Mouse plasma IgM and IgG binding to MDA-LDL after immunization with <i>P. gingivalis</i>.

<p>C57BL/6 mice were immunized with heat-killed <i>P. gingivalis</i> ATCC33277 (Pg; n = 8) and controls received saline (Co; n = 8). Plasma IgM (A) and IgG (B) to MDA-LDL before (pre) and after immunization (post) were determined with chemiluminescence immunoassay. Each C57BL/6 plasma sample (1∶500) was measured in duplicate and an average for each individual was calculated. LDLR^−/− mice were immunized with killed <i>P. gingivalis</i> (3 strains mixed) (Pg; n = 7) and controls received PBS (Co; n = 8). Plasma IgM (C) and IgG (D) to MDA-LDL after the second booster immunization (imm) and after the HFD (end) were determined. Each LDLR^−/− plasma sample (1∶1000) was measured in duplicate and an average for each individual in two repeated assays was calculated. Additionally, mouse plasma IgM binding to CuOx-LDL (E, G) and PC-BSA (F, H) was determined. For C57BL/6 mice (E, F) this was done similarly as described for panel A. Plasma samples of LDLR^−/− mice (G, H) were pooled between three or four mice (1∶1000) for a single assay, in which the mean ± SD within a group is shown.</p

Mouse plasma IgM binding to apoptotic T lymphocytes after <i>P. gingivalis</i> immunization.

<p>C57BL/6 mice were immunized with heat-killed Pg and controls received sterile saline (n = 8 per group). Mouse plasma (1∶70) IgM binding to UV-irradiated Jurkat T cells was measured with flow cytometry. A, B) Apoptotic T cell population (R1) was verified with propidium iodide (PI) staining. C) Plasma IgM binding in gate R2 of preimmune (black) and postimmune (blue) plasma samples, and competition of IgM binding with 250 µg/ml MDA-LDL (green) or native LDL (red). Inset plots (in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034910#pone-0034910-g006" target="_blank">Fig. 6C</a>) represent the secondary antibody control (2°Ab control) and plasma IgM binding to apoptotic cells in a Pg-immunized mouse (post). D) IgM binding to Jurkat cells was determined for each mouse in Pg-immunized and control group as geometric mean value in R2 subtracted by the 2°Ab control. Box-plot graphs represent the distribution of sample means calculated for two repeated assays. **P<0.01 and *P<0.05.</p

Association between human serum IgM to <i>P. gingivalis</i> and MDA-LDL, and competitive binding with recombinant gingipain domains.

<p>Sera from 29 healthy adults were analyzed for IgM (A) and IgG (B) binding to Pg and MDA-LDL by chemiluminescence immunoassay. Associations between antibody levels were analyzed with Spearman rank correlation test. Human sera were pre-incubated with recombinant gingipain domains Rgp44, Rgp15–27, RgpCAT (C, D) in a competitive immunoassay detecting IgM binding to immobilized MDA-LDL. The ratio of serum IgM binding (B/B₀) to MDA-LDL with and without competitor (175 µg/ml) in 29 human serum samples (C) and dose-dependent competition assays of one sample (D). Reciprocal competition assay was performed to analyze human serum IgM binding to Pg antigen competed with MDA-LDL, nLDL and PC-BSA in a representative sample (E). RU, relative units.</p

Antibodies to recombinant gingipain in <i>P. gingivalis</i> immunized mice.

<p>Recombinant proteins of the arginine specific gingipain protease of <i>P. gingivalis</i> were produced in <i>E.coli</i>: two proteins in the hemagglutinin/adhesion domain, Rgp44 and Rgp15–27, and the catalytic domain, RgpCAT, which were used in chemiluminescence immunoassay to determine mouse plasma (1∶500) IgM and IgG binding in <i>P. gingivalis</i> immunized (Pg) and control (Co) groups in both A) C57BL/6 and B) LDLR^−/− mice. A) For C57BL/6 immunized and control mice the samples (n = 8 each) were determined as duplicate before (pre) and after (post) immunization. Box-plots represent the distribution of the means of the sample duplicates. B) Two plasma samples within the group were pooled for immunized (black bars) and control (hatched bars) LDLR^−/− mice and measured in duplicate. Samples were collected before (pre), after the second booster immunization (imm) and at the end of HFD (end). Columns represent the mean ± SD of pooled samples in each group. **P<0.01 and *P<0.05.</p

Weight gain and plasma lipids of LDLR^−/− mice.

*<p>Female LDLR^−/− mice were immunized with killed <i>P. gingivalis</i> (n = 7) and controls (n = 8) received PBS. Mean ± SD is shown.</p>†<p>Plasma lipids were measured from EDTA-plasma samples collected immediately after sacrifice at the end of HFD. Concentration of LDL cholesterol was estimated using the formula of Friedewald.</p

Quantification of atherosclerosis in LDLR^−/− mice immunized with <i>P. gingivalis</i>.

<p>LDLR^−/− mice (n = 7) were immunized without adjuvant with killed <i>P. gingivalis</i> (3 strains mixed) (Pg) followed by high fat diet (HFD). Controls (PBS, n = 8) received PBS. A) The extent of atherosclerotic plaque development was determined after HFD by <i>en face</i> analysis of the Sudan IV -stained aortas. B) Lesions at the aortic origin were measured on histological sections as percentage of plaque area in the aorta cross-sectional area. Representative pictures of aortas and cross-sections are shown for each group. * P<0.05.</p

Identification of <i>P. gingivalis</i> epitopes for anti-MDA-LDL-IgM.

<p>A) Schematic presentation of arg-gingipain (RgpA) functional domains cloned and produced in a recombinant system <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034910#pone.0034910-Inagaki1" target="_blank">[41]</a>. B) Proteins of <i>P. gingivalis</i> were separated on SDS-PAGE (Pg, gel). Fragments recognized by MDmAb (45, 40 and 32 kDa, black arrows) were identified by mass spectrometry as arginine-specific gingipain or hemagglutinin A of <i>P. gingivalis</i>. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034910#pone.0034910.s001" target="_blank">Figures S1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034910#pone.0034910.s002" target="_blank">S2</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034910#pone.0034910.s003" target="_blank">S3</a> contain the Mascot results of the database searches and the MSMS spectrum showing the matching amino acids in the peptide sequence. Three domains of the recombinant arg-specific gingipain, RgpCAT, Rgp44 and Rgp15–27 were produced in <i>E. coli</i> and analyzed for recognition by MDmAb and anti-PC-IgM control antibody (α-PC-mAb). C) Specific binding of MDmAb to recombinant gingipain domains Rgp15–27, Rgp44 and RgpCAT was tested with a competitive immunoassay. D) Reciprocally, soluble MDA-LDL, nLDL and PC-BSA were used as competitors for MDmAb binding to Rgp44. B/B₀ indicates the ratio of IgM binding with and without a competitor. MW, molecular weight. PC-BSA, phosphocholine-conjugated bovine serum albumin.</p

Association of mouse plasma IgA and IgG levels with atherosclerotic plaque area in LDLR^-/- mice immunized with saline, Rgp44, and heat-inactivated <i>Pg</i>.

<p>Plasma IgA and IgG to Rgp44 and <i>Pg</i>-LPS as well as plasma IgA to PCho were determined in triplicate by chemiluminescence immunoassay. The antibody level is expressed as relative light units measured in 100 milliseconds (RLU/100ms). Associations are determined using Spearman rank correlation coefficient (ρ). P-values (p) less than 0.05 are regarded as statistically significant.</p

Mouse plasma IgA levels to Rgp44, <i>Pg</i>, PCho, and <i>Pg</i>-LPS before immunization (week 0), before HFD (week 13), and at the end of the study (week 39) in LDLR^-/- mice immunized with saline, Rgp44, and heat-inactivated <i>Pg</i>.

<p>Each mouse plasma sample was measured in triplicate using chemiluminescence immunoassay. The IgA antibody binding is expressed as mean ± SD in relative light units measured in 100 milliseconds (RLU/100ms). P-values less than 0.05 are regarded as statistically significant (nonparametric Mann-Whitney U test). * p < 0.05, ** p < 0.01, *** p < 0.001.</p

Association of mouse plasma IL-1α, IL-5, IL-10, and IL-22 cytokine levels with atherosclerotic plaque area in LDLR^-/- mice immunized with saline, Rgp44, and heat-inactivated <i>Pg</i>.

<p>Mouse plasma cytokine levels were determined with cytometric bead assay (CBA) from each mouse plasma sample. They are expressed as picograms per milliliter plasma (pg/mL). Associations are determined using Spearman rank correlation coefficient (ρ). P-values (p) less than 0.05 are regarded as statistically significant.</p