Chronic Immune Reactivity Against Persisting Microbial Antigen in the Vasculature Exacerbates Atherosclerotic Lesion Formation

【摘要】  Objective— The purpose of this study was to examine the relative contribution of different immunopathological mechanisms during murine cytomegalovirus (MCMV)-mediated acceleration of atheroma formation in apolipoprotein E–deficient (apoE –/– ) mice.

Methods and Results— To distinguish between the effects of systemic activation and cognate immune reactivity against a pathogen-derived persisting antigen in the vasculature, we used hypercholesterolemic transgenic mice constitutively expressing the β-galactosidase (β-gal) transgene in the cardiovascular system (apoE –/– x SM-LacZ). After infection with β-gal–recombinant MCMV-LacZ, apoE –/–, and apoE –/– x SM-LacZ mice mounted comparable cellular immune responses against the virus. β-gal–specific CD8 + T cells expanded rapidly and remained detectable for at least 100 days in both mouse strains. However, compared with apoE –/– mice, apoE –/– x SM-LacZ mice developed drastically accelerated atherosclerosis. Moreover, atherosclerotic lesions in MCMV-LacZ–infected apoE –/– x SM-LacZ but not apoE –/– mice were associated with pronounced inflammatory infiltrates.

Conclusions— Taken together, our data indicate that chronic immune reactivity against pathogen-derived antigens persisting in the vasculature significantly exacerbates atherogenesis.

This study demonstrates that T cell responses directed against persisting microbial antigen within the vasculature favor the development of an inflammatory environment that is important for the acceleration of atherosclerotic lesion development.

【关键词】  cytomegalovirus atherosclerosis inflammation immunopathology coronary heart disease

Introduction

Chronic inflammatory processes in the vascular wall are crucial for initiation and perpetuation of atherosclerotic lesions. 1 It appears that it is the "infectious burden", ie, the overall impact of repeated or chronic infections with multiple pathogens, 2 that determines the extent of atherosclerosis and clinical prognosis. 3,4 Epidemiological and experimental evidence indicates that herpesviruses represent important viral pathogens that elicit arterial inflammation and may thereby exacerbate atherosclerotic disease. Seroepidemiological studies have shown a link between human cytomegalovirus (HCMV) infection and atherosclerosis. 5,6 Furthermore, detection of HCMV DNA in atherosclerotic lesions of patients with coronary artery disease 7,8 suggests that HCMV infection may impair vascular functions. HCMV infection of cells in the vascular wall may directly contribute to neointima formation through the viral US28 gene product which functions as a chemokine receptor and enhances the migration of smooth muscle cells in response to inflammatory chemokines. 9 Likewise, the chemokine receptor M33 of the murine CMV (MCMV) is essential for MCMV-induced migration of vascular smooth muscle cells 10 indicating that vascular integrity can be altered through virus-intrinsic factors.

In addition, various infection-associated immunopathological mechanisms impinge on the atherosclerotic process. 11 Molecular similarities ("mimicry") between microbial and host proteins, as found in the structurally related human and chlamydial heat shock proteins (HSP60/65), precipitate inflammatory reactions in atherosclerotic lesions. 12 General immune activation with systemic ("bystander") effects on the vascular wall can be triggered by a MCMV infection-associated increase in IFN serum levels leading to the secretion of proinflammatory cytokines such as monocyte chemoattractant protein-1 (MCP-1) by endothelial cells. 13 It is possible that such processes foster the recruitment of monocytes/macrophages and T cells to atherosclerotic lesions. 14 It has been argued that the transient induction of bystander cytokines during the first 2 weeks after MCMV infection of apoE –/– mice may be crucial for MCMV-mediated acceleration of atherosclerosis. 15,16 However, systemic immune activation by generalized herpesvirus simplex virus 1 infection seems not to be sufficient to aggravate lesion formation in hypercholesterolemic apoE –/– mice. 17 It appears that it is rather the specific tropism of herpesviruses for cells of the vascular wall that determines the enhancement of atherogenesis in apoE –/– mice after herpesvirus infection. 17,18 Therefore, an issue central to the role of murine and human cytomegaloviruses in atherogenesis is the relative contribution of the different immunopathological mechanisms ("bystander activation" versus "reactivity against persisting antigens") during the development of atherosclerotic lesions.

In the present study we used a well-defined mouse model of cardiovascular immunopathology 19 to distinguish between MCMV infection-associated systemic immune activation and specific immune reactivity directed against a persisting viral antigen in the vasculature. In SM-LacZ mice, the β-galactosidase (β-gal) antigen is expressed in arterial smooth muscle cells. 20 The peripherally expressed antigen is ignored by T cells unless the antigen is efficiently presented in secondary lymphoid organs. 21 During infection with β-gal recombinant MCMV (MCMV-LacZ) the vascular β-gal transgene in SM-LacZ mice functions therefore as a pathogen-derived antigen that persists in the arterial wall. Infection of hypercholesterolemic apoE –/– x SM-LacZ mice with MCMV-LacZ revealed that virus-induced T cell responses directed against the transgenic β-gal antigen within the vasculature favor the development of an inflammatory environment that is important for the acceleration of atherosclerotic lesion development.

Materials and Methods

Isolation of Arterial Smooth Muscle Cells

Aortas from C57BL/6, SM-lacZ, and Fas-KO mice were cut into small pieces and digested with collagenase type II (2 mg/mL, dissolved in DMEM; Sigma). After incubation for 45 minutes at 37°C, single cell suspensions were prepared by passing through a syringe with a 21G needle. Cells were washed twice with DMEM 10% FCS and plated onto 6-well plates (cell suspension from 1 aorta into 2 wells). After 4-hour incubation at 37°C, nonadherent cells were removed by washing with medium and the remaining cells were cultured further for 10 days. More than 90% of the cells stained positive for smooth muscle actin (not shown).

Immunohistology

Freshly removed organs were immersed in HBSS and snap-frozen in liquid nitrogen (LN 2 ). Frozen tissue sections were cut in a cryostat and fixed in acetone for 10 minutes. Sections were incubated with antibodies against β-gal (MP Biomedicals,), CD8 (clone YTS169.4.2), CD4 (YTS191.1.2), or F4/80 (Biomedicals AG, clone BM8) followed by goat anti-rat Ig (Caltag Labs) and alkaline phosphatase-labeled donkey anti-goat Ig (Jackson ImmunoResearch Labs). Alkaline phophatase was visualized by using AS-BI phosphate/New Fuchsin, and sections were counterstained with hemalum. For the quantitative evaluation of atherosclerotic lesions, 5 to 10 serial cross-sections through the aortic origin, beginning with the appearance of all 3 valve cusps, were stained with Sudan Red, counterstained with hemalum, and measured by using a Leica DM R microscope, Leica DC300 FX camera and Leica IM1000 (version 1.20) computer-aided morphometry software. The average lesion size for each mouse was calculated.

For semiquantitative assessment of inflammatory alterations in atherosclerotic lesions, sections were evaluated in a blinded fashion by 2 observers using the following criteria: grade 0, no infiltration; grade 1, confined minor infiltration (foci of <20 cells) in the perivascular space; grade 2, confined 20 cells) in the perivascular space and/or within the intimal lesion; grade 3, multiple clusters 100 cells) in the perivascular space; 100 cells) in the perivascular space and within the intimal lesion. Average severity has been calculated for MCMV-LacZ–infected hypercholesterolemic mice. Infiltration with CD8+ T cells was enumerated on 3 sections per mouse covering the area of the coronary artery bifurcations.

For β-gal staining in whole tissue mounts, aortic arches were prepared from C57BL/6 and SM-lacZ mice and immersed in PBS 2 mmol/L MgCl 2. After fixation in PBS containing 0.5% glutaraldehyde and 2 mmol/L MgCL 2 for 1 hour, tissues were rinsed in PBS. β-gal activity was revealed by incubation for 2 to 4 hour at 37°C in X-Gal buffer (5 mmol/L potassium ferrocyanide, 5 mmol/L potassium ferricyanide, 2 mmol/L MgCl 2 and 1 mg/mL X-Gal in PBS).

Please see supplemental materials, available online at http://atvb.ahajournals.org, for additional Materials and Methods.

Results

CD8 + T Cell Reactivity in the Course of MCMV Infection

In a first set of experiments, we determined whether MCMV-induced CTL can recognize β-gal epitopes presented by aortic SMC from SM-LacZ mice. SM-LacZ mice constitutively express the β-gal transgene in aortic SMC ( Figure 1 A). Interestingly, β-gal expressing SMC from SM-LacZ could only be recognized and lysed by β-gal 497–504 specific CTL after preincubation with IFN ( Figure 1 B). Recognition of SMC by MCMV-induced CTL could be enhanced by exogenous pulsing with β-gal 497–504 peptide. Furthermore, SMC lacking the Fas death receptor and Fas-competent C57BL/6 SMC could only be lysed by perforin-competent CTL ( Figure 1 C). These data indicate that (1) SMC expressing a protein shared by a cytomegalovirus can be lysed by virus-specific CTL, and (2) that CTL-mediated death of SMC is mainly mediated via perforin-dependent lysis.

Figure 1. Recognition of aortic β-galactosidase transgene by virus-induced CTL. A, X-Gal staining of aortas from C57BL/6 (left) and SM-LacZ (right) mice. B and C, Lysis of arterial SMC from SM-LacZ by MCMV-LacZ specific CTL. Effector CTL were generated from MCMV-LacZ infected C57BL/6 and perforin-deficient (PKO) mice using in vitro restimulation with irradiated β-gal 497–504 peptide pulsed C57BL/6 splenocytes for 5 days. B, Arterial SMC from C57BL/6 or SM-LacZ were incubated for 24 hours in the absence (left panel) or presence (right panel) of IFN before analysis in a standard chromium release assay. C, In vitro restimulated β-gal 497–504 -specific CTL were used as effectors in a standard chromium release assay using arterial SMC from C57BL/6 or Fas-deficient (Fas-KO) mice pulsed with β-gal 497–504 peptide. Data from SMC not treated with IFN are shown; IFN pretreatment resulted in only slightly enhanced killing of peptide-pulsed SMC by C57BL/6 CTL.

The adaptive immune response against MCMV is dominated by CD8 + T cells that are required for the termination of the productive infection and the establishment of latency. 22 MCMV-specific memory CTL responses may show differences in their expansion and contraction patterns, eg, a rapid expansion can be followed by a pronounced contraction phase or by the continuous increase of IFN producing memory CTL. 23,24 We therefore tested in a first set of experiments, the CTL responses directed against the β-gal 497–504 epitope and the immunodominant MCMV M45 985–993 epitope of MCMV. Analysis with MHC class I tetramers on days 6, 50 and 100 post infection revealed that both β-gal 497–504 - and M45 985–993 -specific CD8 + T cells followed a similar pattern in normo- and hypercholesterolemic mice with maximal expansion during the acute response, a pronounced contraction and subsequent establishment of a stable long-term memory cell population ( Figure 2 A).

Figure 2. Kinetics of MCMV-induced CD8 + T cell responses. A, Female C57BL/6, apoE –/–, and apoE –/– x SM-LacZ mice were infected i.v. with MCMV-LacZ, and tetramer analysis was performed at the indicated days after infection. Mean percentages of tetramer-positive cells in the CD8 compartment are indicated (±SEM; n=3). Representative data from 1 of 2 independent experiments are shown. B, Female mice were infected with MCMV-LacZ and intracellular cytokine staining for IFN was performed on splenocytes on day 6 after infection using the indicate peptides. Mean percentages of IFN + CD8 + cells are indicated (±SEM; n 3). C, For ELISPOT analysis, 5 x 10 4 MACS-purified CD8 + splenocytes were incubated with unpulsed, β-gal–, or M45-peptide pulsed dendritic cells, respectively, and numbers of IFN secreting cells were determined. Representative ELISPOT filters are shown; nd, not done. D, Total numbers of β-gal- or M45-specific IFN -producing CD8 + splenocytes as determined by ELISPOT assay at the indicated time points after MCMV-LacZ infection (±SEM; n 3); nd, not done. CTL reactivity measured as tetramer-reactivity and intracellular IFN secretion against both β-gal and M45 epitopes, was not detectable in naive normo- or hypercholesterolemic mice (not shown). Statistical analysis using the nonparametric Kruskal–Wallis test indicated no significant difference between the 4 mouse strains ( P 0.05).

Based on the finding that IFN permits recognition of aortic SMC by MCMV-induced CTL ( Figure 1 B) and because IFN accelerates atherosclerotic lesion development, 25 we determined the ability of virus-specific CD8 + T cells to produce IFN using intracellular cytokine staining ( Figure 2 B) and ELISPOT assays ( Figure 2C and 2 D). The ELISPOT assays were used for measurement of IFN -secreting cells in the memory phase because this assay provides a better resolution and accuracy at lower T cell frequencies. These analyses revealed that neither hypercholesterolemia nor the presence of the β-gal-transgene had a significant impact on the peak expansion of β-gal 497–504 - and M45 985–993 -specific IFN -secreting CD8 + T cells ( Figure 2 B). Furthermore, both β-gal 497–504 - and M45 985–993 -specific memory CTL were able to secrete IFN following in vitro restimulation ( Figure 2C and 2 D). Again, the activity of anti-MCMV CD8 + T cells was not significantly different between hypercholesterolemic and normocholesterolemic mice, and there was no indication that the β-gal transgene in the vasculature had an influence on MCMV-induced CTL responses ( Figure 2C and 2 D). Taken together, the presented transgenic model provides the means to quantify and to phenotypically characterize virus-specific CD8 + T cells which recognize a persisting vascular antigen both under normo- and hypercholesterolemic conditions.

Virus Replication and Lipid Metabolism

It has been described previously that hypercholesterolemia may negatively influence immune reactivity and consequently alters the host-pathogen interaction with delayed clearance of viruses, 26 bacteria, 27 or fungi. 28 We therefore assessed whether hypercholesterolemia in apoE –/– and apoE –/– x SM-LacZ mice influences initial replication and distribution of MCMV in comparison to normocholesterolemic C57BL/6 and SM-LacZ mice. Polymerase chain reaction (PCR)-based quantification of MCMV genome equivalents revealed that spleens and salivary glands were equally well infected in the four mouse strains ( Figure 3 A). Furthermore and in contrast to previous studies in mice, 29 we could not observe a modulation of cholesterol levels in the course of MCMV infection in normo- or hypercholesterolemic mice ( Figure 3 B). Of particular importance for this study is that apoE –/– and apoE –/– x SM-LacZ mice are only distinguishable by constitutive β-gal expression in the cardiovascular system, all other MCMV infection-associated parameters such as T cell responses, viral distribution, and total cholesterol values were comparable.

Figure 3. Early virus distribution and serum cholesterol levels. A, Amount of virus DNA as determined by real-time PCR on day 3 after i.v. infection with 2 x 10 6 pfu MCMV-LacZ. Statistical analysis using the nonparametric Kruskal–Wallis test indicated no significant difference between the different mouse strains (n=4, P 0.05). B, Serum cholesterol values in uninfected and MCMV infected apoE –/– (hatched bars) and apoE –/– x SM-LacZ (gray bars) mice at the indicated time points after infection. Statistical analysis using the nonparametric Kruskal–Wallis test indicated no significant difference between the different mouse strains ( P 0.05, number of mice per group is indicated; groups were of mixed gender with 50% females).

Accelerated Atherogenesis in MCMV-Infected ApoE –/– x SM-LacZ Mice

The impact of infection with β-gal recombinant MCMV-LacZ on atherosclerotic lesion development was assessed in the aortic sinus. The most striking observation was the nearly 200% increase of atherosclerotic lesion formation in apoE –/– x SM-LacZ versus apoE –/– control mice on day 50 after infection ( Figure 4A and 4 B). The acceleration of atherogenesis attributable to chronic virus-driven immune reactivity against the vascular β-gal antigen was less pronounced on day 100 after infection ( Figure 4A and 4 B). However, in accordance with a previous report from Epstein and colleagues, 30 we found only a mild enhancement of atherosclerosis through infection of apoE –/– mice with MCMV on day 100 after infection ( Figure 4 B). These data indicate that it is mainly the chronic immune reactivity against the persisting MCMV antigen in the vasculature that exacerbates atherosclerotic lesion formation.

Figure 4. Acceleration of atherosclerosis in apoE –/– x SM-LacZ mice. A, Aortic sections were stained with Sudan Red to visualize lipid deposition on days 50 and 100 post infection. Average lesion size in apoE –/– and apoE –/– x SM-LacZ is indicated (µm 2 x 10 3 ±SEM). Asterisks indicate neointimal and arrow heads indicate perivascular mononuclear cell accumulations. Sections of female mice are shown. B, Atherosclerotic lesion size in uninfected apoE –/– (3 males and 9 females on day 50; 6 males on day 100) and apoE –/– x SM-LacZ (1 male, 4 females on day 50; 6 males on day 100) mice or MCMV-LacZ–infected apoE –/– (6 males, 8 females on day 50; 2 males and 6 females on day 100) and apoE –/– x SM-LacZ (3 males, 6 females on day 50; 3 males, 10 females on day 100) mice.

In addition to the accelerated lesion formation, MCMV-LacZ–infected apoE –/– x SM-LacZ mice showed significant mononuclear infiltrations both in the neointima ( Figure 4 A, asterisks) and in the perivascular tissue underlying the atherosclerotic lesions ( Figure 4 A, arrow heads). Immunohistological characterization of the vascular inflammatory infiltrates revealed the presence of macrophages and T cells, with a predominance of CD8 + T cells ( Figure 5 ). Semiquantitative in situ analysis revealed that MCMV-LacZ–infected apoE –/– x SM-LacZ showed significantly stronger inflammatory infiltration compared with apoE –/– mice, both on day 50 (1.8±0.5 versus 0.6±0.2; P <0.05) and on day 100 (2.1±0.3 versus 1.1±0.3; P <0.05) after infection. Furthermore, enumeration of CD8 T cells infiltrating neointima and perivascular space revealed that MCMV-LacZ infection of apoE –/– x SM-LacZ mice elicited a more pronounced recruitment of these cells compared with infected apoE –/– mice; both on day 50 (CD8 T cells per slide: 20.9±8.1 (n=13) versus 6.4±1.6 (n=9); P <0.05) and on day 100 (40.1±6.6 (n=8) versus 12.9±4.7 (n=11); P <0.01) after infection. It is thus conceivable that the abundant CD8 + T cells in and around the atherosclerotic lesions in apoE –/– x SM-LacZ mice were recruited to this location as a consequence of the ongoing immune activation during the persistent MCMV infection.

Figure 5. Cellular composition of inflammatory lesions in MCMV-LacZ–infected female apoE –/– and apoE –/– x SM-LacZ mice. Aortic sections were stained on day 100 after infection for lipid deposition (Sudan Red), T cells (CD4 and CD8), and macrophages (F4/80). Representative data from 4 mice per group are shown.

Discussion

The inflammatory nature of atherosclerosis is mediated by various factors. The major pathological mechanisms underlying the onset and perpetuation of the chronic immune activation within the vascular wall include hemodynamic shear stress leading to the expression of proinflammatory genes, 31 accumulation of dendritic cells at predisposed sites, 32 and systemic immune activation via toll-like receptor (TLR) ligands. 33 Understanding and ranking the contribution of the different immunopathological mechanisms mediating disease initiation and progression is thus essential for the development and evaluation of treatment strategies. General immune activation in the course of systemic virus infection may elicit high levels of cytokines in the circulation. In the context of cytomegalovirus infection, IFN can be produced by virus-specific NK and Th cells. 34 It is possible that IFN that is generated in the course of virus infection, either systemically or locally within the inflamed tissue, could promote CTL-mediated SMC lysis by virus-specific CTL through upregulation of MHC I molecules. In human SMC, for example, cytomegalovirus infection can lead to an increased MHC class I expression in smooth muscle cells, hence modulating their immunogenicity. 35 Nevertheless, the results of this investigation demonstrate that chronic immune reactivity against a persisting microbial antigen in the arterial wall is a dominant immunopathological factor during MCMV-accelerated atherosclerosis in hypercholesterolemic apoE –/– mice.

Evidence from experimental and natural infections with herpesviruses supports the notion that general immune activation by a viral infection is less important for the inflammatory processes in the vascular wall. For example, -herpesvirus infection of large arteries is associated with an acute lymphoid panarteritis and chronic obliterating arteriosclerosis in chicken 36 and cattle. 37 Furthermore, murine -herpesvirus 68 ( HV68) exhibits a prominent tropism for medial smooth muscle cells of great elastic arteries 38 and, consequently, enhances atherogenesis in apoE –/– mice. 17,18 In MCMV infection, viral antigens have been reported to be expressed in endothelial and smooth muscle cells of the aorta. 29 However, the presence of MCMV in the aorta is limited to a few weeks after infection 15; a condition under which MCMV only mildly aggravates atherosclerosis in apoE –/– mice. Thus, accumulation of inflammatory cells in the perivascular space and increased development of atherosclerotic lesions heavily infiltrated with inflammatory cells, depends probably on prolonged antigen presentation within the vessel wall, as it is the case in apoE –/– x SM-LacZ mice. Likewise, factors that favor MCMV persistence in the vasculature lead to increased vascular inflammation 39 and atherosclerosis. 29

Activated T cells are a major fraction of the cellular components in human atherosclerotic plaques. 40 Immunohistological analysis revealed that in advanced plaques of apoE –/– mice, CD4 + T cells are prominent in the fibrous cap and subendothelially, whereas CD8 + T cells are sparse. 41 The importance of CD4 + T cells in the amplification of atherogenesis has been demonstrated in adoptive transfer studies where CD4 + T cells from apoE –/– transferred to immunodeficient apoE –/– x scid/scid mice accelerated lesion formation. 42 CD4 T cells are activated during MCMV infection and help to maintain long-term control of MCMV in certain cell types within salivary gland tissues. 43 It is likely that not only CD8 + but also CD4 + MCMV-specific T cells have contributed to the observed amplification of atherosclerotic lesions in apoE –/– x SM-LacZ mice. Furthermore, it may well be that the recently described dendritic cell network present in atherosclerosis-prone sites of the aorta 32 locally presents viral antigens to both CD4 + and CD8 + T cells.

Infections are an important risk factor in atherosclerosis-related diseases such as coronary artery disease or stroke. It is striking that in particular those infectious agents which possess a pronounced tropism for cells of the vascular wall (HCMV or Chlamydia pneumoniae ) are the most prominent in the list of infectious agents that contribute to the "infectious burden". 4,44,45 Taken together with the data presented in this study, it is most likely that long-lasting immune reactivity against antigens of vascular-tropic infectious agents significantly amplifies inflammatory reactions within atherosclerotic lesions. Thus, treatment strategies against atherosclerosis should aim at reducing exaggerated immune reactivity within the atherosclerotic lesion without impairing general immune defense mechanisms against the persisting pathogen.

Acknowledgments

We thank Silvia Behnke and Andre Fitsche for help with immunohistochemistry.

Sources of Funding

The project received support from the Swiss National Science Foundation, the Fritz Thyssen Stiftung, the Novartis Foundation for Biomedical Research and the Jubiläumsstiftung Rentenanstalt.

Disclosures

None.

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作者单位：Research Department, Kantonal Hospital St Gallen, St Gallen, Switzerland.