Gamma Interferon Positively Modulates Actinobacillus actinomycetemcomitans-Specific RANKL+ CD4+ Th-Cell-Mediated Alveolar Bone Destruction I
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感染与免疫杂志 2005年第6期
Division of Periodontics, Eastman Department of Dentistry, and Centre for Oral Biology, Department of Microbiology & Immunology, School of Medicine and Dentistry, the University of Rochester, Rochester, New York, 14620
Department of Microbiology & Immunology, School of Medicine and Dentistry, the University of Western Ontario, London, Ontario, N6A 5C1, Canada
ABSTRACT
Recent studies have shown the biological and clinical significance of signaling pathways of osteogenic cytokines RANKL-RANK/OPG in controlling osteoclastogenesis associated with bone pathologies, including rheumatoid arthritis, osteoporosis, and other osteolytic disorders. In contrast to the inhibitory effect of gamma interferon (IFN-) on RANKL-mediated osteoclastogenesis reported recently, alternative new evidence is demonstrated via studies of experimental periodontitis using humanized NOD/SCID and diabetic NOD mice and clinical human T-cell isolates from diseased periodontal tissues, where the presence of increasing IFN- is clearly associated with (i) enhanced Actinobacillus actinomycetemcomitans-specific RANKL-expressing CD4+ Th cell-mediated alveolar bone loss during the progression of periodontal disease and (ii) a concomitant and significantly increased coexpression of IFN- in RANKL(+) CD4+ Th cells. Therefore, there are more complex networks in regulating RANKL-RANK/OPG signaling pathways for osteoclastogenesis in vivo than have been suggested to date.
INTRODUCTION
Human periodontal disease (i.e., periodontitis) results from the interplay between the specific subgingival microorganisms and the host's immune and inflammatory response (9). Periodontitis is characterized by loss of attachments including the periodontal connective tissues and supporting alveolar bone. Actinobacillus actinomycetemcomitans (A. actinomycetemcomitans) is a G(–) facultative anaerobe strongly associated with human aggressive periodontitis (AgP) and also involved in medical diseases such as urinary tract and hip-joint prosthetics infections, thyroid and brain abscesses, and subacute endocarditis (54). Although several carbohydrate and protein antigens (Ags), fimbriae, and polysaccharides were identified, much less is known about critical Ags of A. actinomycetemcomitans that sensitize T or B cells involved in the periodontal pathogenesis (16).
The recently identified tumor necrosis factor (TNF) family molecule, receptor activator of NF-B ligand (RANKL [or TRANCE, OPGL, or ODF]), its receptor RANK, and the natural antagonist, osteoprotegerin (OPG), have been shown to be the key regulators of bone remodeling and are directly involved in the differentiation, activation, and survival of osteoclasts (OC) and OC precursors (27, 40, 52-53). Previously, we showed that activated CD4+ T cells express RANKL, which can directly trigger osteoclastogenesis and alveolar bone loss associated with periodontitis in vivo (46). Further, blocking RANKL activity via OPG injections results in significantly reduced bone loss in arthritis (25, 49), periodontitis (46, 50), osteoporosis (19, 30), cancer-related bone metastasis (5, 20), and enhanced alveolar bone loss associated with type 1 diabetes in vivo (29). RANKL-RANK signaling is also critical for survival of dendritic cells, lymph node formation, and organogenesis and involved in dendritic cell/T-cell interactions (1, 26). Genetic mutations of RANKL and RANK demonstrate similar phenotypes in OC development with severe osteopetrosis, suggesting that they are essential for osteoclastogenesis during bone remodeling (26, 28, 50). OPG-transgenic mice are osteopetrotic with defective OC activity, and OPG-deficient mice are severely osteoporotic (30, 40, 50). Thus, RANKL, RANK, and OPG are essential for controlling OC development and functions in bone remodeling. These studies have supported the new paradigm of linking adaptive immunity and bone remodeling (termed osteo-immunology) associated with various inflammatory bone disorders (1, 5, 19-20, 25-26, 28-30, 40, 46, 50, 53). More recently, studies have shown that periodontal residen T cells and tissues (i.e., periodontal ligament and fibroblast tissues) can also be induced to express RANKL/OPG under microbe- or microbial product-induced inflammatory conditions in vivo or/and in vitro (15, 32-33, 37), suggesting the broad contributions of cytokine RANKL-RANK/OPG signaling network in periodontal disease.
We have established a humanized mouse model to study human immune-parasite interactions in periodontitis in vivo by using immunodeficient NOD/SCID mice (called: HuPBL-NOD/SCID mice) where microbial A. actinomycetemcomitans-specific human CD4+ T cells are functionally active and their TCR immune repertoire overlaps significantly (83 to 91%) with those of human clinical isolates from active disease sites (14, 44, 46, 47). Interestingly, these microorganism-specific RANKL+CD4+ T cells manifest a mixed Th1-Th2 cytokine profile in active periodontal lesions, in which specific cytokines such as gamma interferon (IFN-) and interleukin-10 (IL-10) are significantly coexpressed with RANKL during osteoclastogenesis in vivo (44). It has been shown that both TNF- and/or IL-1 can work synergistically or independently with RANKL to modulate bone resorption in arthritis and osteoporotic disorders in vitro and in vivo (2, 34, 18, 38, 41, 45, 50). Despite studies that have suggested inhibitory effect of IFN- on RANKL-associated osteoclastogenesis and bone remodeling in vitro and in vivo (11, 13, 42), it has also been shown that IFN-+ Th1 cells are strongly associated with enhanced alveolar bone loss during periodontal infections (3, 29, 22, 43, 51) and that RANKL is often highly coexpressed in Th1 cells (7, 21). Furthermore, there is strong evidence suggesting that this RANKL and IFN- coexpression exists in active arthritic lesions in vivo (4, 6, 35-36, 39) and that deficient IFN- expression significantly reduces the severity of periodontal bone loss in mice after mounting a microbial challenge (3).
To date, it is not clear what the exact role and contribution of IFN- cytokines are in terms of modulating RANKL expression and associated osteoclastogenesis under the inflammatory periodontal lesions "in vivo." We thus hypothesized that a Th1 cytokine, IFN-, can positively modulate RANKL-mediated alveolar bone loss under inflammatory conditions in vivo. This issue was addressed by several approaches: (i) detecting early cytokine expression profile and injecting low-dose hIFN- into A. actinomycetemcomitans-infected HuPBL-NOD/SCID mice followed by monitoring the coexpression of IFN- and RANKL and alveolar bone loss over time in vivo, (ii) analyzing human clinical T-cell isolates extracted from the diseased periodontal tissues of AgP subjects, and (iii) assessing cytokine expression profile in diabetic NOD mice with enhanced alveolar bone loss in vivo. The results show that, in contrast to what has been suggested in recent studies (11, 13, 42), IFN- can indeed positively modulate its coexpression with RANKL in periodontal microorganism-reactive CD4+ Th cells, which can further mediate osteoclastogenesis associated with alveolar bone loss in vivo.
MATERIALS AND METHODS
Human subjects, CD4+ T-cell purification, tissue cultures and reagents. Four consenting AgP subjects (two female and two male; mean age = 21 ± 4) whose clinical diagnosis were confirmed based on clinical, X-ray, and microbiological criteria described previously (14, 44, 45) and two consenting age-matched healthy patients (N1 and N2) (one female and one male; age = 20 and 21) were recruited for the present study. The diseased periodontal tissues obtained from the surgical discards of aggressive periodontitis (AgP) subjects undergoing periodontal surgical treatment, and periodontally healthy tissues from normal control subjects undergoing prescribed periodontal surgeries for different reasons, were collected. These procedures were all approved by the human ethics committees of the University of Western Ontario, London, Ontario, Canada, and the University of Rochester, Rochester, N.Y. Later, hCD4+ T cells were enriched and purified from the above-described surgical discards by collagenase treatment and in vitro direct panning as described previously (14, 44, 46, 47). Meanwhile, to generate mCD4+ T cells, a splenic single-cell suspension was prepared after lysing red blood cells. Then, total splenocytes were passed through a nylon-wool column to enrich T cells, after which mCD4+ T cells were further purified via direct panning on an anti-mCD4 GK1.5 monoclonal antibody (MAb)-coated petri dish (44, 46). The purity of both kinds of CD4+ T cells was 95 to 97% (by fluorescence-activated cell sorter [FACS]) (14, 44, 46, 47).
All primary T-cell cultures in the present study were performed in complete RPMI 1640 medium supplemented with 10% heat inactivated fetal bovine serum (GIBCO, Ontario, Canada), 50 μM 2-mercaptoethanol, 100 μg/ml streptomycin, and 100 U/ml penicillin, and cells were incubated at 37°C in a humidified 5% CO2 incubator as described previously (46). The following reagents and MAbs were purchased from commercial sources: recombinant hIFN- cytokine, anti-mFc-R (CD16/32), anti-mCD4, phosphatidylethanolamine (PE)-conjugated anti-h/m CD4, fluorescein isothiocyanate (FITC)- or antigen-presenting cell (APC)-conjugated goat anti-hFc- and anti-h/m IL-4, PE-, or PerCP-Cy5.5-conjugated anti-h/m IFN- (BD Pharmingen, Toronto, Canada), goat anti-rabbit biotinylated immunoglobulin G (IgG), and streptavidin-PE molecules (Vector Laboratories, Calif.), and recombinant OPG-hu-Fc and OPG-FITC fusion proteins were kindly provided by J. M. Penninger (Austria) (25, 44).
Mice and A. actinomycetemcomitans oral inoculation in vivo. Female BALB/c (H-2d) and NOD/LtJ mice (H-2g7 = Kd, Aad, Abg7, Enull, Db) (4 to 6 weeks old) were purchased from Jax Mice (Bar Harbor, Maine), and the NOD/SCID mice were generated and maintained in our own mouse-breeding suites at both Universities. All mice were housed under specific-pathogen-free environmental conditions in the Animal Care Facility of the University of Rochester, Rochester, New York, and the University of Western Ontario, Ontario, Canada. All animal protocols were conducted based on each institution's guidelines and approved by the Animal Experimentation and Use Committees of the universities.
In addition, NOD mice studied here were monitored for type 1 diabetes by daily monitoring of urine ketone and glucose levels by Diastix strips and a Glucometer Elite XL meter (Bayer, Ontario, Canada) (29). Mice were considered diabetic when whole-blood glucose levels exceeded 200 mg/dl on 2 to 3 consecutive days, with the histological evidence of severe lymphocytic infiltration in pancreatic -islets (insulitis). Typically, 70 to 80% female NOD mice developed diabetes by 16 to 20 weeks (29). Keto-Diastix strips (Bayer, Ontario, Canada) were used for daily monitoring and diagnosis. Diabetic mice were treated for hyperglycemia with Humulin U insulin (Eli Lilly, Indianapolis) (1 to 4 units/day) to maintain urine ketones (0 to 0.5 unit) and glucose at <2 units on the Keto-Diastix strips. All mice were categorized based on their age and the state of diabetes with random pancreatic histology as follows: mice 6 to 8 weeks were considered prediabetic with peri-insulitis, mice >12 to 16 week with blood glucose levels <140 mg/dl that did not develop diabetes were considered nondiabetic, and mice more than 16 to 20 weeks old with blood glucose levels >200 mg/dl and insulitis were considered diabetic (29).
The A. actinomycetemcomitans JP2 (ATCC-29523) strain was originally purchased from the American Type Culture Collection (Rockville, MD) and grown anaerobically (80% N2, 10% H2, 10% CO2) in tryptic soy broth-yeast extract culture broth (Sigma Chemical, St. Louis, Missouri) supplemented with 0.75% glucose and 0.4% NaHCO3. Experimental mice, including BALB/c, NOD/SCID, and NOD strains, were orally inoculated with freshly prepared A. actinomycetemcomitans (109 CFU/100 μl broth mixed with 2% carboxy-methylcellulose in phosphate-buffered saline [PBS]) twice per week for three consecutive weeks as described previously (46). The mice were then sacrificed at different time points between week 3 and week 7 to 8. Age-matched NOD mice without bacterial inoculation served as controls and were kept under the same specific-pathogen-free environmental conditions. For the immunization study, 6-week-old BALB/c mice were immunized intraperitoneally (i.p.) with 20 μg A. actinomycetemcomitans-CagE-homologue Ag (48-49) in colonization factor antigen (CFA) (1:1 ratio) or with CFA alone or were sham immunized 2 weeks before the start of A. actinomycetemcomitans oral inoculation as described above and elsewhere (14, 44, 46, 47). All mice were then sacrificed at the end of week 7.
Quantitative reverse transcriptase PCR (RT-PCR) and FACS analysis. The levels of HuPBL engraftment into NOD/SCID mice obtained in the present study were comparable (30%) to those reported in our previous studies (46, 47). The generation of different sources of tissues, cells, and cDNA samples used in this study were described previously (14, 44, 47). Briefly, at different time points of the experiments, periodontal or/and cervical lymph node (CLN) CD4+ T cells of the mice were purified by positive panning (>95 to 97%) and then subjected to in vitro stimulation with autologously irradiated human monocytes/macrophages (Mo/MQ) or mouse total splenocytes and A. actinomycetemcomitans sonicate Ags (JP2 strain) in 24-well plates (14, 44, 47). After 2, 3, or 4 days in cultures, 2 x 106 to 3 x 106 A. actinomycetemcomitans-reactive CD4+ T cells were collected by Ficoll-gradient centrifugation, followed by fluorescence-activated cell scanning and sorting (FACSVantage, BD Biosciences) for RANKL(+) CD4+ T cells by using OPG-hu-Fc plus 20-conjugate MAb (purity >95 to 97%; CD25+ CD69+) (14, 44, 46). Further purification of the CD4+ T cells studied here by sorting CD69/CD25-positive cells followed by labeling with CFSE (carboxyfluorescein diacetate succinimidyl ester), a FITC-based dye, for a 3-day restimulation assay in the presence of 25 Gy-irradiated human Mo/MQ or mouse splenocytes and A. actinomycetemcomitans-Ags rendered 85 to 90% CFSE-positive T cells in active division, indicating their specific reactivity to A. actinomycetemcomitans Ags (references 14, 29, 44, and 46 and data not shown).
To quantify the cytokine expression levels, total RNA was prepared from an individual pool of 1 x 106 A. actinomycetemcomitans-reactive RANKL(+) periodontal CD4+ Th cells derived from the above-described cultures representing individual groups of A. actinomycetemcomitans-HuPBL-NOD/SCID mice for quantitative RT-PCR analysis (Q-PCR) (14, 44). The first-strand cDNA was prepared from 2 μg of total RNA and then precipitated and diluted in 40 μl of Tris-EDTA (14, 44). cDNA from each sample (2 μl) was aliquoted into PCR tubes containing specific forward and reverse primers of hIFN- and hIL-4 genes as described previously (10, 44). The amplified h-actin gene products were used as the internal control for all PCRs and subsequent quantitation. Based on our previous studies (14, 44), all RT-PCRs were carried out for 30 cycles under the following conditions: 94°C denaturation for 1 min, 60°C annealing for 1 min, and 72°C extension for 1.5 min (RoboCycler 96 Gradient; Stratagene, CA) with an additional 7 min at 72°C after the last cycle. The resulting PCR products were analyzed by electrophoresis in 2% agarose gels for their respective sizes. All PCRs and subsequent quantification were performed at least two to three times from the same cDNA sources to ensure consistent and reproducible measurements. Fluorescent intensities of the amplified PCR products were captured by a UVP digital camera and quantified via image acquisition-and-analysis software (v3.0.2; LabWorks, Upland, CA). The resulting signal intensities were then normalized to the mean values of the internal control h-actin set as 1. Another endogenous control, hTCR-C primers (14, 44), was included to compare the total amounts of transcripts derived from each of 1 x 106 A. actinomycetemcomitans-reactive RANKL(+) CD4+ T cells as described above for Q-PCR analyses. To this end, repeated PCR analyses of individually amplified cytokine transcripts performed two to three times did not change the results obtained, indicating high reproducibility.
In parallel, 1 x 106 to 2 x 106 purified A. actinomycetemcomitans-reactive periodontal CD4+ T cells derived in vitro as described above were harvested and subjected to immunostaining for their cell surface expression of RANKL (OPG-Fc-FITC or APC labeled) (44, 46) and intracellular staining after being fixed by 4% formaldehyde and permeabilized with 0.2% Triton X-100 followed by incubation with IgG-conjugate for the expression of IFN- (PE-coupled or PerCP-Cy5.5: a True-Red fluorochrome conjugate for flow cytometry analysis with Ex at 490 and 675 nm and Em at 695 nm) and IL-4 (FITC coupled) by FACS analysis described previously (29, 44, 46). Briefly, to block nonspecific immunostaining, cells were first incubated with anti-FcR-IgG followed by incubation with OPG-hu-Fc to label RANKL molecules and PerCP-Cy5.5-conjugated rat anti-CD4-MAb to label CD4 molecules on T cells, respectively. Then, samples were washed twice and incubated with goat anti-hFc-IgG-FITC 20-conjugate. The isotypic control was incubated with goat anti-hFc-IgG-FITC. For FACS scanning, cells were gated on live lymphocytes and analyzed for RANKL expression in CD4+ T cells using FACSCalibur and CellQuest software (BD Biosciences) (29).
Measurement of alveolar bone loss via a digital histomorphometry. The mouse jaw samples were defleshed and stained with methylene blue to define the area between the cementum-enamel junction and the alveolar bone crest (44, 46). The surface areas represent measurement of the total amount of alveolar bone loss on the jaws, in square micrometers, which was carried out with a calibrated Leica MZ95 stereo microscope and a Hamamatsu Orca digital camera (44, 46). The jaw images were captured under x16 magnification; the right and left maxillary first two molars (i.e., M1 and M2) perpendicular to the optical light source were scanned and automatically enumerated by using the density-slice features of the OpenLab for full quantitation (29). To ensure reproducible and accurate images captured for quantification, independent jaw images were taken by two calibrated members (MA and XZ) whose results showed consistent measurements for the exposed surfaces between cementum-enamel junction-alveolar bone crest. The results of alveolar bone loss detected in the present study were consistent with our previous findings using the immunocompetent BALB/c, diabetic NOD (29), and humanized NOD/SCID mice where active alveolar bone loss and/or significantly inflamed periodontal lesions did not occur until 6 to 8 weeks post-A. actinomycetemcomitans inoculation (14, 44, 46, 47). Thus, the resulting values for alveolar bone loss are expressed as the means of the surface areas ± standard errors (S.E.) (in square micrometers) of M1 and M2 from each mouse in each group.
Statistical analysis. Statistical analysis was performed using the two-sided Student t test, and the difference between various groups was considered statistically significant when the P value was <0.05.
RESULTS
hIFN-, not hIL-4, is detected in A. actinomycetemcomitans-reactive RAMKL+ Th cells at the early stage (week 3), and increasing hIFN- is associated with enhanced alveolar bone loss by week 7 post-microbial infection in vivo. We previously reported that RANKL and some Th1-Th2 cytokines can be coexpressed in A. actinomycetemcomitans-reactive CD4+ T cells during active alveolar bone loss in HuPBL-NOD/SCID mice between week 6 and 8 post-microbial infection in vivo (44). Therefore, in order to establish an early temporal relationship between RANKL and expression of key Th1-Th2 (i.e., IFN- and IL-4) cytokines prior to active alveolar bone destruction in vivo, we studied their expression by quantitative PCR (Q-PCR) using diseased periodontal tissues of four different groups of A. actinomycetemcomitans-infected HuPBL-NOD/SCID mice (10 to 12 mice per donor group) whose autologous HuPBL samples were obtained from 4 AgP subjects (mean age, 21 ± 4), individually. Interestingly, the results of Q-PCR showed that there were significantly higher expression of hIFN- than hIL-4 in A. actinomycetemcomitans-reactive RANKL(+) periodontal CD4+ T cells at as early as week 3 after restimulating these cells for 2 to 4 days in vitro (Fig. 1A). Similar results were also detected at the end of week 7, based on the results of FACS analysis (Fig. 1B). These data indicate (i) that IFN- does coexpress with RANKL in microorganism-reactive periodontal hCD4+ Th cells in vivo, consistent with our previous study (44) and (ii) that increasing coexpression of IFN- in the microorganism-reactive RANKL(+) periodontal hCD4+ Th cells is associated with increased alveolar bone loss over time, as significantly higher alveolar bone loss occurred post-A. actinomycetemcomitans infection between week 6 to 8 in the current human-mouse chimeras (44, 46). To confirm the impact of increasing hIFN- on A. actinomycetemcomitans-specific RANKL(+) periodontal Th1 cells and alveolar bone remodeling, a low-level dose of rh-IFN- (200 ng/mouse) or PBS (sham control) was i.p. injected into A. actinomycetemcomitans-HuPBL-NOD/SCID mice (n = five per group) once a week for the first 3 weeks. These mice were sacrificed at various time points until week 7 to 8 for assessing the coexpression of IFN- and RANKL by Q-PCR and FACS and the mean alveolar bone loss via a digital imaging for histomorphometry. The results of studying samples collected at the end of week 7 clearly showed that increasing IFN- in the environment had led to significantly increased A. actinomycetemcomitans-reactive RANKL(+) periodontal CD4+ Th cells coexpressing IFN- in vivo compared to those seen with PBS-injected sham-control A. actinomycetemcomitans-HuPBL-NOD/SCID mice (Fig. 2A and B). Meanwhile, injection of rh-IFN- also yielded significantly higher alveolar bone loss in vivo compared to the sham-control results (P = 0.017; Fig. 2C and D). These data confirm the above hypothesis that a specific Th1 cytokine, hIFN-, can indeed positively modulate RANKL-medicated osteoclastogenesis in vivo where there is an associated increase of periodontal microorganism-reactive RANKL(+) CD4+ Th cells coexpressing hIFN- in the diseased tissues.
Higher IFN- expression is strongly associated with the diseased human periodontal tissues and significant RANKL-mediated alveolar bone loss in diabetic NOD mice in vivo. Since other studies have suggested an opposite effect of IFN- on bone remodeling (11, 13, 42), we though it was necessary to seek further evidence by using different approaches or models. A. actinomycetemcomitans-reactive CD4+ T cells collected from periodontal tissues and CLN were purified, respectively (14, 44, 46), from (i) surgical discard samples of three AgP subjects who were diagnosed to have active A. actinomycetemcomitans infection (14, 44, 47) and (ii) diabetic NOD mice which were orally infected with freshly prepared A. actinomycetemcomitans and monitored for up to 8 weeks for assessing "enhanced" alveolar bone loss after comparison to the prediabetic and nondiabetic NOD mice (data not shown) (29). Further, it is worth mentioning that the "enhanced" alveolar bone destruction observed and detected in "diabetic" NOD mice post-A. actinomycetemcomitans oral inoculation by week 8 is significantly associated with the severity of autoimmune insulititis (i.e., lymphocytic infiltration in -islets) where significantly increased A. actinomycetemcomitans-specific CD4+ T cells proliferation and associated RANKL expression are noted (29). Since a NOD mouse is the analogue of human type 1 diabetes, hence, "diabetic" NOD mice can serve as a good model to study the molecular interactions between microbe-induced immune responses and exacerbated alveolar bone loss in vivo. As expected, there was significantly higher coexpression of IFN- in A. actinomycetemcomitans-reactive RANKL(+) periodontal CD4+ Th cells in clinical human T-cell isolates (Fig. 3A) (Q-PCR) and in "diabetic" NOD mice where there was concomitantly higher alveolar bone loss (for RANKL+ murine IFN-+ (mIFN-+) at 22 to 36% in "nondiabetic" and 49 to 56% in "diabetic" NOD mice, see FACS in Fig. 3B) (29). In contrast, there were no significant differences regarding IL-4 expression in RANKL+ CD4+ Th cells in both cases. These results strongly suggest that coexpression of IFN- in microorganism-reactive RANKL(+) periodontal Th1 cells is significantly associated with alveolar bone destruction in vivo, based on all three different lines of evidence (Figs. 1 to 3).
Microbial Ag (CagE)-specific RANKL+ IFN--expressing Th cells are strongly associated with increasing alveolar bone loss in vivo. Finally, to examine whether the above-described event of cytokine regulation in response to A. actinomycetemcomitans challenge can also be induced by specific microbial Ag in vivo, we employed an A. actinomycetemcomitans-associated CagE homologue (in short, CagE) (48, 49) recognized by human CD4+ T cells as a model Ag for testing via an immunization strategy. Based on our previous study, CagE is a critical microbial virulence factor associated with human CD4+ T-cell-mediated destructive immunity for alveolar bone loss and induction of apoptosis of different human cell types in the periodontal tissues (46, 48-49). Therefore, BALB/c mice were i.p. immunized with CagE Ag (group 3; n = 5 per group) prior to oral infection with A. actinomycetemcomitans (for 3 weeks), which resulted in a significantly enhanced alveolar bone loss by week 7 to 8 in vivo (Fig. 4A and B). The controls included A. actinomycetemcomitans oral inoculation with sham-treated mice (group 1) and CFA-immunized BALB/c mice (group 2; Fig. 4A and B). Meanwhile, the same protocol also resulted in a significantly increased coexpression of mIFN- in A. actinomycetemcomitans-reactive RANKL(+) periodontal CD4+ Th cells in CagE-immunized BALB/c mice by week 7 to 8, suggesting that modulation of RANKL-mediated osteoclastogenesis by mIFN- can be induced by the whole microorganism and a single microbial Ag (i.e., CagE) in vivo. Together, the above data are in concordance with our hypothesis that IFN-, a Th1 signature cytokine, can positively modulate RANKL-mediated osteoclastogenesis in A. actinomycetemcomitans-induced periodontal bone loss in human-mouse chimeras, clinical periodontal T-cell isolates of AgP patients, and diabetic NOD mice during periodontal disease progression in vivo.
DISCUSSION
The present report clearly describes the positive role of IFN- (for human and mouse) in modulating RANKL+ Th cell-mediated alveolar bone loss under inflammatory conditions post-microbial challenge in vivo. Although IFN- has been shown to directly inhibit RANKL-mediated osteoclastogenesis in vitro and in animal studies (11, 13, 42), possibly via an STAT1-dependent promotion of TRAF6 degradation in the OC precursors pool, opposite findings also exist (4, 6-7, 21, 35-36, 39) in which microbial Ag (i.e., an outer membrane protein of A. actinomycetemcomitans)-reactive CD4+Th1-cells capably of mediating periodontal bone destruction also coexpressed IFN- (22, 43, 51).
Moreover, the results of the present study are consistent with the findings by Baker et al. (3) that IFN- knockout mice manifest significantly reduced alveolar bone loss after Porphyromonas gingivalis oral challenge in vivo, pinpointing the critical role of IFN- in modulating alveolar bone destruction in the diseased periodontal tissues and/or cells. At present, the in vivo molecular mechanism(s) of this phenomenon as described above remains unclear. However, it may not be attributed solely to the proteosome degradation pathway(s) or signaling (42), as there are no significant differences regarding the TRAF6 transcripts and proteins in the periodontal tissue samples between IFN--treated or sham-treated A. actinomycetemcomitans-HuPBL-NOD/SCID mice (data not shown here). In addition, IFN- might mediate its downstream signaling effects independently of RANKL-induced osteoclastogenesis pathways under various inflammatory conditions (i.e., in the presence of TNF- and IL-1) in vivo. One such possible interaction may be related to signaling via other non-TRAF6 or non-STAT1 family molecules in the NF-B and/or JNK pathways (11, 24, 41-42). Alternatively, there are regulatory interactions mediated by non-T-cell sources (i.e., B and NK cells) (8, 23) or different cytokines (i.e., TNF- and IL-1) (2, 34, 18, 38, 41, 45) that may, at least in part, contribute to the above phenomenon, as it has been shown that B and NK cells can express RANKL and IFN-, respectively, associated with bone remodeling (8, 23). On the same token, it requires further study to seek whether periodontal resident in tissues or cells can potentially influence RANKL-mediated osteoclastogenesis in vivo (15, 32, 33, 37). Lastly, it is known that IFN- can up-regulate the expression of major histocompatibility complex class II and other accessory molecules on the antigen-presenting cells, leukocytes, and mesenchymal cells, which may further recruit other signaling molecules and/or immune effectors associated with bone remodeling (12, 17, 31). Ultimately, the fine balance between IFN- and RANKL under various inflammatory conditions may also contribute to determining the outcome of their coexpression on Th1 cells for osteoclastogenesis associated with bone remodeling in vivo.
In summary, the present study provides new lines of evidence describing a positive coexpression relationship and interactions between IFN- and RANKL-mediated osteoclastogenesis for alveolar bone loss in periodontal microorganism-specific CD4+T-cell subpopulation(s) in vivo. Our study strongly suggests that there are complex networks of cytokine regulation associated with RANKL-RANK/OPG signaling pathways for osteoclastogenesis in vivo that are much more complicated than what we currently have explored and understood. Further study of these critical signaling networks is essential for better understanding and developing new treatment protocols when dealing with inflammatory bone disorders such as human periodontitis.
ACKNOWLEDGMENTS
We thank X. Zhang and M. Alnaeeli for their help in the work.
This work was supported by grants to Y.-T.A.T. from the Ministry of Health of Ontario, Canada; the Canadian Institute of Health Research (CIHR), Canada (MOP-37960); the University of Rochester; and the National Institutes of Health of the United States (DE12969 and DE14473).
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Department of Microbiology & Immunology, School of Medicine and Dentistry, the University of Western Ontario, London, Ontario, N6A 5C1, Canada
ABSTRACT
Recent studies have shown the biological and clinical significance of signaling pathways of osteogenic cytokines RANKL-RANK/OPG in controlling osteoclastogenesis associated with bone pathologies, including rheumatoid arthritis, osteoporosis, and other osteolytic disorders. In contrast to the inhibitory effect of gamma interferon (IFN-) on RANKL-mediated osteoclastogenesis reported recently, alternative new evidence is demonstrated via studies of experimental periodontitis using humanized NOD/SCID and diabetic NOD mice and clinical human T-cell isolates from diseased periodontal tissues, where the presence of increasing IFN- is clearly associated with (i) enhanced Actinobacillus actinomycetemcomitans-specific RANKL-expressing CD4+ Th cell-mediated alveolar bone loss during the progression of periodontal disease and (ii) a concomitant and significantly increased coexpression of IFN- in RANKL(+) CD4+ Th cells. Therefore, there are more complex networks in regulating RANKL-RANK/OPG signaling pathways for osteoclastogenesis in vivo than have been suggested to date.
INTRODUCTION
Human periodontal disease (i.e., periodontitis) results from the interplay between the specific subgingival microorganisms and the host's immune and inflammatory response (9). Periodontitis is characterized by loss of attachments including the periodontal connective tissues and supporting alveolar bone. Actinobacillus actinomycetemcomitans (A. actinomycetemcomitans) is a G(–) facultative anaerobe strongly associated with human aggressive periodontitis (AgP) and also involved in medical diseases such as urinary tract and hip-joint prosthetics infections, thyroid and brain abscesses, and subacute endocarditis (54). Although several carbohydrate and protein antigens (Ags), fimbriae, and polysaccharides were identified, much less is known about critical Ags of A. actinomycetemcomitans that sensitize T or B cells involved in the periodontal pathogenesis (16).
The recently identified tumor necrosis factor (TNF) family molecule, receptor activator of NF-B ligand (RANKL [or TRANCE, OPGL, or ODF]), its receptor RANK, and the natural antagonist, osteoprotegerin (OPG), have been shown to be the key regulators of bone remodeling and are directly involved in the differentiation, activation, and survival of osteoclasts (OC) and OC precursors (27, 40, 52-53). Previously, we showed that activated CD4+ T cells express RANKL, which can directly trigger osteoclastogenesis and alveolar bone loss associated with periodontitis in vivo (46). Further, blocking RANKL activity via OPG injections results in significantly reduced bone loss in arthritis (25, 49), periodontitis (46, 50), osteoporosis (19, 30), cancer-related bone metastasis (5, 20), and enhanced alveolar bone loss associated with type 1 diabetes in vivo (29). RANKL-RANK signaling is also critical for survival of dendritic cells, lymph node formation, and organogenesis and involved in dendritic cell/T-cell interactions (1, 26). Genetic mutations of RANKL and RANK demonstrate similar phenotypes in OC development with severe osteopetrosis, suggesting that they are essential for osteoclastogenesis during bone remodeling (26, 28, 50). OPG-transgenic mice are osteopetrotic with defective OC activity, and OPG-deficient mice are severely osteoporotic (30, 40, 50). Thus, RANKL, RANK, and OPG are essential for controlling OC development and functions in bone remodeling. These studies have supported the new paradigm of linking adaptive immunity and bone remodeling (termed osteo-immunology) associated with various inflammatory bone disorders (1, 5, 19-20, 25-26, 28-30, 40, 46, 50, 53). More recently, studies have shown that periodontal residen T cells and tissues (i.e., periodontal ligament and fibroblast tissues) can also be induced to express RANKL/OPG under microbe- or microbial product-induced inflammatory conditions in vivo or/and in vitro (15, 32-33, 37), suggesting the broad contributions of cytokine RANKL-RANK/OPG signaling network in periodontal disease.
We have established a humanized mouse model to study human immune-parasite interactions in periodontitis in vivo by using immunodeficient NOD/SCID mice (called: HuPBL-NOD/SCID mice) where microbial A. actinomycetemcomitans-specific human CD4+ T cells are functionally active and their TCR immune repertoire overlaps significantly (83 to 91%) with those of human clinical isolates from active disease sites (14, 44, 46, 47). Interestingly, these microorganism-specific RANKL+CD4+ T cells manifest a mixed Th1-Th2 cytokine profile in active periodontal lesions, in which specific cytokines such as gamma interferon (IFN-) and interleukin-10 (IL-10) are significantly coexpressed with RANKL during osteoclastogenesis in vivo (44). It has been shown that both TNF- and/or IL-1 can work synergistically or independently with RANKL to modulate bone resorption in arthritis and osteoporotic disorders in vitro and in vivo (2, 34, 18, 38, 41, 45, 50). Despite studies that have suggested inhibitory effect of IFN- on RANKL-associated osteoclastogenesis and bone remodeling in vitro and in vivo (11, 13, 42), it has also been shown that IFN-+ Th1 cells are strongly associated with enhanced alveolar bone loss during periodontal infections (3, 29, 22, 43, 51) and that RANKL is often highly coexpressed in Th1 cells (7, 21). Furthermore, there is strong evidence suggesting that this RANKL and IFN- coexpression exists in active arthritic lesions in vivo (4, 6, 35-36, 39) and that deficient IFN- expression significantly reduces the severity of periodontal bone loss in mice after mounting a microbial challenge (3).
To date, it is not clear what the exact role and contribution of IFN- cytokines are in terms of modulating RANKL expression and associated osteoclastogenesis under the inflammatory periodontal lesions "in vivo." We thus hypothesized that a Th1 cytokine, IFN-, can positively modulate RANKL-mediated alveolar bone loss under inflammatory conditions in vivo. This issue was addressed by several approaches: (i) detecting early cytokine expression profile and injecting low-dose hIFN- into A. actinomycetemcomitans-infected HuPBL-NOD/SCID mice followed by monitoring the coexpression of IFN- and RANKL and alveolar bone loss over time in vivo, (ii) analyzing human clinical T-cell isolates extracted from the diseased periodontal tissues of AgP subjects, and (iii) assessing cytokine expression profile in diabetic NOD mice with enhanced alveolar bone loss in vivo. The results show that, in contrast to what has been suggested in recent studies (11, 13, 42), IFN- can indeed positively modulate its coexpression with RANKL in periodontal microorganism-reactive CD4+ Th cells, which can further mediate osteoclastogenesis associated with alveolar bone loss in vivo.
MATERIALS AND METHODS
Human subjects, CD4+ T-cell purification, tissue cultures and reagents. Four consenting AgP subjects (two female and two male; mean age = 21 ± 4) whose clinical diagnosis were confirmed based on clinical, X-ray, and microbiological criteria described previously (14, 44, 45) and two consenting age-matched healthy patients (N1 and N2) (one female and one male; age = 20 and 21) were recruited for the present study. The diseased periodontal tissues obtained from the surgical discards of aggressive periodontitis (AgP) subjects undergoing periodontal surgical treatment, and periodontally healthy tissues from normal control subjects undergoing prescribed periodontal surgeries for different reasons, were collected. These procedures were all approved by the human ethics committees of the University of Western Ontario, London, Ontario, Canada, and the University of Rochester, Rochester, N.Y. Later, hCD4+ T cells were enriched and purified from the above-described surgical discards by collagenase treatment and in vitro direct panning as described previously (14, 44, 46, 47). Meanwhile, to generate mCD4+ T cells, a splenic single-cell suspension was prepared after lysing red blood cells. Then, total splenocytes were passed through a nylon-wool column to enrich T cells, after which mCD4+ T cells were further purified via direct panning on an anti-mCD4 GK1.5 monoclonal antibody (MAb)-coated petri dish (44, 46). The purity of both kinds of CD4+ T cells was 95 to 97% (by fluorescence-activated cell sorter [FACS]) (14, 44, 46, 47).
All primary T-cell cultures in the present study were performed in complete RPMI 1640 medium supplemented with 10% heat inactivated fetal bovine serum (GIBCO, Ontario, Canada), 50 μM 2-mercaptoethanol, 100 μg/ml streptomycin, and 100 U/ml penicillin, and cells were incubated at 37°C in a humidified 5% CO2 incubator as described previously (46). The following reagents and MAbs were purchased from commercial sources: recombinant hIFN- cytokine, anti-mFc-R (CD16/32), anti-mCD4, phosphatidylethanolamine (PE)-conjugated anti-h/m CD4, fluorescein isothiocyanate (FITC)- or antigen-presenting cell (APC)-conjugated goat anti-hFc- and anti-h/m IL-4, PE-, or PerCP-Cy5.5-conjugated anti-h/m IFN- (BD Pharmingen, Toronto, Canada), goat anti-rabbit biotinylated immunoglobulin G (IgG), and streptavidin-PE molecules (Vector Laboratories, Calif.), and recombinant OPG-hu-Fc and OPG-FITC fusion proteins were kindly provided by J. M. Penninger (Austria) (25, 44).
Mice and A. actinomycetemcomitans oral inoculation in vivo. Female BALB/c (H-2d) and NOD/LtJ mice (H-2g7 = Kd, Aad, Abg7, Enull, Db) (4 to 6 weeks old) were purchased from Jax Mice (Bar Harbor, Maine), and the NOD/SCID mice were generated and maintained in our own mouse-breeding suites at both Universities. All mice were housed under specific-pathogen-free environmental conditions in the Animal Care Facility of the University of Rochester, Rochester, New York, and the University of Western Ontario, Ontario, Canada. All animal protocols were conducted based on each institution's guidelines and approved by the Animal Experimentation and Use Committees of the universities.
In addition, NOD mice studied here were monitored for type 1 diabetes by daily monitoring of urine ketone and glucose levels by Diastix strips and a Glucometer Elite XL meter (Bayer, Ontario, Canada) (29). Mice were considered diabetic when whole-blood glucose levels exceeded 200 mg/dl on 2 to 3 consecutive days, with the histological evidence of severe lymphocytic infiltration in pancreatic -islets (insulitis). Typically, 70 to 80% female NOD mice developed diabetes by 16 to 20 weeks (29). Keto-Diastix strips (Bayer, Ontario, Canada) were used for daily monitoring and diagnosis. Diabetic mice were treated for hyperglycemia with Humulin U insulin (Eli Lilly, Indianapolis) (1 to 4 units/day) to maintain urine ketones (0 to 0.5 unit) and glucose at <2 units on the Keto-Diastix strips. All mice were categorized based on their age and the state of diabetes with random pancreatic histology as follows: mice 6 to 8 weeks were considered prediabetic with peri-insulitis, mice >12 to 16 week with blood glucose levels <140 mg/dl that did not develop diabetes were considered nondiabetic, and mice more than 16 to 20 weeks old with blood glucose levels >200 mg/dl and insulitis were considered diabetic (29).
The A. actinomycetemcomitans JP2 (ATCC-29523) strain was originally purchased from the American Type Culture Collection (Rockville, MD) and grown anaerobically (80% N2, 10% H2, 10% CO2) in tryptic soy broth-yeast extract culture broth (Sigma Chemical, St. Louis, Missouri) supplemented with 0.75% glucose and 0.4% NaHCO3. Experimental mice, including BALB/c, NOD/SCID, and NOD strains, were orally inoculated with freshly prepared A. actinomycetemcomitans (109 CFU/100 μl broth mixed with 2% carboxy-methylcellulose in phosphate-buffered saline [PBS]) twice per week for three consecutive weeks as described previously (46). The mice were then sacrificed at different time points between week 3 and week 7 to 8. Age-matched NOD mice without bacterial inoculation served as controls and were kept under the same specific-pathogen-free environmental conditions. For the immunization study, 6-week-old BALB/c mice were immunized intraperitoneally (i.p.) with 20 μg A. actinomycetemcomitans-CagE-homologue Ag (48-49) in colonization factor antigen (CFA) (1:1 ratio) or with CFA alone or were sham immunized 2 weeks before the start of A. actinomycetemcomitans oral inoculation as described above and elsewhere (14, 44, 46, 47). All mice were then sacrificed at the end of week 7.
Quantitative reverse transcriptase PCR (RT-PCR) and FACS analysis. The levels of HuPBL engraftment into NOD/SCID mice obtained in the present study were comparable (30%) to those reported in our previous studies (46, 47). The generation of different sources of tissues, cells, and cDNA samples used in this study were described previously (14, 44, 47). Briefly, at different time points of the experiments, periodontal or/and cervical lymph node (CLN) CD4+ T cells of the mice were purified by positive panning (>95 to 97%) and then subjected to in vitro stimulation with autologously irradiated human monocytes/macrophages (Mo/MQ) or mouse total splenocytes and A. actinomycetemcomitans sonicate Ags (JP2 strain) in 24-well plates (14, 44, 47). After 2, 3, or 4 days in cultures, 2 x 106 to 3 x 106 A. actinomycetemcomitans-reactive CD4+ T cells were collected by Ficoll-gradient centrifugation, followed by fluorescence-activated cell scanning and sorting (FACSVantage, BD Biosciences) for RANKL(+) CD4+ T cells by using OPG-hu-Fc plus 20-conjugate MAb (purity >95 to 97%; CD25+ CD69+) (14, 44, 46). Further purification of the CD4+ T cells studied here by sorting CD69/CD25-positive cells followed by labeling with CFSE (carboxyfluorescein diacetate succinimidyl ester), a FITC-based dye, for a 3-day restimulation assay in the presence of 25 Gy-irradiated human Mo/MQ or mouse splenocytes and A. actinomycetemcomitans-Ags rendered 85 to 90% CFSE-positive T cells in active division, indicating their specific reactivity to A. actinomycetemcomitans Ags (references 14, 29, 44, and 46 and data not shown).
To quantify the cytokine expression levels, total RNA was prepared from an individual pool of 1 x 106 A. actinomycetemcomitans-reactive RANKL(+) periodontal CD4+ Th cells derived from the above-described cultures representing individual groups of A. actinomycetemcomitans-HuPBL-NOD/SCID mice for quantitative RT-PCR analysis (Q-PCR) (14, 44). The first-strand cDNA was prepared from 2 μg of total RNA and then precipitated and diluted in 40 μl of Tris-EDTA (14, 44). cDNA from each sample (2 μl) was aliquoted into PCR tubes containing specific forward and reverse primers of hIFN- and hIL-4 genes as described previously (10, 44). The amplified h-actin gene products were used as the internal control for all PCRs and subsequent quantitation. Based on our previous studies (14, 44), all RT-PCRs were carried out for 30 cycles under the following conditions: 94°C denaturation for 1 min, 60°C annealing for 1 min, and 72°C extension for 1.5 min (RoboCycler 96 Gradient; Stratagene, CA) with an additional 7 min at 72°C after the last cycle. The resulting PCR products were analyzed by electrophoresis in 2% agarose gels for their respective sizes. All PCRs and subsequent quantification were performed at least two to three times from the same cDNA sources to ensure consistent and reproducible measurements. Fluorescent intensities of the amplified PCR products were captured by a UVP digital camera and quantified via image acquisition-and-analysis software (v3.0.2; LabWorks, Upland, CA). The resulting signal intensities were then normalized to the mean values of the internal control h-actin set as 1. Another endogenous control, hTCR-C primers (14, 44), was included to compare the total amounts of transcripts derived from each of 1 x 106 A. actinomycetemcomitans-reactive RANKL(+) CD4+ T cells as described above for Q-PCR analyses. To this end, repeated PCR analyses of individually amplified cytokine transcripts performed two to three times did not change the results obtained, indicating high reproducibility.
In parallel, 1 x 106 to 2 x 106 purified A. actinomycetemcomitans-reactive periodontal CD4+ T cells derived in vitro as described above were harvested and subjected to immunostaining for their cell surface expression of RANKL (OPG-Fc-FITC or APC labeled) (44, 46) and intracellular staining after being fixed by 4% formaldehyde and permeabilized with 0.2% Triton X-100 followed by incubation with IgG-conjugate for the expression of IFN- (PE-coupled or PerCP-Cy5.5: a True-Red fluorochrome conjugate for flow cytometry analysis with Ex at 490 and 675 nm and Em at 695 nm) and IL-4 (FITC coupled) by FACS analysis described previously (29, 44, 46). Briefly, to block nonspecific immunostaining, cells were first incubated with anti-FcR-IgG followed by incubation with OPG-hu-Fc to label RANKL molecules and PerCP-Cy5.5-conjugated rat anti-CD4-MAb to label CD4 molecules on T cells, respectively. Then, samples were washed twice and incubated with goat anti-hFc-IgG-FITC 20-conjugate. The isotypic control was incubated with goat anti-hFc-IgG-FITC. For FACS scanning, cells were gated on live lymphocytes and analyzed for RANKL expression in CD4+ T cells using FACSCalibur and CellQuest software (BD Biosciences) (29).
Measurement of alveolar bone loss via a digital histomorphometry. The mouse jaw samples were defleshed and stained with methylene blue to define the area between the cementum-enamel junction and the alveolar bone crest (44, 46). The surface areas represent measurement of the total amount of alveolar bone loss on the jaws, in square micrometers, which was carried out with a calibrated Leica MZ95 stereo microscope and a Hamamatsu Orca digital camera (44, 46). The jaw images were captured under x16 magnification; the right and left maxillary first two molars (i.e., M1 and M2) perpendicular to the optical light source were scanned and automatically enumerated by using the density-slice features of the OpenLab for full quantitation (29). To ensure reproducible and accurate images captured for quantification, independent jaw images were taken by two calibrated members (MA and XZ) whose results showed consistent measurements for the exposed surfaces between cementum-enamel junction-alveolar bone crest. The results of alveolar bone loss detected in the present study were consistent with our previous findings using the immunocompetent BALB/c, diabetic NOD (29), and humanized NOD/SCID mice where active alveolar bone loss and/or significantly inflamed periodontal lesions did not occur until 6 to 8 weeks post-A. actinomycetemcomitans inoculation (14, 44, 46, 47). Thus, the resulting values for alveolar bone loss are expressed as the means of the surface areas ± standard errors (S.E.) (in square micrometers) of M1 and M2 from each mouse in each group.
Statistical analysis. Statistical analysis was performed using the two-sided Student t test, and the difference between various groups was considered statistically significant when the P value was <0.05.
RESULTS
hIFN-, not hIL-4, is detected in A. actinomycetemcomitans-reactive RAMKL+ Th cells at the early stage (week 3), and increasing hIFN- is associated with enhanced alveolar bone loss by week 7 post-microbial infection in vivo. We previously reported that RANKL and some Th1-Th2 cytokines can be coexpressed in A. actinomycetemcomitans-reactive CD4+ T cells during active alveolar bone loss in HuPBL-NOD/SCID mice between week 6 and 8 post-microbial infection in vivo (44). Therefore, in order to establish an early temporal relationship between RANKL and expression of key Th1-Th2 (i.e., IFN- and IL-4) cytokines prior to active alveolar bone destruction in vivo, we studied their expression by quantitative PCR (Q-PCR) using diseased periodontal tissues of four different groups of A. actinomycetemcomitans-infected HuPBL-NOD/SCID mice (10 to 12 mice per donor group) whose autologous HuPBL samples were obtained from 4 AgP subjects (mean age, 21 ± 4), individually. Interestingly, the results of Q-PCR showed that there were significantly higher expression of hIFN- than hIL-4 in A. actinomycetemcomitans-reactive RANKL(+) periodontal CD4+ T cells at as early as week 3 after restimulating these cells for 2 to 4 days in vitro (Fig. 1A). Similar results were also detected at the end of week 7, based on the results of FACS analysis (Fig. 1B). These data indicate (i) that IFN- does coexpress with RANKL in microorganism-reactive periodontal hCD4+ Th cells in vivo, consistent with our previous study (44) and (ii) that increasing coexpression of IFN- in the microorganism-reactive RANKL(+) periodontal hCD4+ Th cells is associated with increased alveolar bone loss over time, as significantly higher alveolar bone loss occurred post-A. actinomycetemcomitans infection between week 6 to 8 in the current human-mouse chimeras (44, 46). To confirm the impact of increasing hIFN- on A. actinomycetemcomitans-specific RANKL(+) periodontal Th1 cells and alveolar bone remodeling, a low-level dose of rh-IFN- (200 ng/mouse) or PBS (sham control) was i.p. injected into A. actinomycetemcomitans-HuPBL-NOD/SCID mice (n = five per group) once a week for the first 3 weeks. These mice were sacrificed at various time points until week 7 to 8 for assessing the coexpression of IFN- and RANKL by Q-PCR and FACS and the mean alveolar bone loss via a digital imaging for histomorphometry. The results of studying samples collected at the end of week 7 clearly showed that increasing IFN- in the environment had led to significantly increased A. actinomycetemcomitans-reactive RANKL(+) periodontal CD4+ Th cells coexpressing IFN- in vivo compared to those seen with PBS-injected sham-control A. actinomycetemcomitans-HuPBL-NOD/SCID mice (Fig. 2A and B). Meanwhile, injection of rh-IFN- also yielded significantly higher alveolar bone loss in vivo compared to the sham-control results (P = 0.017; Fig. 2C and D). These data confirm the above hypothesis that a specific Th1 cytokine, hIFN-, can indeed positively modulate RANKL-medicated osteoclastogenesis in vivo where there is an associated increase of periodontal microorganism-reactive RANKL(+) CD4+ Th cells coexpressing hIFN- in the diseased tissues.
Higher IFN- expression is strongly associated with the diseased human periodontal tissues and significant RANKL-mediated alveolar bone loss in diabetic NOD mice in vivo. Since other studies have suggested an opposite effect of IFN- on bone remodeling (11, 13, 42), we though it was necessary to seek further evidence by using different approaches or models. A. actinomycetemcomitans-reactive CD4+ T cells collected from periodontal tissues and CLN were purified, respectively (14, 44, 46), from (i) surgical discard samples of three AgP subjects who were diagnosed to have active A. actinomycetemcomitans infection (14, 44, 47) and (ii) diabetic NOD mice which were orally infected with freshly prepared A. actinomycetemcomitans and monitored for up to 8 weeks for assessing "enhanced" alveolar bone loss after comparison to the prediabetic and nondiabetic NOD mice (data not shown) (29). Further, it is worth mentioning that the "enhanced" alveolar bone destruction observed and detected in "diabetic" NOD mice post-A. actinomycetemcomitans oral inoculation by week 8 is significantly associated with the severity of autoimmune insulititis (i.e., lymphocytic infiltration in -islets) where significantly increased A. actinomycetemcomitans-specific CD4+ T cells proliferation and associated RANKL expression are noted (29). Since a NOD mouse is the analogue of human type 1 diabetes, hence, "diabetic" NOD mice can serve as a good model to study the molecular interactions between microbe-induced immune responses and exacerbated alveolar bone loss in vivo. As expected, there was significantly higher coexpression of IFN- in A. actinomycetemcomitans-reactive RANKL(+) periodontal CD4+ Th cells in clinical human T-cell isolates (Fig. 3A) (Q-PCR) and in "diabetic" NOD mice where there was concomitantly higher alveolar bone loss (for RANKL+ murine IFN-+ (mIFN-+) at 22 to 36% in "nondiabetic" and 49 to 56% in "diabetic" NOD mice, see FACS in Fig. 3B) (29). In contrast, there were no significant differences regarding IL-4 expression in RANKL+ CD4+ Th cells in both cases. These results strongly suggest that coexpression of IFN- in microorganism-reactive RANKL(+) periodontal Th1 cells is significantly associated with alveolar bone destruction in vivo, based on all three different lines of evidence (Figs. 1 to 3).
Microbial Ag (CagE)-specific RANKL+ IFN--expressing Th cells are strongly associated with increasing alveolar bone loss in vivo. Finally, to examine whether the above-described event of cytokine regulation in response to A. actinomycetemcomitans challenge can also be induced by specific microbial Ag in vivo, we employed an A. actinomycetemcomitans-associated CagE homologue (in short, CagE) (48, 49) recognized by human CD4+ T cells as a model Ag for testing via an immunization strategy. Based on our previous study, CagE is a critical microbial virulence factor associated with human CD4+ T-cell-mediated destructive immunity for alveolar bone loss and induction of apoptosis of different human cell types in the periodontal tissues (46, 48-49). Therefore, BALB/c mice were i.p. immunized with CagE Ag (group 3; n = 5 per group) prior to oral infection with A. actinomycetemcomitans (for 3 weeks), which resulted in a significantly enhanced alveolar bone loss by week 7 to 8 in vivo (Fig. 4A and B). The controls included A. actinomycetemcomitans oral inoculation with sham-treated mice (group 1) and CFA-immunized BALB/c mice (group 2; Fig. 4A and B). Meanwhile, the same protocol also resulted in a significantly increased coexpression of mIFN- in A. actinomycetemcomitans-reactive RANKL(+) periodontal CD4+ Th cells in CagE-immunized BALB/c mice by week 7 to 8, suggesting that modulation of RANKL-mediated osteoclastogenesis by mIFN- can be induced by the whole microorganism and a single microbial Ag (i.e., CagE) in vivo. Together, the above data are in concordance with our hypothesis that IFN-, a Th1 signature cytokine, can positively modulate RANKL-mediated osteoclastogenesis in A. actinomycetemcomitans-induced periodontal bone loss in human-mouse chimeras, clinical periodontal T-cell isolates of AgP patients, and diabetic NOD mice during periodontal disease progression in vivo.
DISCUSSION
The present report clearly describes the positive role of IFN- (for human and mouse) in modulating RANKL+ Th cell-mediated alveolar bone loss under inflammatory conditions post-microbial challenge in vivo. Although IFN- has been shown to directly inhibit RANKL-mediated osteoclastogenesis in vitro and in animal studies (11, 13, 42), possibly via an STAT1-dependent promotion of TRAF6 degradation in the OC precursors pool, opposite findings also exist (4, 6-7, 21, 35-36, 39) in which microbial Ag (i.e., an outer membrane protein of A. actinomycetemcomitans)-reactive CD4+Th1-cells capably of mediating periodontal bone destruction also coexpressed IFN- (22, 43, 51).
Moreover, the results of the present study are consistent with the findings by Baker et al. (3) that IFN- knockout mice manifest significantly reduced alveolar bone loss after Porphyromonas gingivalis oral challenge in vivo, pinpointing the critical role of IFN- in modulating alveolar bone destruction in the diseased periodontal tissues and/or cells. At present, the in vivo molecular mechanism(s) of this phenomenon as described above remains unclear. However, it may not be attributed solely to the proteosome degradation pathway(s) or signaling (42), as there are no significant differences regarding the TRAF6 transcripts and proteins in the periodontal tissue samples between IFN--treated or sham-treated A. actinomycetemcomitans-HuPBL-NOD/SCID mice (data not shown here). In addition, IFN- might mediate its downstream signaling effects independently of RANKL-induced osteoclastogenesis pathways under various inflammatory conditions (i.e., in the presence of TNF- and IL-1) in vivo. One such possible interaction may be related to signaling via other non-TRAF6 or non-STAT1 family molecules in the NF-B and/or JNK pathways (11, 24, 41-42). Alternatively, there are regulatory interactions mediated by non-T-cell sources (i.e., B and NK cells) (8, 23) or different cytokines (i.e., TNF- and IL-1) (2, 34, 18, 38, 41, 45) that may, at least in part, contribute to the above phenomenon, as it has been shown that B and NK cells can express RANKL and IFN-, respectively, associated with bone remodeling (8, 23). On the same token, it requires further study to seek whether periodontal resident in tissues or cells can potentially influence RANKL-mediated osteoclastogenesis in vivo (15, 32, 33, 37). Lastly, it is known that IFN- can up-regulate the expression of major histocompatibility complex class II and other accessory molecules on the antigen-presenting cells, leukocytes, and mesenchymal cells, which may further recruit other signaling molecules and/or immune effectors associated with bone remodeling (12, 17, 31). Ultimately, the fine balance between IFN- and RANKL under various inflammatory conditions may also contribute to determining the outcome of their coexpression on Th1 cells for osteoclastogenesis associated with bone remodeling in vivo.
In summary, the present study provides new lines of evidence describing a positive coexpression relationship and interactions between IFN- and RANKL-mediated osteoclastogenesis for alveolar bone loss in periodontal microorganism-specific CD4+T-cell subpopulation(s) in vivo. Our study strongly suggests that there are complex networks of cytokine regulation associated with RANKL-RANK/OPG signaling pathways for osteoclastogenesis in vivo that are much more complicated than what we currently have explored and understood. Further study of these critical signaling networks is essential for better understanding and developing new treatment protocols when dealing with inflammatory bone disorders such as human periodontitis.
ACKNOWLEDGMENTS
We thank X. Zhang and M. Alnaeeli for their help in the work.
This work was supported by grants to Y.-T.A.T. from the Ministry of Health of Ontario, Canada; the Canadian Institute of Health Research (CIHR), Canada (MOP-37960); the University of Rochester; and the National Institutes of Health of the United States (DE12969 and DE14473).
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