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Cardiovascular Research 2002 56(2):269-276; doi:10.1016/S0008-6363(02)00544-8
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Copyright © 2002, European Society of Cardiology

Chlamydophila pneumoniae (Chlamydia pneumoniae) accelerates the formation of complex atherosclerotic lesions in Apo E3-Leiden mice

R Ezzahiria,*, H.J.M.G Nelissen-Vranckenb, H.A.J.M Kurversa, F.R.M Stassenb, I Vliegenb, G.E.L.M Graulsb, M.M.L van Pula,b, P.J.E.H.M Kitslaara and C.A Bruggemanb

aDepartment of Surgery, Cardiovascular Research Institute Maastricht, Maastricht University, PO Box 5800, 6202 AZ Maastricht, The Netherlands
bDepartment of Medical Microbiology, Cardiovascular Research Institute Maastricht, Maastricht University, PO Box 5800, 6202 AZ Maastricht, The Netherlands

rajaa_ezzahiri{at}hotmail.com

* Corresponding author. Tel.: +31-43-387-6644; fax: +31-43-387-6643.

Received 15 March 2002; accepted 21 June 2002


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: Atherosclerosis is an inflammatory process and is characterised by the presence of T-lymphocytes in the lesions. To study the role of Chlamydophila pneumoniae (C. pneumoniae) in this process and the effect of infection on T-cell influx, we infected Apo E3-Leiden mice with C. pneumoniae and investigated the effect on lesion development and T-cell influx in atherosclerotic lesions at different time points post infection (pi). Methods: Nine week old mice, fed an atherogenic diet, were either mock-infected or infected with C. pneumoniae and sacrificed at 1, 6 and 9 months pi. Longitudinal sections of the aortic arches of the mice were stained with hematoxylin–eosin for atherosclerotic lesion type and lesion area analysis, or with rabbit-anti-CD3+ to detect the presence of T-cells in the atherosclerotic lesions. T-cell influx was expressed as number of T-lymphocytes/lesion area. Results: At 1 month pi, type 1, 2 and 3 lesions were present. At other time points pi, more complex lesion types 4, 5a and 5b were also present. Although infection did not influence the total lesion number or area, we observed an effect of C. pneumoniae infection on lesion type. Infection resulted in a significant shift in lesion formation from type 3 to type 4 (P=0.022) at 6 months pi, and from type 4 to type 5a (P=0.002) at 9 months pi. T-cells were observed at every time point pi. At 1 month pi, a significant increase in T-cell influx in the C. pneumoniae-infected atherosclerotic lesions was observed (P=0.0005). Conclusion: This study shows that C. pneumoniae infection enhances the inflammatory process by increasing T-lymphocytes in the plaque and accelerates the formation of complex lesions.

KEYWORDS Atherosclerosis; Immunology; Infection/inflammation; Leukocytes; Macrophages

This article is referred to in the Editorial A.C. Van der Wal (pages 178–180) in this issue.


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Cardiovascular complications due to atherosclerosis are the major causes of morbidity and mortality in the Western world. Besides several well known risk factors, such as smoking and hypercholesterolemia [1], infections have been suggested to play an important role in the development of atherosclerosis over the last decades [2–4]. Infectious agents such as Cytomegalovirus, Chlamydophila pneumoniae (C. pneumoniae), Helicobacter pylori, and bacteria causing dental infections have all been linked to atherosclerosis [1,5–7]. For C. pneumoniae the association has been demonstrated in sero-epidemiological, experimental, and clinical studies [4,8–15]. However, the mechanism by which C. pneumoniae contributes to atherosclerosis has not been clarified. To understand this mechanism, various animal models, mostly rabbits and mice, have been used [11–14,16]. The progression of atherosclerosis has been reported in New Zealand White rabbits [15], as well as in low-density lipoprotein receptor (LDLR) knockout mice [11] inoculated with C. pneumoniae. In all of these studies, an effect of C. pneumoniae infection was only documented on lesion size. However, little is known about the effect of infection on the development and/or progression of lesion types. Since lesion type is an important indicator of the severity of atherosclerosis, we studied the effect of C. pneumoniae infection on the type of atherosclerotic lesion by grading the lesions according to the guidelines of the American Heart Association [17,18].

By understanding the atherogenicity of C. pneumoniae, insight should be gained into the ability of this micro-organism to cause local infection and inflammation of the vascular wall as characterised by the influx of leukocytes, consisting mainly of monocytes and T-lymphocytes. When T-lymphocytes are activated, they can act as a powerful source of pro-atherogenic cytokines, thereby contributing to the progression of atherosclerotic lesion formation from fatty streaks into complex atherosclerotic plaques [19]. Immunohistochemical studies have demonstrated the presence of T-lymphocytes in human [20,21] and animal [22] atherosclerotic plaques. Activation of T-lymphocytes in the plaque can be established by antigens such as ox-LDL [23–25]. However, C. pneumoniae has also been demonstrated to be a good candidate for activating T-cells cultured from the peripheral blood of patients with atherosclerosis as well as T-cells cultured from carotid endarterectomy tissue [20]. Recently, it has even been demonstrated that C. pneumoniae can infect lymphocytes of human peripheral blood and mouse spleens as well as a T-lymphocyte cell line [26].

In this study, the role of C. pneumoniae in atherosclerotic lesion progression was studied using Apo E3-Leiden mice. Mice were infected with C. pneumoniae and sacrificed at different time points post infection (pi) to analyse lesion progression in the aortic arch and main branchpoints. To understand the possible mechanism by which C. pneumoniae accelerates atherosclerotic lesion progression, we studied inflammation of the vascular wall by determining the influx of T-lymphocytes into the atherosclerotic lesion.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 Mice
Sixty-four transgenic Apo E3-Leiden mice on a C57Bl/6J background were bred at the Central Animal Facility at Maastricht University [27]. For the study, both males and females were used and randomly divided over all groups. All mice were specific-pathogen-free (SPF) and were housed under standard conditions.

This study was approved by the Institutional Committee for the Welfare of Laboratory Animals of Maastricht University.

2.2 C. pneumoniae
C. pneumoniae strain TWAR 2043 (ATCC VR-1355) was cultured in HEp-2 cells as previously described [28]. Bacterial titers were determined by titration in HEp-2 cells and staining with monoclonal antibody RR 402 (Dako, Denmark) followed by fluorescein-isothiocyanate (FITC)-conjugated rabbit-anti-mouse (Dako). Titers were expressed as inclusion-forming units per milliliter (IFU/ml).

2.3 Experimental design
At the age of 5 weeks, the mice received a high-fat cholesterol (HFC) diet containing 15% cacao butter, 0.5% cholate, 1% cholesterol, 40.5% sucrose, 10% corn starch, 1% corn oil, and 4.7% cellulose (Hopefarms, Woerden, The Netherlands). Since it has been demonstrated that multiple infections, the age of the animal at the time of inoculation and the interval between inoculations are all critical factors in mice for enabling C. pneumoniae to promote atherosclerosis [11,14,29,30], we decided to inoculate our mice twice with C. pneumoniae using different time intervals between inoculations. All mice received the first infection by intraperitoneal injection of 6x107 IFU C. pneumoniae at the age of 9 weeks (Fig. 1). Control mice were injected with sterile phosphate-buffered saline (PBS, mock infection). Since no differences in the dissemination of C. pneumoniae after intraperitoneal or intranasal inoculation have been reported [31], we selected intraperitoneal infection for our mice. In group 1, mice received a second inoculation at the age of 11 weeks and were sacrificed at the age of 13 weeks, 1 month after the first infection (Fig. 1). In group 2, mice were inoculated for the second time at the age of 29 weeks and were sacrificed at the age of 33 weeks, 6 months after the first infection. Finally, mice in group 3 received a second inoculation at the age of 41 weeks and were sacrificed at the age of 45 weeks, 9 months after the first infection.


Figure 1
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  		Fig. 1 Design of animal experiment, showing the different mice groups analysed in this study, the inoculation time points and time points of sacrifice.

 
2.4 Tissue handling
At sacrifice, the mice were anesthetized with a weight-adjusted dose of pentobarbital [32] (Nembutal®, Sanofi Sante, Maassluis, The Netherlands) and blood was collected from the left ventricular apex for assessment of lipid profiles and anti-C. pneumoniae antibody titers. The arterial tree was perfused with PBS followed by 1.85% PBS-buffered formaldehyde, both containing 0.1 mg/ml sodium-nitroprusside (Merck, Darmstadt, Germany), through a catheter placed in the left ventricular apex. The arterial tree was removed, fixed overnight in 3.7% PBS-buffered formaldehyde and embedded in paraffin. Four micrometer thick longitudinal sections were consecutively cut, stained and analysed [18].

2.5 Evaluation of atherosclerotic lesions
Since all lesion types were found in the aortic arch [18], lesion analysis was restricted to this segment of the arterial tree. Only aortic arches with an intact morphology (brachiocephalic trunk, left and right common carotid artery and left subclavian artery) were used.

Longitudinal, coronal, consecutive sections of the entire arch were prepared. Every fifth section was hematoxylin–eosin (HE)-stained to evaluate lesion type, size and number. Since atherosclerotic lesion development progresses over time, thereby increasing the size of the aortic arch, more sections were collected from the aortic arches of groups 2 (33 weeks of age) and 3 (45 weeks of age) than from arches of group 1 (13 weeks of age). Five HE-stained sections from each aortic arch were analysed in group 1, while in groups 2 and 3, 11–15 sections per aortic arch were analysed. The type of atherosclerotic lesion was determined according to the guidelines of the American Heart Association (Table 1) [17]. The size of the lesions was determined using a microscope coupled to a computer-assisted morphometry system (Quantimet 570, Leica, The Netherlands). For every lesion, the mean area was calculated and total lesion area was calculated as the sum of all mean areas. All sections were analysed by impartial investigators (inter-observer variability <10%), who were blinded with respect to the presence or absence of C. pneumoniae infection as well as to the time point of sacrifice.


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  		Table 1 Classification of atherosclerotic lesion types according to the Stary classification defined in 1995

 
2.6 Detection and evaluation of T-lymphocytes in atherosclerotic lesions
In order to detect T-lymphocytes in the atherosclerotic lesions, a rabbit-anti-CD3+ polyclonal antibody was used (Dako). After a blocking step with bovine serum albumin (BSA)/PBS 2% for aspecific binding, slides were incubated with the CD3+ polyclonal antibody for 60 min at a dilution of 1:200, followed by a second-step incubation with a biotinylated swine-anti-rabbit IgG (1:1000, Dako) for 30 min. Sections were then labelled with an alkaline phosphatase coupled ABC reagent (Dako) for 30 min. Alkaline phosphatase activity was visualised using Fast-red (Sigma, St. Louis, MO, USA). Rabbit-anti-rat IgG (Dako) was used as a negative control. For positive controls, tissue sections from mouse spleens were used. T-lymphocytes in the atherosclerotic lesion were counted and expressed as the number of T-lymphocytes in the plaque divided by the total plaque area. The investigator was blinded to the presence or absence of C. pneumoniae infection as well as to the time point of sacrifice.

2.7 Detection of C. pneumoniae antigens and anti-C. pneumoniae antibodies
In order to detect C. pneumoniae antigens, a Chlamydia genus-specific mouse monoclonal antibody, CF-2 (Washington Research Foundation, Seattle, WA, USA), was used [33]. This monoclonal is directed against chlamydial lipopolysacharide. For each mouse, a representative section with the highest lesion number and the most advanced lesion type, as analysed by the HE staining, was used for C. pneumoniae detection. The primary antibody was probed with goat-anti-mouse IgG conjugated to peroxidase (Dako, Glostrup, Denmark). To visualise the antibody binding, 3,3'-diaminobenzodine (DAB, Sigma) was used. Normal mouse ascitic fluid (Sigma–Aldrich, Zwijndrecht, The Netherlands) was used as negative control. Tissue sections from mock-infected mice were used as controls. For positive controls, sections of C. pneumoniae-infected HEp-2 cells were used, which were embedded in agarose, fixed overnight in 3.7% PBS-buffered formaldehyde and embedded in paraffin.

Anti-C. pneumoniae antibodies were measured with the indirect micro-immunofluorescence (MIF) technique using mouse plasma sample dilutions of 1:10 and 1:100 on antigen-coated slides (Labsystems, Helsinki, Finland). Goat-anti-mouse IgG conjugated to FITC (Sigma) was used as the secondary antibody. The presence of anti-C. pneumoniae antibodies was determined by two independent observers.

2.8 Assessment of lipid profile
Total plasma cholesterol and triglyceride concentrations were determined by a standard cholesterol oxidase method performed on a Beckman Synchron CX System [12].

2.9 Statistical analysis
The difference in lesion type between the C. pneumoniae and mock group was compared and analysed with the Chi-square test. Differences in lesion number and size were analysed with the Mann–Whitney U-test. To determine differences in T-cell influx in the lesions the Mann–Whitney U-test was used. Plasma lipids of the C. pneumoniae- and mock-infected groups were compared using Student’s two-tailed t-test. P<0.05 was considered statistically significant. Data are expressed as mean±S.E.M.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
To investigate the effect of C. pneumoniae infection on atherosclerotic lesion type, number and total lesion size, 64 Apo E3-Leiden mice were infected, sacrificed and analysed. Seven mice died before reaching the point of sacrifice, leaving 57 mice for analysis. The cause of death was unknown.

3.1 C. pneumoniae infection leads to more severe atherosclerotic lesions
Atherosclerotic lesions were found in the C. pneumoniae-infected group and in the mock-infected group. The type of lesion was analysed according to the histological criteria given in Table 1, which are based on the Stary classification defined in 1995 [17].

At 1 month pi, types 1, 2 and 3 lesions were found. The more advanced lesion types, 4, 5a and 5b, were present at 6 and 9 months pi and consisted of a fibrous cap surrounding a necrotic central core. The presence of these lesion types at these time points is in agreement with a previous report [18]. Type 6 lesions were never observed in any of the mice at any given time point pi.

The effect of C. pneumoniae infection on atherosclerotic lesion type is presented in Table 2. The complexity of the atherosclerotic lesion type was found to increase over time when lesion types of 1 month pi were compared with lesion types of 6 and 9 months pi. At 6 months pi, all lesion types, except type 1 lesions, were present, while at 9 months pi, types 1 and 2 lesions were absent. When the distribution of the lesion types in the C. pneumoniae-infected and mock-infected group was analysed, a significant shift in lesion type was observed from type 3 to type 4 at 6 months pi (P<0.05, Fig. 2), while at 9 months pi, a significant shift was seen from type 4 to type 5a (P<0.01, Fig. 3).


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  		Table 2 Atherosclerotic lesion types in C. pneumoniae- and mock-infected mice

 

Figure 2
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  		Fig. 2 Lesion types in the C. pneumoniae and mock groups 6 months post infection. The values are presented as the mean frequency±S.E.M. at which that type of lesion was present. *P=0.022.

 

Figure 3
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  		Fig. 3 Lesion types in the C. pneumoniae and mock groups 9 months post infection. The values are presented as the mean frequency±S.E.M. at which that type of lesion was present. *P=0.002.

 
3.2 C. pneumoniae infection has no effect on lesion size and number of lesions
The HE-stained sections used for classification of atherosclerotic lesion types were also used for determination of lesion area and number of lesions. Data on the total lesion area are presented in Table 2. No statistically significant differences in lesion area could be detected when the C. pneumoniae-infected and mock-infected groups were compared at the different time points.

The number of atherosclerotic lesions was analysed in all HE-stained sections. The number of lesions in the C. pneumoniae-infected and mock-infected groups was calculated at different time points pi. Data on lesion number are presented in Table 3. There were no significant differences in total lesion number between C. pneumoniae-infected and mock-infected mice at any time point pi.


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  		Table 3 Total lesion number in C. pneumoniae- and mock-infected groups at different time points pi. Data are expressed as mean±S.E.M.

 
3.3 C. pneumoniae infection results in an increased T-cell influx in atherosclerotic lesions
The effect of C. pneumoniae infection on the influx of T-cells was determined at every time point pi in atherosclerotic lesions of the C. pneumoniae-infected mice and the mock-infected mice. More specifically, at 1 month pi a significant increase in T-cell influx in the C. pneumoniae-infected atherosclerotic lesions was observed. Infection resulted in a significant increase in T-cells from 0.29 cells/µm2 in the mock group to 2.63 cells/µm2 in the C. pneumoniae group (P=0.0005, Fig. 4, 5Go). At 6 and 9 months pi, no effect of C. pneumoniae infection on the influx of T-cells was observed.


Figure 4
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  		Fig. 4 Number of T-cells in atherosclerotic lesions at 1 month pi. Data are presented as number of T-cells divided by total plaque area. *P=0.0005.

 

Figure 5
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  		Fig. 5 T-lymphocyte staining showing positive cells (arrows) in an atherosclerotic lesion of a C. pneumoniae-infected mouse 1 month pi.

 
3.4 Lipid profiles
At 1, 6 and 9 months pi, blood was collected from C. pneumoniae- and mock-infected mice for determination of plasma cholesterol and triglyceride levels (Table 4). When the C. pneumoniae and the mock groups were compared at one time point pi, no statistically significant difference was found in cholesterol and triglyceride plasma levels, suggesting that C. pneumoniae infection does not affect plasma lipid levels in the long term (data not shown).


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  		Table 4 Cholesterol and triglyceride levels in the C. pneumoniae- and mock-infected groups at different time points pi. Data are expressed as mean±S.E.M.

 
3.5 C. pneumoniae infection
All C. pneumoniae-infected mice seroconverted after inoculations with C. pneumoniae, indicating that all our inoculated mice had indeed been successfully infected. Anti-C. pneumoniae antibodies were found in all these mice at plasma dilutions of 1:10 and 1:100. No anti-C. pneumoniae antibodies were detected in plasma of mock-infected mice.

C. pneumoniae antigen was detected by immunohistochemistry staining in about 30% of the aortic arches of the infected animals at the time points 1, 6 and 9 months pi. C. pneumoniae antigen was mainly found in the subendothelial layer of the atherosclerotic lesions of these mice, as shown in Fig. 6. Since the percentage of positive C. pneumoniae antigen was equally divided over all lesion types, no preference of C. pneumoniae antigen for a specific atherosclerotic lesion type was observed (data not shown). As expected, mock-infected mice were all negative.


Figure 6
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  		Fig. 6 Chlamydia pneumoniae staining. Chlamydia pneumoniae was detected in a type 3 lesion of an infected mouse, 1 month post infection. Staining is concentrated in the subendothelial layer of the atherosclerotic lesion (see arrows). Magnification 200x.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
Infection with C. pneumoniae has been associated with the process of atherosclerosis [4,9], but whether infection influences the initiation or progression of atherosclerosis, or whether it plays a role in plaque (in)stability, has not been clarified. Most previously published experimental studies on C. pneumoniae infections have reported on the effect of infection on atherosclerotic lesion size [11,14,15,29]. No reports on the effect of C. pneumoniae infection on lesion type have been published. Since the type of atherosclerotic lesion may be a more important indicator for morbidity and mortality than lesion size or number, we evaluated the incidence of different atherosclerotic lesion types. In this study we demonstrate that C. pneumoniae infection leads to an accelerated formation of complex atherosclerotic lesions. At 6 and 9 months pi, an acceleration in lesion type development was seen, whereas at 1 month pi, no effect of C. pneumoniae infection on early lesion formation was observed. This is an intriguing finding, as it contributes to the suggestion that C. pneumoniae infection may promote progression [11,14–16,29] of the atherosclerotic process rather than its initiation [33]. The effect of C. pneumoniae on lesion formation seems to be the result of a long-standing process, since its effect is only found at later time points, 6 and 9 months pi. This suggests that an antigen-specific immune activation early after infection may contribute to the progression of atherosclerosis [1,6,9]. Since C. pneumoniae has been shown to infect macrophages and use them as a transport vehicle for dissemination [31] it may be speculated that C. pneumoniae enters the vascular wall in this way. Having entered the vascular wall, C. pneumoniae-infected macrophages may act as antigen-presenting cells, thereby attracting T-cells. Indeed, in the present study we demonstrated a markedly enhanced T-cell influx shortly after the first infection. Since activated T-cells are a powerful source of pro-atherogenic and pro-inflammatory cytokines, such as IFN-{gamma}, IL-2 and TNF-{alpha} [34], this may explain the observed acceleration of plaque development in the long term. On the other hand, T-cells themselves may also function as a transport vehicle for C. pneumoniae. Recently, it has been shown that C. pneumoniae can infect and multiply in T-cells [26]. Furthermore, a significantly higher proportion of C. pneumoniae-positive T-cells were observed in patients with coronary artery disease compared with healthy blood donors [35]. Future co-localisation studies may shed some light on where C. pneumoniae is exactly located in the vascular wall of our infected mice.

Besides atherosclerotic lesion type, we also determined the effect of C. pneumoniae infection on lesion size and number and found no difference between the C. pneumoniae- and mock-infected mice. This corresponds to findings in previous studies that also found no effect of C. pneumoniae infection on lesion size [36,37]. In contrast, other studies reporting an increase in lesion size after infection with C. pneumoniae have also been published [11,14]. The difference between these other studies and ours may result from differences in the C. pneumoniae strain, the inoculation schedule or the type of animal model used. LDLR knockout [11] and Apo E knockout mice [14,36,37] were the mice strains used by other groups. In the present study, Apo E3-Leiden mice were used [18]. It is known that different mouse strains vary in susceptibility to C. pneumoniae infection and the formation of atherosclerotic lesions [38]. Of the latter studies, only one reports elevated lipid levels after C. pneumoniae infection in LDLR knockout mice [11]. However, in the present study and in previous reports [14,36,37], no differences between infected and non-infected groups were observed in cholesterol or triglyceride levels, excluding this as a predominant factor.

In humans, the first association between C. pneumoniae infection, myocardial infarction and chronic heart disease was shown in patients with anti-C. pneumoniae antibodies by Saikku et al. [4]. Later, several other studies supported this finding, suggesting a correlation between C. pneumoniae infection and cardiovascular disease [8,9,39,40]. However, the correlation between the presence of anti-C. pneumoniae antibodies and C. pneumoniae antigen in atherosclerotic lesions is questionable [12,15,33,39,41]. A similar finding was also observed in our study, since all infected mice were positive for anti-C. pneumoniae antibodies, while C. pneumoniae antigen was detected in only one-third of the aortic arches. This discrepancy may be explained by the notion that immunohistochemical staining has a limited sensitivity for detecting C. pneumoniae in vascular tissue. A second explanation could be that anti-C. pneumoniae antibody in plasma and C. pneumoniae antigen in aortic tissue do not necessarily have to be present at the same time [36].

In summary, this study demonstrates that C. pneumoniae infections result in an acceleration of the formation of complex atherosclerotic lesions in Apo E3-Leiden mice, either by increasing the influx of activated T-cells to the vascular wall by infected and antigen-presenting macrophages, or by direct activation of T-cells.

Time for primary review 25 days.


   Acknowledgements
 
This study was supported by a grant from Pfizer BV, Capelle aan de Ijssel, The Netherlands. We thank L. Kupers, Limburg University, Diepenbeek, Belgium, for assistance with animal experiments, P. Terporten, Department of Medical Microbiology, Maastricht University, The Netherlands, for assistance with the statistical analysis of the data, and C. Vink, Department of Medical Microbiology, for a critical review of the manuscript and valuable discussions.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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