon priming of 10BiT mice with P.g., there was an increase in the percentages (Fig. 2A) and numbers (Fig. 2B) of CD4+Thy1.1+ T cells. Moreover, increased numbers of CD8+Thy1.1+ T cells and CD11b+Thy1.1+ cells were also detected (Fig. 2B). Stimulation of splenocytes from naive or P.g.-primed 10BiT mice, showed an increase in the percentages of Thy1.1+ CD4 T cells (Fig. 2C and F), CD8 T cells (Fig. 2C), and CD11b+ cells (Fig. 2D). These results suggest that in addition to CD4 T cells, CD8 T cells and CD11b+ cells are potential sources of IL-10 during P.g. infection. With the use of intracellular cytokine staining, we confirmed further that blocking of the IL-10R resulted in an increased capacity of CD4 and CD8 T cells to produce IFN-γ (Fig. 2E). Examination of IFN-γ and Thy1.1 expression on CD4 T cells showed that during P.g. stimulation of naive CD4 T cells, there is a small percentage of IFN-γ+ cells (∼0.6%), of which >80% of this population (∼0.5%) also expressed Thy1.1 (Fig. 2F), suggesting the induction of IFN-γ+/IL-10+ CD4 T cells following exposure to P.g. Interestingly, blockade of the IL-10R resulted in an increase in Thy1.1+ CD4, CD8, and CD11b+ cells (Fig. 2C and D) and IL-10 production (see Fig. 4B), likely to compensate for the decrease in receptor availability.

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P.g. priming and stimulation induce IL-10-competent CD4 and CD8 T cells and CD11b+ cells.

WT and 10BiT mice were primed with P.g., as described previously. Eight days following priming, splenocytes were stained ex vivo for CD4, CD8, or CD11b and Thy1.1 (A and B) or stimulated further with P.g. ± α-IL-10R and then stained with the previous antibodies and/or IFN-γ (C–F). (A) Flow cytometry dot-plots gated on CD4 T cells showing percentages of cells expressing Thy1.1. (B) Bar graphs representing the numbers of CD4, CD8, or CD11b+ cells expressing Thy1.1 ex vivo following infection. Results are expressed as the mean ± sem of two to three individual mice from four independent experiments. (C–F) Expression of Thy1.1 or IFN-γ on T or CD11b+ cells following stimulation with P.g. ± α-IL-10R. Graphs represent percentages of CD4 and CD8 (C and E) or CD11b+ cells (D) expressing Thy1.1 (C and D) following stimulation for 5 days or IFN-γ (E) following stimulation for 3 days. These time-points represented the peak levels of Thy1.1 expression and of IFN-γ production, respectively. (F) Flow cytometry dot-plots gated on CD4 T cells showing percentages of Thy1.1 and IFN-γ following stimulation of splenocytes for 5 days with P.g. ± α-IL-10R. Results are expressed as the mean ± sem of three to four individual mice from one of two to four independent experiments. Significant differences were ***P < 0.001, **P < 0.01, and *P < 0.05.

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nTregs do not inhibit the IFN-γ response to P.g.

(A) CD4+CD25+ and CD4+CD25− T cells were purified from splenocytes of WT naive mice, and equal numbers of each population were cultured alone or in combination with irradiated, CD4-depleted feeder cells. Cells were stimulated with P.g. ± α-IL-10. (B and C) Total CD4 T cells or CD4+CD25− T cells were purified from splenocytes of naive WT or TLR2−/− mice and were cultured with irradiated, CD4-depleted feeder cells of the appropriate genotype. Cells were stimulated with P.g. ± α-IL-10R. Culture supernatants were harvested 5 days poststimulation and assessed for the production of IFN-γ (A and C) or IL-10 (B) by ELISA. Results are expressed as the mean ± sem of duplicate or triplicate cultures from one of three independent experiments. Significant differences were ***P < 0.001, **P < 0.01, and *P < 0.05.

IL-10 production by CD4 T cells stimulated with P.g. is dependent on TLR2/1 signaling

Although P.g. stimulation resulted in IL-10 production by naive and sensitized CD4 T cells, we wanted to focus on understanding the role of IL-10 in regulating the naive CD4 T cell response to P.g. as it represented the initial interplay between naive CD4 T cells and the bacteria. Whereas studies have shown that TLR4 has minimal effect on the outcome of P.g. infection, TLR2 signaling sustains the infection process [29, 30]. To determine whether the production of IL-10 by naive CD4 T cells following P.g. stimulation was dependent on TLR2 signaling, we compared the levels of IL-10 in P.g.-stimulated CD4 T cells from naive WT, TLR2−/−, and TLR4−/− mice. Our results showed that IL-10 production by naive, P.g.-stimulated CD4 T cells was dependent on TLR2 but not TLR4 (Fig. 3A) and that TLR2 played a more substantial role in modulating IL-10 production when expressed on APCs rather than on CD4 T cells (data not shown). As TLR2 heterodimerizes with TLR1 or TLR6 [48], we next examined the role of TLR1 and TLR6 on the production of IL-10 by P.g.-stimulated CD4 T cells. Our results showed that IL-10 production was dependent on TLR1 but not TLR6 signaling, indicating that signaling via TLR2/1 is necessary for IL-10 production. Interestingly, neutralization of IL-10 showed that production of IFN-γ by naive CD4 T cells following P.g. stimulation was also dependent on TLR2/1 signaling (Fig. 3A and B), suggesting that the bacterial antigen(s) that induced the IL-10 and the IFN-γ response used the TLR2/1 signaling pathway.

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IL-10 production by naive CD4 T cells following P.g. stimulation is dependent on TLR2/1 signaling.

Purified CD4 T cells from splenocytes of naive WT, TLR2−/−, or TLR4−/− (A) or WT, TLR1−/−, or TLR6−/− mice (B) were cultured with equal numbers of irradiated CD4-depleted feeders of the appropriate genotype and stimulated with P.g. ± α-IL-10. Culture supernatants were harvested 5 days poststimulation and assayed for IL-10 or IFN-γ by ELISA. Results are expressed as the mean ± sem of triplicate cultures from two to five independent experiments. Significant differences were ***P < 0.001, **P < 0.01, and *P < 0.05.

CD4+CD25+ nTregs do not inhibit IFN-γ production following P.g. stimulation

Several types of Tregs have been described, including natural and inducible CD4+CD25+ T cells, IL-10-producing Tr-1 cells, and TGF-β-producing Th-3 cells [49, 50]. To determine whether nTregs mediated the inhibition of IFN-γ production by naive CD4 T cells, purified CD4+CD25+ and CD4+CD25− T cells from naive WT mice were cultured alone or in combination with feeder cells and stimulated with P.g. Absence of nTreg from CD4+CD25− T cell cultures did not enhance IFN-γ production by these cells upon P.g. stimulation (Fig. 4A). In addition, upon blocking of the IL-10R, an increase in IFN-γ production was detected in CD4+CD25− T cell cultures, and this increase was not inhibited by the addition of CD4+CD25+ T cells (Fig. 4A). This suggested that nTregs do not play a role in the inhibition of IFN-γ production by effector T cells in response to P.g. Moreover, IL-10 was produced only by P.g.-stimulated cultures that contained CD4+CD25− T cells and not by those with CD4+CD25+ nTregs (data not shown). To further verify these results, total purified CD4 T cells from naive WT mice were stimulated with P.g., alongside purified CD4+CD25− T cells. No difference was detected in the IL-10 levels produced between total CD4 and purified CD4+CD25− T cells (Fig. 4B). In addition, no difference in IFN-γ levels was detected in the culture supernatants of total CD4 or purified CD4+CD25− T cells stimulated with P.g. in the absence or presence of the IL-10R antibody (Fig. 4C). These data suggest that inhibition of IFN-γ by CD4 T cells was mediated by IL-10-producing CD4+CD25− T cells and not by nTregs. As expected, production of IL-10 or of IFN-γ following blocking of IL-10R signaling in purified CD4+CD25− T cells was dependent on TLR2 signaling (Fig. 4B and C).

Blocking of IL-10 signaling during P.g. stimulation of CD4+CD25− T cells results in a TLR2-dependent increase in T-bet and CD69 expression

IFN-γ production by CD4 T cells is controlled by the expression of the transcription factor T-bet [51, 52]. As blocking of IL-10 signaling resulted in an increase in the production of IFN-γ by CD4 T cells, we wanted to investigate whether blocking of the IL-10R would lead to the up-regulation of T-bet in CD4+CD25− T cells. P.g.-stimulated, naive CD4+CD25− T cells from WT mice showed no up-regulation of T-bet; however, blocking of the IL-10R resulted in a significant increase in T-bet expression (approximately threefold; Fig. 5A and C). This increase was accompanied by an increase in the early activation marker, CD69 (Fig. 5A and E), and some increase, although insignificant, in CD25 expression (Fig. 5B and F), suggesting that blocking IL-10 signaling together with P.g. stimulation resulted in an increased activation of CD4 T cells. The increase in T-bet expression and the up-regulation of CD69 were TLR2-dependent (Fig. 5A).

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Up-regulation of T-bet and CD69 on naive CD4 T cells upon blocking of IL-10 during P.g. stimulation is dependent on TLR2.

CD4+CD25− T cells were purified from splenocytes of naive WT or TLR2−/− mice and were cultured with irradiated, CD4-depleted feeder cells of the appropriate genotype. Cells were stimulated with P.g. ± α-IL-10R, harvested 36 h poststimulation, and stained with CD4, CD69, and T-bet (A, C, and E) or CD4, CD25, and Foxp3 (B, D, and F). (A and B) Flow cytometry dot-plots gated on CD4 T cells. Numbers represent percentages of cells in each quadrant. (C–F) Graphs showing the percentages of CD4 T cells expressing T-bet (C), Foxp3 (D), CD69 (E), and CD25 (F). Results represent mean ± sem of duplicate cultures from one of two to three independent experiments. Significant differences were ***P < 0.001, and **P < 0.01.

Recently, it has become evident that Foxp3-expressing Tregs can develop from non-Foxp3-expressing or CD4+CD25− T cells [37, 53]. Thus, we next wanted to determine whether stimulation of CD4+CD25− T cells with P.g. would result in up-regulation of Foxp3. Following stimulation with P.g., there was a slight but insignificant increase in Foxp3 expression compared with unstimulated CD4+CD25− T cells (Fig. 5B and D), suggesting that P.g. stimulation did not result in the development of Foxp3-expressing Tregs, further supporting our results (Fig. 4) that Tregs do not play a prominent, inhibitory role during the initial immune response to P.g. Overall, our findings revealed that the increase in IFN-γ production during blockade of IL-10R signaling in CD4+CD25− T cells stimulated with P.g. was a result of the ability of these cells to up-regulate T-bet in a TLR2-dependent manner.

CD4 T cells up-regulate PD-1 expression in response to P.g. in a TLR2-independent and IL-10 partially dependent manner

Activation and proliferation of T cells can be controlled by accessory signaling molecules, some of which are inhibitory, such as PD-1 [54, 55]. To determine whether PD-1 was involved in the CD4 T cell response to P.g., we assessed the expression of PD-1 ex vivo on CD4 T cells in P.g.-primed WT mice. Our results showed that there was a significant increase in the expression of PD-1 on CD4 T cells isolated from P.g.-primed mice when compared with naive control mice (Fig. 6A). However, no difference was detected with CD8 T cells (Fig. 6A).

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PD-1 expression is up-regulated on CD4 T cells in response to P.g. in an IL-10 partially dependent manner.

(A) WT mice were primed with P.g., as described in Fig. 1. Splenocytes were harvested 8 days following infection and stained ex vivo with CD4, CD8, and PD-1 antibodies. Numbers represent percentages of CD4 or CD8 T cells that express PD-1. Results are expressed as the mean ± sem of three independent experiments. (B–G) Total splenocytes (B and E–G) or equal numbers of purified CD4+CD25− T cells and irradiated CD4-depleted feeder cells of the appropriate genotype (C and D) from WT (B–D), TLR2−/− (C and D), or 10BiT (E–G) naive mice were stimulated with P.g. ± α-IL-10R. Cells were harvested 2 days poststimulation unless otherwise indicated and stained with CD4, PD-1, and Thy1.1 (B and E–G) or CD4, CD69, and PD-1 (C and D). (B) The graph shows the percentage of CD4+PD-1+ T cells. Results represent the mean ± sem of two to four mice from one of four independent experiments. (C and D) Flow cytometry histograms (C) and graphs (D) showing the expression of PD-1 on activated CD4+CD69+ T cells. Numbers in C indicate the percentages (black) and MFI (gray) of PD-1 expression. Results represent one of two independent experiments. (E) Flow cytometry dot-plots showing the expression of PD-1 and Thy1.1 gated on CD4 T cells. Numbers represent the percentages of cells in each quadrant. (F and G) Flow cytometry histograms and graphs showing the MFI of PD-1 expression on Thy1.1+ and Thy1.1− CD4 T cells. (G) Results represent the mean ± sem of two to four mice from one of four independent experiments. Significant differences were ***P < 0.001, **P < 0.01, and *P < 0.05.

To determine whether IL-10 signaling had any effect on PD-1 expression by CD4 T cell, we next examined the expression of PD-1 on total CD4 T cells and on purified CD4+CD25− T cells from WT naive mice following in vitro P.g. stimulation. Our results showed that PD-1 expression was up-regulated on total CD4 T cells (Fig. 6B) and activated CD4+CD25− T cells (Fig. 6C and D). This increase in PD-1 percentage was partially dependent on IL-10 signaling (Fig. 6B). We also noticed an increase in MFI of PD1 upon stimulation with P.g. (Fig. 6G); however, this increase was completely dependent on IL-10, suggesting that whereas there might be a factor other than IL-10 that controls the expression of PD-1, such would play a lesser role in regulating PD-1 expression. Interestingly, the up-regulation of PD-1 was not dependent on TLR2 signaling (Fig. 6C and D), suggesting that the antigen influencing PD-1 expression is not a TLR2 agonist.

As PD-1 up-regulation was partially dependent on IL-10, we next determined whether IL-10-competent cells expressed PD-1. By examining CD4 T cells from naive 10BiT mice, we observed that IL-10-competent cells (Thy1.1+) were at least 60% PD-1+ (Fig. 6E). However, PD-1 expression increased on Thy1.1− cells 2 days following P.g. stimulation, and as Thy1.1 expression increased on CD4 T cells, up-regulation of PD-1 expression on Thy1.1+ cells was also seen 5 days following stimulation (Fig. 6E). Comparison of the MFI of PD-1 expression in Thy1.1+ and Thy1.1− CD4 T cells showed that Thy1.1+ cells had a higher MFI of PD-1 compared with that of Thy1.1− cells (Fig. 6F and G). The MFI of PD-1 was increased further upon stimulation with P.g. on both cell populations (Fig. 6G). These results show that although not all cells expressing PD-1 following P.g. stimulation are capable of producing IL-10, cells that are IL-10-competent express a higher level of PD-1, suggesting that these cells may exercise a higher suppressive activity than non-IL-10-competent cells to inhibit the IFN-γ response.

PD-L1 expression is up-regulated on CD11b+ cells in response to P.g

PD-1 binds to PD-L1 expressed on APCs, such as macrophages and DCs, resulting in the regulation of T cell responses [54–56]. As PD-1 was up-regulated on CD4 T cells following P.g. stimulation, we next determined whether PD-L1 was also up-regulated on CD11b+ cells. P.g. stimulation of WT naive splenocytes resulted in an increased expression of PD-L1 on CD11b+ cells, but unlike the up-regulation of PD-1 on CD4 T cells, this was not dependent on IL-10 signaling (Fig. 7A). Assessment of CD11b+ cells from 10BiT naive mice demonstrated that Thy1.1+ cells were at least 70% PD-L1+ (Fig. 7B), yet following P.g. stimulation, PD-L1 expression on Thy1.1+ and Thy1.1− was increased further (Fig. 7B). Similar to CD4 T cells, our results showed that Thy1.1+CD11b+ cells had a higher MFI of PD-L1 compared with Thy1.1− cells (Fig. 7C and D), indicating that whereas not all CD11b+ cells expressing PD-L1 are capable of producing IL-10 following P.g. stimulation, IL-10-competent cells expressed the highest levels of PD-L1. Overall, these results suggest that in addition to IL-10 production, up-regulation of the PD-L1/PD-1 inhibitory signaling complex might be another mechanism used by P.g. to suppress the CD4 T cell response.

An external file that holds a picture, illustration, etc. Object name is zgb0011357580007.jpg
PD-L1 expression is up-regulated on CD11b+ cells in response to P.g.

Total splenocytes from naive WT (A) or 10BiT (B–D) mice were stimulated with P.g. ± α-IL-10R. Cells were harvested 2 days poststimulation and stained with CD11b, PD-L1, and Thy1.1. (A) The graph shows the percentage of CD11b+PD-L1+ cells. Results represent the mean ± sem of three to four mice from one of three independent experiments. (B) Flow cytometry dot-plots showing the expression of PD-L1 and Thy1.1 gated on CD11b+ cells. Numbers represent the percentages of cells in each quadrant. (C and D) Flow cytometry histograms and graphs showing the MFI of PD-L1 expression on Thy1.1+ and Thy1.1− CD11b+ cells. (D) Results represent the mean ± sem of three to four mice from one of three independent experiments. Significant differences were ***P < 0.001, and **P < 0.01.

IL-10 production is dependent on FimA

Several studies have shown that fimbriae, one of the main virulence factors of P.g., are a potent TLR2 agonist [29, 57, 58]. As our results showed that IL-10 production by CD4 T cells is dependent on TLR2 signaling, we assessed the involvement of the major fimbrial protein, FimA, in the induction of the IL-10 response. With the use of two different P.g. 33277 strains deficient in FimA (FimA mutant and YPF1), we showed that IL-10 production by total splenocytes and purified CD4 T cells was dependent on the expression of FimA by P.g. (Fig. 8A). Moreover, upon neutralization of IL-10, IFN-γ production was completely or partially dependent on FimA expression by splenocytes or CD4 T cells, respectively (Fig. 8A). The IFN-γ detected could be a result of the presence of another antigen(s) in the FimA mutant strains that can induce the production of IFN-γ in the purified CD4 T cells upon blocking IL-10 signaling. Failure to detect IFN-γ in the splenocyte cultures may be a result of a lesser proportion of CD4 T cells present in these cultures. Expression of PD-1 and PD-L1 following stimulation with P.g. FimA deficient strains on CD4 T and CD11b+ cells, respectively, showed that up-regulation of PD-1 and PD-L1 occurs independently of FimA (Fig. 8B and C), suggesting that this antigen does not use the PD-L1/PD-1 pathway for the potential inhibition of CD4 T cell responses.

An external file that holds a picture, illustration, etc. Object name is zgb0011357580008.jpg
IL-10 production following P.g. stimulation is dependent on FimA.

Total splenocytes (A–C) or isolated CD4 T cells and irradiated CD4-depleted feeder cells (A) from naive WT mice were stimulated with P.g., FimA mutant, or YPF1 ± α-IL-10. (A) Culture supernatants were harvested 5 days following stimulation and assessed for IL-10 and IFN-γ production by ELISA. Results are expressed as the mean ± sem of triplicate cultures from one of two or three independent experiments. (B and C) Cells were harvested 2 days following stimulation and assessed for the expression of CD4 and PD-1 (B) or CD11b and PD-L1 (C). Graphs represent percentages of CD4+PD-1+ (B) or CD11b+PD-L1+ (C) cells. Results represent the mean ± sem of three to four mice from one of three independent experiments. Significant differences were ***P < 0.001, **P < 0.01, and *P < 0.05.

DISCUSSION

In this manuscript, we investigated the role of IL-10 in modulating the T cell response during the initial systemic exposure of the immune system to P.g., the mechanisms involved in the regulation of IL-10 production, and a P.g. virulence factor that elicits such a response. Our results showed that CD4 and CD8 T cells and CD11b+ cells produced IL-10 in response to P.g. and that this response was mediated by FimA via TLR2 signaling. IL-10 production by CD4 T cells was accompanied by the up-regulation of PD-1. Furthermore, blocking of IL-10 signaling resulted in a substantial increase in IFN-γ production by CD4 and CD8 T cells, the up-regulation of CD69, and an increase in T-bet expression by CD4 T cells. Our results also highlight that the systemic exposure to P.g. can indeed inhibit the proper activation of T cells and suppress their function.

Although IL-10 has been known to inhibit IFN-γ production by T cells [47], here, we have delineated for the first time that IL-10 produced during the initial systemic exposure of the host cells to P.g. results in the inhibition of the IFN-γ response by T cells, and this inhibition was observed upon secondary bacterial challenge. However, our results showed that neutralization of IL-10 in naive splenocytes and CD4 and CD8 T cells was more effective in augmenting an IFN-γ response than in P.g.-primed cells (>15-fold vs. sixfold, respectively), suggesting that the establishment of an anti-inflammatory milieu created by the production of IL-10 is critical for the initial infection and subsequently for the development of disease, as bias toward IL-10 production is maintained, albeit to a lesser extent, in P.g.-sensitized cells. Whereas other studies have described IL-10 as a protective cytokine against periodontitis [23, 24], we believe that the role of IL-10 is much more dynamic, and the timing of the production of IL-10 can be absolutely critical in determining the outcome of the disease. These previous studies used the IL-10 knockout mouse model, which although useful in dissecting the fundamental role of IL-10, this model may not accurately reflect the events that take place during the infection process in a host that is still capable of producing IL-10 and where the levels of IL-10 produced change during the course of infection. This point is illustrated by the fact that the IL-10 knockout mouse model develops spontaneous inflammation in multiple sites [23, 59, 60], thus not accurately reproducing the interplay among IL-10, inflammatory cytokines such as IFN-γ and TNF-α, and T cells during infection. We believe that early, high levels of IL-10 can suppress the immune response by inhibiting the activity of robust inflammatory cytokines, reducing the capacity of neutrophils and macrophages to kill the bacteria [21], and increasing the activation and proliferation of B cells [61, 62], ultimately leading to a lesser protective humoral, rather than a more protective cellular immune response. The failure to mount a protective cellular response, together with the internalization of P.g. by the host cell, shifts the infection process to a delayed hypersensitivity-type reaction, characterized by an increased production of inflammatory mediators and a failure of the host to clear the bacteria. This condition likely further increases inflammation, which could contribute to the eventual loss of alveolar bone [5]. Whereas it remains to be proven, this theory is supported by several studies that showed that immunization with antigens from P.g., which lead to a robust initial IFN-γ response and hence, a decrease in IL-10, resulted in better clearance of the bacteria following infection [26, 28, 46].

Interestingly, although P.g. can be internalized, no role has been demonstrated for CD8 in the protection or detriment of periodontitis. Gemmell et al. [19] showed that there are more than 1000 genes that are down-regulated in CD8 T cells following P.g. immunization, suggesting that their cytotoxic function is inhibited. Along these lines, we demonstrate for the first time that naive or primed CD8 T cells exposed to P.g. produce significant amounts of IL-10, which upon neutralization, resulted in an increase in IFN-γ production by CD8 T cells at levels that are higher than those by CD4 T cells (Fig. 2E). In recent years, a number of studies have described IFN-γ+/IL-10+ CD4 T cells that possess suppressive functions and are detected in chronic infections with pathogens such as Leishmania major, Mycobacterium tuberculosis, and Toxoplasma gondii [63–65]. Whether these IFN-γ+/IL-10+ CD4 T cells that were observed in our studies (Fig. 2F) inhibit adequate CD8 T cell responses to the bacteria, as suggested in other models of chronic infections, is an intriguing question that will require further investigations.

Our results show that CD11b+ cells produce IL-10 and could potentially inhibit the IFN-γ response by T cells. These results lend support to the previously suggested notion that myeloid cells use the IL-10 signaling pathway and potentially inhibit T cell responses through the inhibition of IL-12 [27]. Whereas the exact type of CD11b+ cells that produce IL-10 during P.g. infection needs to be determined, one could predict that these cells are myeloid-derived suppressor cells or CD11b+GR-1+ macrophages [66, 67], as these cells have been shown to inhibit inflammatory T cell responses, allowing the persistence of specific infections [68, 69]. Whereas we examined the correlation between Gr-1 and Thy1.1 expression in the present study, our results were inconclusive (data not shown). Further studies are thus required to determine the phenotype of the cell population involved in P.g. infections.

The exact role of Tregs in periodontitis is not quite understood. Our finding show that CD4+CD25+ nTregs did not play a role in abrogating the production of IFN-γ by CD4 T cell to P.g. in vitro or in vivo, as the depletion of nTregs prior to infection with P.g. did not increase the levels of IFN-γ (data not shown). Moreover, the expression of Foxp3 on purified CD4+CD25− T cells upon P.g. stimulation was relatively weak, similar to the observations made by Ernst et al. [70]. This low level of Foxp3 expressed by CD4+CD25− T cells following P.g. stimulation can be explained by a lack of IL-2, as IL-2 is needed to maintain Foxp3 expression [71, 72], and T cells isolated from periodontitis patients have been shown to produce less IL-2 upon stimulation [18].

Studies examining chronic viral infections, such as HIV, hepatitis B virus, or CMV in humans and murine lymphocytic choriomeningitis, as well as chronic cutaneous leishmaniasis, have shown that the PD-L1/PD-1 signaling pathway plays an important role in inhibiting CD4 and CD8 T cell responses in these infections [54, 73–79]. To our knowledge, no correlation between PD-L1/PD-1 expression and P.g. infection or periodontitis has been documented to this date. In this manuscript, we show for the first time that priming or stimulation of the host cells with P.g. results in the up-regulation of PD-L1 or PD-1 expression on CD11b+ cells or CD4 T cells, respectively. Thus, we propose that in addition to IL-10, PD-L1/PD-1 is another mechanism used by P.g. to suppress the T cell response, thus possibly contributing to bacterial persistence in the host. The exact mechanism by which P.g. stimulation leads to the up-regulation of PD-L1/PD-1 remains to be delineated, but our results suggest that it occurs independent of FimA or TLR2 signaling and that there is only a partial dependency between PD-1 expression and IL-10. This partial dependency on IL-10 is in agreement with a study by Said et al. [80], demonstrating that there is a positive correlation between PD-1 expression on monocytes and serum IL-10 in viremic HIV patients. This study suggested that PD-L1/PD-1 signaling maybe playing a role in inhibiting T cell responses required to clear the HIV infection in these patients. Perhaps the differential regulation involved in the production of IL-10 and in the up-regulation of PD-L1/PD-1 observed in our studies suggests that both mechanisms may be used separately and redundantly by P.g. to ensure its survival following infection of the host. Further studies are required to prove this possibility.

In conclusion, we have provided novel evidence that IL-10 production, by naive cells, upon early systemic exposure to the bacteria, is a mechanism by which P.g., through its FimA, uses TLR2 signaling to inhibit the IFN-γ response by T cells. We have demonstrated further, for the first time, a correlation between IL-10 production and the PD-L1/PD-1 inhibitory signaling pathway during P.g. stimulation and propose that such is a novel mechanism by which P.g. can evade the host's immune response. These results are of importance, as they augment the idea that the tight regulation between IL-10 and IFN-γ production early during P.g. infection can ultimately control the infection and result in tissue healing or can lead to a chronic persistence of the bacteria and eventually, to an excessive inflammation that destroys the periodontal structures. The findings of the present work provide relevant information that can be used for the design of strategies to combat P.g. infections.

ACKNOWLEDGMENTS

This work was supported by Grant DE14215 from NIDCR (J.K.), and D.E.G was supported in part by National Institute of Allergy and Infectious Diseases training grant T32 AI007051 and NIDCR training grant T32 DE017607 from the U.S. National Institutes of Health. We thank Amit Ashtekar for technical assistance with P.g. priming of mice. We thank Richard Lamont for providing us with P.g. FimA-deficient strains.

SEE CORRESPONDING EDITORIAL ON PAGE 3

−/−
knockout
10BiT
IL-10 reporter mice
α-IL-10
anti-IL-10
FimA
Major fimbrial protein A of P. gingivalis
Foxp3
forkhead box protein p3
MFI
mean fluorescence intensity
NIDCR
National Institute of Dental and Craniofacial Research
nTreg
naturally occurring regulatory T cell
PD-1
programmed death 1
PD-L1
programmed death ligand 1
P.g.
Porphyromonas gingivalis and Porphyromonas gingivalis ATCC 33,277
T-bet
T box transcription factor TBX21
Treg
regulatory T cell
YPF1
Fim A mutant strain of P. gingivalis

AUTHORSHIP

D.E.G. performed all experiments and collected and analyzed all data. C.L.M. edited the manuscript and provided insightful critiques and technical assistance. C.T.W. contributed the 10BiT mice. D.E.G., S.M.M., and J.K. wrote the manuscript.

REFERENCES

1. Van Dyke T. E., Sheilesh D. (2005) Risk factors for periodontitis. J. Int. Acad. Periodontol. 7, 3–7 [PMC free article] [PubMed] [Google Scholar]
2. Slots J., Bragd L., Wikstrom M., Dahlen G. (1986) The occurrence of Actinobacillus actinomycetemcomitans, Bacteroides gingivalis and Bacteroides intermedius in destructive periodontal disease in adults. J. Clin. Periodontol. 13, 570–577 [PubMed] [Google Scholar]
3. Socransky S. S., Haffajee A. D., Cugini M. A., Smith C., Kent R. L., Jr., (1998) Microbial complexes in subgingival plaque. J. Clin. Periodontol. 25, 134–144 [PubMed] [Google Scholar]
4. Lamont R. J., Jenkinson H. F. (1998) Life below the gum line: pathogenic mechanisms of Porphyromonas gingivalis. Microbiol. Mol. Biol. Rev. 62, 1244–1263 [PMC free article] [PubMed] [Google Scholar]
5. Gemmell E., Yamazaki K., Seymour G. J. (2007) The role of T cells in periodontal disease: homeostasis and autoimmunity. Periodontol. 2000 43, 14–40 [PubMed] [Google Scholar]
6. Gaffen S. L., Hajishengallis G. (2008) A new inflammatory cytokine on the block: re-thinking periodontal disease and the Th1/Th2 paradigm in the context of Th17 cells and IL-17. J. Dent. Res. 87, 817–828 [PMC free article] [PubMed] [Google Scholar]
7. Seymour G. J. (1991) Importance of the host response in the periodontium. J. Clin. Periodontol. 18, 421–426 [PubMed] [Google Scholar]
8. Teng Y. T. (2006) Protective and destructive immunity in the periodontium: part 2—T-cell-mediated immunity in the periodontium. J. Dent. Res. 85, 209–219 [PubMed] [Google Scholar]
9. Katz J., Sambandam V., Wu J. H., Michalek S. M., Balkovetz D. F. (2000) Characterization of Porphyromonas gingivalis-induced degradation of epithelial cell junctional complexes. Infect. Immun. 68, 1441–1449 [PMC free article] [PubMed] [Google Scholar]
10. Kuramitsu H. K. (1998) Proteases of Porphyromonas gingivalis: what don't they do? Oral Microbiol. Immunol. 13, 263–270 [PubMed] [Google Scholar]
11. Oleksy A., Banbula A., Bugno M., Travis J., Potempa J. (2002) Proteolysis of interleukin-6 receptor (IL-6R) by Porphyromonas gingivalis cysteine proteinases (gingipains) inhibits interleukin-6-mediated cell activation. Microb. Pathog. 32, 173–181 [PubMed] [Google Scholar]
12. Potempa J., Banbula A., Travis J. (2000) Role of bacterial proteinases in matrix destruction and modulation of host responses. Periodontol. 2000 24, 153–192 [PubMed] [Google Scholar]
13. Zhang J., Dong H., Kashket S., Duncan M. J. (1999) IL-8 degradation by Porphyromonas gingivalis proteases. Microb. Pathog. 26, 275–280 [PubMed] [Google Scholar]
14. Seymour G. J., Powell R. N., Davies W. I. (1979) Conversion of a stable T-cell lesion to a progressive B-cell lesion in the pathogenesis of chronic inflammatory periodontal disease: an hypothesis. J. Clin. Periodontol. 6, 267–277 [PubMed] [Google Scholar]
15. Baker P. J., Dixon M., Evans R. T., Dufour L., Johnson E., Roopenian D. C. (1999) CD4+ T cells and the proinflammatory cytokines γ interferon and interleukin-6 contribute to alveolar bone loss in mice. Infect. Immun. 67, 2804–2809 [PMC free article] [PubMed] [Google Scholar]
16. Baker P. J., Howe L., Garneau J., Roopenian D. C. (2002) T cell knockout mice have diminished alveolar bone loss after oral infection with Porphyromonas gingivalis. FEMS Immunol. Med. Microbiol. 34, 45–50 [PubMed] [Google Scholar]
17. Sosroseno W., Bird P. S., Gemmell E., Seymour G. J. (2002) The role of CD4+ cells in vivo on the induction of the immune response to Porphyromonas gingivalis in mice. J. Periodontol. 73, 1133–1140 [PubMed] [Google Scholar]
18. Seymour G. J., Cole K. L., Powell R. N., Lewins E., Cripps A. W., Clancy R. L. (1985) Interleukin-2 production and bone-resorption activity in vitro by unstimulated lymphocytes extracted from chronically-inflamed human periodontal tissues. Arch. Oral Biol. 30, 481–484 [PubMed] [Google Scholar]
19. Gemmell E., Drysdale K. E., Seymour G. J. (2006) Gene expression in splenic CD4 and CD8 cells from BALB/c mice immunized with Porphyromonas gingivalis. J. Periodontol. 77, 622–633 [PubMed] [Google Scholar]
20. Guyodo H., Meuric V., Pottier L., Martin B., Faili A., Pers J. O., Bonnaure-Mallet M. (2011) Colocalization of Porphyromonas gingivalis with CD4+ T cells in periodontal disease. FEMS Immunol. Med. Microbiol. 64, 175–183 [PubMed] [Google Scholar]
21. Moore K. W., de Waal Malefyt R., Coffman R. L., O'Garra A. (2001) Interleukin-10 and the interleukin-10 receptor. Annu. Rev. Immunol. 19, 683–765 [PubMed] [Google Scholar]
22. Filippi C. M., von Herrath M. G. (2008) IL-10 and the resolution of infections. J. Pathol. 214, 224–230 [PubMed] [Google Scholar]
23. Al-Rasheed A., Scheerens H., Rennick D. M., Fletcher H. M., Tatakis D. N. (2003) Accelerated alveolar bone loss in mice lacking interleukin-10. J. Dent. Res. 82, 632–635 [PubMed] [Google Scholar]
24. Sasaki H., Okamatsu Y., Kawai T., Kent R., Taubman M., Stashenko P. (2004) The interleukin-10 knockout mouse is highly susceptible to Porphyromonas gingivalis-induced alveolar bone loss. J. Periodontal. Res. 39, 432–441 [PubMed] [Google Scholar]
25. Sasaki H., Hou L., Belani A., Wang C. Y., Uchiyama T., Muller R., Stashenko P. (2000) IL-10, but not IL-4, suppresses infection-stimulated bone resorption in vivo. J. Immunol. 165, 3626–3630 [PubMed] [Google Scholar]
26. Herminajeng E., Sosroseno W., Bird P. S., Seymour G. J. (2001) The effects of interleukin-10 depletion in vivo on the immune response to Porphyromonas gingivalis in a murine model. J. Periodontol. 72, 1527–1534 [PubMed] [Google Scholar]
27. Sasaki H., Suzuki N., Kent R., Jr., Kawashima N., Takeda J., Stashenko P. (2008) T cell response mediated by myeloid cell-derived IL-12 is responsible for Porphyromonas gingivalis-induced periodontitis in IL-10-deficient mice. J. Immunol. 180, 6193–6198 [PubMed] [Google Scholar]
28. Sosroseno W., Herminajeng E., Herwiyanti S., Ghufron M. (2000) Effects of cytokines on Porphyromonas gingivalis-induced opsonophagocytosis of a murine macrophage cell line. Eur. J. Oral Sci. 108, 561–563 [PubMed] [Google Scholar]
29. Wang M., Shakhatreh M. A., James D., Liang S., Nishiyama S., Yoshimura F., Demuth D. R., Hajishengallis G. (2007) Fimbrial proteins of Porphyromonas gingivalis mediate in vivo virulence and exploit TLR2 and complement receptor 3 to persist in macrophages. J. Immunol. 179, 2349–2358 [PubMed] [Google Scholar]
30. Burns E., Bachrach G., Shapira L., Nussbaum G. (2006) Cutting edge: TLR2 is required for the innate response to Porphyromonas gingivalis: activation leads to bacterial persistence and TLR2 deficiency attenuates induced alveolar bone resorption. J. Immunol. 177, 8296–8300 [PubMed] [Google Scholar]
31. Netea M. G., Van der Meer J. W., Kullberg B. J. (2004) Toll-like receptors as an escape mechanism from the host defense. Trends Microbiol. 12, 484–488 [PubMed] [Google Scholar]
32. Kobayashi R., Kono T., Bolerjack B. A., Fukuyama Y., Gilbert R. S., Fujihashi K., Ruby J., Kataoka K., Wada M., Yamamoto M. (2011) Induction of IL-10-producing CD4+ T-cells in chronic periodontitis. J. Dent. Res. 90, 653–658 [PMC free article] [PubMed] [Google Scholar]
33. Hu S. W., Huang C. H., Huang H. C., Lai Y. Y., Lin Y. Y. (2006) Transvascular dissemination of Porphyromonas gingivalis from a sequestered site is dependent upon activation of the kallikrein/kinin pathway. J. Periodontal. Res. 41, 200–207 [PubMed] [Google Scholar]
34. Boggess K. A., Madianos P. N., Preisser J. S., Moise K. J., Jr., Offenbacher S. (2005) Chronic maternal and fetal Porphyromonas gingivalis exposure during pregnancy in rabbits. Am. J. Obstet. Gynecol. 192, 554–557 [PubMed] [Google Scholar]
35. Seymour G. J., Taubman M. A., Eastcott J. W., Gemmell E., Smith D. J. (1997) CD29 expression on CD4+ gingival lymphocytes supports migration of activated memory T lymphocytes to diseased periodontal tissue. Oral Microbiol. Immunol. 12, 129–134 [PubMed] [Google Scholar]
36. Kawai T., Shimauchi H., Eastcott J. W., Smith D. J., Taubman M. A. (1998) Antigen direction of specific T-cell clones into gingival tissues. Immunology 93, 11–19 [PMC free article] [PubMed] [Google Scholar]
37. Maynard C. L., Harrington L. E., Janowski K. M., Oliver J. R., Zindl C. L., Rudensky A. Y., Weaver C. T. (2007) Regulatory T cells expressing interleukin 10 develop from Foxp3+ and Foxp3− precursor cells in the absence of interleukin 10. Nat. Immunol. 8, 931–941 [PubMed] [Google Scholar]
38. Katz J., Black K. P., Michalek S. M. (1999) Host responses to recombinant hemagglutinin B of Porphyromonas gingivalis in an experimental rat model. Infect. Immun. 67, 4352–4359 [PMC free article] [PubMed] [Google Scholar]
39. Katz J., Leary R. M., Ward D. C., Harmon C. C., Michalek S. M. (1992) Humoral response to Porphyromonas (Bacteroides) gingivalis in rats: time course and T-cell dependence. Infect. Immun. 60, 3579–3585 [PMC free article] [PubMed] [Google Scholar]
40. Katz J., Ward D. C., Michalek S. M. (1996) Effect of host responses on the pathogenicity of strains of Porphyromonas gingivalis. Oral Microbiol. Immunol. 11, 309–318 [PubMed] [Google Scholar]
41. Zhang P., Yang Q. B., Balkovetz D. F., Lewis J. P., Clements J. D., Michalek S. M., Katz J. (2005) Effectiveness of the B subunit of cholera toxin in potentiating immune responses to the recombinant hemagglutinin/adhesin domain of the gingipain Kgp from Porphyromonas gingivalis. Vaccine 23, 4734–4744 [PubMed] [Google Scholar]
42. Love R. M., McMillan M. D., Park Y., Jenkinson H. F. (2000) Coinvasion of dentinal tubules by Porphyromonas gingivalis and Streptococcus gordonii depends upon binding specificity of streptococcal antigen I/II adhesin. Infect. Immun. 68, 1359–1365 [PMC free article] [PubMed] [Google Scholar]
43. Capestany C. A., Tribble G. D., Maeda K., Demuth D. R., Lamont R. J. (2008) Role of the Clp system in stress tolerance, biofilm formation, and intracellular invasion in Porphyromonas gingivalis. J. Bacteriol. 190, 1436–1446 [PMC free article] [PubMed] [Google Scholar]
44. Gaddis D. E., Michalek S. M., Katz J. (2011) TLR4 signaling via MyD88 and TRIF differentially shape the CD4+ T cell response to Porphyromonas gingivalis hemagglutinin B. J. Immunol. 186, 5772–5783 [PMC free article] [PubMed] [Google Scholar]
45. Houri-Haddad Y., Soskoine W. A., Shapira L. (2001) Immunization to Porphyromonas gingivalis enhances the local pro-inflammatory response to subcutaneous bacterial challenge. J. Clin. Periodontol. 28, 476–482 [PubMed] [Google Scholar]
46. Yonezawa H., Kato T., Kuramitsu H. K., Okuda K., Ishihara K. (2005) Immunization by Arg-gingipain A DNA vaccine protects mice against an invasive Porphyromonas gingivalis infection through regulation of interferon-γ production. Oral Microbiol. Immunol. 20, 259–266 [PubMed] [Google Scholar]
47. Fiorentino D. F., Zlotnik A., Vieira P., Mosmann T. R., Howard M., Moore K. W., O'Garra A. (1991) IL-10 acts on the antigen-presenting cell to inhibit cytokine production by Th1 cells. J. Immunol. 146, 3444–3451 [PubMed] [Google Scholar]
48. Hajjar A. M., O'Mahony D. S., Ozinsky A., Underhill D. M., Aderem A., Klebanoff S. J., Wilson C. B. (2001) Cutting edge: functional interactions between Toll-like receptor (TLR) 2 and TLR1 or TLR6 in response to phenol-soluble modulin. J. Immunol. 166, 15–19 [PubMed] [Google Scholar]
49. Shevach E. M. (2009) Mechanisms of Foxp3+ T regulatory cell-mediated suppression. Immunity 30, 636–645 [PubMed] [Google Scholar]
50. Sojka D. K., Huang Y. H., Fowell D. J. (2008) Mechanisms of regulatory T-cell suppression—a diverse arsenal for a moving target. Immunology 124, 13–22 [PMC free article] [PubMed] [Google Scholar]
51. O'Shea J. J., Paul W. E. (2010) Mechanisms underlying lineage commitment and plasticity of helper CD4+ T cells. Science 327, 1098–1102 [PMC free article] [PubMed] [Google Scholar]
52. Zhu J., Paul W. E. (2008) CD4 T cells: fates, functions, and faults. Blood 112, 1557–1569 [PMC free article] [PubMed] [Google Scholar]
53. Chen W., Jin W., Hardegen N., Lei K. J., Li L., Marinos N., McGrady G., Wahl S. M. (2003) Conversion of peripheral CD4+CD25− naive T cells to CD4+CD25+ regulatory T cells by TGF-β induction of transcription factor Foxp3. J. Exp. Med. 198, 1875–1886 [PMC free article] [PubMed] [Google Scholar]
54. Keir M. E., Francisco L. M., Sharpe A. H. (2007) PD-1 and its ligands in T-cell immunity. Curr. Opin. Immunol. 19, 309–314 [PubMed] [Google Scholar]
55. Riley J. L. (2009) PD-1 signaling in primary T cells. Immunol. Rev. 229, 114–125 [PMC free article] [PubMed] [Google Scholar]
56. Keir M. E., Butte M. J., Freeman G. J., Sharpe A. H. (2008) PD-1 and its ligands in tolerance and immunity. Annu. Rev. Immunol. 26, 677–704 [PMC free article] [PubMed] [Google Scholar]
57. Hajishengallis G., Tapping R. I., Harokopakis E., Nishiyama S., Ratti P., Schifferle R. E., Lyle E. A., Triantafilou M., Triantafilou K., Yoshimura F. (2006) Differential interactions of fimbriae and lipopolysaccharide from Porphyromonas gingivalis with the Toll-like receptor 2-centred pattern recognition apparatus. Cell. Microbiol. 8, 1557–1570 [PubMed] [Google Scholar]
58. Hajishengallis G., Wang M., Liang S. (2009) Induction of distinct TLR2-mediated proinflammatory and proadhesive signaling pathways in response to Porphyromonas gingivalis fimbriae. J. Immunol. 182, 6690–6696 [PMC free article] [PubMed] [Google Scholar]
59. Kuhn R., Lohler J., Rennick D., Rajewsky K., Muller W. (1993) Interleukin-10-deficient mice develop chronic enterocolitis. Cell 75, 263–274 [PubMed] [Google Scholar]
60. Tournoy K. G., Kips J. C., Pauwels R. A. (2000) Endogenous interleukin-10 suppresses allergen-induced airway inflammation and nonspecific airway responsiveness. Clin. Exp. Allergy 30, 775–783 [PubMed] [Google Scholar]
61. Champaiboon C., Yongvanitchit K., Pichyangkul S., Mahanonda R. (2000) The immune modulation of B-cell responses by Porphyromonas ginginvalis and interleukin-10. J. Periodontol. 71, 468–475 [PubMed] [Google Scholar]
62. Rousset F., Garcia E., Defrance T., Peronne C., Vezzio N., Hsu D. H., Kastelein R., Moore K. W., Banchereau J. (1992) Interleukin 10 is a potent growth and differentiation factor for activated human B lymphocytes. Proc. Natl. Acad. Sci. USA 89, 1890–1893 [PMC free article] [PubMed] [Google Scholar]
63. Anderson C. F., Oukka M., Kuchroo V. J., Sacks D. (2007) CD4+CD25-Foxp3- Th1 cells are the source of IL-10-mediated immune suppression in chronic cutaneous leishmaniasis. J. Exp. Med. 204, 285–297 [PMC free article] [PubMed] [Google Scholar]
64. Nagase H., Jones K. M., Anderson C. F., Noben-Trauth N. (2007) Despite increased CD4+Foxp3+ cells within the infection site, BALB/c IL-4 receptor-deficient mice reveal CD4+Foxp3-negative T cells as a source of IL-10 in Leishmania major susceptibility. J. Immunol. 179, 2435–2444 [PubMed] [Google Scholar]
65. Chen J., Liu X. S. (2009) Development and function of IL-10 IFN-γ-secreting CD4+ T cells. J. Leukoc. Biol. 86, 1305–1310 [PubMed] [Google Scholar]
66. Sica A., Bronte V. (2007) Altered macrophage differentiation and immune dysfunction in tumor development. J. Clin. Invest. 117, 1155–1166 [PMC free article] [PubMed] [Google Scholar]
67. Fairweather D., Cihakova D. (2009) Alternatively activated macrophages in infection and autoimmunity. J. Autoimmun. 33, 222–230 [PMC free article] [PubMed] [Google Scholar]
68. Tacke R. S., Lee H. C., Goh C., Courtney J., Polyak S. J., Rosen H. R., Hahn Y. S. (2012) Myeloid suppressor cells induced by hepatitis C virus suppress T-cell responses through the production of reactive oxygen species. Hepatology 55, 343–353 [PMC free article] [PubMed] [Google Scholar]
69. Vollbrecht T., Stirner R., Tufman A., Roider J., Huber R. M., Bogner J. R., Lechner A., Bourquin C., Draenert R. (2012) Chronic progressive HIV-1 infection is associated with elevated levels of myeloid-derived suppressor cells. AIDS 26, F31–F37 [PubMed] [Google Scholar]
70. Ernst C. W., Lee J. E., Nakanishi T., Karimbux N. Y., Rezende T. M., Stashenko P., Seki M., Taubman M. A., Kawai T. (2007) Diminished forkhead box P3/CD25 double-positive T regulatory cells are associated with the increased nuclear factor-κB ligand (RANKL+) T cells in bone resorption lesion of periodontal disease. Clin. Exp. Immunol. 148, 271–280 [PMC free article] [PubMed] [Google Scholar]
71. Crispin J. C., Tsokos G. C. (2009) Transcriptional regulation of IL-2 in health and autoimmunity. Autoimmun. Rev. 8, 190–195 [PMC free article] [PubMed] [Google Scholar]
72. Lan R. Y., Selmi C., Gershwin M. E. (2008) The regulatory, inflammatory, and T cell programming roles of interleukin-2 (IL-2). J. Autoimmun. 31, 7–12 [PubMed] [Google Scholar]
73. Barber D. L., Wherry E. J., Masopust D., Zhu B., Allison J. P., Sharpe A. H., Freeman G. J., Ahmed R. (2006) Restoring function in exhausted CD8 T cells during chronic viral infection. Nature 439, 682–687 [PubMed] [Google Scholar]
74. Day C. L., Kaufmann D. E., Kiepiela P., Brown J. A., Moodley E. S., Reddy S., Mackey E. W., Miller J. D., Leslie A. J., DePierres C., Mncube Z., Duraiswamy J., Zhu B., Eichbaum Q., Altfeld M., Wherry E. J., Coovadia H. M., Goulder P. J., Klenerman P., Ahmed R., Freeman G. J., Walker B. D. (2006) PD-1 expression on HIV-specific T cells is associated with T-cell exhaustion and disease progression. Nature 443, 350–354 [PubMed] [Google Scholar]
75. Peng G., Li S., Wu W., Tan X., Chen Y., Chen Z. (2008) PD-1 upregulation is associated with HBV-specific T cell dysfunction in chronic hepatitis B patients. Mol. Immunol. 45, 963–970 [PubMed] [Google Scholar]
76. Liang S. C., Greenwald R. J., Latchman Y. E., Rosas L., Satoskar A., Freeman G. J., Sharpe A. H. (2006) PD-L1 and PD-L2 have distinct roles in regulating host immunity to cutaneous leishmaniasis. Eur. J. Immunol. 36, 58–64 [PubMed] [Google Scholar]
77. Krishnan A., Zhou W., Lacey S. F., Limaye A. P., Diamond D. J., La Rosa C. (2010) Programmed death-1 receptor and interleukin-10 in liver transplant recipients at high risk for late cytomegalovirus disease. Transpl. Infect. Dis. 12, 363–370 [PMC free article] [PubMed] [Google Scholar]
78. Brooks D. G., Ha S. J., Elsaesser H., Sharpe A. H., Freeman G. J., Oldstone M. B. (2008) IL-10 and PD-L1 operate through distinct pathways to suppress T-cell activity during persistent viral infection. Proc. Natl. Acad. Sci. USA 105, 20428–20433 [PMC free article] [PubMed] [Google Scholar]
79. Konkel J. E., Frommer F., Leech M. D., Yagita H., Waisman A., Anderton S. M. (2010) PD-1 signalling in CD4+ T cells restrains their clonal expansion to an immunogenic stimulus, but is not critically required for peptide-induced tolerance. Immunology 130, 92–102 [PMC free article] [PubMed] [Google Scholar]
80. Said E. A., Dupuy F. P., Trautmann L., Zhang Y., Shi Y., El-Far M., Hill B. J., Noto A., Ancuta P., Peretz Y., Fonseca S. G., Van Grevenynghe J., Boulassel M. R., Bruneau J., Shoukry N. H., Routy J. P., Douek D. C., Haddad E. K., Sekaly R. P. (2010) Programmed death-1-induced interleukin-10 production by monocytes impairs CD4+ T cell activation during HIV infection. Nat. Med. 16, 452–459 [PMC free article] [PubMed] [Google Scholar]
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