Figure 1.
(A) Mesio-distal view on a random section. Red, yellow, gray and green represent the enamel, dentin, bone and the residual bone area above the reference line, respectively. (B) Ncr1+/+ (WT) and Ncr1gfp/gfp (KO) mice (n = 8 in each group) were challenged orally three times at 2-day intervals with inoculums of F. nucleatum and of P. gingivalis (2×108 CFU in 0.2 ml of PBS and 2% carboxymethylcellulose). Six weeks later, the jaws were harvested and the residual alveolar bone volume (mm3×10−3) was measured. Error bars represent SD. Data represent percentage of bone loss ± SD and are average of two independent experiments. ** p<0.01. The difference in bone loss between the KO and the WT mice following P. gingivalis inoculation was not statistically significant, p<0.09.
Figure 2.
Infiltration of cells into challenged chambers.
Immune cells in the chambers were analyzed by FACS 2 hours (A) and 24 hours (B) following bacteria inoculation. The intrinsic GFP labeling marks mainly NK cells. Staining was performed with Phycoerythrin conjugated anti-DX5 and anti-NKG2D mAbs. Staining with NK1.1 was visualized by using Cy-5 conjugated strepavidin. The cell percentage and cell numbers is indicated in each quadrant. One representative experiment out of four is shown.
Figure 3.
Survival of bacteria in chamber exudates.
Viable CFU from chamber exudates were determined, 2 h and 24 h following inoculation of P. gingivalis, F. nucleatum or E. coli. Each symbol represents an individual mouse. One representative experiment out of four is shown. The y axis represents number of colonies (logarithmic scale).
Figure 4.
Cytokine profiles in challenged chambers.
TNF-α levels were determined by ELISA in chamber exudates of Ncr1+/+ (WT) and Ncr1gfp/gfp (KO) mice at baseline (0), 2 h and 24 h following bacterial challenge. Data represent absorbance at 650 nm ± SD and are average of three different experiments. ** p<0.01.
Figure 5.
The infiltration of immune cells into the F. nucleatum challenged chambers is NCR1-independent.
The presence of immune cells; (A) lymphocytes and (B) DC (identified by CD11c) and macrophages (identified by F4/80) in the chambers were analyzed by FACS, 2 hours following the injection of F. nucleatum. The mAb used for staining is indicated in the Y axis. IC is isotype control. The percentage of the various cells and the cell numbers are indicated in each quadrant. One representative experiment out of four is shown.
Figure 6.
In vivo response to heat-treated F. nucleatum bacteria.
(A) FACS analysis of chamber exudates, 2 hours and 24 hours following challenge with heat-killed F. nucleatum. Staining was performed with Phycoerythrin conjugated anti-DX5 mAb. The percentages of GFP+ NK cells are indicated. One representative experiment out of three is shown. (B) TNF-α level in chamber exudates of Ncr1+/+ (WT) and Ncr1gfp/gfp (KO) was determined by ELISA, 2 and 24 hours following challenge with viable and heat-killed F. nucleatum. Data represent absorbance at 650 nm ± SD and are average of two different experiments. * p<0.05, ** p<0.01.
Figure 7.
Direct binding of NCR1 and NKp46 to F. nucleatum.
F. nucleatum (A), P. gingivalis (B), uninfected 721.221 cells (C), and 721.221 cells infected with influenza PR8 virus (D), were stained with NCR1-Ig (left) and with NKp46-Ig (right) at two different dosages; 5 µg, blue line and 1 µg, red line. Staining was visualized using a Phycoerythrin conjugated anti-human Ig antibody. The filled grey histograms represent control staining with the NKp46D1-Ig fusion protein. The Median Fluorescence Intensity (MFI) values of the D1-Ig, NCR1-Ig and NKp46 staining are indicated in each of the histograms. One representative experiment out of five is shown. (E) F. nucleatum (upper) and P. gingivalis (lower) bacteria were stained with various activating NK cell receptors fused to Ig (indicated above the quadrants). Figure shows one representative experiment out of three performed.
Figure 8.
The F. nucleatum ligand is sensitive to heat, proteinase K and pronase treatment.
Viable untreated, or treated (treatments are indicated above the histograms) F. nucleatum bacteria were stained with 5 µg of NCR1-Ig (upper) and of NKp46-Ig (lower). Filled grey histograms represent the staining with the NKp46D1-Ig. The blue histograms represent the staining of the untreated F. nucleatum and the red histograms represent the staining of the treated F. nucleatum. One representative experiment out of three is shown.
Figure 9.
(A) Basal IL-2 secretion of BW-NCR1, BW-NKp46 and parental BW cells. (B) BW-NKp46 transfected cells were cultured for 48 h in presence of plate bound (dosages indicated in the X axis) anti NKp46 mAb and control mAb. (C) 5×104 BW-NKp46 transfected cells were cultured for 48 hours, with treated or untreated F. nucleatum and with 721.221 cells infected with PR8 influenza virus in the presence and in the absence of anti-NKp46 mAb. IL-2 secretion was determined by ELISA. Data represent absorbance at 650 nm ± SD and are average of two different experiments * p<0.05, ** p<0.01.
Figure 10.
NCR1-dependent secretion of TNF-α following interaction with F. nucleatum.
NK cells isolated from Ncr1+/gfp (Het) and from Ncr1gfp/gfp (KO) mice were cultured with F. nucleatum and with P. gingivalis bacteria for 48 hours. TNF-α secretion was determined by ELISA. Data represent absorbance at 650 nm ± SD and are average of two different experiments. *** p<0.001.