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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2005;25:2308.)
© 2005 American Heart Association, Inc.

Vascular Biology

Toll-Like Receptor 2 Mediates Persistent Chemokine Release by Chlamydia pneumoniae–Infected Vascular Smooth Muscle Cells

Xin Yang; Daniel Coriolan; Kelly Schultz; Douglas T. Golenbock; Debbie Beasley

From the Molecular Cardiology Research Institute and Department of Medicine (X.Y., D.C, K.S., D.B.), Tufts–New England Medical Center, Boston, and the Division of Infectious Diseases and Immunology (D.T.G.), University of Massachusetts Medical School, Worcester, Mass.

Correspondence to Debbie Beasley, PhD, New England Medical Center, Box 8486, 750 Washington St, Boston, MA, 02111. E-mail Dbeasley{at}tufts-nemc.org

	Abstract

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Objective— The intracellular bacterium Chlamydia pneumoniae is present in many atherosclerotic lesions, where it could promote inflammation. This study determined whether monocyte chemoattractant protein 1 (MCP-1) release is stimulated in vascular smooth muscle cells (VSMCs) that are exposed to or infected by C pneumoniae and whether toll-like receptor 2 (TLR2) or TLR4 mediate these effects.

Methods and Results— TLR2 mRNA was expressed constitutively and was upregulated by C pneumoniae exposure in mouse aortic SMC and was inducible by C pneumoniae and TLR3 and TLR4 agonists in human coronary artery SMCs. Exposure to inactivated or viable extracellular C pneumoniae evoked a robust increase in MCP-1 release and activated nuclear factor-B and extracellular signal–regulated kinase 1/2 in wild-type and TLR4 signaling–deficient mouse aortic SMCs but not in TLR2-deficient SMCs, probably because of TLR2-mediated recognition of a chlamydial antigen. Brief exposure to viable C pneumoniae led to active infection of VSMCs, shown by chlamydial protein synthesis, and caused a persistent (>48-hour) MCP-1 release that was also TLR2 dependent.

Conclusions— The results show that VSMCs express functional TLR2 and that TLR2 mediates both a persistent activation of chemokine release in C pneumoniae–infected VSMCs and its acute stimulation by extracellular C pneumoniae. Therefore, TLR2 expressed in VSMCs may promote inflammation within the arterial wall.

TLR2 is expressed constitutively in mouse VSMCs, its expression is inducible in human VSMCs, and it mediates both persistent stimulation of MCP-1 release in SMCs infected with C pneumoniae and acute stimulation by extracellular C pneumoniae. Activation of TLR2 expressed in VSMCs may promote inflammation within the arterial wall.

Key Words: monocyte chemoattractant protein-1 • nuclear factor-B • extracellular signal–regulated kinase 1/2 • heat shock protein 60 • lipopolysaccharide

	Introduction

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Inflammation is a key component of atherosclerosis, the earliest stages of which are characterized by invasion of the intima by mononuclear phagocytes.¹ Growing evidence indicates that infectious agents, including Chlamydia pneumoniae, a Gram-negative obligate intracellular bacterium, may promote arterial inflammation. C pneumoniae is frequently found in atherosclerotic lesions residing within vascular smooth muscle cells (VSMCs) and endothelial cells as well as in macrophages, but it is rare or absent in normal arteries.^2–6 In animal models of respiratory infection, the organism localizes to the arterial wall and promotes atherogenesis by acting in concert with traditional risk factors such as hyperlipidemia,^7–13 raising the possibility that C pneumoniae may affect vascular cell phenotype and function directly. In vitro studies support the hypothesis that C pneumoniae can promote cellular changes similar to those occurring during atherogenesis, including increased lipid accumulation in macrophages¹⁴ and proliferation of human VSMCs.¹⁵ Despite substantial evidence supporting a pathogenic role of C pneumoniae in atherosclerosis, little is known about the molecular mechanisms by which it may alter vascular cell function.

Effective defense against invading microbes requires a sensitive pathogen recognition system. The toll-like receptor (TLR) family includes at least 10 transmembrane pathogen-recognition receptors, which are expressed by immune cells and are thought to be essential in microbial detection and host cell activation. Each TLR consists of an intracellular domain with homology to the type I interleukin-1 receptor and a leucine-rich extracellular domain that provides ligand specificity.¹⁶ For example, TLR2 is activated by a repertoire of microbial cell wall components, including lipoproteins and peptidoglycans, mycobacterial lipoarabinomannan, and yeast zymosan.¹⁷ In contrast, TLR4 is a key signal-transducing receptor for enterobacteria-derived lipopolysaccharide (LPS).^18,19 Recognition of intact bacteria and microbes is more complex and may involve recognition of multiple molecular components by different TLRs. Extracellular C pneumoniae can activate dendritic cells via TLR2- and TLR4-dependent pathways,²⁰ but this intracellular organism may activate different pathogen-recognition receptors when localized in intracellular vesicles. In addition, C pneumoniae appears to express genes encoding a type III secretion system,²¹ suggesting that chlamydial proteins could reach the cytoplasm of host cells, where they could potentially activate cytoplasmic pathogen-recognition receptors such as Nod1, as suggested by a study with human endothelial cells.²²

Considering the role of inflammation in atherogenesis and that microbes such as C pneumoniae localize to the arterial wall, TLR expression by VSMCs may be relevant to vascular disease. However, little is known about the role of TLRs in VSMC activation. Human arterial SMCs express a functional TLR4 signaling complex linked to chemokine and proinflammatory cytokine release,²³ and C pneumoniae stimulates their proliferation via TLR4-dependent mechanisms.¹⁵ In addition, a recent study raised the possibility that VSMCs express functional TLR2.²⁴ However, it is currently unknown whether microbes having potential relevance to atherosclerosis can promote inflammation via TLR2 signaling in VSMCs.

Accordingly, in this study we sought to determine whether extracellular or intracellular C pneumoniae stimulates chemokine release from VSMCs and to elucidate the cellular receptors involved by using SMCs derived from mice deficient in either TLR2 or TLR4 signaling. We studied monocyte chemoattractant protein-1 (MCP-1), a critical promoter of monocyte migration into the subendothelial space,^25,26 and found that TLR2 is expressed by VSMCs and that it mediates persistent VSMC activation by intracellular C pneumoniae infection. Thus, VSMCs may contribute to microbe-induced inflammation by intra-arterial release of monocyte-recruiting chemokines.

	Methods

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A detailed description of the methods can be found online at http://atvb.ahajournals.org.

	Results

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C pneumoniae Stimulates TLR2 Expression in Mouse Aortic SMCs and Human Coronary Artery SMCs
Aortic SMCs derived from wild-type C57BL/6 mice expressed both TLR2 and TLR4 mRNA constitutively (Figure 1A). TLR2 mRNA levels were markedly increased 5 hours after exposing mouse aortic SMCs to C pneumoniae, whereas TLR4 mRNA levels were unchanged, as indicated by reverse transcription–polymerase chain reaction (RT-PCR) analysis (Figure 1A). Real-time RT-PCR analysis confirmed that C pneumoniae did not alter TLR4 mRNA expression (not shown) but increased TLR2 mRNA 8-fold (Figure 1B). Although human coronary artery SMCs did not constitutively express TLR2 mRNA, it was induced after exposure to the selective TLR4 and TLR3 agonists Escherichia coli LPS and double-stranded RNA (polyinosinic-polycytidylic acid), respectively, or to C pneumoniae (Figure 1C and 1D). Pretreatment of human SMCs with LPS for 24 hours did not further augment C pneumoniae–induced TLR2 mRNA expression (Figure 1D).

View larger version (34K): [in this window] [in a new window]   Figure 1. C pneumoniae stimulates TLR2, but not TLR4, mRNA expression in mouse SMCs and induces TLR2 mRNA expression in human coronary artery SMCs. A and B, Mouse aortic SMCs were incubated with C pneumoniae (multiplicity of infection [MOI]=10) or control (CON) lysate for 5 hours, and total RNA was prepared. cDNA was amplified for 30 cycles and products were separated by electrophoresis and stained with ethidium bromide (A). Relative TLR2 mRNA levels as determined by real-time RT-PCR (n=4 replicates per group), *P<0.001 vs mock infected (B). C and D, Human coronary artery SMCs were incubated with or without dsRNA [poly(I:C); 10 µg/mL] or E. coli LPS and soluble CD14 (each at 100 ng/mL) for 6 hours (C) or with or without LPS/CD14 for 24 hours followed by exposure to C pneumoniae (MOI=10) or control lysate for 6 hours (D). Other abbreviations are as defined in text.

C pneumoniae Stimulates MCP-1 Release via a TLR2-Dependent Pathway
SMCs exposed to control mock inoculant released low but detectable levels of MCP-1. Exposure to C pneumoniae strongly and dose-dependently stimulated MCP-1 release in SMCs from both wild-type and TLR4 signaling–deficient mice but had no effect on MCP-1 release in TLR2–/– SMCs (Figure 2). The results support a primary role for TLR2 in C pneumoniae–induced MCP-1 release in mouse aortic SMCs.

View larger version (20K): [in this window] [in a new window]   Figure 2. C pneumoniae stimulates MCP-1 release in wild-type (WT) and TLR4 signaling–deficient but not TLR2–/– SMCs. Aortic SMCs were isolated from TLR4-WT (C3H/OuJ), TLR4-mutant (C3H/HeJ), TLR2+/+ (C57BL/6), and TLR2–/– mice. SMCs were serum deprived and incubated for 6 hours in media with C pneumoniae at the indicated MOI or with control lysate (CON), and supernatant MCP-1 levels were determined. Results shown are mean±SE of 6 cultures, representative of 3 experiments. *P<0.001 vs CON. Other abbreviations are as defined in text.

C pneumoniae Stimulates NF-B via a TLR2-Dependent Pathway
MCP-1 expression is regulated at the transcriptional level by nuclear factor (NF)-B, which is strongly activated by TLRs in many cell types. We tested whether C pneumoniae stimulated NF-B via TLR-dependent mechanisms by comparing its stimulation of NF-B–dependent luciferase reporter activity in SMCs from wild-type and TLR signaling–deficient mice. C pneumoniae stimulated NF-B–dependent luciferase expression similarly in TLR4 wild-type and TLR4 signaling–deficient mouse aortic SMCs (Figure 3), but the marked C pneumoniae–induced NF-B activation seen in TLR2+/+ SMCs was virtually absent in TLR2–/– SMCs, indicating that C pneumoniae activates NF-B primarily via TLR2 activation.

View larger version (12K): [in this window] [in a new window]   Figure 3. C pneumoniae activates NF-B in wild-type (WT) and TLR4 signaling–deficient but not TLR2–/– SMCs. SMCs from respective mouse strains were transfected with NF-B–luciferase reporter plasmid, serum deprived, and incubated for 6 hours with C pneumoniae (MOI=10) or control lysate (CON). Luciferase activity was expressed relative to the level in CON SMCs. Values are mean±SE of 15 replicates from 3 experiments. *P<0.002 vs CON. +P<0.001 vs TLR2+/+, +CP. Other abbreviations are as defined in text.

Activation of ERK1/2 by C pneumoniae Involves TLR2-Dependent and -Independent Pathways
MCP-1 expression is also regulated by extracellular signal–regulated kinase (ERK) 1/2, which is activated by TLR engagement in some cell types. To determine whether C pneumoniae activates ERK1/2 in mouse aortic SMCs, we compared the levels of phospho-ERK1/2 in SMCs exposed to C pneumoniae or mock inoculant for 1.5 or 5 hours. In SMCs from wild-type and TLR4 signaling–deficient mice, phospho-ERK1/2 levels were significantly increased by C pneumoniae exposure at both time points (Figure 4). In contrast, TLR2–/– SMCs failed to exhibit any C pneumoniae–induced phospho-ERK1/2 response at 1.5 hours but did exhibit a response similar to that of TLR2+/+ SMCs at 5 hours. The results suggest that TLR2 is required for early-phase but not late-phase ERK1/2 activation.

View larger version (30K): [in this window] [in a new window]   Figure 4. Exposure to C pneumoniae elicits sustained activation of ERK1/2: ERK1/2 is activated at late (5 hours) but not early (1.5 hours) time points in TLR2–/– SMCs. SMCs from indicated mouse strains were serum deprived, incubated for 30 minutes with C pneumoniae (CP; MOI=10) or control lysate (CON) and analyzed for phospho- (p-) ERK1/2 and total ERK1/2 (ERK1/2). Shown are representative blots and quantitative analyses of p-ERK1/2 expression in CP-exposed relative to CON SMCs. Values are mean±SE of 3 experiments. *P<0.03 vs CON. +P<0.05 vs TLR2+/+, +CP. WT indicates wild type; other abbreviations are as defined in text.

Early C pneumoniae–Induced MCP-1 Release Does Not Require Viable Organisms and Is Mimicked by Chlamydial hsp60
The early activation of MCP-1 release by C pneumoniae could be triggered by direct recognition of a chlamydial antigen or alternatively may require active intracellular chlamydial infection. To test these alternative hypotheses, we compared the activities of viable and heat (56°C)- or UV light–inactivated C pneumoniae. Nonviable and viable C pneumoniae stimulated MCP-1 release similarly during the first 6 hours after exposure (Table I, available online at http://atvb.ahajournals.org) and also activated ERK1/2 similarly at 1.5 and at 5 hours (not shown). These results indicate that early induction of MCP-1 release does not require intracellular C pneumoniae replication or metabolism but is likely mediated by recognition of antigens already present on C pneumoniae. Molecular components of C pneumoniae known to activate immune cells include LPS²⁷ and heat shock protein 60 (hsp60).²⁸ Heating the C pneumoniae to 100°C reduced their MCP-1 stimulating activity by &50% (Table I), suggesting that both heat-labile antigens and heat-resistant LPS may contribute to the stimulatory activity of C pneumoniae. Indeed, exposure to recombinant chlamydial hsp60 for 6 hours dose-dependently induced MCP-1 release (Figure I, available online at http://atvb.ahajournals.org), suggesting that non-LPS antigens contribute to C pneumoniae–induced MCP-1 release. Chlamydial hsp60 (5 µg/mL) markedly stimulated MCP-1 release in wild-type and TLR4 signaling–deficient SMCs but failed to do so in TLR2-deficient SMC, indicating that the effect of hsp60, like that of C pneumoniae, was TLR2-dependent (Figure I). These results support the hypothesis that chlamydial hsp60 and also heat-stable chlamydial antigens can induce rapid TLR2-dependent MCP-1 release in mouse aortic SMCs.

Role of TLR2 in C pneumoniae Infection–Dependent MCP-1 Release
To determine whether intracellular, metabolically active C pneumoniae stimulate MCP-1 release in VSMCs, we tested whether brief exposure of mouse aortic SMCs to C pneumoniae would lead to active infection accompanied by increased MCP-1 release. VSMCs were exposed to C pneumoniae for 2 hours, then washed thoroughly to remove the inoculant, and MCP-1 release was determined 48 to 72 hours later, an expected period of active chlamydial metabolism and growth within SMCs. C pneumoniae was metabolically active in SMCs 48 to 72 hours after inoculation with viable organisms, as indicated by the presence of abundant C pneumoniae hsp60 (CP-hsp60) mRNA and protein (Figure 5A and 5B). In contrast, SMCs exposed to inactivated organisms contained only negligible levels of CP hsp-60 mRNA and protein. The lack of significant CP-hsp60 mRNA and protein in SMCs exposed to inactivated organisms was not simply due to impairment of their uptake by SMCs, because shortly (1 hour) after the inoculation period, cell-associated CP-hsp60 protein levels were similar in SMCs exposed to viable or inactivated C pneumoniae (Figure 5B). Also, the level of CP-hsp60 protein was markedly increased 72 hours after inoculation with viable C pneumoniae, compared with the low level found after only 1 hour, confirming that CPs were synthesizing new hsp60 protein. MCP-1 release remained elevated 48 to 72 hours after inoculation with viable but not UV-inactivated C pneumoniae (Figure 5C). This late, infection-dependent induction of MCP-1 release in SMCs exposed to viable C pneumoniae was absent in TLR2–/– SMCs, indicating that it also was TLR2- dependent.

View larger version (40K): [in this window] [in a new window]   Figure 5. Active C pneumoniae infection of mouse aortic SMCs stimulates persistent MCP-1 release. Serum-deprived SMCs were inoculated with viable C pneumoniae (CP; MOI=25) for 2 hours and then washed extensively. A, Chlamydial hsp60 and ß-actin mRNAs were detected by RT-PCR analysis. Abundant hsp60 mRNA was seen 48 hours after inoculation with viable CP but not inactivated CP (CP-UV) or mock inoculant (CON). B, Western blots of chlamydial hsp60 protein. Abundant chlamydial hsp60 was present 72 hours after inoculation with CP but not CP-UV or mock inoculant. Low but similar levels of hsp60 were seen 1 hour after exposure to either CP or CP-UV but were absent in CON. C, MCP-1 release was increased in TLR2+/+ SMCs 48 to 72 hours after exposure to viable CP but not CP-UV. The response was absent in TLR2–/– SMC *P<0.001 vs CON. Other abbreviations are as defined in text.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
These findings show that C pneumoniae is a potent activator of MCP-1 release from mouse aortic SMCs. Exposure to C pneumoniae evoked both rapid effects that did not require viable organisms and persistent responses that occurred only after exposure to viable organisms during a period of new chlamydial protein synthesis. Furthermore, both the rapid effects of C pneumoniae exposure and the persistent responses induced by C pneumoniae infection were found to be primarily mediated by TLR2.

The finding that C pneumoniae strongly induces MCP-1 release by arterial SMCs is highly relevant to its proposed role in atherosclerosis, because MCP-1 is thought to be an important mediator of monocyte migration into the intima during atherogenesis. For example, mice deficient in either MCP-1 or its receptor exhibit reduced levels of mononuclear phagocyte accumulation within the artery wall and smaller lesions evoked by a range of pathogenic stimuli, including hypercholesterolemia and arterial injury.^29,30 Intra-arterial monocyte/macrophage accumulation and arterial lesion size were also reduced by pharmacological inhibition of MCP-1 activity in a monkey model of arterial injury.³⁰ In addition, MCP-1 is present in macrophage-rich areas of human atherosclerotic plaques, consistent with a role in the human disease process.^25,26 The present results raise the possibility that C pneumoniae, when present in arterial lesions, may promote recruitment of monocytes into the vessel wall by stimulating VSMCs to release MCP-1, thereby promoting inflammation.

Exposure to C pneumoniae activated NF-B and ERK1/2 predominantly via TLR2 at early time points, whereas ERK1/2 activation was not TLR2-dependent at a later time (5 hours). The late phase of C pneumoniae–induced ERK1/2 activation may involve TLR4 signaling, consistent with previous findings that C pneumoniae can activate cells via TLR4. SMCs derived from human saphenous veins do not express TLR2, and C pneumoniae stimulates their proliferation via a mechanism that is at least partially dependent on TLR4.¹⁵ Also, C pneumoniae rapidly activates dendritic cells via a mechanism that is primarily TLR2-dependent but that also involves TLR4 to a minor extent.²⁰ Alternatively, the later phase of C pneumoniae–induced ERK1/2 activation could involve a receptor other than TLR2 or TLR4. Potentially relevant in this regard is the finding that the cytoplasmic pattern-recognition receptor Nod1 is required for C pneumoniae–induced interleukin-8 production in human endothelial cells.²² Accordingly, it will be of interest to determine whether Nod1 or alternative receptors are involved in the later phase of C pneumoniae–induced ERK1/2 activation.

The finding that inactivated C pneumoniae sufficed for the early phase of SMC activation suggests that it is mediated by TLR2-dependent recognition of 1 or more preexisting chlamydial antigens and does not require new products synthesized by viable organisms. Candidate mediators of the TLR2-dependent effect of C pneumoniae include LPS and hsp60. The outer membrane of C pneumoniae contains a form of LPS that is thought to have only weak TLR4 agonist activity, because its lipid A moiety has only 5, relatively longer-chain fatty acids rather than the classic 6 shorter-chain fatty acids found in the highly-active LPS forms of other Gram-negative bacteria.³¹ Here we found that C pneumoniae failed to stimulate MCP-1 release in TLR2–/– SMCs, despite their expression of functional TLR4, indicating that the LPS associated with intact C pneumoniae is unable to significantly activate TLR4. On the other hand, our findings do support the existence of a heat-stable C pneumoniae antigen that stimulates MCP-1 release, raising the possibility that chlamydial LPS contributes to C pneumoniae–induced chemokine release by activating TLR2. In contrast with LPS derived from enterobacteria such as E. coli, which is known to activate cells via TLR4,^18,19 LPS from several nonenterobacterial species, including C trachomatis and Porphyromonas gingivalis,^32,33 may activate cells via TLR2. On the other hand, the activity of C pneumoniae observed in the present study was partially heat-labile, suggesting that it may additionally involve lipoprotein or protein constituents such as hsp60. A potential role of chlamydial hsp60 is supported by our finding that the stimulation of MCP-1 release by recombinant chlamydial hsp60 was TLR2-dependent. Identification of the specific chlamydial antigens responsible for TLR2-dependent activation of SMC responses will require further investigation, but the present results suggest that multiple classes of chlamydial antigens possess such activity, even in the absence of active intracellular infection.

Mouse aortic SMCs are susceptible to C pneumoniae infection, as shown by the presence of chlamydial mRNA and hsp60 protein in SMCs 48 to 72 hours after exposure to viable bacteria. SMCs were infected by simple exposure to a relatively low level of C pneumoniae, contrasting with previous studies that used special procedures to infect vascular cells, such as centrifugation of the host cells with C pneumoniae–containing culture media or coculture with C pneumoniae–infected macrophages.^34–37 In the present study, we avoided using methods involving centrifugation or coculture to preclude any potential confounding effects of these procedures on SMC activation. Also, to maintain C pneumoniae viability and infectivity, we used partially purified organisms rather than organisms purified by density gradient centrifugation, because the latter procedure may impair the infectivity of C pneumoniae. This may account for our success in infecting VSMCs with C pneumonia by using only brief exposure to relatively low concentrations of the organism.

Although the early phase of SMC activation and MCP-1 release by chlamydial antigens did not require intracellular infection, we did find that active infection was essential for a persistent cellular response and furthermore, that persistent activation of VSMCs by C pneumoniae infection is mediated by TLR2. This TLR2 dependence may be direct, such that C pneumoniae antigens produced during the infection activate TLR2. Alternatively, C pneumoniae–infected VSMCs may produce a native TLR2 ligand, such as endogenous hsp60,³⁸ which in turn activates MCP-1 release. Further studies will be required to distinguish between these 2 alternative mechanisms.

Our findings provide new support for the hypothesis, first proposed in 1988,³⁹ that C pneumoniae infection may promote atherogenesis, and also support a possible contributing role of VSMCs. C pneumoniae antigens are frequently found in late atherosclerotic lesions and sometimes in early lesions of young adults,^40,41 suggesting a potential role in both early and late stages of the disease. Despite the fact that C pneumoniae are generally difficult to culture in vitro, viable organisms have been cultured from atherosclerotic lesions,^42–45 indicating that vascular cells are suitable hosts for this pathogen. Infection with C pneumoniae via the respiratory tract accelerated arterial lesion formation in hyperlipidemic rabbits and mice in many,^7–9,46,47 although not all, studies,^48,49 suggesting an atherosclerosis-promoting role for C pneumoniae under certain conditions. The proatherogenic effect of C pneumoniae appears to be specific to this organism, because a strain of C trachomatis that infects the murine respiratory tract and causes pneumonia did not exacerbate hypercholesterolemia-induced atherosclerosis, as did C pneumoniae.¹² After inoculation of the respiratory tract, C pneumoniae persists longer in the aortic lesions of apolipoprotein E–deficient mice than in normal arteries of normocholesterolemic mice,¹¹ suggesting a relation between persistence of the organism within the aorta and lesion formation. Our finding that C pneumoniae directly activates chemokine release from SMCs, considered together with several converging lines of evidence, supports the hypothesis that C pneumoniae is not an "innocent bystander" but rather, may play a contributing role in atherogenesis.

Much interest has focused on the adaptive role of TLRs as key mediators of the innate immune response, inducing synthesis of antimicrobial peptides and cytokines that promote adaptive immunity. However, TLR activation does not necessarily enhance antimicrobial defense and may not always be beneficial to the host. Rather, in certain situations, TLR activation may induce inflammatory responses that lead to chronic, pathogenic conditions in host tissues. For example, TLR2 deficiency had no effect on the course or resolution of infection when mice were inoculated vaginally with the murine biovar of C trachomatis,⁵⁰ a species that also causes reproductive tract infections in humans. However, TLR2 deficiency did attenuate the chronic oviductal inflammation that followed resolution of the infection and also diminished C trachomatis–induced proinflammatory cytokine expression in fibroblasts studied in vitro,⁵⁰ consistent with a deleterious proinflammatory role for TLR2. Similarly, TLR2 contributes to the early inflammatory response to Streptococcus pneumoniae in mice but not to antibacterial defense.⁵¹ These results support the hypothesis that TLR2 may play a deleterious role after some chronic infections by promoting chronic inflammation. In this regard, it will be of interest to determine whether TLR2 promotes resistance to C pneumoniae infection in mouse VSMCs in vitro and in vivo.

Although the subject remains controversial, there is considerable interest in the pathogenic roles of microbes in atherogenesis, based in part on evidence that bacterial and viral products are present in some atherosclerotic lesions. Chronic infections may promote atherosclerosis via multiple mechanisms, including direct effects of microbial products on circulating leukocytes and endothelial cells, as well as on VSMCs within the vascular wall. We have recently shown that both human and mouse VSMCs express an efficient TLR4 signaling complex and that exposure to E. coli LPS promotes a proinflammatory VSMC phenotype by activating TLR4 signaling.²³ The present study demonstrates that C pneumoniae can also activate arterial SMCs via another member of the TLR family, TLR2. Although TLR2 is reportedly not expressed constitutively in VSMCs within normal human blood vessels or advanced atherosclerotic plaques,⁵² our results show that human coronary artery SMCs do express TLR2 when exposed to microbial products, raising the possibility that VSMCs express TLR2 at earlier stages of atherogenesis. Together, these findings raise the important question of whether activation of both TLR2 and TLR4 by multiple microbial antigens may contribute to clinical observations that both the extent of atherosclerosis and the risk of premature death are associated with infectious burden or number of infectious pathogens in patients.⁵³

	Acknowledgments

 
This work was supported by NIH grants HL47569 (D.B.), GM54060 (D.T.G.), and AI52455 (D.T.G.). We thank Dr Jeffrey B. Tatro for critical review of the manuscript.

Received December 17, 2004; accepted September 9, 2005.

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