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

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

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

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

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 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 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

Detection and quantitation of lesion areas from <i>en face</i> descending aorta of Apob^tm2SgyLdlr^tm1Her/J mice fed a high-fat diet after immunization with each peptide antigen versus control mice immunized with KLH only.

<p>(A) Representative stained <i>en face</i> descending aorta from mice infected with <i>Cpn</i>. (B) Percentage of lesion-occupied area versus total area. (C) Percentage reduction of the lesion. Data represent mean ± SEM. *<i>P<</i>0.05; ** <i>P</i><0.01; ***<i>P<</i>0.001.</p

Figure 7

<p>(A–D) Plasma concentrations of cytokines in Apob^tm2SgyLdlr^tm1Her/J mice versus controls after immunization with peptide antigens. (E–H) Concentrations of cytokines in the supernatant of splenocytes stimulated with Apob^tm2SgyLdlr^tm1Her/J Mice fed with a high-fat diet after immunization with peptide antigens versus infected controls (N = 6 mice). *<i>P<</i>0.05; **<i>P<</i>0.01; ***<i>P<</i>0.001.</p

Immunization of <i>Chlamydia pneumoniae</i> (<i>Cpn</i>)-Infected Apob^tm2SgyLdlr^tm1Her/J Mice with a Combined Peptide of <i>Cpn</i> Significantly Reduces Atherosclerotic Lesion Development

<div><p>Objective</p><p>To investigate the antigenic effect of a peptide containing two epitopes of <i>Chlamydia pneumoniae</i> (<i>Cpn</i>) on atherosclerotic lesion formation in mice infected with <i>Cpn</i>.</p><p>Materials and Methods</p><p>Six-week-old Apob^tm2SgyLdlr^tm1Her/J mice were immunized using a repetitive immunization multiple-sites strategy with KLH-conjugated peptides derived from the major outer membrane protein and the putative outer membrane protein 5 of <i>Cpn</i>. Mice were fed a high-fat diet and infected with <i>Cpn</i> twice during the 10-week diet period. Lesions were evaluated histologically; local and systemic immune responses were analyzed by immunohistochemistry of aorta samples and cytokine measurements in plasma samples and splenocyte supernatants.</p><p>Results</p><p>Mice immunized with the combined <i>Cpn</i> peptide showed a greater reduction in lesion size compared to mice immunized with either epitope alone [54.7% vs 39.8% or 41.72%] and was also associated with a significant decrease in lesion area in descending aortas compared with those in controls (88.9% for combined Cpn peptide, 81.9% for MOMP peptide and 75.7% for Omp5, respectively). This effect was associated with a shift in the cellular composition of plaques towards decreased inflammatory cell and increased regulatory T-cell content. Additionally, the effect was also connected with decreased secretion of proinflammatory cytokines and increased production of anti-inflammatory cytokines demonstrated in plasma and in supernatant on stimulated spleen cells.</p><p>Conclusions</p><p>Atherosclerotic lesion formation may be promoted by <i>Cpn</i> infection in the presence of a high-fat diet, and reduced by immunization with the combined <i>Cpn</i> peptide. The combined peptide has more potential than either epitope alone in reducing atherosclerotic lesion development through Treg expansion.</p></div