Chlamydia pneumoniae Infection and Inflammatory Diseases

Abstract

Chlamydia pneumoniae, an obligate intracellular bacterial pathogen, has long been investigated as a potential developmental or exacerbating factor in various pathologies. Its unique lifestyle and ability to disseminate throughout the host while persisting in relative safety from the immune response has placed this obligate intracellular pathogen in the crosshairs as a potentially mitigating factor in chronic inflammatory diseases. Many animal model and human correlative studies have been performed to confirm or deny a role for C. pneumoniae infection in these disorders. In some cases, antibiotic clinical trials were conducted to prove a link between bacterial infections and atherosclerosis. In this review, we detail the latest information regarding the potential role that C. pneumoniae infection may have in chronic inflammatory diseases.

I. INTRODUCTION

Chlamydia pneumoniae is an obligate intracellular bacterial pathogen that infects the respiratory tract. The majority of individuals are exposed to C. pneumoniae throughout their lifetimes with an antibody prevalence of 50% by age 20 and 80% by 60–70 years old.¹ Although C. pneumoniae infection is predominantly asymptomatic or mild, it can result in the development of acute upper and lower respiratory illness including bronchitis, pharyngitis, sinusitis, and pneumonia.¹ C. pneumoniae infection and its relationship to chronic inflammatory diseases remains a controversial topic. A mounting body of evidence shows that not only is C. pneumoniae involved in respiratory infection, it also contributes to the pathogenesis of a range of inflammatory diseases including, but not limited to, atherosclerosis, arthritis, asthma, lung cancer, and chronic obstructory pulmonary disease as well as neurological disorders, namely, Alzheimer’s disease, multiple sclerosis, and schizophrenia (Fig. 1). In this review, we investigate the latest findings regarding the role of C. pneumoniae in the development and/or exacerbation of chronic inflammatory diseases.

FIG. 1:

C. pneumoniae infection and inflammatory disease. In addition to pneumonia, C. pneumoniae infection may contribute to a range of inflammatory diseases including asthma and lung cancer. Dissemination of C. pneumoniae from the lung throughout the body can possibly lead to atherosclerosis, arthritis, and neurological diseases. Some evidence suggests that C. pneumoniae may also be associated with biliary cirrhosis, diabetes, and Bechet’s disease.

II. C. PNEUMONIAE INFECTION AND IMMUNE RESPONSE

C. pneumoniae undergoes a biphasic life cycle alternating between morphologically distinct and functional forms (Fig. 2). The infectious and metabolically inert elementary body (EB) attaches to and enters cells via endocytosis, whereby it inhibits phagolysosome fusion. The EB then matures into a noninfectious metabolically active reticulate body (RB) that is separated from the cytosol within nonlysosomal inclusions. Inside these inclusion bodies, C. pneumoniae creates an intracellular niche, whereby it can modify host cell pathways, replicate, and form new infectious EBs that are released from the cell.

FIG. 2:

Innate immune response directed against C. pneumoniae. C. pneumoniae EBs are endocytosed, escaping lysosome fusion to create an intracellular niche (inclusion), in which they form RBs and replicate. C. pneumoniae EBs are detected by TLRs on the cell surface, and C. pneumoniae RBs are detected intracellularly by NOD1 and NOD2. Furthermore, cellular stress induced by C. pneumoniae infection such as enhanced ROS production results in activation of the NLRP3 inflammasome. Together, activation of these innate pathways results in the induction of inflammatory cytokines, chemokines, and cell adhesion molecules that act downstream to clear infection. Immune evasion by C. pneumoniae can result in persistent infection.

C. pneumoniae can infect a range of different cells types. In regard to respiratory infection, C. pneumoniae initially infects lung epithelial cells and alveolar macrophages. Infection can then spread to infiltrating immune cells such as monocytes, macrophages, monocyte-derived dendritic cells (DCs), lymphocytes, and neutrophils. Failure to eradicate C. pneumoniae can lead to chronic infection, during which C. pneumoniae enters a state of quiescence with intermittent periods of replication. Persistence of the RB within cells may occur during long periods of time due to its ability to hide from the host immune system within inclusion bodies. Through the lower respiratory tract, chronically infected monocytes may disseminate systemically throughout the body,² leading to infection at distal sites including blood vessels, joints, and the central nervous system (CNS). At these distal sites, C. pneumoniae can infect endothelial cells, smooth muscle cells (SMCs), microglial cells, astrocytes, and neurons.

The innate immune response to C. pneumoniae is initiated through the detection of C. pneumoniae antigens by receptors specialized in bacterial sensing, such as Toll-like receptors (TLRs) and nucleotide-binding oligomerization domain-like receptor (NOD) 1 and NOD2, and by triggering the NOD-like receptor family pyrin-domain-containing 3 (NLRP3) inflammasome pathway (Fig. 2). Activation of these pathways results in the up-regulation of proinflammatory cytokines and chemokines including interferon-γ (IFN-γ); tumor necrosis factor (TNF)-α; interleukin (IL)-6, −1β, −8, −12, and −23; monocyte chemoattractant protein-1 (MCP-1); macrophage-induced gene (MIG); regulated-on-activation normal T-cell expressed and secreted (RANTES); intracellular adhesion molecule (ICAM)-1; vascular cell adhesion molecule (VCAM)-1; and E selectin. Cells of the innate immune response, including macrophages, monocyte-derived DCs, plasmacytoid DCs, and neutrophils, are important for the eradication of infection.^3–6 Furthermore, subsequent activation of the adaptive immune response, including CD8⁺ and CD4⁺ T cells, is also critical to resolve infection.⁵

A number of C. pneumoniae antigens have been implicated in the activation of the innate immune response. The outer membrane of the C. pneumoniae EB consists of lipopolysaccharides (LPS), the highly expressed chlamydial heat-shock protein 60 (hsp60), major outer-membrane protein, cytoseine-rich protein (CRP), and outer-membrane protein A. The immune response elicited by LPS and hsp60 are the best characterized. The detection of these antigens by TLR4 (LPS, hsp60) and TLR2 (hsp60) results in the activation of epithelial, endothelial, and antigen-presenting cells and consequently triggers the inflammatory immune response. In regard to the TLR family, TLR2 appears to be the most important for resolution of infection; however, although TLR4 also contributes to the innate immune response, it is not essential for the clearance of bacteria.⁷ The myeloid differentiation primary response 88 (MyD88)-dependent pathway is crucial for mediating the TLR response to C. pneumoniae, resulting in the production of inflammatory cytokines that in turn activate the cell-mediated immune response, predominantly T-helper (Th)1, that is required for clearance of infection.⁸

Intracellular receptors NOD1 and NOD2, which recognize peptidoglycan components, are also important players in the immune response to C. pneumoniae. This pathway, mediated by receptor interacting protein 2, is important for inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production, IL-6, IFN-γ, chemokine C-X-C motif ligand 1 (CXCL1), and macrophage inflammatory protein 1 production, resulting in neutrophil recruitment and bacterial clearance.⁶

The NLRP3 inflammasome can detect cellular stress induced by C. pneumoniae infection, resulting in caspase-1 activation and production of IL-1β and IL-18 following priming by the TLR2/MyD88 pathway.^4,9 IL-1β secretion, through the NLRP3 pathway, is essential for bacterial clearance during early stages of infection.⁴ Activation of the NLRP3 inflammasome may occur via C. pneumoniae reactive oxygen species (ROS) induction, K⁺ efflux, and lysosomal damage.¹⁰ Indeed, our laboratory has shown that K⁺ efflux and ROS can result in cytosolic oxidized mitochondrial DNA release that in turn can activate NLRP3.¹¹ This pathway may also occur in response to C. pneumoniae infection, because it has been reported that C. pneumoniae can induce mitochondrial damage in alveolar macrophages.⁴ The exact mechanism of C. pneumoniae-induced mitochondrial damage, however, is not well characterized.

Activation of the cell-mediated immune response is important to control C. pneumoniae infection.⁵ CD8⁺ T cells regulate bacterial growth during early infection (at d 14 and 28), likely through the production of IFN-γ and TNF-α. At early time points, CD4⁺ T cells are not required for control of infection, but they do have an important role at later time points (60 d after infection) and upon reinfection.⁵ Interestingly, at early time points, CD4⁺ T cells actually promote bacterial growth in the absence of CD8⁺ T cells. This is thought to be the result of CD4⁺ T cells favoring a Th2 (not a Th1) response, in the absence of CD8⁺ T cells.⁵ In line with this, IFN-γ is essential for the resolution of infection, as evidenced by a significant bacterial load increased in IFN-γ receptor (Ifngr)^−/− mice.⁵ This effect is mediated in part through IFN-γ–induced iNOS expression and NO production, which is important for killing and inhibiting bacterial growth.⁵ Because exposure to C. pneumoniae results predominantly in asymptomatic or mild infection, the development of chronic cases of C. pneumoniae infection and inflammatory disease likely requires additional host genetic or environmental factors that result in failure to eliminate infection or predispose to the relevant disease.

III. DETECTION OF C. PNEUMONIAE

C. pneumonia–specific antibody detection by immunofluorescence is the standard method for serological diagnosis of C. pneumoniae infection.¹² The kinetics of immunoglobulin (Ig) M, G, and A secretion are taken into account when determining acute or chronic infection. IgM levels peak at 2–3 wk after initial infection, become undetectable at 2–3 mo, and are not induced following subsequent infection. In contrast, IgG typically peaks at 6–8 wk after initial infection and are rapidly induced following subsequent infections (1–2 wk). Chronic infection is somewhat more difficult to determine and requires the detection of persistent IgG levels, which is complicated by the fact that IgG has a half-life of weeks to months and may therefore be present for some time following acute infection. It has been proposed that IgA levels may provide a better indication of chronic infection, but according to Dowell et al.,¹² the use of IgG and A serological markers alone should not be used. Polymerase chain reaction (PCR) methods are also used for the detection of C. pneumoniae infection within tissues and cell types.^12,13 Identification of C. pneumoniae messenger RNA (mRNA) by real-time (RT)-PCR can also be used to determine whether C. pneumoniae is in a metabolically activated state.¹⁴ Other methods include bacterial culture, immunocytochemistry, and electron microscopy.¹⁵

IV. DISEASES

A. Atherosclerosis

Atherosclerosis, a chronic inflammatory disease characterized by the accumulation of lipids and fibrosis within large arteries, is the leading cause of heart disease and stroke. Increasing evidence shows a relationship between the pathogenesis of atherosclerosis and infectious agents including C. pneumoniae, Porphyromonas gingivitis, Helicobacter pylori, influenza A virus, hepatitis C virus, cytomegalovirus, and human immunodeficiency virus.^16,17

Initiation of atherosclerosis involves the accumulation of low-density lipoproteins (LDLs) in the intima, the innermost layer of the artery that consists of an elastic membrane covered by a monolayer of endothelial cells. Oxidation and other modifications of LDLs within this subendothelial matrix initiate an inflammatory response. Stimulation of endothelial cells results in the expression of adhesion molecules, growth factors, and cytokines. Monocytes migrate through the endothelium, proliferate, differentiate into macrophages, and take up oxidized LDLs (oxLDLs) to form foam cells. Foam cell necrotic death enhances inflammation, resulting in the recruitment and proliferation of T cells, B cells, and DCs. Furthermore, inflammatory cytokines and mediators act on vascular SMCs, causing them to migrate and proliferate within the subendothelial matrix. SMC production of the extracellular matrix within this region forms a fibrotic plaque together with extracellular lipids covered by a fibrous cap. Disease progression can lead to rupture of this fibrous cap, heart disease, and stroke. Human studies, mouse models, and in vitro data point to an important role of C. pneumoniae infection at all stages of disease, from initiation through plaque rupture by acting through a range of cell types.

A large number of human studies have determined this association of C. pneumonia with atherosclerosis. A case-control meta-analysis using standardized serological criteria from 16 studies showed a significant increase in seroprevalence (C. pneumoniae-specific IgA and G) in atherosclerotic patients. Association of seropositivity to inflammatory makers in atherosclerotic patients was also evidenced using meta-analyses demonstrating that seropositive patients had significantly greater high-sensitivity C-reactive protein, IL-6, and fibrinogen levels.¹⁸ In addition to serological data, PCR, immunohistochemistry, and electron microscopy techniques have found evidence of C. pneumoniae within atherosclerotic tissues.^13,14,19 Furthermore, when detected, C. pneumoniae and human hsp60 are localized to plaque macrophages in the majority of atherosclerotic tissue isolated from patients.²⁰ Finally, C. pneumoniae isolated from atherosclerotic lesions has been cultured in vitro.^21,22 These data therefore provide strong evidence that C. pneumoniae infection may play a part in the prevalence and/or severity of atherosclerosis.

Animal models provide further evidence that C. pneumoniae is involved in the pathogenesis of atherosclerosis. New Zealand white rabbits infected with C. pneumoniae can develop atherosclerosis with²³ and without²⁴ a cholesterol-enriched diet. Apolipoprotein-E knockout mice (Apoe^−/−) and LDL receptor (LDLR) knockout mice (Ldlr^−/−) are prone to the development of atherosclerosis and are commonly used to study this disease. Murine studies of C. pneumonia–induced atherosclerosis performed in wild-type (WT), Ldlr^−/−, or Apoe^−/− mice indicate that persistent C. pneumoniae infection of the aorta and C. pneumoniae-mediated enhancement of atherosclerosis in mice require high serum lipid levels. It was shown that Ldlr^−/− mice present with enhanced atherosclerotic lesion areas following 9 mo of once-monthly intranasal inoculations or 6 mo of twice-monthly intranasal inoculations only when fed a high-fat diet.^25,26 Furthermore, direct infection of the aorta of Ldlr^−/− mice fed a high-fat diet resulted in enhanced collar-induced atherosclerosis accompanied by increased expression of MCP-1 and ICAM-1 within the arterial wall.²⁷ Apoe^−/− mice fed a high-fat diet present with enhanced atherosclerosis following single²⁸ or multiple^29,30 intranasal inoculations of C. pneumoniae before being fed a high-fat diet, accompanied by enhanced inflammation. This includes increased expression of cytokines MCP-1, IL-12p40, IL-6, IFN-γ, and TNF-α.^31,32 Furthermore, enhanced infiltration of monocytes, macrophages, monocyte-derived DCs, plasmacytoid DCs (pDCs), and T cells are observed in the atherosclerotic lesion.^28,30,31

Some murine studies did not find an association of C. pneumoniae infection with atherosclerosis.³³ However, this discrepancy may be due to study design or the use of different C. pneumoniae strains. Studies in which no association was found used the Kajaani 7 strain³³ or a Finnish C. pneumoniae isolate,³⁴ as compared to the AR39,^29,35 TWAR 2043 (American Type Culture Collection [ATCC] VR-1355),^30,36 IOL-207,³⁷ or CM-1 strains²⁸ that were used in studies that found significant associations.

The first step involved in C. pneumonia–associated enhancement of atherosclerosis requires dissemination from the infected lung to the aorta. This occurs through chronically infected monocytes and macrophages that migrate from the lung and take residence within the arterial wall.² Replication and release of C. pneumoniae within the arterial wall spreads infection to proximal endothelial cells and SMCs. As shown in carotid atherosclerotic lesions resected from patients, C. pneumoniae infects endothelial cells of the intima as well as macrophages and SMCs within the atherosclerotic plaque but is absent from surrounding tissues.^14,15,21 C. pneumoniae can also be detected within the medial smooth muscle layer underlying the atherosclerotic plaque.³⁸ In vitro studies also confirm direct C. pneumoniae infection of these cell types.^39–45 Two virulence factors associated with C. pneumoniae (LPS and hsp60) that activate TLRs contribute to many of the pathogenic effects associated with C. pneumonia–mediated atherosclerosis.^{20,42,44,46–51} In line with this, C. pneumoniae inflammatory responses are dependent on TLRs and the Myd88-dependent pathway. This is observed in the Apoe^−/− models wherein C. pneumoniae infection of Apoe/Tlr2, Apoe/Tlr4, and Apoe/Myd88 double knockout mice resulted in reduced inflammation and development of atherosclerosis, when compared to C. pneumonia–infected Apoe^−/− mice.³¹ Interestingly, acute infection and enhanced inflammation can still be detected within the aorta of normocholesterolemic mice following intranasal C. pneumoniae inoculation, indicating that C. pneumoniae infection of the artery does not necessarily require a high-fat diet.^35,52

The expression of inflammatory cytokines induced by C. pneumoniae likely contributes to C. pneumonia–mediated atherosclerosis. IFN-γ, which is expressed by Th1 cells and macrophages following C. pneumoniae infection, is found at high levels within atherosclerotic tissues and has long been implicated in the pathogenesis of atherosclerosis. This is evident within atherosclerosis mice models, in which knockout of the IFN-γ receptor reduces disease, and increased expression of IFN-γ enhances disease.⁵³ Regarding the effects on macrophages, it can induce the expression of TNF-α, IL-6, and MMPs; increase lipid uptake; and enhance ROS.⁵³ It can also act on endothelial cells to induce cell adhesion molecule expression and increase immune cell accumulation within the atherosclerotic plaque.⁵³ TNF-α also has an important role, as evidenced by knockout of TNF-α receptor 1 (TNF-R1) p55, which results in reduced numbers and size of C. pneumonia–mediated atherosclerotic lesions in mice fed a high-fat diet.⁵⁴ Recently, a role for Th17 cells in atherosclerosis was identified. IL-17A knockout in Apoe^−/− mice attenuates C. pneumonia–mediated atherosclerosis as measured by plaque lipid content and lesion size.³² Other cytokines induced by C. pneumoniae, including MCP-1 and IL-1, −18, −6, and −12, have all been shown to enhance disease in murine models of atherosclerosis.⁵⁵ However, experiments showing the knockout of these cytokines, or their receptors, in a C. pneumonia–mediated model of atherosclerosis are unavailable.

Initiation of disease through the accumulation, modification, and uptake of lipids by monocytes or macrophages to produce inflammatory foam cells is enhanced in the presence of C. pneumoniae. Murine models show that although C. pneumoniae has no effect on serum lipid levels, it enhances the accumulation of lipids within the aorta even in normocholesterolemic mice after repeated C. pneumoniae infections.⁵⁶ C. pneumonia–infected monocytes oxidize LDL in a dose-dependent manner that is mediated by hsp60.⁵⁷ Furthermore, C. pneumoniae stimulates platelet production of ROS, likely through an LPS-induced pathway and induction of iNOS and lipoxygenase. This is associated with enhanced oxidization of LDLs.⁴⁶ oxLDL is highly inflammatory, causing injury to endothelial cells, formation of foam cells, and the migration and proliferation of immune cells and SMCs within the intima.⁵⁸ Overall, these events create a highly inflammatory environment that can lead to the development and progression of disease.

C. pneumoniae infection of monocytes and macrophages also enhances foam cell formation directly in the presence of high-LDL concentrations through an LPS-induced pathway.^42,47 Studies found that perturbed intracellular macrophage cholesterol homeostasis arises from C. pneumonia–induced expression of acetyl-CoA acetyltransferase 1 and reduced expression of adenosine triphosphate (ATP)-binding cassette subfamily A member 1 and subfamily G member 1 via a Jun amino-terminal kinase/peroxisome proliferator-activated receptor-γ pathway that facilitates cholesterol accumulation and foam cell formation.⁵⁹ The contribution of perturbed macrophage lipid metabolism for C. pneumonia–mediated atherosclerosis is also evident with knockout of liver X receptor α in Apoe^−/− mice, which results in enhanced foam cell formation and atherosclerosis when compared to Apoe^−/− mice.³¹ In addition to altered metabolism, C. pneumoniae infection can induce macrophage uptake of nonoxidized LDL. Because macrophages typically only take up oxLDL, this mechanism leads to enhanced foam cell formation independent of LDL oxidization and the classical LDLR pathway.⁴¹

The endothelial layer is critical for maintenance of vascular function. C. pneumoniae can modify endothelial cell function and layer integrity by indirect mechanisms, such as through enhanced oxLDL or inflammatory molecules, as well as by C. pneumoniae antigen stimulation and direct infection of endothelial cells.⁴⁰ Destabilized vascular integrity is evident by reduced endothelial relaxation in C. pneumonia–infected mice dependent on NO.^37,60 C. pneumoniae also activates mitogen-activated protein kinase (MAPK) and nuclear factor–κB pathways within endothelial cells,^39,61 consequently up-regulating expression of chemokines and adhesion molecules that enhance leukocyte rolling, adhesion, and transendothelial migration.^39,61 Chemokines expressed by infected endothelial cells include IL-8, MIG, IFN-γ inducible protein (IP)-10, RANTES, and MCP-1,^40,62,63 and adhesion molecules include ICAM-1, VCAM-1, and E selectin.^39,61,62 C. pneumoniae infection of primary aortic endothelial cells also results in enhanced production of granulocyte macrophage colony stimulating factor (GM-CSF) in a TLR2-, TLR4-, and MyD88-dependent manner that may promote enhanced DC proliferation within the atherosclerotic lesions.³¹ Furthermore, C. pneumonia–infected endothelial cells display enhanced apoptosis and necrosis⁶⁴ that may also exacerbate inflammation through danger-associated molecular pattern signaling pathways.

C. pneumoniae can also stimulate platelet activation, which has proatherogenic properties,^48,65 likely through an LPS-dependent pathway and resulting in up-regulation of P selectin, platelet aggregation, and release of ATP.⁴⁸ In addition, C. pneumoniae triggers platelets to release chemokine ligands (CCLs) including CCL3, 5, and 7 and CXCL8. This may recruit inflammatory cells to the lesions, thus exacerbating disease.⁶⁶

C. pneumonia–mediated endothelial dysfunction also has an important role in promoting SMC migration and proliferation through the expression of soluble factors.⁴³ These may include heparin-binding epidermal growth factor and platelet-derived growth factor subunit B, which increase in response to endothelial C. pneumoniae infection and have previously been shown to modify SMC proliferation.⁶³ Direct infection of SMCs also promotes their migration and proliferation within the lesion. In vitro studies demonstrate that C. pneumonia–infected rat vascular SMCs enhanced migration in a TLR2-dependent manner.⁶⁷ Rabbit-infected vascular SMCs show enhanced proliferation in response to C. pneumoniae through hsp60 and the extracellular regulated kinase-1 and −2 pathways; this is further enhanced in the presence of oxLDL.^44,51 Furthermore, in vitro studies with human vascular SMCs show that C. pneumoniae EBs induce hsp60, and this activates TLR4-mediated p44/p42 MAPK activation and SMC proliferation.⁴⁹

C. pneumoniae may also play a part in destabilization of atherosclerotic plaque. Ldlr^−/−Apoe^−/− mice present with a reduced fibrous cap area relative to total lesion size, a characteristic of rupture-prone plaques. This is accompanied by increased matrix metalloproteinase (MMP)-2 and MMP-9 expressions, likely mediators of fibrous cap thinning through breakdown of the extracellular matrix.³⁶ C. pneumoniae enhancement of MMP production (including MMP-7 and −9) can occur through direct infection of monocytes and macrophages and is regulated by hsp60.²⁰ SMCs may also express MMPs to destabilize the plaque, because culture of SMCs with soluble factors taken from supernatants of C. pneumonia–infected monocytes can induce MMP-2 expression.^20,50,68 SMCs and endothelial cells may also contribute to plaque instability following direct infection, because in vitro studies show increased tissue factor, plasminogen activator inhibitor-1, and IL-6 expression following infection, all of which are implicated in thrombosis and reduced plaque instability.⁴⁵ Finally, the T-cell response generated against C. pneumoniae may be involved in enhanced plaque instability. One study analyzed the T-cell population of unstable atherosclerotic plaques and found that T cells with antigen specificity for C. pneumoniae predominantly exhibited a Th1 response (94%).⁶⁹ Indeed, IFN-γ, produced by Th1 cells, can inhibit SMCs proliferation and collagen synthesis and enhance MMP expression, thus contributing to plaque instability.⁷⁰

A number of large-scale trials investigating the effects of antichlamydial antibiotics, including azithromycin, gatifloxacin, or clarithromycin, for the treatment of atherosclerosis have been performed.^71–74 These studies failed to provide conclusive evidence that antibiotics are an effective treatment and have led to controversy as to whether C. pneumoniae actually has a role in the initiation/exacerbation of atherosclerosis. However, a number of factors have been hypothesized to explain why these trials failed, such as the timing of antibiotic treatment in the context of disease, antibiotic treatment regime, effectiveness of antibiotics in eliminating persistent C. pneumoniae infection, complications of other pathogenic factors, and use of other approved clinical treatment regimes masking the effectiveness of antibiotics.⁷⁵ For example, these studies were performed on patients with advanced atherosclerosis, which would not take into account C. pneumonia–mediated initiation and early progression of disease. The trials also used a single antibiotic, which may not be as effective in clearing bacteria as combination treatment, particularly in regard to persistent infection.^76–78 Therefore, the results of these trials cannot rule out a role for C. pneumoniae in the initiation/exacerbation of disease. Additional trials are needed to address these complicating factors and conclusively determine the effectiveness of antibiotic treatment for atherosclerosis.

Investigations into the effectiveness of antibiotic treatment in animal models of C. pneumonia–mediated atherosclerosis have yielded mixed results. Antibiotic trials of C. pneumonia–infected rabbits on a high-fat diet show reduction in atherosclerosis development following azithromycin treatment during a 7-wk time course started after the final infection.²³ In another study, azithromycin treatment of C. pneumonia–infected rabbits begun at an early time point (5 d after initial infection) showed reduced incidence of atherosclerosis, but the same treatment regime begun at a later time point (2 wk after final infection) did not.⁷⁹ The analysis of antibiotic treatment in the Apoe^−/− mouse model of C. pneumonia–induced atherosclerosis showed that azithromycin begun 2 wk after infection did not have an effect.⁸⁰ Additional investigation using combination antibiotic treatment is needed. Furthermore, the timing of antibiotic treatment in the context of disease must be further explored to enhance our understanding of the effectiveness of antibiotic treatment for atherosclerosis.

B. Asthma

Asthma is an allergic disease of the lung characterized by a Th2 response, high IgE titers, and increased eosinophil infiltration. The pathogenesis of allergic asthma has been associated with bacterial and viral infections. As evidenced through case-control and cohort studies, C. pneumoniae has been associated with initiation and exacerbation of asthma. Early studies indicated the association of C. pneumoniae Ig levels with wheezing and adult-onset asthma.⁸¹ Succeeding studies found that this was due to chronic C. pneumoniae infection, and wheezing induced by C. pneumoniae commenced before the development of asthma.^82,83 The majority of subsequent studies support these findings. Serological studies found that increased C. pneumoniae IgG levels were associated with incidence of chronic allergic asthma, recent asthma, and the severity of chronic asthma.^84,85 C. pneumoniae IgA levels were also found to be associated with asthma.⁸⁶ Furthermore, the titer of C. pneumonia–specific IgE levels in asthmatic patients significantly correlated with asthma severity.⁸⁷ C. pneumoniae was also found to be associated with chronic asthma, and patients that tested positive for mycoplasma and/or C. pneumoniae have increased mast-cell infiltration of the lung.⁸⁸ There is also evidence for an association of C. pneumoniae with the development, incidence, and exacerbation of acute and chronic asthma in children. C. pneumoniae was associated with increased wheezing in pediatric asthma, which improved with antibiotic treatment.⁸⁹ In addition, C. pneumonia–specific IgE levels were found to be greater in asthmatic children with C. pneumoniae infection.⁹⁰ Another study found that chronic infection, as measured by PCR, as well as increased IgA levels, were associated with increased incidence of asthmatic exacerbations in children.⁹¹ Compared with asthmatic patients treated with low-dose steroids, increased titers of C. pneumonia–specific antibodies, 74.1% more IgG, and 70.1% more IgA were observed in asthmatic patients receiving a high-dose steroid treatment.⁹²

C. pneumoniae is thought to promote the development of asthma by inducing a Th2 immune response; however, the specific mechanisms involved in this process are still unknown. This effect is evidenced by high IgE titers and enhanced mast-cell infiltration in asthmatic patients with C. pneumoniae infection.^87,88,90 Murine models also shed light on the mechanisms by which C. pneumoniae induces asthma. Mice sensitized with the human serum albumin (HSA) allergen 5 d after low-dose C. pneumoniae infection, and challenged with HSA 2 wk later, developed lung inflammation characterized by enhanced eosinophil infiltration as well as increased goblet cells and HSA-specific IgE levels.^93,94 This allergic response is dependent on DC activation through the MyD88 pathway.⁹³ TLR4, but not TLR2, deficiency prevented the development of the allergic response.⁹⁴ In fact, TLR2 deficiency allowed allergen sensitization but at a later time point, which was not observed in WT mice.⁹⁴ Those disparities were due to a differential regulatory T (T_reg)–cell response, with TLR4 deficiency enhancing and TLR2 deficiency reducing T_reg-cell amounts in the lung after infection.⁹⁴ The allergic response, however, was only observed with low-dose C. pneumoniae infection; high-dose infection increased T_reg cells and pDCs in the lung, which in turn act to suppress sensitization.⁹⁴ Other studies investigating chlamydia family member Chlamydia muridarum indicate that infection induces airway hyper-responsiveness in mice through reduced expression of the IL-13 decoy receptor IL-13Rα2, thus enhancing IL-13 and signal transducer and activator of transcription–6 signaling.⁹⁵ Further studies indicate that reduced expression of IL-13Rα2 is a consequence of programmed death ligand (PDL)-1, expressed on lung DCs and monocytes, and its receptor PD-1 expressed on T cells following infection.⁹⁶ These proteins have previously been shown to induce a Th2 response and enhance airway hyper-responsiveness and lung inflammation.⁹⁷ Whether these pathways are also important for C. pneumoniae induction of allergic airway disease remains to be elucidated.

C. Cancer

The development of cancer has been long associated with a number of pathogens, both causative and opportunistic.⁹⁸ A number of case-control and cohort studies have looked for association of C. pneumoniae with lung cancer, although with somewhat varied results. A meta-analysis⁹⁹ of 12 studies was performed based on detection of IgA or IgG within patient or control blood according to standardized serological criteria,¹², with adjustment for confounding effects of lung cancer risk factors and timing of blood sampling (before or after disease onset). This identified a significant association of C. pneumoniae infection with lung cancer risk, although sampling of blood before lung cancer diagnosis resulted in a weaker association than sampling after diagnosis. Overall association of C. pneumoniae with lung cancer was found to increase with stricter IgA cutoff levels (IgA ≥ 16, odds ratio [OR] = 1.22, and 95% confidence interval [CI] = 1.06–1.41 compared to IgA ≥ 64, OR = 2.35, and 95% 1.88–2.93). These findings are also in line with a meta-analysis conducted by Littman et al.¹⁰⁰ Enhanced infection after disease onset indicates that C. pneumoniae infection during cancer is opportunistic. However, given that a significant association was found for infection before disease onset, this also indicates a causal relationship.

The mechanism of C. pneumonia–mediated lung cancer development is not well defined, but lessons from other bacterial species associated with cancer development provide insight as to how this may occur. Bacterial infection of the epithelium leads to the production of chemokines, which promotes immune cell activation and tissue infiltration that result in the production of inflammatory mediators such as TNF- α, IL-1β, IFN-γ, IL-6, and IL-8. Expression of these inflammatory mediators can enhance ROS production, resulting in tissue damage, DNA damage, increased cell turnover, and DNA mutations that promote uncontrolled cell proliferation.¹⁰¹ Indeed, C. pneumoniae infection can induce expression of these inflammatory mediators and enhance ROS production, as discussed previously.

D. Arthritis

Persistent bacterial infection within the joint can lead to the development of reactive arthritis. Many studies have found that both C. trachomatis and C. pneumoniae are associated with reactive and other forms of arthritis, although a stronger association exists for C. trachomatis. In regard to C. pneumoniae, disease arises following dissemination of infected lung monocytes and macrophages that take residence within the joint and induce inflammation. The association of C. pneumoniae with arthritis has been through detection of C. pneumoniae serologically as well as within the synovial membrane and synovial fluid, using immunofluorescence, PCR, electron microscopy, and bacterial culture. In one study, serological evidence of C. pneumoniae was found in five of 70 patients, and synovial lymphocytes from these patients demonstrated C. pneumonia–mediated proliferation in vitro.¹⁰² Furthermore, three of these patients presented with upper respiratory tract infection before disease onset, indicating causality. Another study found serological evidence of C. pneumoniae infection in four of 35 patients with arthritis, and three of these patients experienced lower respiratory illness before disease onset.¹⁰³ C. trachomatis was not identified in these patients, suggesting that C. pneumoniae was the causative agent.¹⁰³ In addition, three of these patients were positive for human leukocyte antigen (HLA)-B27, a risk factor for arthritis, indicating that C. pneumoniae may work synergistically with host genetic factors to promote disease. This is further supported by findings that the APOe4 allele, a risk factor for late-onset arthritis, is associated with enhanced C. pneumoniae within the joint, as measured by PCR.¹⁰⁴ Additional studies using PCR demonstrated that C. pneumoniae was present within 12.7% and 7.9% of joint tissue and synovial fluid from arthritic patients, respectively.¹⁰⁵ Furthermore, a study using RT-PCR to detect C. pneumoniae mRNA, the presence of which indicates a metabolically active state, determined positive results for ten patients who were also found to be positive by PCR. These patients had various forms of arthritis including Reiter’s syndrome, undifferentiated oligoarthritis, reactive arthritis, rheumatoid arthritis, and osteoarthritis.¹⁰⁶ Another study also found, using PCR, RT-PCR, or bacterial culture, that C. pneumoniae was present within the synovial fluid or peripheral blood mononuclear cells (PBMCs) in synovitis–acne–pustulosis–hyperostosis–osteitis syndrome, psoriatic arthritis, undifferentiated oligoarthritis, and ankylosing spondylitis.¹⁰⁷

E. Neurological Diseases

C. pneumoniae infection has also been associated with a number of neurological diseases including Alzheimer’s disease, multiple sclerosis, and schizophrenia. These arise following transmigration of infected monocytes, disseminated from the lung, across the blood–brain barrier.^108,109 Mediated by infection of the human brain microvascular endothelial cells, this results in up-regulation of VCAM-1 and ICAM-1.¹⁰⁸ Within the CNS, C. pneumoniae infection is likely to spread, thus inducing an inflammatory response and mediating disease. This is supported by studies that show C. pneumoniae can also infect brain microglial cells, astrocytes, and neuronal cells.^109–111

1. Alzheimer’s Disease

Alzheimer’s disease is a neurodegenerative disorder leading to dementia, usually occurring in later life. Alzheimer’s is characterized by intracellular neurofibrillary tangles and extracellular amyloid deposits that induce inflammation, leading to neurite and brain atrophy and impaired synaptic signaling.¹¹² Many studies now support a role for pathogens in promoting Alzheimer’s disease, which include herpes simplex virus 1, hepatitis C virus, cytomegalovirus, toxoplasma, and C. pneumoniae.^113,114 Studies investigating a role for C. pneumoniae in Alzheimer’s patients have been variable. However, a meta-analysis of case-control studies indeed indicates a positive association (OR = 5.66, 95% CI = 1.83–17.51).¹¹⁵ Furthermore, C. pneumoniae has been cultured from the brains of Alzheimer’s disease patients.¹¹⁶

Murine models demonstrate that respiratory inoculation with a C. pneumoniae isolate from Alzheimer’s diseased brain can result in amyloid-β deposits in the neuronal cells of the murine brain for up to 3 mo after infection, progressively increasing over time.¹¹⁷ It is likely that C. pneumoniae initiates or exacerbates inflammatory events within the brain, thus contributing to disease. Such mechanisms may occur via induction of oxidative stress or increased production of inflammatory cytokines by activated microglial cells, which can influence amyloid-β expression or directly contribute to neurodegeneration.¹¹⁸

2. Multiple Sclerosis

Multiple sclerosis is a chronic autoimmune disease of the CNS that is characterized by demyelination of axons, resulting in progressive neurological deterioration.¹¹⁹ A number of pathogens are associated with the development of multiple sclerosis including Epstein-Barr virus, human herpes virus 6, human endogenous retrovirus, Staphylococcus aureus, Mycoplasma pneumoniae, and C. pneumoniae.¹²⁰ Studies investigating the association of C. pneumoniae and multiple sclerosis have also generated varying results, likely due to study design and detection methods. However, a meta-analysis of 26 studies found that multiple sclerosis patients have increased C. pneumoniae levels, as measured by PCR, within their cerebrospinal fluid (OR = 3.216, 95% CI = 1.204–8.585).¹²¹ In contrast, no significant association was found for C. pneumoniae Ig levels within the serum or cerebrospinal fluid.¹²¹

Animal models suggest that C. pneumoniae may have a role in initiating multiple sclerosis. Studies in a rat model demonstrate that a C. pneumoniae peptide that shares a seven–amino acid motif with a myelin basic protein epitope can induce a Th1 response, resulting in severe experimental autoimmune encephalomyelitis (EAE).¹²² Furthermore, mild symptoms of EAE could be induced following immunization of rats with sonicated C. pneumoniae together with complete Freund’s adjuvant.¹²² Another study using the EAE model in mice showed that the disease was exacerbated with intraperitoneal administration of C. pneumonia; this was attenuated with antibiotic treatment.¹²³

3. Schizophrenia

Schizophrenia is a mental illness characterized by hallucination, delusions, paranoia, and depressive symptoms.¹²⁴ In the pathogenesis of disease, evidence exists for a role of pathogen infection, particularly during brain development.¹²⁵ Pathogens associated with schizophrenia include herpes virus, influenza, cytomegalovirus, Borna disease virus, Toxoplasma gondii, cytomegalovirus, Toxoplasma gondii, Chlamydia psittaci, and C. pneumoniae.¹²⁵ A number of studies by Fellerhoff et al. have found an association of C. pneumoniae with schizophrenia.^126–128 A meta-analysis of the two earlier studies by Fellerhoff et al. identified a significant positive association of C. pneumoniae with schizophrenia (OR = 6.34, CI 95% = 2.83–14.19, p < 0.001).¹²⁵ These studies measured C. pneumoniae within PBMCs by PCR.¹²⁶ The latter study by Fellerhoff et al. identified four times as much chlamydial DNA within the frontal cortex in schizophrenia patients as compared to controls.¹²⁷ A study by another group failed to find an association, but this was hypothesized to be due to the high prevalence of C. pneumoniae within healthy controls of the Korean population studied.¹²⁹

F. Other Diseases

C. pneumoniae has also been implicated in other inflammatory diseases including Behcet’s disease, primary biliary cirrhosis, and diabetes. Evidence, however, is scarce, as outlined below, and additional studies are required to provide further support for a role of C. pneumoniae in these diseases.

Behcet’s disease is an autoimmune systemic vasculitis characterized by recurrent episodes of oral aphtous ulcers, genital ulcers, skin lesions, ocular lesions, and other manifestations involving vascular, gastrointestinal, and neurological systems. Evidence suggests that C. pneumoniae may be involved in the pathogenesis of Behcet’s disease, because patients have increased C. pneumoniae IgA and IgG levels compared to controls,¹³⁰ and hsp60 has been found in gastrointestinal lesions associated with this disease.¹³¹

Primary biliary cirrhosis is an autoimmune disease of the liver. Pathogens are hypothesized to induce inflammation within the liver and trigger disease. A number of studies have yielded mixed results regarding the involvement of C. pneumoniae in the pathogenesis of disease. One study found the C. pneumoniae antigen and RNA in the liver of patients.¹³² Another study found that C. pneumoniae IgM levels were increased in patients compared to controls; however, there was no difference compared to posthepatitis cirrhosis, thus generating inconclusive results.¹³³ Another study found no association of C. pneumoniae seropositivity with disease, although there was a positive correlation with disease severity.¹³⁴ Murine studies of infection have found the presence of C. pneumoniae within the liver.^2,135 Additionally, C. pneumoniae has been shown to replicate in murine Kupffer cells, resulting in TNF-α production.¹³⁵

The pathogenesis of type-2 diabetes, characterized by insulin resistance, has been linked to bacterial infections including C. pneumoniae. However, association studies are complicated because patients with diabetes are more prone to infection. Nevertheless, one study found that C. pneumoniae seropositivity was linked to insulin resistance in healthy middle-aged men, and this correlation increased with increased C. pneumoniae Ig levels.¹³⁶ Murine studies show that C. pneumoniae can enhance insulin resistance and inflammation in obese mice, which is dependent on TNF-α.¹³⁷ Murine studies also indicate that C. pneumoniae infection can increase IL-1β levels in the pancreas, a pathogenic factor associated with type-2 diabetes.¹³⁸ Furthermore, C. pneumoniae infection of mast cells promotes reduced β-cell ATP and insulin production and enhanced β-cell destruction.¹³⁸

V. CONCLUSION

Given the complexities regarding disease development and/or exacerbation, it is not surprising that it is extremely difficult to definitively correlate or establish a causal relationship between C. pneumoniae infection and the various diseases described here. Whereas mouse studies have generally established that C. pneumoniae infection can exacerbate or accelerate most of these pathologies, human studies can only infer on the basis of circumstantial evidence. Additionally, negative results from poorly designed clinical trials have led to a reduction in enthusiasm for these types of studies. However, a better understanding of the possible mechanisms by which a persistent bacterial infection might affect chronic inflammatory conditions has reopened the idea linking C. pneumoniae infection and the development of various diseases. Although there has been no “smoking gun” evidence for this association, it is clear from the sheer number of positive studies, and the multitude of diseases affected, that where there is smoke there may be fire, and further investigations are warranted to understand the role of C. pneumoniae infection in disease development and the potential interventions that may be designed to reduce and/or eliminate this possible risk.

ABBREVIATIONS:

Apoe

Apoplipoprotein

CCL

chemokine ligands

DC

dendritic cells

hsp60

heat-shock protein 60

IFN-γ

interferon γ

ICAM-1

intracellular adhesion molecule-1

Ig

immunoglobulin

LDL

low-density lipoprotein

LPS

lipopolysaccharides

MCP-1

monocyte chemoattractant protein

MMP

matrix metalloproteinase

MyD88

myeloid differentiation primary response 88

NLRP3

NOD-like receptor family pyrin-domain-containing 3

NOD

nucleotide-binding oligomerization domain-like receptor

PCR

polymerase chain reaction

ROS

reactive oxygen species

RT-PCR

real-time PCR

SMC

smooth muscle cell

Th

T helper

TLR

Toll-like receptor

TNF-α

tumor necrosis factor α

VCAM-1

vascular cell adhesion molecule 1