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

<p>Calculated values.</p

Figure 1

<p><b>1a.</b> Agarose gel electrophoresis of PCR products (first stage, lanes 2–6; second stage, lanes 8–12). Lanes 2 and 3 using genomic DNA as a template from two Apob^tm2SgyLdlr^tm1Her/J mice infected with <i>Cpn</i> bacteria; lanes 5 and 6 using genomic DNA from two non-infected Apob^tm2SgyLdlr^tm1Her/J mice; lanes 8, 9, 11 and 12 using first-stage PCR products 2, 3, 5 and 6 as a template, respectively. Lanes 1 and 7 show the Phi174 DNA/HaeIII maker (Promega). <b>1b.</b> Chlamedia LPS antibody (MCA 2718, AbD serotec) stained lesion sites in aortic sinus. <i>Chlamedia</i> LPS antibody (MCA 2718, AbD serotec) was used as the first antibody (10 mg/ml), anti-mouse IgG-FITC, developed in sheep, was used as a second antibody. Green represents <i>Chlamedia</i> LPS and blue represents cell nnucleuses stained with 4′,6-diamidino-2-phenylindole (DAPI) (Vector Lab, Peterborogh, UK) [N = 6 mice].</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

Assessment of interleukin-10-producing T cells and tumor necrosis factor-α expression in the lesions of Apob^tm2SgyLdlr^tm1Her/J mice fed a high-fat diet after immunization with peptide antigens.

<p>(A) Representative photomicrographs showing dual-immunohistochemical staining for IL-10 (red) and CD4 (green). (B) Percentage of IL-10-positive area co-localized with CD4⁺ area. (C) Relative ratio of immunohistochemical stained area (green) for TNF-α in the lesion versus total lesion area. (D) Percentage reduction of TNF-α-positive area versus that in the control mice (N = 6 mice).</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

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

Detection and quantitation of the lesion areas in the 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 (both infected and non-infected).

<p>(A) Representative photomicrographs of lesions observed in atherosclerotic aortas as analyzed with elastin/van Gieson staining. (B) Percentage of luminal surface occupied by lesions in the aortic sinus versus control mice immunized with KLH (ratio of lesion areas [µm²] versus total areas [µm²] [N = 6 mice]). (C) Reduction of lesion size shown as percentage of the lesion area versus that in control mice (infected). Data represent mean ± SEM.</p

Figure 5

<p>(A–D) Assessment of inflammation-associated cells in the lesions of Apob^tm2SgyLdlr^tm1Her/J mice fed a high-fat diet after immunization with peptide antigens. (A) Representative photomicrographs showing immunohistochemical staining of CD68 (green) and CD11c (red) markers, respectively. Percent occupied lesion (vs. infected control) for (B) CD68 and (C) CD11c. Magnification: 400×. Data represent the mean value ± SEM. (D) Assessment of inflammation-associated cells as percentage of CD11c areas co-localized with CD68 area (N = 6 mice). (E–G) Assessment of CD4⁺ Treg cells in the lesions of Apob^tm2SgyLdlr^tm1Her/J mice fed on a high-fat diet after immunization with peptide antigens. (E) Representative photomicrographs showing immunohistochemical staining of CD4⁺ (green) and Foxp3⁺ Treg (red) cells. Magnification 800×. (F) Observation of CD4⁺ occupied lesion area (N = 6 mice). (G) Assessment of Treg cells as percentage of Foxp3⁺ areas co-localized with CD4⁺ area (N = 6 mice). (H) Representative flow cytometric plots for CD4⁺CD25⁺Foxp3⁺ (Treg) cell population in spleen cells. Spleen cells from mice immunized with either <i>Cpn</i> peptides or KLH (control) were performed using a Treg detection kit (Ailtenyi Biotec, Surrey, UK) according to manufacturer's protocols. (I) Bar chart presentation of flow cytometric analysis. Data represent mean±SEM of data from 3 independent samples.</p

Assessment 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 control (KLH–immunized) mice (A,B,F) and peptide-immunized mice when the MOMP- peptide (A,C,F), the Omp5-pepide (A,D,F), and the combined peptide (A,E,F) were used as antigens. 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 expressed as mean of 6 analyses ± SEM.</p