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Published in final edited form as: Cell Immunol. 2015 Jan 30;294(1):25–32. doi: 10.1016/j.cellimm.2015.01.007

A single infection with Chlamydia pneumoniae is sufficient to exacerbate atherosclerosis in ApoE deficient mice

Rosalinda Sorrentino a,b,2, Atilla Yilmaz c,2, Katja Schubert c, Timothy R Crother a, Aldo Pinto b, Kenichi Shimada a, Moshe Arditi a,1, Shuang Chen a,1
PMCID: PMC4391498  NIHMSID: NIHMS659760  PMID: 25666507

Abstract

Several studies have demonstrated a strong link between Chlamydia pneumoniae (Cp) infection and atherosclerosis progression/exacerbation. Here, we try to understand whether a single administration of Cp could exacerbate atherosclerosis. Apoe−/− mice were intranasally infected with Cp followed by a high fat diet. Mice were sacrificed at different time points after Cp infection to monitor the development of the atheroma. Cp infection increased lipid content in the aortic sinus of Apoe−/− mice starting from 8 weeks. This was associated with increased numbers of active myeloid Dendritic cells and plasmacytoid DCs which were co-localized with T-cells in the atherosclerotic plaque. The serum levels of IFN-γ showed a Th1-like environment typical of atherosclerosis. In conclusion, we demonstrate that one dose of Cp. could exacerbate atherosclerotic lesion development, triggering innate immune cell accumulation early on that allowed the involvement of Th1-like cells in the exacerbation of the atherosclerotic plaque at later time points.

Keywords: Atherosclerosis, C. pneumoniae, innate immunity

1. Introduction

The role of chronic arterial inflammation and immune effector cells and their products in the development and progression of atherosclerosis is now widely accepted[1, 2]. However, the extent to which chronic inflammation contributes to atherosclerotic development and how pathogens might influence this process is much less clear. More often, serological, histological, and experimental evidence has pointed to a significant role for Chlamydia pneumoniae (C. pneumoniae), an obligate intracellular Gram-negative bacteria, in the development of atherosclerosis[3–5]. There is a strong link between Chlamydia pneumoniae infection and atherosclerosis progression/exacerbation [4–7]. In support, C. pneumoniae has been isolated from the coronary arteries of patients with acute coronary syndrome [8] and from carotids and aortas[9, 10]. Exploring the mechanisms underlying the atherogenic exacerbation after C. pneumoniae infection can provide new mechanistic insights into the pathogenesis of atherosclerosis, and a promising avenue for novel therapies for chronic cardiovascular inflammatory disorders.

C. pneumoniae infection-induced acceleration of atherosclerosis in the Apoe−/− (C57BL/6) mice has been well established by our [11] and several other groups [5, 12] Our previous data showed that hypercholesterolemic mice infected with C. pneumoniae presented higher lipid content in the aortic sinus and aortas than in uninfected mice [5]. This was Toll-like receptor (TLR)-2- and TLR4-dependent [5], which are pattern recognition receptors for innate immune cell activation [2]. Because innate immune cells express TLRs, it is plausible that the acceleration of atherosclerosis by C. pneumoniae is due to the recognition of the bacterium or molecules derived from the pathogen by TLRs on macrophages and dendritic cells (DCs). DCs are found to populate the aortic sinus of atherosclerotic patients [13]. It was postulated that the interaction between the resident DCs and T cells that migrate from the adventitia to the intima is critical for the focal inflammation in the aortic sinus that contributes to atherosclerotic plaque formation and stability [1]. The arrival of C. pneumoniae from the circulation to the inflamed tissue can both induce the first signs of inflammation that can be the basis of plaque formation, and exacerbate the already existing inflammatory process in the aortic sinus [11].

Despite reasonable molecular and serologic evidence, antibiotic trials failed to improve clinical outcomes. The main arguments countering the negative results focus on C. pneumoniae being refractory to the antibiotics, the patients in antibiotic trails already reached an irreversible stage, and the bacterium might be acting by a “hit-and-run” mechanism [14]. Indeed, independent studies provide direct evidence that C. pneumoniae establishes persistent infection in vivo, which is refractory to antibiotic intervention [15]. The lack of protective effect in these antibiotic trials calls for further investigations into its pathological mechanisms.

Thus, the aim of our present study was to understand the implication of C. pneumoniae in atherosclerotic acceleration in Apoe−/− mice using a single inoculation intranasally instead of the typical chronic infection model with multiple inoculations. We found that even a single inoculation of C. pneumoniae accelerated the accumulation of lipids into the aortic sinus at early time points. This was associated with the presence of activated myeloid DCs (mDCs) and by the presence of plasmacytoid DCs (pDCs). The presence of active mDCs and pDCs in the atherosclerotic plaques of C. pneumoniae-infected mice suggest a Th1-like environment in the lesions, further pointing out the relevance of these immune cells in the atherosclerotic process. We now show for the first time the time course of C. pneumoniae-induced acceleration of atherosclerosis in the ApoE-deficient hypercholesterolemic mouse model.

2. Materials and Methods

2.1 Ethics Statement

All animal experiments were performed according to the guidelines and approved protocol (IACUC Protocol #2096) of the Cedars-Sinai Medical Center Institutional Animal Care and Use Committee. Cedars-Sinai Medical Center is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC International) and abides by all applicable laws governing the use of laboratory animals. Laboratory animals are maintained in accordance with the applicable portions of the Animal Welfare Act and the guidelines prescribed in the DHHS publication, Guide for the Care and Use of Laboratory Animals.

2.2 Experimental Design

Homozygous Apoe−/− mice were generated as described previously [16]. Mice were fed with a high fat diet containing 0.15% cholesterol (Harlan Teklad) starting at 8 wk of age before infection and continuing until sacrifice (Figure 1A). Mice were maintained under specific pathogen-free conditions. Male and female Apoe−/− mice were used, but the vast majority of the animals in each group were male (>80% in each group investigated). All experiments were approved by the Cedars-Sinai Medical Center Institutional Animal Care and Use Committee and performed according to institutional guidelines.

Figure 1.

Figure 1

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C. pneumoniae progressively induces acceleration of atherosclerosis in Apoe−/− mice. A. Apoe−/− mice were infected intranasally with C. pneumoniae (5×104 IFU/mouse) once and fed with high fat diet until sacrifice at 8, 12, and 16 weeks after the bacterial inoculation. Lipid Contents were increased starting at 8 weeks (B and B′) up to 16 weeks (D and D′). Oil Red O staining was performed and data was expressed as percentage of lipid content relative to the total area in the aortic sinus to show plaque formation. Data are presented as mean values ± SEM, n=7/group. Statistically significant differences are denoted by * and ** indicating p<0.05 and p<0.01, respectively, as determined by Student’s t test.

2.3 C. pneumoniae infection

C. pneumoniae strain CM-1 (American Type Culture Collection) was propagated in Hep-2 cells as previously described [11]. Eight weeks old mice were anesthetized with isoflurane before intranasal application of 5 × 104 inclusion-forming units (IFU) of C. pneumoniae suspended in sucrose-phosphate-glutamate buffer (40 μl per nostril) per mouse. The intranasal administration of the buffer alone was performed as a negative control. Following infection, mice were fed on high fat diet and sacrificed at either 8-12-16 weeks later (Figure 1A).

2.4 Assessment of atherosclerotic lesions in the aorta and aortic sinus

Mice were anesthetized with isoflurane before the hearts were excised. Hearts were embedded in OCT compound (Tissue-Tek; Sakura), and cross-sections of the aortic sinus were stained with Oil Red O. Lesions areas were quantified with Image-Pro Plus (Media Cybernetics). Image analysis was performed by a trained observer blinded to the genotypes of mice as previously described [16]. The lesion area and lipid-stained areas in the aortic sinus were measured. The lesion size in the aortic sinus was expressed as “aortic sinus lesion area”, and the plaque composition in the aortic sinus was expressed as “% lipid content aortic sinus” (percentage of plaque area staining with lipid). Five serial sections per animal were analyzed with the resulting data were averaged and presented as average aortic sinus lesion area.

Immunohistochemical staining and quantification of immune cells in the aortic sinus: Frozen heart sections were analyzed for infiltration of activated DCs using rat anti-mouse MIDC-8 Ab (Serotec) and a catalyzed signal amplification (CSA) kit from DakoCytomation as we described previously[17]. An isotype control (IgG2a; Serotec) was used to demonstrate specificity of staining. The rat mAb MIDC-8, which binds a still unidentified Ag within intracellular granules of mature myeloid DCs[18, 19], has been used in multiple studies as a specific marker of mature (activated) myeloid DCs[18–20], including in murine atherosclerosis in Apoe-null mice [21]. The stained DCs (MIDC8+) were determined in three representative aortic sinus Digital images were taken at a magnification of x300 with a charge coupled device camera (Nikon DXM 1200) of sections of the aortic root. Stained cells were quantified by computer-assisted histomorphometry (Image J). For each analysis, the color threshold for stained cells was manually adjusted in the images until the computerized detection matched visual interpretation. The numbers of immunostained cells were digitally counted in the defined area (0.20 μm2) of the corresponding sections. For each cell type, the mean cell number was calculated out of the corresponding three consecutive sections for each animal. Microscopic analyses were performed independently by two different investigators, and intra- and coefficients of variabilities were less than 10%.

Similarly, pDCs and T cells were identified by PDCA1 Ab (MiltenyiBiotec, Germany) and CD3 Ab (Serotec, Germany), respectively. Additionally, Tregs were stained by FoxP3 Ab. The numbers of activated DCs, pDCs, and T cells were quantified using Image-Pro Plus software (Media Cybernetics)

2.5 Lipid profiles

Total cholesterol concentrations were determined in duplicate by using a colorimetric assay (infinity cholesterol reagent’ Sigma Diagnostics). Triglyceride concentrations were determined by using the L-type triglyceride H assay according to the manufacturer’s instructions (Wako Chemicals). These assays were performed on serum obtained from blood withdrawn at the time of sacrifice from mice that had undergone an overnight fast.

2.6 Serum levels of cytokines

Serum concentrations of IFN-γ and IL-10,(eBiosciences) were detected by means of ELISA according to the manufacturer’s instructions.

2.7 Statistical analysis

Results are expressed as means ± SEM. Changes observed in treated groups compared with controls were analyzed using Student’s t test or Mann-Whitney where appropriate. p values less than 0.05 were considered statistically significant. *p<0.05, **p<0.01, ***p<0.001.

3. Results

3.1 C. pneumoniae exacerbates atherosclerotic lesions in a time-dependent manner

In our previous study, we demonstrated that C. pneumoniae infection accelerates atherosclerotic lesions in Apoe−/− mice [11]. However, this traditional experimental model was based on repeated infections with C. pneumoniae for three consecutive weeks into mice followed by high fat diet. In this study, we aimed to evaluate whether a single intranasal administration of this bacteria could still promote atherosclerotic exacerbation in a hypercholesterolemic mouse model. Accordingly, ApoE−/− mice (8 weeks of age) were intranasally infected with 5×104 IFU/ml of C. pneumoniae (Figure 1A), put on a high fat diet, and then sacrificed at different time points to monitor the kinetics of development of the atherosclerotic plaque. The percentage of lipid content in the lesion area of the aortic sinus was increased in Apoe−/− mice treated with C. pneumoniae compared to mock-treated mice at 8 (Figure 1B, B′ and B″), 12 (Figure 1C, C′ and C″), and 16 weeks (Figure 1D, 1D′ and D″). The lipid content increased approximately 30% over each time period for both groups. However, the lipid content increased 74% (Figure 1B′) and 80% (Figure 1C′) after the treatment of Apoe−/− mice with C. pneumoniae compared to mock injection. Similarly, the same data was obtained with mice treated with C. pneumoniae and then sacrificed at 16 and more weeks (Fig. 1D′), agreeing with previous studies despite only infecting the mice one time with C. pneumoniae. Serum cholesterol and plasma triglyceride concentrations were similar in C. pneumoniae-infected or uninfected mice (Table 1) as already reported[11]. Thus C. pneumoniae-induced exacerbation of atherosclerotic plaques was progressively observed in Apoe−/− mice after a single exposure to the bacterium, with detectable acceleration as early as 8 weeks after of high fat diet. We did not observe C. pneumoniae inclusions in the aortic sinus at any time point post infection (data not shown).

Table 1.

Total cholesterol and Triglycerides level in serum (mg/dI)

TG (mg/dL) TC(mg/dL)
Cp− Cp+ Cp− Cp+
8 wks 61.07 63.8 1266.5 1129.8
12 wks 70.99 77.29 1278.3 1240.4
>16 wks 82.3 115.79 1437.3 1448.7
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3.2 Innate Immune Cells contribute to C. pneumoniae-induced exacerbation of atherosclerotic plaques

We have reported that C. pneumoniae induces acceleration of atherosclerosis in a TLR/MyD88-dependent manner[11]. As we have previously published, C. pneumoniae infection significantly increased the macrophage accumulation in aortic root plaques [22]. To further understand the role of the innate immunity in C. pneumoniae infection-induced acceleration, we analyzed the kinetics of the accumulation of activated myeloid Dendritic cells (mDCs) and plasmacytoid Dendritic cells (pDCs) into the aortic sinus of C. pneumoniae-infected and uninfected Apoe−/− mice. We performed immunohistochemical staining using MIDC-8 Ab to quantitatively measure numbers of mature, activated mDCs (15). Infection of Apoe−/− mice with C. pneumoniae increased MIDC-8 positive cells in the aortic sinus (Figure 2A). This significant increase was progressively observed at 8 (Figure 2B), 12 (Figure 2C), and 16 weeks (Figure 2D) after C. pneumoniae infection compared to mock inoculation. These results suggest that some of the effects of C. pneumoniae-induced acceleration of the atherosclerotic plaque is mediated through increased dendritic cells present immediately after infection in the plaques. We also assessed for pDCs using PDCA-1 as a marker (Figure 3A). We observed a significant increase in pDC numbers at 8 weeks (Figure 3B) and 16 weeks (Figure 3D) after C. pneumoniae infection compared to uninfected Apoe−/− mice while they trended towards an increase at 12 weeks. Both mDCs and pDCs were recruited to the aortic sinus and their localization started from the intima at 8 weeks up to the adventitia at 12 weeks after C. pneumoniae infection (Figure 2A and Figure 3A). Taken together, these results further support the hypothesis that mDCs and pDCs both play important roles in C. pneumoniae-induced inflammation accelerates the atherosclerotic plaque formation. at the aortic sinus.

Figure 2.

Figure 2

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C. pneumoniae infection progressively increased the number of active mDCs in the aortic sinus of Apoe−/− mice. A. MIDC-8-positive staining in the aortic sinus of C. pneumoniae infected (CP+) and non-infected (CP−) Apoe−/− mice. Isotype control did not show any positive staining. B, C, D. Quantification of active mDCs in infected and non-infected Apoe−/− mice after 8 weeks (B), 12 weeks (C) and more than 16 weeks (D) after C. pneumoniae or mock inoculation. Data are presented as mean values ± SEM, n=6/group. Statistically significant differences are denoted by * indicating p<0.05 as determined by Student’s t test.

Figure 3.

Figure 3

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C. pneumoniae infection progressively increased the number of pDCs in the aortic sinus of Apoe−/− mice. A. PDCA-1-positive staining in the aortic sinus of C. pneumoniae infected (CP+) and non-infected (CP−) Apoe−/− mice. Isotype control did not show any positive staining. B, C, D. Quantification of pDCs in infected and non-infected Apoe−/− mice after 8 weeks (B), 12 weeks (C) and more than 16 weeks (D) after C. pneumoniae or mock inoculation. Data are presented as mean values ± SEM, n=6/group. Statistically significant differences are denoted by * indicating p<0.05 as determined by Student’s t test.

3.3 Influence of the adaptive immune system in C. pneumoniae-induced exacerbation of atherosclerotic plaques

Atherosclerotic plaques are populated by T cells and the migration of these cells from the adventitia to the intima[13, 17] determines the role of these cells in the pathology. 8 weeks after high fat diet C. pneumoniae-infected mice had a significant increase in T-cells present in the aortic sinus lesions compared to mock infected controls (Figure 4A). T-cells are also significantly increased in the infected group at weeks 8 and 16 (Figure 4B, D). While there were no significant differences between the groups at weeks 12, both infected groups did trend towards an increase in T-cells (Figure 4C). Moreover, these cells were distributed over the whole aortic sinus starting at 8 weeks up to 16 weeks (Figure 4A). These data correlate well with the presence of mDCs (Figure 2) and pDCs (Figure 3) into the aortic sinus and more specifically in the intima of the aortic sinus.

Figure 4.

Figure 4

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C. pneumoniae infection progressively increased the number of CD3+ T cells in the aortic sinus of Apoe−/− mice. A. CD3-positive staining in the aortic sinus of C. pneumoniae infected (CP+) and non-infected (CP−) Apoe−/− mice. Isotype control did not show any positive staining. B, C, D. Quantification of T cells in infected and non-infected Apoe−/− mice after 8 weeks (B), 12 weeks (C) and more than 16 weeks (D) after C. pneumoniae or mock inoculation. Data are presented as mean values ± SEM, n=6/group. Statistically significant differences are denoted by * indicating p<0.05 as determined by Student’s t test.

Additionally, C. pneumoniae infection increased the number of FoxP3+ cells (Regulatory T-cells (Tregs) in the aortic sinus starting at 8 weeks post infection compared with non-infected Apoe−/− mice (Figure 5B). These data correlated with the increased influx of CD3+ T cells into the aortic sinus of C. pneumoniae treated ApoE−/− mice (Figure 4). The amount of Treg cells was significantly increased at 16 weeks post infection compared with control mice (Figure 5A and 5D), indicating a possible response by the immune system to curb inflammation. To confirm the nature of T cells in C. pneumoniae-infected Apoe−/− mice and to understand their systemic impact, we analyzed the serum levels of IFN-γ and IL-10. C. pneumoniae infection resulted in an increase in IFN-γ (Figure 6A) and IL-12 (Figure 6C), but not of IL-10 (Figure 6B), suggesting a Th1-like environment in infected mice, as already reported.

Figure 5.

Figure 5

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C. pneumoniae infection increased the number of FoxP3+ T cells in the aortic sinus of Apoe−/−mice. A. FoxP3-positive staining in the aortic sinus of C. pneumoniae infected (CP+) and non-infected (CP−) Apoe−/− mice. Isotype control did not show any positive staining. B, C, D. Quantification of Treg cells in infected and non-infected Apoe−/− mice after 8 weeks 1(B), 12 weeks (C), and 16 weeks (D) after C. pneumoniae or mock inoculation. Data are presented as mean values ± SEM, n=6/group. Statistically significant differences are denoted by * indicating p<0.05 as determined by Student’s t test.

Figure 6.

Figure 6

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C. pneumoniae infection increased circulating levels of IFN-γ (A), IL-12 (C) but not of IL-10 (B). Data are presented as mean values ± SEM, n=6/group. Statistically significant differences are denoted by * indicating p<0.05 as determined by Student’s t test.

4. Discussion

A large number of studies from independent laboratories have demonstrated the presence of C. pneumoniae within human atherosclerotic tissue by detection of antigen and/or DNA[15]. Growing evidence has linked the development/progression of atherosclerosis to various infectious agents, including C. pneumoniae[1]. In this study, we report that C. pneumoniae progressively induces atherosclerotic plaque exacerbation even after only a single administration of the bacterium. Consistent with our published data [11], C. pneumoniae infection led to an increase in lipid content in the atherosclerotic plaques of Apoe−/− mice. Atheroma exacerbation was associated with greater numbers of activated DCs and increased accumulation of pDCs, T cells, and Tregs into the lesion area of C. pneumoniae-infected Apoe−/− mice compared to non-infected littermates. Our previous studies were conducted on 8 week old Apoe−/− mice, which were infected three times (weekly) with C. pneumoniae intranasally, followed by a high fat diet for 4 months [11]. In this study, instead, Apoe−/− mice were infected only once and then sacrificed at different time points in order to observe the effect of a single administration of the bacterium on the progression of atherosclerosis and to monitor the immune cell accumulation to the atherosclerotic plaques.

Several investigators proposed that the extent of the infectious burden from all pathogenic sources is a key step for their impact on the atherosclerotic disease process[23]. It has been strongly suggested that infections from certain pathogens, such as C. pneumoniae[3–5, 11] and herpes viruses (HSV-1, EBV, and CMV) are associated with atherosclerosis[2]. These pathogens are all obligate intracellular organisms that are able to elicit a persistent lifelong immune response described as the ‘Pathogen burden’ and are associated with a high risk of cardiovascular diseases[23]. Here we demonstrated that even a single low dose of C. pneumonia-induced lung infection could still lead to atherosclerotic plaque exacerbation in the presence of hypercholesterolemia in a time-dependent manner. Although other studies on infection models demonstrated the effect of repeated administration of different pathogens on atherosclerosis development[5–7], this study demonstrates for the first time, that an acute C. pneumoniae infection following a single inoculation of Apoe−/− mice is able to accelerate the atherosclerotic process by inducing an increase in lipid content associated with an increased number of activated DCs and the presence of pDCs in the aortic sinus.

The innate immune system is fundamentally required for the induction and the progression of atherosclerosis in mice[1, 2] and humans[24]. In particular, we observed that C. pneumoniae-induced acceleration of atherosclerosis was accompanied by increased numbers of activated DCs accumulating in the plaques of infected Apoe−/− mice compared to the non-infected littermates. Concomitantly, T cells were similarly detected at 8 weeks and the migration of these cells from the adventitia to the intima, where DCs were located, implied the activation of T cells by DCs. This phenomenon was also reported in human atheroma plaques[17, 25]. Indeed, there is growing evidence that immune cells can be recruited not only from the vascular lumen into the intima (inside-out) but also via the vasa vasorum of the adventitia through the neovascularization (outside-in) Since the lipid content in plasma was not different between the infected and uninfected groups, any increase in DCs in the atheroma is likely due to the infection itself as the activation of DCs by C. pneumoniae has been widely described by our group and others[5–7, 11].

Molecular motifs deriving from C. pneumoniae are detected by Toll-like receptors (TLRs). In our previous study we showed that TLR2 and TLR4 can mediate the pro-atherogenic effects of C. pneumoniae infection. Apoe/TLR2, Apoe/TLR4 double as well as Apoe/MyD88 knockout mice were protected from C. pneumoniae-induced atherosclerosis exacerbation. Moreover, we showed that C. pneumoniae induced a dose-dependent release of GM-CSF from infected primary aortic cells as well as from the aortic tissue[11]. GM-CSF plays a key role for DC differentiation[26] and migration[26] to the lesions of Ldlr−/− mice. These latter studies found that Ldlr/GM-CSF double knockout mice had diminished lesion size and decreased DCs accumulation in the atherosclerotic plaques [26]. In agreement with prior studies, our current study revealed the presence of DCs at early time points after C. pneumoniae infection, implying a fundamental role for DCs in C. pneumoniae-induced atherosclerosis acceleration.

Importantly, pDCs, were also increased in the atherosclerotic plaques after C. pneumoniae infection. These cells were located in the intima closely with mDCs and to T cells, however, the role of pDCs in atherosclerosis remains controversial. Some authors reported that pDCs populated human atherosclerotic plaques [27] and facilitated lesion exacerbation in an type I IFN-dependent manner[28, 29]. The mechanism underlying pDC-induced atherosclerosis exacerbation was ascribed to the release of TNF-alpha related apoptosis ligand (TRAIL), which is implicated in apoptosis[28, 29]. The release of TRAIL by pDCs favored atheroma formation. In contrast, others support the hypothesis that pDCs suppress atherosclerotic lesion formation[30] via the dampening of T-cell proliferation and activity [30]. Our paper is the first to our knowledge to show that C. pneumoniae infection allows pDCs to migrate towards the inflamed aortic sinus of Apoe−/− mice compared to the uninfected littermates. These cells were increased in the aortic sinus starting from 8 weeks up to 16 weeks after C. pneumoniae infection. In conjunction with the higher serum levels of IFN-γ compared to IL-10 levels, we propose that the collaboration between active mDCs and pDCs favors a Th1-like environment after C. pneumoniae infection. In this scenario pDCs may contribute as interferon-producing cells to induce Th1-skewing that is typical of atherosclerotic lesions[2]. Moreover, it is still not known whether C. pneumoniae can directly or indirectly be sensed by pDCs. In a basic infection mouse model, we also found that C. pneumoniae can induce the accumulation of inflammatory pDCs to the lung[31] suggesting a potential recognition of C. pneumoniae-derived motifs by pDCs. To date, pDCs can modulate adaptive immunity by either inducing a pro-inflammatory or an anti-inflammatory scenario. Type I and II IFNs and IL-12 production are at the basis of the pro-inflammatory activity of pDCs [27]. However, several mechanisms have been proposed for pDC-mediated immune-suppression under pathological conditions [32]. Both cell-cell contact and the humoral arm participate in pDC-induced tolerance. pDCs can promote indoleamine-2,3-dyoxigenase activity[33] and the release of suppressive factors[33]. Moreover, the direct contact of pDCs with Treg can also alter the pro-inflammatory scenario to an immune-suppressive environment[30, 34]. We did find a large increase in Treg numbers in the aortic sinus at 16 weeks, indicating a possible attempt by the immune system to curb inflammation. A caveat to these studies is that we used anti-pDCA1 to identify pDCs in the lesions. However, during inflammation, pDCA1 can be upregulated in other immune cells, although to a lesser degree that pDCs ([35]). Thus it is possible that other immune cell types may be positively staining in the lesions for pDCA. Although, our data still need further investigation to prove a direct role of pDCs in our experimental model, it is likely that in the first place pDCs induce a Th1-like bias that over time is attempted to be controlled by an influx of Tregs.

In conclusion, this study shows that a single administration of C. pneumoniae is still able to accelerate atherosclerotic lesions via the activation of both innate and adaptive immune cells with a Th1 skew. Moreover, the acceleration of atherosclerotic lesions is progressively observed at early time points, implying that the recognition of the bacterium by the innate immune arm leads to an adaptive immune response that contributes to the systemic inflammation, which facilitates atherosclerosis progression. It is also possible that the recognition of the bacterium at the level of the aortic sinus can contribute to the activation of the resident innate immune cells that then amplifies a systemic inflammation that accelerates the atherosclerotic process.

Highlights.

  • A single administration of Chlamydia pneumoniae exacerbates atherosclerosis development in Apoe−/− mice.

  • Myeloid Dendritic cells, plasmacytoid DCs, and T cells are increased in the lesion of this Chlamydia pneumoniae infected model.

  • The exacerbation of atheroma development is mediated by a Th1-like environment after Chlamydia pneumoniae infection.

Acknowledgments

This work was supported by National Institutes of Health Grants HL66436 (to MA); HL 111483 (to SC) and AHA 2060145 (to SC). We thank Sun, P. for technical assistance.

Abbreviations

C. pneumoniae

Chlamydia pneumonia

mDCs

myeloid dendritic cells

pDCs

plasmacytoid dendritic cells

Footnotes

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