Transient increase in atherosclerotic plaque macrophage content following Streptococcus pneumoniae pneumonia in ApoE-deficient mice

Figures

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  Figure 1. Significant mortality but no significant effect on atherosclerosis burden in male Paigen fed ApoE-/- mice following Type 1 S. pneumoniae infection. Cumulative survival (A) after intratracheal instillation of 107 cfu serotype 1 S. pneumoniae (n=8) or mock infection with PBS (n=5) (p=0.037 logrank test). No significant difference in lesion area in the aortic sinus (B). ns, not significant.

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  Figure 2. Microbiological outcomes in 10 week old female C57BL/6J mice 24 hours after intranasal instillation of S. pneumoniae. Bacteria in blood (A) and lung homogenates (B) following infection with 107 cfu of type 1, 4 or 14 pneumococci (n=4).

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  Figure 3. Characterization of microbiological and inflammatory features of Western diet-fed male ApoE-/- mice 24 hours after infection with 5 x105 cfu type 4 S. pneumoniae by intranasal instillation. Bacteria in blood (A) and lung homogenates (B) following infection with S. pneumoniae (n=9). Differential BAL fluid cell count expressed as % neutrophils (C) following infection or mock infection with PBS (n=8-9, **p<0.005, unpaired t-test with Welch’s correction). Total BAL fluid cell count (D) following infection or mock infection with PBS (n=8-9, *p<0.05).

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  Figure 4. Pneumococcal pneumonia results in increased atherosclerotic plaque macrophage content 2 weeks post pneumonia but no effect on other markers of plaque vulnerability. Male ApoE-/- mice aged 11-12 weeks were fed a Western diet for 8 weeks prior to intranasal instillation of 5x105 cfu serotype 4 (TIGR4) S. pneumoniae or mock infection with PBS. Mice received three doses of s.c. 100 mg/kg amoxicillin 12 hourly commenced 24 h after infection/mock infection and were maintained on chow diet post infection. Aortic sinus lesion area (A), expressed as a % of sinus CSA, in control mice and S. pneumoniae infected mice. Plaque macrophage (B), collagen (C) and smooth muscle actin (D) content expressed as % area of positive staining/total lesion area. Plaque necrotic core area expressed as % of total plaque area (E). Plaque cell replication (F) expressed as % of plaque cells staining positive for Ki67. Data were analysed using unpaired t test (A–C, E, F) or Mann-Whitney U test (D) (n=11-12, *p<0.05).

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  Figure 5. Aortic sinus sections 2 weeks post infection and mock infection. Representative images of MVG, MAC-3, Picrosirius Red (PCS) and smooth muscle actin (SMA) stained aortic sinus sections from S. pneumoniae infected (n=12) and PBS mock infected (n=11) ApoE-/- mice, using the optimised model as described in Figure 4 . Scale bars represent 400 µm.

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  Figure 6. Individual aortic sinus atherosclerotic plaque sections 2 weeks post infection and mock infection. Representative images of MAC 3 (A, B) and haematoxylin and eosin (C) stained atherosclerotic plaque sections from S. pneumoniae infected (n=12) and PBS mock infected (n=11) ApoE-/- mice, using the optimised model as described in Figure 4 . Scale bars represent 100 µm.

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  Figure 7. Pneumococcal pneumonia had no effect on aortic sinus plaque size or composition 8 weeks post pneumonia. Quantification of aortic sinus plaque area (A), plaque macrophage (B), collagen (C), smooth muscle (D) content and necrotic core area (E) in infected and control ApoE-/- mice (n=11-12).

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  Figure 8. Volcano plot comparing plaque macrophage gene expression in infected mice as compared to mock infected mice. All microarray probes, both coding and non-coding, are represented. Volcano plot compares log2 (fold change) to log10 p value. Green dots represent differentially expressed genes as identified by FDG unadjusted p value < 0.05 and log2 fold change >1. Red dots represent probes with unadjusted p value < 0.05, log2 fold change ≤1 and orange dots p value ≥ 0.05, log2 fold change >1.

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  Figure 9. Pneumococcal infection leads to downregulation of Huwe1 and Itch expression in plaque macrophages. Gene expression (normalised to housekeeping gene 18s) of (A) Huwe1, (B) Itch and (C) Anapc1 in LCM derived plaque macrophages from infected mice compared with mock infected mice (*p<0.05, n=7).

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  Figure 10. Atorvastatin had no effect on aortic sinus plaque size or composition 2 weeks post pneumonia. Quantification of aortic sinus plaque area (A), plaque macrophage (B), collagen (C), smooth muscle (D) content and necrotic core area (E) (n=12). Representative images (F) of MAC-3 immunostained aortic sinus sections from a S. pneumoniae infected ApoE-/- mouse (n=12) following atorvastatin therapy and an infected control mouse (n=12) given methylcellulose only. Scale bars represent 400 µm.