Microbial carriage state of peripheral blood dendritic cells (DCs) in chronic periodontitis influences DC differentiation, atherogenic potential

Abstract

The low-grade oral infection chronic periodontitis (CP) has been implicated in coronary artery disease risk, but the mechanisms are unclear. In this study, a pathophysiological role for blood dendritic cells (DCs) in systemic dissemination of oral mucosal pathogens to atherosclerotic plaques was investigated in humans. The frequency and microbiome of CD19(-)BDCA-1(+)DC-SIGN(+) blood myeloid DCs (mDCs) were analyzed in CP subjects with or without existing acute coronary syndrome and in healthy controls. FACS analysis revealed a significant increase in blood mDCs in the following order: healthy controls < CP < acute coronary syndrome/CP. Analysis of the blood mDC microbiome by 16S rDNA sequencing showed Porphyromonas gingivalis and other species, including (cultivable) Burkholderia cepacia. The mDC carriage rate with P. gingivalis correlated with oral carriage rate and with serologic exposure to P. gingivalis in CP subjects. Intervention (local debridement) to elicit a bacteremia increased the mDC carriage rate and frequency in vivo. In vitro studies established that P. gingivalis enhanced by 28% the differentiation of monocytes into immature mDCs; moreover, mDCs secreted high levels of matrix metalloproteinase-9 and upregulated C1q, heat shock protein 60, heat shock protein 70, CCR2, and CXCL16 transcripts in response to P. gingivalis in a fimbriae-dependent manner. Moreover, the survival of the anaerobe P. gingivalis under aerobic conditions was enhanced when within mDCs. Immunofluorescence analysis of oral mucosa and atherosclerotic plaques demonstrate infiltration with mDCs, colocalized with P. gingivalis. Our results suggest a role for blood mDCs in harboring and disseminating pathogens from oral mucosa to atherosclerosis plaques, which may provide key signals for mDC differentiation and atherogenic conversion.

FIGURE 1

Increased blood mDC frequency in chronic periodontitis and acute coronary syndrome A. Representative scattergrams from flow cytometry analysis of CD1c+ (BDCA-1) (x-axis) DC-SIGN+ (y-axis) blood mDCs at baseline in healthy control (CTL) subjects (n=25), chronic periodontitis (CP) subjects (n=25) and subjects with acute coronary syndrome and CP (ACS+CP) (n= 15). B. Mean numbers (panel 1) and percentages (panel 2) of blood mDCs (mDCs/20,000 PBMCs) and total numbers of PBMCs/ml (panel 3) in CP, ACS/CP relative to CTL. ** p<0.05, Students t-test. C. Serum ELISA levels of FLT-3L, soluble TNFR-1, TNFR-2 (ng/ml) in all three patient groups performed in triplicate for each patient sample. *Significant difference in ACS+CP groups relative to CP and CTL (p<0.05, Students t-test).

FIGURE 2

P. gingivalis carriage state and frequency of blood mDCs, before and after induced bacteremia. A. Image series from scanning laser confocal microscopy (panels 1–7, z-stack series of 1μm slices) of CD1c⁺ (BDCA-1⁺) blood mDCs from oral carriage positive CP patient, permeablized and cytocentrifuged on slides and probed with AEZαMfa1-PE, followed by Vectashield ^™ mounting media. P. gingivalis (red) in mDC is shown by white arrow. B. Epifluorescence deconvolution image analysis of blood mDCs from a healthy donor pulsed in vitro with CFSE-labeled P. gingivalis 381 (green) at an MOI of 1 for 3 hours. MDCs were counterstained with DAPI (blue) and RPE-conjugated DC-SIGN (red). C. Significant increase in P. gingivalis 16s rDNA content of blood mDCs from CP subjects (n=6) 24 hours after local debridement (S&RP). MDCs were analyzed for 16s rDNA of P. gingivalis, quantified as in the supplementary methods. (*p=0.02, paired T-test). D. Representative scattergrams from flow cytometry analysis of CD1c⁺ (BDCA-1⁺), CD209 (DC-SIGN⁺) blood mDCs before (0hr) and 24 hours after local debridement (S&RP) of CP patients as described in materials and methods. E. Significant increase in mean number (panel 1) and percentages (panel 2) of blood mDCs 24 hours after S&RP (per 30,000 PBMCs) (**p<0.05, Paired t-test).

FIGURE 3

P. gingivalis induces mDC differentiation, survives within mDCs and induces atherogenic mDC phenotype A. Pre-DCs monocytes from healthy controls were cultured in triplicate with growth factors GM-CSF/IL-4 +/− P. gingivalis 381 at MOIs of 0.1, 0.5, and 1. The mean number of cells appearing in the MoDC gate (CD1c⁺DC-SIGN⁺) per μl ± S.E. is shown. Phenotype of immature MoDCs was further confirmed by evidence of downregulation of CD14 and low to no expression of CD83 and CCR7 (not shown). *Significant difference between control + GF (plus growth factors [GF]) and MOI of 0.1 + GF (ANOVA, p< 0.05). Data are representative of results of assay repeated three separate times. B. Performed as in A., but without growth factors. Significant differences were noted in mean # MoDCs per μl in MOI 0.1-GF vs. MOI 0.5 - GF at 2, 3 days, and in MOI 0.1 - GF, MOI 0.5 - GF vs control and MOI 1-GF at 2 days (ANOVA, p< 0.05). C. Wild type Pg381 was incubated in mDC buffer alone (filled circle) or with mDCs (filled square), or with mDCs pre-treated with cytochalasin D (filled triangle), or control PMNs (inverted filled triangle) at a multiplicity of infection of 100 for from 0, 6, 24 hours. Internalization of CFSE-labeled P. gingivalis was confirmed by epifluorescence microscopy. Cells were lysed and viable bacteria recovered on enriched anaerobic 5% blood agar plates in triplicate under anaerobic conditions (10% H2, 5% CO2 in nitrogen) at a 1:10 dilution at 35°C for 14 days after which Log₁₀ cfu/ml were determined. Experiment was repeated three separate times and data are representative of consistent results D. MoDCs were pulsed with wild type Pg 381, its mfa-1 minor fimbriae deficient strain (MFI) or fimA major fimbriae deficient strain (DPG-3) or no Pg (DC ctl) for 18 h at a MOI of 1:25. Secretion of MMP-9 in pg/ml were assessed by ELISA. The data are the mean ± S.D. of triplicate assays. E. MoDCs were pulsed in triplicate with wild type Pg381, its fimbriae-less mutant MFB or no Pg (CTL) at a 25:1 MOI for 3 hrs and uptake of CFSE-labeled Pg monitored by FACS analysis (not shown). qRT- PCR (BioRad) was used to determine expression levels, normalized to β-actin and expressed as fold- changes in mRNA. PCR primers were designed using PRIMER3 Sofware (77). For relative quantification of transcript expression, Ct values were obtained for each gene and data were analyzed using the Excel (Microsoft) macro GENEX v1.10 (Gene Expression analysis for iCycle iQ®Real-time PCR Detection System, v1.10, 2004, Bio-Rad Laboratories.

FIGURE 4

P. gingivalis infected mDCs in oral submucosa of CP patient A. Representative gingival tissue section (7μM thick) from a CP patient stained with hematoxylin and eosin (H&E) (20x). B. Section was stained with FITC-conjugated mouse anti-human CD1c (BDCA-1) and RPE-conjugated mouse anti-human CD209 (DC-SIGN) and image captured at 100x. C. Shown at ~500x final enlargement (optical and digital) is detail view of individual of CD1c+ mDC/LC in epithelium and D. DC-SIGN+ mDCs in lamina propria (200x). E. FITC-conjugated mouse anti-human CD209 and Alexa Fluor 594 conjugated to mfa-1 antibody AEZαMfa1 using commercial DyLight^™ microscale antibody labeling kit in CP oral mucosal tissues (100x). Shown in panels 1–3 (at 200x) are detail of areas of mfa-1-DC-SIGN colocalization.

FIGURE 5

P. gingivalis infected mDCs in atherosclerotic plaque of CP patient A. Representative post- mortem coronary artery 7μM tissue section from ACS/CP patient stained with H&E, image obtained with enhanced light microscopy (Nikon E600) (4x). B. CAD tissue sections were stained with FITC-conjugated CD1c+, C. RPE-conjugated DC-SIGN+ and D. DC-SIGN-mfa-1 dual staining using FITC-conjugated DC-SIGN+ and Alexa Fluor 594 conjugated-mfa-1. Prior to staining, all tissue sections were blocked with 5% BSA in PBS, along with anti-human FcR block reagent. Appropriate controls included isotype matched antibodies and pre-immune antibodies (not shown). All sections were mounted with VectaShield mounting medium containing DAPI. Images were acquired with a Zeiss LSM 510 META NLO Two-Photon Laser Scanning Confocal Microscope System.