Investigation of antigen-specific regulatory function in antigen-immunized mice.

<p>Inhibition of CD4⁺CD25^- effector T cell proliferation by CD4⁺CD25⁺ regulatory T-cells isolated from the spleen of control (GST-Den–immunized) and construct-immunized mice when AHHC, RHHC and RPHC were used as antigens. (A) Quantitative analyses of proliferation of CD4⁺CD25^- effector T cells in the presence of Treg cell by flow cytometry is shown. (B) Proliferation of effector cells isolated from immunized mice alone is indicated in the leftmost bar of each group. Addition of Treg cells to T effector cells at different ratios was also shown. Data are given as mean of 3 analyses ± SEM.</p

Levels of constructed protein-induced IgG, IgG1, and IgG2c antibodies in the sera of <i>B6;129S-Ldlr</i>^{<i>tm1Her</i>}<i>Apob</i>^{<i>tm2Sgy</i>}<i>/J</i> mice at 2 weeks and 12 weeks respectively, after the first immunization and in controls (GST-Den-immunized mice).

<p>The mean optical densities (ODs) and SEM obtained from plasma samples of constructs AHHC, RHHC, and RPHC-immunized mice on Cpn peptide (C)-, hHSP60^153-163 (H)-, hHSP60^303-312 (H)-, C5aR-peptide (R)-, ApoB peptide (A)-, PAR-1(P)-coated ELISA plates are shown. (A) IgG at the dilution ratio: 1:100. (B) IgG1 at the dilution ratio: 1:6250. (C) IgG2c at the dilution ratio: 1:50. (D) Cross reaction between human ApoB peptide-induced antiserum and Cpn peptide. (E) Cross reaction between human ApoB peptide- and hHSP60^303-312 peptide-induced antiserum and their antigens. (F) Cross reaction between ApoB peptide-induced antiserum and antigens of either PAR-1 peptide or C5aR peptide. (G) Cross reaction between C5aR peptide-induced antiserum and antigens of either ApoB peptide or PAR-1 peptide. Antiserum used for cross-reaction was taken at week 8.</p

Detection and quantitation of the lesion areas in the aorta of <i>B6;129S-Ldlr</i>^{<i>tm1Her</i>}<i>Apob</i>^{<i>tm2Sgy</i>}<i>/J</i> mice fed on a high-fat diet after immunization with constructs versus controls (GST-Den).

<p>(A) Photomicrograph of lesions observed in atherosclerotic aortas as analyzed with elastin/van Gieson staining. (B) Scatter plot showing mean of lesion area in the aortic sinus of mice immunized with constructs compared with those in controls (GST-Den). (N = 6–9 mice). Error bars = SEM. (C) Percentage of reduction in lesion size in the aortic sinus (the reduction of control [GST-Den] group was set at zero). (D) Representative photomicrographs and quantitative analysis of collagen (Sirius Red coloration under polarized light) in atherosclerotic aortas in individual mice. (E) Quantification of collagen content at lesion area in the aorta of <i>B6;129S-Ldlr</i>^{<i>tm1Her</i>}<i>Apob</i>^{<i>tm2Sgy</i>}<i>/J</i> mice (N = 7 mice). NS: not significant. (F) Representative Oil Red O-stained <i>en face</i> descending aorta from mice. (G) Percentage of lesion-occupied area versus total area of descending aortas in individual mice (N = 6 mice) of the different experimental groups. The mean lesion size and the difference in lesion size between the experimental groups are shown. (H) Percentage of reduction in lesion size in descending aortas (the reduction of control [GST-Den] group was set at zero).</p

Evaluation of monocyte differentiation into macrophages.

<p>(A and B) PBMCs differentiation stimulated by recombinant constructs (1 μg/ml) as assessed by analyses of CD206 expression by flow cytometry and inhibition of differentiation in the presence of respective construct-induced antibodies. (C and D) Inhibition of differentiation by respective and other construct induced antibodies. (E and F) Individual peptide as a stimulator (1 μg/ml) of monocyte differentiation as analysed by CD206 expression. Data are expressed as average values of 3 analyses.</p

Assessment of inflammation-associated cells in the lesions of <i>B6;129S-Ldlr</i>^{<i>tm1Her</i>}<i>Apob</i>^{<i>tm2Sgy</i>}<i>/J</i> mice mice fed a high-fat diet after immunization with constructs AHHC, RHHC, and RPHC.

<p>(A) Photomicrographs showing IHC staining of CD68 (green) and CD11c (red) markers (scale bar: 100 μm and 12.5μm for magnified ones). (B) Scatter plot showing anti-CD68-stained area in lesion versus total lesion area; Data are given as the mean of 6 mice. (C) Scatter plot showing anti-CD11c-stained area in lesion versus total lesion area; Data are given as the mean of 6 mice. (D) Co-localization of CD68⁺ and CD11c⁺ areas (derived from Fig 3B and 3C). (E) Photomicrographs showing IHC staining of CD4⁺ T-cells (green) and Foxp3⁺ Treg cells (red) (scale bar: 100 μm and 12.5μm for magnified ones). (F) Scatter plot showing anti-Foxp3-stained area versus anti-CD4⁺ stained area in lesion (N = 6). (G) Representative analysis of Foxp3 expression by CD4⁺ T cells in lymph nodes from construct-immunized mice fed on a high-fat diet as assessed using flow cytometry. (H) Percentage of Foxp3⁺ cells among CD4⁺ spleen cells as analyzed by flow cytometry. Data are expressed as mean of 3 analyses ± SEM. Differences between groups are shown.</p

Evaluation of expression of smooth muscle alpha actins, vascular cell adhesion molecule (VCAM)1 and matrix metalloproteinase 9 (MMP9) in lesion site.

<p>(A) Photomicrographs showing IHC staining for smooth muscle alpha actin (red), vascular cell adhesion molecule VCAM1(green) (scale bar: 100 μm and 12.5μm for magnified ones). (B) anti-SMC stained area. (C) Scatter plot showing means of anti-VCAM-1 stained area (N = 6). (D) Photomicrographs showing IHC staining for MMP9 (green) (scale bar: 100 μm and 12.5μm for magnified ones). (E) Scatter plot showing means of anti-MMP9 stained area (N = 6).</p

Assessment of IL-10-producing T cells, TNF-α expression in the lesions and cytokine levels in <i>B6;129S-Ldlr</i>^{<i>tm1Her</i>}<i>Apob</i>^{<i>tm2Sgy</i>}<i>/J</i> mice fed a high-fat diet after immunization with constructs AHHC, RHHC, and RPHC.

<p>(A) Photomicrographs showing dual-IHC staining for IL-10 (red) and CD4 (green) (scale bar: 100 μm and 12.5μm for magnified ones). (B) Scatter plot showing mean of IL-10-positive area co-localized with CD4⁺ area (%) (N = 6). (C) Photomicrographs showing IHC staining for TNF-α (green) of lesions (scale bar: 100 μm and 12.5μm for magnified ones). (D) Scatter plot showing mean of anti- TNF-α-stained area in the lesion versus total lesion area (N = 6). (E-H) Cytokine levels measured in plasma. (I-L) Cytokine levels measured in the supernatant of splenocytes stimulated with ConA. (M) Representative analysis of CD4⁺IL-4⁺ T-cells in splenocytes from construct-immunized mice fed on a high-fat diet as assessed by flow cytometer. (N) Percentages of IL-4⁺ expressing CD4⁺ spleen cells. Data are expressed as mean of 3 analyses ± SEM. Differences between groups are shown. (O) Representative analysis of IL-17A expression by CD4⁺ T-cells in splenocytes from construct-immunized mice fed on a high-fat diet as assessed by flow cytometry. (P) Levels of IL-17A expression in spleen cells. Data are expressed as mean of 3 analyses ± SEM. Differences between groups are shown. (Q) Representative analysis of CD4⁺IL-2⁺ T-cells in splenocytes from construct-immunized mice fed on a high-fat diet as assessed by flow cytometer. (R) Levels of IL-2⁺ expressing CD4⁺ in spleen cells. Data are expressed as mean of 3 analyses ± SEM. Differences between groups are shown.</p

Evaluation of the content of TLR4 and MyD88 contents at the lesion sites.

<p>(A) Photomicrographs showing IHC staining for TLR4 (red) (scale bar: 100 μm and 12.5μm for magnified ones). (B) Scatter plot showing means of anti-TLR4-stained area (N = 6). (C) Photomicrographs showing IHC staining for MyD88 (red) (scale bar: 100 μm and 12.5μm for magnified ones). (D) Scatter plot showing means of anti-MyD88-stained area (N = 6).</p

Figure 2

<p>(A–C) Concentration of <i>Cpn</i> peptide-induced IgG antibodies and KLH controls in the sera of Apob^tm2SgyLdlr^tm1Her/J mice at 2 and 12 weeks after the first immunization. The mean optical densities (ODs) were obtained from plasma samples of each peptide-immunized mouse on relevant peptide-coated ELISA plates. Dilution ratio: 1∶100. (D, E) Concentrations of <i>Cpn</i> peptide-induced IgG1 and IgG2a antibodies in the sera of Apob^tm2SgyLdlr^tm1Her/J mice at 2 weeks after the first immunization. Dilution ratio: 1∶400 for IgG1 and 1∶50 for IgG2a. (F, G) Cross-reaction between the combined <i>Cpn</i> peptide and ApoB peptide (the data of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081056#pone-0081056-g002" target="_blank">Fig. 2F</a> were from individual samples, the data of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081056#pone-0081056-g002" target="_blank">Fig. 2G</a> were from pooled samples).</p

Plasma lipid concentrations in mice after being fed a high-fat diet for 10 weeks.

<p>Calculated values.</p