Genital Chlamydia infection in hyperlipidemic mouse models exacerbates atherosclerosis

Abstract

Background and aims: Atherosclerosis is a chronic inflammatory disease, and recent studies have shown that infection at remote sites can contribute to the progression of atherosclerosis in hyperlipidemic mouse models. In this report, we tested the hypothesis that genital Chlamydia infection could accelerate the onset and progression of atherosclerosis.

Methods: Apolipoprotein E (Apoe^-/-) and LDL receptor knockout (Ldlr^-/-) mice on a high-fat diet were infected intra-vaginally with Chlamydia muridarum. Atherosclerotic lesions on the aortic sinuses and in the descending aorta were assessed at 8-weeks post-infection. Systemic, macrophage, and vascular site inflammatory responses were assessed and quantified.

Results: Compared to the uninfected groups, infected Apoe^-/- and Ldlr^-/- mice developed significantly more atherosclerotic lesions in the aortic sinus and in the descending aorta. Increased lesions were associated with higher circulating levels of serum amyloid A-1, IL-1β, TNF-α, and increased VCAM-1 expression in the aortic sinus, suggesting an association with inflammatory responses observed during C. muridarum infection. Genital infection courses were similar in Apoe^-/-, Ldlr^-/-, and wild type mice. Further, Apoe^-/- mice developed severe uterine pathology with increased dilatations. Apoe-deficiency also augmented cytokine/chemokine response in C. muridarum infected macrophages, suggesting that the difference in macrophage response could have contributed to the genital pathology in Apoe^-/- mice.

Conclusions: Overall, these studies demonstrate that genital Chlamydia infection exacerbates atherosclerotic lesions in hyperlipidemic mouse and suggest a novel role for Apoe in full recovery of uterine anatomy after chlamydial infection.

Keywords: Atherosclerosis; Chlamydia; Inflammation; Uterine pathology.

Fig 1.

Aortic lesions were increased in Apoe^−/− mice with intra vaginal infection of C. muridarum. Apoe^−/− mice (N=10) were infected with 3 × 10⁵ IFU vaginally and were fed high fat diet starting one week after infection. Mice were sacrificed 8-weeks post infection (A). Aortic sinus (N=6) and descending aorta (N=10) were stained using oil red O to visualize plaques and average lesion area plotted. Aortic sinus from a representative mouse from each group is shown and (B) lesion area in aortic sinus (C) for all mice determined. Descending aorta from a representative mouse from each group is shown (D) and lesion area in descending aorta (E) for all mice determined. Significance determined by ‘t’ test. Figure represents one of two independent experiments.

Fig 2.. Aortic lesions were increased in Ldlr^−/− mice infected with C. muridarum.

Ldlr^−/− mice (N=10) were infected with 3 × 10⁵ IFU vaginally and were fed high fat/high cholesterol diet starting one week after infection (as shown in Figure 1A). Mice were sacrificed 8-weeks post infection. Aortic sinus (N=5) was stained using oil red O to visualize plaques. Aortic sinus (A) from a representative mouse from each group is shown and average lesion area (B) plotted. En face analysis of descending aorta from a representative mouse from each group is shown (C). Uninfected Ldlr^−/− mice fed a high-fat/high cholesterol diet was used as control. Significance determined by ‘t’ test.

Figure 3.. Increased levels of SAA, TNF-α, and IL-1β in the plasma of infected hyperlipidemic mice.

Apoe^−/− mice (N=10) or Ldlr^−/− were infected with 3 × 10⁵ IFU genitally, and one week after infection mice were fed hyperlipidemic diet. Mice were sacrificed 8-weeks post infection (7 weeks on a high fat diet). Plasma from uninfected mice were used as controls. Plasma levels of SAA-1 (A), TNF-α (B) in Apoe^−/− mice, and IL-1β in Apoe^−/− (C), and Ldlr^−/− (D) were determined as described in methods. Significance determined by ‘t’ test.

Fig 4.

Increased VCAM-1 expression and macrophages in Chlamydia-infected mice. Representative aortic sinus sections from uninfected or Chlamydia-infected Apoe^−/− (A, B) and or Ldlr^−/− (C, D) were stained with anti-mouse VCAM-1 IgG to detect VCAM-1 protein expression at the lesion site. Arrows indicate VCAM-1 positive staining. (E), Macrophages in aortic sections from uninfected and Chlamydia-infected or Apoe^−/− mice was determined as described the Methods section. Under similar conditions, aortic sections incubated with an isotype-matched rat IgG control were minimal. Representative of five aortic sinus sections from uninfected and chlamydia-infected Apoe^−/− or Ldlr^−/− mice are presented (100 × magnification).

Fig 5.. Severe uterine dilatation in Apoe^−/− mice in comparison with WT mice.

(A), Representative photograph of genital tracts from infected WT and Apoe^−/− mice (N=10). (B) H&E staining of longitudinal sections of genital tracts WT and Apoe^−/− mice (N=5), and (C) histopathological scoring for immune cells and dilatation (N=5). (D) Dilatation scores from an independent experiment where 5 mice from each group were fed high fat diet or fed a regular chow diet. Statistics were determined by non-parametric two-tailed Mann Whitney test.

Fig 6.

Augmented inflammatory responses in Chlamydia infected Apoe^−/− macrophages. BMDM from Apoe^−/− and Ldlr^−/− were infected with C. muridarum (1 MOI) for 8 h, and expression of inflammatory cytokine and chemokine responses was determined by qRTPCR. Uninfected macrophages were used as controls. Values are mean ± SD of triplicates. Representative two independent experiments are presented. Cytokine expression in Chlamydia infected Apoe^−/− BMDM was compared to infected Ldlr^−/− BMDM. Significance determined by ‘t’ test.