Activation of Invariant NK T Cells in Periodontitis Lesions Free

Periodontitis is one of the most prevalent chronic inflammatory diseases in humans, with a prevalence of 30% in the United States population (1). Untreated periodontitis leads to destruction of periodontal tissues and supporting bone, ultimately resulting in tooth loss (2, 3). In addition to its detrimental role in the oral cavity, periodontal infections have been causally linked to atherosclerosis and other systemic diseases (4). Periodontitis is a biofilm-mediated disease, and several oral bacteria that colonize the tooth surfaces, including Gram-negative anaerobic species, have been identified as causative agents (3). The two main categories of destructive periodontal disease are chronic and aggressive periodontitis (5, 6). Aggressive periodontitis is characterized by rapid attachment loss and destruction, often at young age and with familiar predisposition (7, 8). Infections with certain serotypes of the facultative aerobic Gram-negative pathogen Aggregatibacter actinomycetemcomitans have been causally linked to aggressive periodontitis (9, 10). Chronic periodontitis, in contrast, is described as slowly progressing inflammatory loss of periodontal tissues associated with moderate to heavy deposits of bacterial plaque and calculus (7). A principal pathogen in chronic periodontitis is the anaerobic, Gram-negative Porpyromonas gingivalis (11). Specifically, no histopathological differences between these two chronic inflammatory subforms of periodontal disease are available to date (12). In this study, we assessed the role of type I NKT cells, a cell population with critical properties in guiding immune responses against infection, in both forms of periodontitis, and delineate the mechanisms of their activation.

NKT cells are a population of lymphocytes with unique activation and effector properties, which bridge innate and adaptive immunity. The majority of NKT cells, termed type I or invariant NKT (iNKT) cells, are CD1d restricted and express a semi-invariant TCR using the segments Vα14 and Jα18 in mice and Vα24 and Jα18 in humans. Type I NKT cells recognize lipid Ags presented in nonpolymorphic CD1d molecules, which are predominantly expressed on APCs (dendritic cells [DCs], macrophages, and B cells) (13).

Interactions among DCs, expressing CD1d molecules, and type I NKT cells have intensively been studied (14, 15). Presentation of CD1d–lipid complexes by DCs initiates a positive feedback. In particular, stimulation of DCs by interactions between CD40L (CD154) expressed on type I NKT cells and CD40 molecules on DCs leads to functional maturation and IL-12 production in DCs (16–18). This in turn induces the secretion of proinflammatory cytokines, including IFN-γ, by type I NKT cells. The secretion of IL-4, which is used as readout for an anti-inflammatory cytokine profile of NKT cells, is independent of the costimulatory axis between NKT cells and DC (18). Hence, type I NKT cells contribute to host defense against viral and bacterial pathogens.

Lipid Ags derived from certain bacteria (e.g., Sphingomonas and Borrelia burgdorferi) (19–21) have been described. However, other pathogens (e.g., viruses) do not even contain lipids or conceivably do not contain CD1d-presentable lipids and thus might not be recognized by NKT cells. Nature has evolved different receptors, including the group of TLRs, to detect conserved pathogen-associated molecular patterns (PAMPs). Upon ligation of TLR4 or TLR9, which recognize LPS and unmethylated CpG DNA sequences, respectively, endogenous glycolipids are generated in DCs and loaded onto CD1d molecules, which then trigger the secretion of IFN-γ by type I NKT cells (22). This process requires the expression of type I IFNs, IFN-α, or IFN-β by activated DCs. Under normal conditions, the glycosphingolipid (GSL) isoglobotrihexosylceramide (iGb₃) is constantly degraded in lysosomes. TLR ligation inhibits activity of the rate-limiting enzyme in iGb₃ turnover, α-galactosidase A (α-GalA) and permits the intracellular accumulation and CD1d binding of iGb₃. Thus, TLR9-stimulated DC trigger IFN-γ production in type I NKT cells (23).

In this work, we show a pronounced infiltration of type I NK T cells in aggressive, but not chronic, periodontitis lesions in vivo and DC-mediated activation of these cells in vitro by aggressive periodontitis-associated A. actinomycetemcomitans, but not by P. gingivalis. Furthermore, we demonstrate that in contrast to A. actinomycetemcomitans infection, P. gingivalis challenge does not result in a type I IFN response or presentation of endogenous glycolipids, thereby preventing the activation of type I NKT cells by bacterial-challenged DCs. Addition of exogenous IFNs to DCs challenged with P. gingivalis rescued the production of proinflammatory cytokines by type I NKT cells and thus may circumvent the apparent immune evasion by P. gingivalis.

All animal experiments were covered by approvals from the Government of the State of Nordrhine-Westphalia (permit number 8.87-51.04.20.10.042). Design and procedures of the study involving human biopsies were approved by the Columbia University Medical Center Institutional Review Board (AAAB0869).

C57BL/6J wild-type (WT) mice were purchased from The Jackson Laboratory (Bar Harbor, ME), MyD88-deficient mice were a kind gift of Heike Weighardt (University of Düsseldorf, Düsseldorf, Germany). Mice were housed under specific pathogen-free conditions and used for experiments at age 6–8 wk. Mice lacking the type I IFN receptor (IFNAR) were a kind gift of Beatrix Schumak (University of Bonn, Bonn, Germany). Animal experiments were performed in accordance to protocols approved by the Institutional Animal Care and Use Committee.

A. actinomycetemcomitans Y4 (8) was cultured on blood-agar plates at 37°C/5% CO₂. P. gingivalis strain 381 (24) was cultured under anaerobic conditions on blood agar plates. Bacterial suspensions in DMEM medium without antibiotics were adjusted to 10⁹ CFU/ml using a spectrophotometer at 600 nm and established growth curves.

DCs were generated from mouse bone marrow (BM) in the presence of GM-CSF, as described (25). In brief, BM cells were cultured in complete DMEM medium supplemented with 20 ng/ml rGM-CSF (Immunotools, Friesoythe, Germany), replaced every other day. On day 6, BM-derived DCs (BMDC) were detached with trypsin-EDTA (PAA Laboratories, Pasching, Austria) and washed in DMEM/5% FCS without antibiotics. Where indicated, DCs from BMDC cultures further enriched by MACS using pan-DC microbeads (Miltenyi Biotec, Bergisch-Gladbach, Germany) to a purity >90%. BMDC were pulsed with α-galactosylceramide (200 ng/ml; Kirin, Gunma, Japan) or vehicle (Tween-20) in medium for 3 h at 37°C. For infections, BMDC were cultured in DMEM/5% FCS without antibiotics in the presence of A. actinomycetemcomitans or P. gingivalis at indicated multiplicity of infection (MOI). After 24 h of culture, DCs were washed three times in complete medium before responder NKT cells were added.

Single-cell suspensions from spleens were prepared by standard techniques. Liver mononuclear cells (MNC) were isolated as previously described (26). Briefly, livers were perfused with PBS, minced, and type I NKT cells were enriched by centrifugation in a two-step Percoll gradient. Enriched populations typically contained 20–30% iNKT cells.

Where indicated, CD1d-restricted NKT cells were further enriched by MACS. Hepatic MNC suspensions were stained with PBS57-loaded, PE-conjugated CD1d-tetramers from the National Institutes of Health tetramer facility at Emory University Vaccine Center (Atlanta, GA), washed, and stained with anti-PE microbeads before selection on miniMACS columns (Miltenyi Biotec). Enriched preparations contained >70% CD1d-restricted NKT cells and <3% conventional T cells as determined by flow cytometry (Supplemental Fig. 1A). Type NKT cells were stimulated in the presence of either bacterial infected or α-galactosylceramide–pulsed BMDC at a DC/NKT ratio of 1:10. After 24 and 48 h, cell-culture supernatants were harvested and used for cytokine analysis. Cytokine-specific ELISA assays (IL-4, IL-17/A, IFN-γ, IL-12 [eBioscience, San Diego, CA]; mouse IFN-β ELISA [Hoelzel Diagnostika, Cologne, Germany]) of cell-culture supernatants were performed following the manufacturers' instructions.

α-GalA enzyme activity in BMDC was determined as described previously (27). In brief, following treatment, DCs were lysed in lysosomal assay buffer. Protein concentrations were measured using the Lowry method. A total of 10 μg lysates were incubated in 96-well black flat-bottom plates in a total volume of 100 μl supplemented with fluorogenic α-GalA substrate 3-methylumbelliferyl-α-d-galactospyranoside (5 mM) and N-acetylgalactosamine (10 mM) for 2 h at 37°C. Decreases in fluorescence intensities were assessed at 360 and 455 nm for excitation and emission (Infinite M200 fluorometer; Tecan, Crailsheim, Germany). Enzyme activities were normalized to values of untreated BMDC.

A total of 15 subjects with moderate to severe generalized chronic periodontitis and 10 subjects with generalized aggressive periodontitis were recruited among the patients referred for periodontal therapy to the Clinic for Postdoctoral Periodontics, Columbia University College of Dental Medicine. Eligible patients: 1) were at least 25 y old; 2) had a minimum of 22 teeth present; 3) had no past history of systematic periodontal therapy other than occasional prophylaxis provided by the referring general dentist; 4) had received no systemic antibiotics or anti-inflammatory drugs for at least 6 mo; 5) harbored a minimum of four teeth with radiographic bone loss; 6) did not suffer from diabetes mellitus; 7) did not suffer from any of the systemic conditions or genetic disorders that entail a diagnosis of “periodontitis as a manifestation of systemic diseases”; 8) were not pregnant; and 9) were not current users of tobacco products or nicotine replacement medication. Signed informed consent was obtained prior to enrollment. Design and procedures of the study were approved by the Columbia University Medical Center Institutional Review Board (AAAB0869).

The diagnosis of generalized chronic or aggressive periodontitis was independently performed by two periodontists.

Identification of donor sites and harvesting of gingival tissue samples was performed as earlier described (13). In brief, a diseased interproximal papilla showed bleeding on probing, probing pocket depth ≥4 mm, and clinical attachment level ≥3 mm, whereas a healthy papilla demonstrated no bleeding on probing, probing pocket depth ≤4 mm, and clinical attachment level ≤2 mm. All tissue specimens were collected during periodontal surgery. In a paired design, each study subject contributed with one diseased tissue sample and one healthy tissue sample.

Total RNA was extracted from the gingival tissue biopsies, as described previously (28). In brief, the biopsies were homogenized in TRIzol reagent (Invitrogen, Carlsbad, CA) and further subjected to a spin-column cleanup (RNeasy Mini Kit; Qiagen, Hilden, Germany). RNA quantity and quality was assessed by Nanodrop spectrophotometer and Agilent Bioanalyzer (Agilent Technologies). Total RNA was reverse-transcribed using Superscript III (Invitrogen).

Aliquots of cDNA reactions were amplified using Sensimix SYBR No-Rox master mix (Bioline, Eberswalde, Germany; Eppendorf RealPlex S machine [Eppendorf, Hamburg, Germany]) using primers specific for the Vα24Jα18 junction (sense, 5′-GAT ATA CAG CAA CTC TGG ATG-3′; antisense, 5′-GAG TTC CTC TTC CAA AGT ATA GCC-3′) previously described (14).

cDNA prepared from a human iNKT cell line was amplified using Vα24Jα18 primers using Phusion polymerase (Fermentas, St. Leon-Roth, Germany) and cloned into pGemTeasy vector (Promega, Gainsville, FL). Dilutions of the sequence-verified cloned plasmid were included in quantitative PCR (qPCR) runs as standard curve. Copy numbers were calculated of sample values normalized against the housekeeping gene β-actin (sense, 5′-ACA GAG CCT CGC CTT TGC CG-3′; antisense, 5′-TGG GCC TCG TCG CCC ACA TA-3′).

Whole-genome gene expression profiles from all biopsies were analyzed using Affymetrix HG-U133plus 2.0 microarrays (Affymetrix). In brief, 10 μg total RNA was reverse transcribed and biotin labeled, fragmented, and hybridized to arrays following the manufacturer's instructions. Data were analyzed using R/Bioconductor and limma. Data were background-corrected, RMA normalized, log-transformed, and assessed for differentially expressed probes using paired statistics. Probes differentially regulated in aggressive and chronic periodontitis (affected versus clinically healthy biopsies) were identified using a Venn diagram. Experimental details and results following the Minimum Information About a Microarray Experiment standards are available at the Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE16134) under accession number GSE16134.

mRNA expression was analyzed by real-time PCR using primers specific for IFN-α (sense, 5′-CTT CCA CAG GAT CAC TGT GTA CCT-3′; antisense, 5′-TTC TGC TCT GAC CAC CTC CC-3′), IFN-β (sense, 5′-CTG GAG CAG CTG AAT GGA AAG-3′; antisense, 5′-CTT CTC CGT CAT CTC CAT AGG G-3′), glyceronephosphate O-acyltransferase (sense, 5′-CCA TGG ACG TTC CTA GCT CC-3′; antisense, 5′-ACT TGA TGT CCC CTG GCT TG-3′), ceramide glucosyltransferase (sense, 5′-CCG TAT AGC AAG CTC CCT GG-3′; antisense, 5′-AAG CCT TGT CTG TCG GCT AC-3′) and normalized against GAPDH (sense, 5′-AAC TTT GGC ATT GTG GAA GG-3′; antisense, 5′-ACA CAT TGG GGG TAG GAA CA-3′) as housekeeping gene.

A total of 100 ng TRIzol (Invitrogen) isolated and spin-column–cleaned total RNA was subjected to a single round of in vitro transcription and biotin labeling (Total Prep RNA amplification kit; Ambion, Austin, TX). cRNA was hybridized on Mouse WG-6 v2 Expression BeadChips (Illumina, San Diego, CA) according to standard protocols.

Expression data were exported as unnormalized sample and control probe profiles from the Illumina BeadStudio software and analyzed using R/Bioconductor and limma. Data were quality weighed (16), background-corrected, quantile normalized, log-transformed (17), and explored for differentially expressed genes with a false discovery rate (FDR) <0.05 (18) using Bayesian statistics. Transcriptomic datasets were assessed for similarity using hierarchical clustering. A heat map illustrating the differential regulation of probes in A. actinomycetemcomitans– and P. gingivalis–infected DCs versus untreated controls was constructed using the CIMminer program at http://discover.nci.nih.gov/, a development of the Genomics and Bioinformatics Group, Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute (19). Probes differentially regulated by A. actinomycetemcomitans and P. gingivalis infection in DCs were identified using a Venn diagram. Differential regulation of signaling pathways was performed using the signaling pathway impact analysis algorithm (20). IFN-stimulated transcripts in the different experimental groups were identified using the Interferome database (29). Experimental details and results following the Minimum Information About a Microarray Experiment standards are available at the Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE41383) under accession number GSE41383.

Statistical analyses not involving microarrays were performed using GraphPad Prism V (GraphPad Software, San Diego, CA). Parametric testing for two groups was performed using unpaired, two-tailed t tests; three or more groups were analyzed using one-way ANOVA with post hoc Newman–Keuls tests. Nonparametric testing was performed using Wilcoxon signed-rank test (two groups) and Kruskal–Wallis tests (three or more groups). The p values < 0.05 were considered significant.

Given the proposed role of type I NKT cells in bacterial infections, we asked whether type I NKT cells infiltrate periodontal lesions. We extracted RNA from paired diseased and clinically healthy gingival tissue specimens (average periodontal probing depth healthy sites 2.25 ± 0.058 mm, diseased sites 7.67 ± 1.78 mm, average probing depth difference 5.44 ± 1.81 mm) obtained from patients diagnosed with aggressive and chronic periodontitis, respectively. cDNAs were analyzed for the presence of type I NKT cells. To this end, from paired biopsies, we amplified transcripts of the Vα24-Jα18 rearranged TCR, which is selectively carried on type I NKT cells. Enumeration of type I NKT cells according to TCR mRNA transcripts has been described previously (30, 31).

Numbers of type I NKT cells present in unaffected (healthy) periodontal tissue were not significantly different between both patient groups (p = 0.11).

Eight of 10 patients with aggressive periodontitis showed increasing numbers of type I NKT cells in diseased compared with healthy biopsies. Overall, in patients with aggressive periodontitis, type I NKT cells infiltrated the tissue lesions at significantly higher levels (p < 0.01; mean fold influx of Vα24-Jα18⁺ cells: 41.59) (Fig. 1A). In contrast, in only 5 of 15 patients with chronic periodontitis, we found type I NKT increases >5-fold; thus, the overall type I NKT cell infiltration in this group did not reach statistical significance (Fig. 1B, p < 0.09; mean fold influx of Vα24-Jα18⁺ cells: 3.73).

To establish the determinants and consequences of increased NKT cell influx in aggressive periodontitis lesions, we then assessed the whole-genome transcriptomic profiles of the used biopsies for transcripts related to NKT cell biology. We found increased expression of several chemokines in aggressive periodontitis lesions, as compared with the clinically healthy pairs, that may explain the increased influx of type I NKT cells in these lesions (Supplemental Table I). Specifically, we observed a significant induction of the chemokine CCL8 and its receptor CCR5 (Supplemental Table II) that is well documented to be strongly expressed on type I NKT cell surfaces (32). When assessing the transcriptomic data for evidence of NKT cell activation, we found the proinflammatory product of activated type I NKT cells, IFN-γ, to be significantly induced in aggressive, but not chronic, lesions. Furthermore, IL-17A and -B mRNA was significantly induced in aggressive, but not chronic periodontitis lesions. Because type I NKT cells require for activation mature DCs presenting CD1d molecules, we subsequently assessed the expression of the maturation marker CD86 and CD1d and found both to be significantly induced in aggressive, but not chronic, periodontitis (Supplemental Table I). Based on these data, we then proceeded to further characterize the activation of type I NKT cells by periodontal pathogens in vitro.

We assessed whether A. actinomycetemcomitans, causally related to aggressive periodontitis, and P. gingivalis, a major pathogen in chronic periodontitis, were able to stimulate DCs to activate type I NKT cells in vitro. DCs differentiated from BM of healthy WT mice were infected with either A. actinomycetemcomitans or P. gingivalis at different MOIs for 24 h. Cells were washed and used as stimulator cells for type I NKT cells enriched from livers of WT mice. DCs infected with A. actinomycetemcomitans stimulated the secretion of proinflammatory cytokine IFN-γ (48 h) in an MOI-dependent manner. Similar results were seen in response to other A. actinomycetemcomitans strains (data not shown). In contrast, levels of the anti-inflammatory cytokine IL-4 in culture supernatants did not increase above background levels (Fig. 2A, black bars). Contrary to this, DCs infected with P. gingivalis neither stimulated IL-4 nor IFN-γ release in type I NKT cells (Fig. 2A, right panels). Furthermore, infection of purified DCs with A. actinomycetemcomitans but not P. gingivalis stimulated significantly elevated levels of IL-17A protein in purified type I NKT cells (Supplemental Fig. 1). To test whether activation of type I NKT cells by bacteria-infected DCs is mediated by CD1d, groups of DCs were incubated with CD1d blocking Abs. Blockade of CD1d molecules at MOI 50 and 100 significantly abrogated the production of IFN-γ by NKT cells (Fig. 2A, shaded bars). This indicates that the activation of type I NKT cells by A. actinomycetemcomitans–infected DCs requires expression of CD1d molecules.

Furthermore, we explored whether A. actinomycetemcomitans and P. gingivalis differ in the ability to induce functional maturation of DCs. To this end, BMDCs were infected with A. actinomycetemcomitans or P. gingivalis at an MOI of 10 or cultured in the presence of LPS used as positive control. As shown in Fig. 2C, LPS upregulates the expression of the maturation marker CD86 compared with unstimulated controls (34.2 versus 23.5%). Levels of CD86 expression as well as the frequency of CD86-expressing DCs after A. actinomycetemcomitans infection (35.9%) were comparable to values of LPS control. In contrast, and corroborating the lack of type I NKT cell activation, infection with P. gingivalis did not upregulate CD86 on BMDCs (Fig.2B). These data demonstrate a clear lack of activation of DCs and type I NKT cells in response to challenge with P. gingivalis.

To gain further insight into the mechanisms of type I NKT cell activation in response to periodontal bacteria, we asked whether TLR signaling in DCs is required to promote type I NKT cell activation. BMDCs generated from WT mice and mice homo- and heterozygous for the adaptor molecule MyD88, involved in signaling by most TLRs, were infected with A. actinomycetemcomitans and subsequently used as stimulators for freshly isolated type I NKT cells. WT DCs infected with A. actinomycetemcomitans stimulated a strong IFN-γ secretion in type I NKT cells. In contrast, IFN-γ levels to DCs hemizygous for MyD88 were reduced to ∼50% of those induced by WT DC. DCs lacking both MyD88 alleles were unable to activate type I NKT cells in response A. actinomycetemcomitans challenge (Fig. 3A).

Furthermore, we stained type I NKT cells (gated as CD3⁺ α-GC–loaded CD1d-tetramer⁺ NK1.1⁺) for the expression of CD25 protein. CD25 is expressed by type I NKT cells and further upregulated upon activation. Type I NKT cells stimulated with A. actinomycetemcomitans–infected WT DCs MOI-dependently upregulated the expression of CD25 (Fig. 3B, triangles). In contrast, no upregulation of CD25 was detectable on type I NKT cells cocultured with infected MyD88-deficient DCs (Fig. 3B, squares). Similar results were observed using DCs generated from MyD88 hemizygous mice (Fig. 3B, circles)

These data indicate that TLR signaling is required for the activation of type I NKT cells by DCs challenged with A. actinomycetemcomitans.

The observation of the apparent inability of P. gingivalis to activate type I NKT cells via DCs (in contrast to A. actinomycetemcomitans) prompted us to evaluate whether this effect was reflected in genome-wide transcriptomic profiles of dendritic cells infected with P. gingivalis or A. actinomycetemcomitans, as compared with uninfected controls.

Indeed, the genes differentially regulated by P. gingivalis infection were markedly different from those regulated in A. actinomycetemcomitans infections (Fig. 4A, Supplemental Table I). Unsupervised hierarchical clustering revealed that expression profiles of P. gingivalis–infected DCs were in fact more similar to uninfected controls than A. actinomycetemcomitans–infected DCs that cluster completely differently (Fig. 4B). A Venn diagram of genes differentially regulated in P. gingivalis and A. actinomycetemcomitans–infected cells showed that both pathogens share a certain proportion of commonly regulated genes. Still, P. gingivalis challenge leads to an apparently dampened inflammatory reaction, as indicated by the far less pronounced induction of ILs and IFNs (Table I). However, and in line with the aforementioned analyses, A. actinomycetemcomitans infection triggered exclusive differential regulation of 3830 genes, whereas P. gingivalis challenge only leads to the exclusive regulation of less than one tenth, 336 genes (Fig. 4C, Supplemental Table I).

Interferome database analysis of the differentially regulated genes demonstrated that A. actinomycetemcomitans infection lead to the exclusive differential regulation (FDR <0.05) of 450 transcripts that are known to be triggered by IFN stimulation, whereas P. gingivalis challenge merely elicited differential regulation of 34 IFN-regulated genes (Fig. 4D). Importantly, most of the genes triggered by A. actinomycetemcomitans are known to be regulated by type I IFNs (Fig. 4E). When evaluating the impact of P. gingivalis and A. actinomycetemcomitans infection on signaling pathways in DCs, we found that A. actinomycetemcomitans lead to a highly significant activation of the Ag-processing and presentation pathway, whereas this pathway was not significantly regulated by P. gingivalis (Supplemental Table I).

These data indicate that challenge with P. gingivalis, in contrast to A. actinomycetemcomitans, does not trigger a type I IFN response, leading to an impaired IL induction in BMDCs. Therefore, the Ag-processing machinery necessary for activation of NKT cells is seemingly not activated in response to P. gingivalis infection.

Based on our microarray data showing a lack of a type I IFN response in P. gingivalis–infected versus A. actinomycetemcomitans–infected DCs, we subsequently sought to further characterize the production of type I IFNs. First, we stimulated DCs with A. actinomycetemcomitans or P. gingivalis and analyzed IFN-α and IFN-β mRNA expression. IFN-α mRNA expression levels did not change significantly between the tested groups (Fig. 5A). In contrast, infection with A. actinomycetemcomitans induced a significant induction of IFN-β mRNA expression in DCs (Fig. 5B). Consistent with this, we observed a significant induction of IFN-β protein secretion by DCs infected with A. actinomycetemcomitans (Fig. 5C). However, no significant increase of IFN-β protein nor its mRNA level was detectable after P. gingivalis infection (Fig. 5B, 5C).

In response to TLR ligation, the endosomal GSL turnover in DCs is disrupted, leading to their accumulation and presentation in CD1d molecules (23). We explored whether A. actinomycetemcomitans and P. gingivalis were able to downregulate the rate-limiting enzyme of GSL turnover, α-GalA, in infected DCs. DCs were infected with A. actinomycetemcomitans or P. gingivalis or were incubated with rIFN-α or IFN-β. First, we evaluated the enzymatic activity of α-GalA in DCs after 6 h of incubation. Compared to α-GalA activity in uninfected BMDC, A. actinomycetemcomitans infection lead to a 50% decrease in α-GalA activity. In contrast, P. gingivalis–infected DCs showed slightly elevated activity of this enzyme compared with untreated DCs (Fig. 5D). Notably, the type I IFNs IFN-α and IFN-β as well as LPS were also able to reduce the activity of α-GalA in DCs (Fig. 5D).

We next investigated whether type I IFNs, which are upregulated upon bacterial infection, directly inhibited α-GalA as analyzed by its mRNA expression in DCs after 24 h of infection. Corroborating the marked differences in α-GalA enzymatic activity observed between A. actinomycetemcomitans and P. gingivalis infection, A. actinomycetemcomitans challenge led to a downregulation of α-GalA mRNA to ∼40%. In contrast, P. gingivalis infection did not induce a clear change in α-GalA mRNA levels, nor did IFN-α or IFN-β significantly change α-GalA mRNA levels (Fig. 5E).

We extended this analysis on genes, namely glyceronephosphate O-acyltransferase and ceramide glucosyltransferase, which had previously been reported in the context of lipid loading on CD1d (33, 34). Neither infection with A. actinomycetemcomitans or P. gingivalis nor stimulation with LPS regulated the expression of these two genes (Fig. 5F, 5G), indicating the effect of A. actinomycetemcomitans infection to be specific for α-GalA expression and activity.

These data indicate that A. actinomycetemcomitans infection leads to an induction of IFN-β in DCs. Corroborating our finding that the activation of type I NKT cells by A. actinomycetemcomitans is TLR dependent, we observed a disruption of GSL turnover in A. actinomycetemcomitans–infected DCs. Infection with P. gingivalis, however, did not induce type I IFNs in DCs. The TLR-dependent GSL turnover disruption could not be observed in P. gingivalis–infected DCs.

Whether type I IFNs are acting on DCs in an autocrine fashion or are necessary for the activation of type I NKT cells is unknown. To address this question, we performed coculture experiments in which DCs were infected in the presence or absence of a blocking Ab against the IFNAR before type I NKT cells were added as responder cells. Parallel to this, blocking Abs were added during the stimulation of responder type I NKT cells. DCs infected with A. actinomycetemcomitans for 24 h in the absence of Abs (Fig. 6A, bar 2) or in presence of isotype controls (Fig. 6A, bar 3) triggered significant amounts of IFN-γ released from responding type I NKT cells. Addition of IFNAR-blocking Abs with A. actinomycetemcomitans abrogated the ability of DCs to stimulate type I NKT cells (Fig. 6A, bar 4). However, when IFNAR-neutralizing Abs were added simultaneously to type I NKT cells, the IFN-γ release remained unaffected (Fig. 6A, bar 5). Corroborating these results, DCs generated from BM of mice lacking the type I IFN receptor and infected with A. actinomycetemcomitans failed to stimulate type I NKT cells to secrete IFN-γ (Fig. 6B). These data suggest that type I IFNs (IFN-α, IFN-β) secreted from bacterial-infected DCs, rather than acting on type I NKT cells, are necessary to achieve DC stimulation of type I NKT cells.

To confirm that the absence of type I IFN production in P. gingivalis–infected DCs is in fact responsible for the observed inability of P. gingivalis–infected DCs to activate type I NKT cells, we performed rescue experiments.

First, we tested whether exogenous type I IFNs added during the infection of DCs would rescue the ability of P. gingivalis–infected DCs to stimulate type I NKT cells. BMDCs were infected with either A. actinomycetemcomitans or P. gingivalis at an MOI of 10 in the presence or absence of rIFN-α in the culture medium. After 24 h, DCs were washed, and type I NKT cells were added as responder cells. Confirming data shown in Figs. 2 and 4, A. actinomycetemcomitans–infected DCs triggered the production of IFN-γ in type I NKT cells, whereas DCs infected with P. gingivalis failed to do so (Fig. 7A, bars 1 and 2). Importantly, addition of exogenous type I IFNs (IFN-α, IFN-β) during the infection with P. gingivalis was sufficient to promote the downstream activation of type I NKT cells (Fig. 7, bars 3 and 4, 7 and 8). Addition of exogenous type I IFNs simultaneously to type I NKT cells comparably rescued the IFN-γ production by type I NKT cells.

To test whether the inability of P. gingivalis–infected DCs is due to a lack of IL-12 secretion, cell-culture supernatants were tested for their IL-12 content. As expected from maturation data shown in Fig. 2C, infection with A. actinomycetemcomitans results in the release of IL-12 by DCs (Fig. 7B). Addition of exogenous IFN-α to DC cultures resulted in reduced release of IL-12, probably due to DC exhaustion. In contrast, infection with P. gingivalis did not result in IL-12 release (Fig. 7B, shaded bars). The addition of IFN-α to P. gingivalis/DC cultures significantly increased the IL-12 secretion of DC to levels comparable to those elicited by A. actinomycetemcomitans infections.

The addition of exogenous IFNs was sufficient to restore the immunogenic cytokine secretion of type NKT cells in response to challenge with P. gingivalis.

In this study, we describe clear differences in type I NKT cell infiltration in chronic and aggressive periodontitis lesions (Fig. 1). Type I NKT cells specifically infiltrated lesions of the aggressive subtype. Albeit numbers of paired periodontal biopsies available to this study were limited due to the restrictions imposed on the resection of clinically healthy gingiva, significant differences could be observed in the disease–health comparison in these cohorts. We furthermore observed a trend (p = 0.11) toward lower type I NKT cell numbers in clinically healthy aggressive versus chronic lesions. If confirmed, this finding could demonstrate an inherent difference in innate immune cell repertoire even in unchallenged gingiva from both disease entities. We analyzed the frequency of type I NKT cells, measuring the presence of Vα24-Jα18 TCR transcripts selectively expressed by type I NKT cells. To date, no working Ab specifically staining type I NKT cells in tissue sections is available, which conceivably might provide additional insight into the localization of NKT in periodontal lesions. Furthermore, we also found significant differences in transcripts linked to DC-mediated NKT cell activation. Specifically, chemokines that bind chemokine receptors expressed by type I NKT cells were exclusively significantly regulated in aggressive periodontitis lesions in vivo, possibly explaining the increased influx of type I NKT cells in this disease subtype. Furthermore, markers of DC-mediated activation, such as CD86 and CD1d, were induced in aggressive periodontitis lesions only.

Therefore, we investigated the DC-mediated type I NKT cell responses to the periodontal pathogens A. actinomycetemcomitans and P. gingivalis in vitro. DCs infected with A. actinomycetemcomitans at different MOIs dose-dependently stimulated a vigorous secretion of the proinflammatory cytokine IFN-γ in type I NKT cells (Fig. 2A). Strikingly, DCs infected with P. gingivalis did not elicit any detectable cytokine secretion in type I NKT cells (Fig. 2). We did not detect elevated concentrations of anti-inflammatory IL-4 in cocultures of infected DCs and type I NKT cells, corroborating earlier data showing that upon TLR ligation, DCs selectively stimulate the production of IFN-γ in type I NKT cells (16, 22, 23, 35, 36).

The cytokine secretion by type I NKT cells depended on the expression of CD1d molecules on the surface of infected DCs. This indicates that the impact of DCs that were inherently, albeit in small numbers, present in our type I NKT cell preparations was negligible in these experiments. In line with the aforementioned in vivo and in vitro results, we found that infection of DCs with A. actinomycetemcomitans, but not P. gingivalis, upregulated the expression of CD86 costimulatory molecules on DC surfaces. Whereas A. actinomycetemcomitans stimulated strong IL-12 secretion by DCs, challenge with P. gingivalis only led to minimal IL-12 protein production (Fig. 7). Whole-genome expression analyses of DCs infected with A. actinomycetemcomitans and P. gingivalis indicate that P. gingivalis infection, in contrast to A. actinomycetemcomitans, resulted in a lack of type I IFN response.

The observed mild induction of IL-12 mRNA in P. gingivalis–infected DCs did apparently not result in protein translation. Thus, our data are not in contrast to results by Zhou and coworkers (37), who demonstrated human macrophage activation by live P. gingivalis and isolated fimbriae, as assessed by TNF-α secretion and upregulation of IL-12 and NF-κB mRNA. Importantly, and in line with our data, neither live P. gingivalis nor isolated fimbriae stimulated IFN-γ secretion by macrophages. Other studies only analyzed the effects of cell-wall extracts on human monocyte-derived DCs (38–40). To our knowledge, our study is first to show a lack of DC maturation in response to infection with live P. gingivalis.

Recognition of conserved PAMPs by TLRs is a key element of innate immunity. Several lines of evidence exist for activation of NKT cells by glycolipids presented in DCs recognizing PAMPs via their TLRs (16, 22, 23, 35, 36, 41). Ligation of TLR9 on DCs was demonstrated to result in production of IFN-γ by NKT cells in vitro (22). Presentation of de novo synthesized charged glycolipids on CD1d molecules was required for this process (22). TLR activation and downstream NKT cell activation within MNC preparations containing both cell types required secretion of IFN-β by DCs and functional expression of the type I IFN receptor IFNAR on type I NKT cells (22). However, the question whether infected DCs require IFNAR expression has not been analyzed. In addition, for a number of bacterial species, lipid Ags have been described that are able to bind CD1d molecules and stimulate type I NKT cells, including Salmonella and Borrelia (19–21). Our result strongly suggest that disruption of the glycolipid turnover by α-GalA is critical to the ability of A. actinomycetemcomitans–infected DCs to stimulate type I NKT cells. In view of this requirement, it appears unlikely that A. actinomycetemcomitans contains lipid-stimulating type I NKT cells.

We analyzed the requirements of TLR and type I IFN signaling in response to infection with periodontal pathogens. Our data indicate that the stimulatory capacity of A. actinomycetemcomitans–infected DCs depends on MyD88 expression, which is the central signaling molecule downstream of most TLR family members, including TLR2, -4, and -9 (42). MyD88 gene–deficient DCs exhibited a gene dose-dependent lack of stimulatory capacity for type I NKT cells (Fig. 3). Corroborating our data, Iweala and coworkers (43) recently found that Salmonella vaccination in MyD88-deficient mice skewed the effector functions of type I NKT cells.

P. gingivalis expresses several cell-wall components mediating its virulence in vivo (44). In particular, P. gingivalis fimbriae and LPS possess immune evasive properties. Hajishengallis and coworkers (44) showed that P. gingivalis fimbriae bind to the chemokine receptor CXCR4 expressed on mature DCs, leading to elevated intracellular cAMP levels that antagonize the TLR-mediated NF-κB activation in DCs. This constitutes one of conceivably several nonexclusive mechanisms that enable P. gingivalis to evade the immune system. Indeed, we show a notably reduced inflammatory response in P. gingivalis versus A. actinomycetemcomitans–infected DCs that could be explained by active immune evasion by P. gingivalis.

We investigated whether the lack of NKT cell stimulation by P. gingivalis–infected DCs is due to a lack of type I IFN expression in infected DCs. Infection of murine DCs with A. actinomycetemcomitans induced type I IFNs (Fig. 5C) and several transcripts involved in type I IFN signaling and DC maturation (Table I, Fig. 4D). In stark contrast, P. gingivalis was unable to upregulate the expression of type I IFNs in DCs (Fig. 5). The activation of type I NKT cells by A. actinomycetemcomitans infection required the expression of the IFNAR and functional TLR signaling in DCs. Interestingly, A. actinomycetemcomitans infection led to the strong upregulation of IFN-β production, whereas transcription levels of IFN-α remained unchanged. The molecular basis of this observation is still elusive. However, A. actinomycetemcomitans is known to secrete the immune-suppressive factor and thereby inhibit protein synthesis in leukocytes, as shown for inhibition of IgG and IgM production in B cells as well as IL-2 translation in T cells (45). Along this line, Gobl et al. (46) using human monocytes showed that IFN-α but not -β secretion requires de novo protein synthesis. To our knowledge, there are no data available as to whether this is also holds true for murine DCs. However, because murine DCs retain the ability to upregulate other proteins (i.e., IL-12, CD86), this remains under further investigation.

Recently, it was demonstrated that combinations of IFN-β and presentation of self-glycolipids from TLR9-stimulated DCs were required for the downstream activation of type I NKT cells (22). It was, however, still unclear whether type I IFNs exert additional autocrine effects on DCs. To address this question, we blocked the IFNAR during the infection of DCs and while coincubating DCs and NKT cells. Using this experimental approach, we clearly demonstrate that expression of the IFNAR is required on the surface of infected DC and indispensable for responding type I NKT cells. This in turn suggests, the stimulatory capacity of A. actinomycetemcomitans–infected DCs to type I NKT cell is attributed to the ability of A. actinomycetemcomitans to signal via TLRs and elicit the production of both IL-12 and type I IFNs by DCs. Darmoise et al. (23) previously showed that the activation of type I NKT cells by α-GalA–deficient DCs despite their enhanced basal stimulatory capacity still depends on their IL-12 production. Whether autocrine IFN-β secretion and IFNAR signaling besides downmodulating of endosomal glycolipid turnover also acts on the production of IL-12 remains further investigation.

Taken together, we show that infiltration by type I NKT cells is a feature of aggressive, but not chronic periodontitis. In line with these in vivo observations, we found that the aggressive periodontitis-associated pathogen A. actinomycetemcomitans–triggered type I NKT cell activation and proinflammatory IFN-γ secretion via DCs in vitro. In contrast, P. gingivalis, a principal pathogen in chronic periodontitis, results in a dampened immune response in DCs characterized by a lack of type I IFN production and a subsequent lack of type I NKT cell activation. Strikingly, the ability of P. gingivalis to accumulate endogenous GSLs in DCs and leading to the activation of type I NKT cells was restored by addition of exogenous type I IFNs. This indicates that immune evasion actively mediated by P. gingivalis may be responsible for the less pronounced activation of the inflammatory repertoire in chronic than in aggressive periodontitis. The activation of type I NKT cells with subsequent production of abundant IFN-γ triggered by A. actinomycetemcomitans possibly aggravates tissue destruction in aggressive periodontitis (47).

We thank Ryan T. Demmer (Columbia University, New York, NY) for logistical help with patient samples, Heike Weighardt (University of Düsseldorf, Düsseldorf, Germany) for providing MyD88 knockout mice, and Beatrix Schumak (University of Bonn) for IFNAR knockout mice. We also thank Bettina Jux for critically reading the manuscript.

The authors have no financial conflicts of interest.