A Hidden Organism, Chlamydia in the Age of Atherosclerosis

Abstract

Atherosclerosis is a chronic inflammatory disease. It is still the leading cause of mortality and morbidity in the world. Inflammation in the vessels plays the most important role in the pathogenesis of atherosclerosis. Many studies have been emphasized that Chlamydia pneumoniae triggers inflammation in the vessels and associated with atherosclerosis. It is stated that most of the chlamydial infections are asymptomatic and around 40% of adult individuals are infected. Chlamydia has different subgroups. It was thought to be a virus due to its intracellular pathogenicity, but it was included in the bacteria genus because it contains DNA and RNA chromosomes and has enzymatic activity. Chlamidya can easily be transmitted through the respiratory tract and sexual transmission. Seroepidemiological and pathological studies of atherosclerotic plaques showed the presence of Chlamydia in the plaque. This section will provide relationship between Chlamydia and atherosclerosis on the recent researces and current information will be discussed.

1. Introduction

Cardiovascular diseases are the leading cause of morbidity and mortality in developed societies. The World Health Organization ranked coronary heart disease first and stroke fourth on its list of major life-threatening diseases. More than 40% of deaths from non-communicable diseases in the community are due to cardiovascular causes.

Cardiovascular disease can manifest in myriad ways, including heart attack, stroke, peripheral artery disease, organ ischemia and necrosis, and aortic and peripheral arterial aneurysms. The main pathogenetic mechanism underlying cardiovascular diseases is atherosclerosis. When evaluated in terms of cost and loss of labor, atherosclerosis is a malignant condition that requires enormous resource allocation and affects all aspects of humanity. Atherosclerosis is progressive and affects all arteries in the body to a varying extent. It is extremely common, occurring in almost all individuals in the population. The main factors associated with a higher rate of atherosclerosis are chronic inflammation, genetic predisposition, advanced age, male sex, hyperlipidemia, hypertension, smoking, diabetes, sedentary lifestyle, and obesity.

Studies conducted in different parts of the world have shown that atherosclerosis begins in childhood. Autopsy studies conducted in Japan demonstrated early atherosclerosis and fatty streaks in the aortas of 29% of infants younger than 12 months and in the coronary arteries of 3.1% of children aged 1–9 years [1]. Similarly, autopsy studies in the USA have shown that the prevalence of fatty streaks in the coronary arteries is 50% between the ages of 2 and 15 years and increases to 85% between the ages of 21 and 39 years. Atherosclerotic plaque formation, a more advanced form of fatty streaking, was observed in 8% of children aged 2–15 years and 69% of adults aged 26–39 years [2].

Atherosclerosis starts with endothelial dysfunction and progresses with subintimal thickening and smooth muscle cell proliferation. The process is characterized with medial thickening due to macrophage infiltration, scavenger cell accumulation, and plaque formation. Lipid deposition is a common vascular condition in which inflammatory and infective processes trigger each other.

Studies on atherosclerosis risk factors have shown that organic pathogenic microorganisms contribute to the inflammatory process. These include Chlamydia pneumoniae, Helicobacter pylori, influenza A virus, hepatitis C virus, cytomegalovirus, and HIV. Chronic inflammation induced by microorganisms can cause many chronic diseases, malignancies, autoimmune diseases, and inflammatory atherosclerosis [3, 4]. Some microorganisms have been directly linked to atherosclerotic disease because they can be isolated and cultured from plaques, whereas the presence of other organisms has been demonstrated by biochemical tests [5, 6]. Microorganisms influence the atherosclerosis process by triggering inflammation; inducing endothelial cell damage, macrophage-derived foam cell formation, and vascular smooth muscle cell proliferation; and stimulating the immune system at all of these stages.

Electron microscope images obtained from atheromatous plaques showed that their central nucleus consists of a lipid-rich structure containing lipid clusters, vesicles, and microorganisms mimicking the appearance of a lipid cluster [3]. In analyses of these microorganisms, C. pneumoniae is the causative bacteria most frequently associated with atherosclerosis.

This chapter examines the relationship between the atherosclerotic process and C. pneumoniae through a review of the relevant literature.

2. Chlamydia pneumoniae

The most important discovery related to the classification of Chlamydia was made by Moulder et al. in 1964. Chlamydia spp. were initially perceived as viruses because they were smaller than normal bacterial size and had life cycles in the host cell. However, they were included in the bacteria class because they have the ability to survive outside the cell. As such, they are defined as “compulsory intracellular bacteria” [3]. Chlamydia are generally larger than the average virus, but smaller than bacteria and human cells [3].

The genus Chlamydia is in the family Chlamydiaceae of the order Chlamydiales. Based on their antigenic structures, intracellular inclusions, and diseases they cause, the genus consists of four species: C. pecorum, C. psittaci, C. trachomatis, and C. pneumoniae. Except for C. pecorum, all can cause disease in humans. C. trachomatis is sexually transmitted and causes ocular trachoma, lymphogranuloma venerum, and neonatal infections. Infection is common in homosexuals [7]. C. psittaci causes a systemic disease often characterized by pneumonia. C. psittaci is also seen in birds and pets. Infection is common in occupational groups that come into contact with birds.

C. pneumoniae, previously known as TWAR, causes respiratory tract infections such as pneumonia, bronchitis, sinusitis, and pharyngitis [8]. It was first isolated from the conjunctival swab of a child with trachoma in Taiwan in 1965 and was named TW-183. The role of C. pneumoniae as a human pathogen was definitively determined in 1983 with the first respiratory tract isolate, named AR-39, obtained from a throat swab sample of a patient with pharyngitis in the USA. The name TWAR (TW+AR) comes from these first conjunctival and respiratory strains. In 1989, C. pneumoniae was identified as a unique species by electron microscopy morphological studies and DNA sequence analysis of TWAR [9]. Unlike C. trachomatis, C. pneumoniae is not sexually transmitted but is spread through respiratory secretions. Unlike C. psittaci, it does not cause disease in birds or animals. In addition to respiratory tract infections, C. pneumoniae is associated with atherosclerosis and cardiovascular diseases.

One in ten cases of community-acquired pneumonia is caused by C. pneumoniae, and studies have shown that the seroprevalence of C. pneumonia in adults is 80%. It has also been shown to cause diseases marked by chronic inflammatory processes, such as chronic obstructive pulmonary disease, asthma, lung cancer, Alzheimer’s disease, arthritis, and atherosclerosis [10, 11, 12, 13, 14, 15]. Chlamydia have a biphasic life cycle and are obligate intracellular bacteria. They are morphologically and structurally similar to gram-negative bacteria, with a three-layered, lipopolysaccharide-rich outer membrane. They require ATP from the host cell to develop and proliferate. Chlamydia were classified as bacteria because they contain both DNA and RNA, reproduce by division, have a cell membrane similar to gram-negative bacteria, and are susceptible to antibiotics [16].

Bacteria of the genus Chlamydia reproduce by forming incubation bodies in the cytoplasm of the cells they infect. The life cycle of C. pneumoniae is divided between two forms, the elementary body (EB) and reticulate body (RB). The EB form is metabolically inactive and is the extracellular form that is transmitted between hosts. It infects the respiratory tract through inhalation and attaches to the mucosal surfaces. The EB enters the host cell via endocytosis, where it transforms into the RB form. EBs are approximately 350 nanometers in diameter. After entering the host cell and becoming activated, they increase in size to a diameter of 800–1000 nm.

The RB form is metabolically active and exploits the host cell’s metabolism. This transformation takes place within the first 24 hours after infection. It replicates within the host cell and then lyses that cell, spreading as newly formed EBs and propagating transmission. The RB form is protected from the endocytic-lysosomal degradation system of the host cell and can remain there for years. This feature enables it to persist in the body and cause a chronic inflammatory process [17, 18]. Its intracellular location enhances its ability to transform into a resistant and recurrent form [8, 19]. The bacteria infects the lung tissue and is taken up by monocytes and macrophages. However, instead of being eliminated they continue to thrive there and spread to the rest of the body via the circulation. Chlamydia that invades the arterial wall as a result of endothelial dysfunction contributes to the atherosclerosis process [20]. Its demonstrated presence in atheromatous plaques and smooth muscle cells, as well as in macrophage and foam cells, is the main feature that distinguishes C. pneumoniae from other microorganisms [21, 22].

2.1 Discovery of Chlamydia

2.1.1 Production in culture

Ramirez et al. [23] reported the first case of C. pneumoniae that could be isolated from a coronary artery plaque and cultured in vitro in 1996. Jackson et al [24]. demonstrated the presence of Chlamydia by immunohistochemical, PCR, or electron microscopy in 75% of 25 patients with carotid endarterectomy, whereas culture was positive in only one patient. A small proportion of atherosclerotic plaques with Chlamydia presence confirmed using other diagnostic methods have been successfully cultured [25, 26]. Karlsson et al. [27] demonstrated the presence of C. pneumoniae immunohistochemically in 20 of 26 abdominal aortic aneurysm tissue specimens, but were able to isolate Chlamydia in culture media in 10 cases. Chlamydia are reported to be difficult bacteria to culture because of their biphasic life cycle. It is accepted that in vitro culture has low sensitivity in demonstrating the presence of Chlamydia in tissue examinations [28].

2.1.2 Serological investigations

While some of the studies using serological tests showed a positive relationship between C. pneumoniae infection and coronary atherosclerosis, some studies reported that this relationship could not be accepted as sufficient evidence for the etiology of atherosclerosis [29, 30, 31].

Some studies revealed a relationship between C. pneumoniae-specific IgG and IgA positivity and atherosclerosis development, whereas other researchers did not observe this relationship [32, 33, 34, 35]. Serological tests are based on the detection of anti-chlamydial antibodies (IgA, IgG) in blood samples. However, these antibodies are indicators of the immune response rather than active infection. They also show cross-reactivity with other Chlamydia species. Danesh et al. [36] conducted a meta-analysis of 14 prospective studies including a total of 3619 patients and reported that there was no relationship between C. pneumoniae antibodies and atherosclerotic heart disease. This is similar to the presence of antituberculosis antibodies in serological tests after tuberculosis vaccination. The presence of antibodies may not mean there is active infection. In addition, it was shown that serological tests were negative even though the presence of Chlamydia could be demonstrated in the atherosclerotic plaques of immunodeficient individuals [19]. In general, pathogenic processes that cause an inflammatory response (such as smoke exposure, hypertension, hyperlipidemia, malignancy, and hyperglycemia) are known to cause errors in serological tests, which reduces their specificity. As a result, it is accepted that serological studies are not useful and are insufficient in the detection of Chlamydia.

2.1.3 Techniques for demonstrating Chlamydia in atheromatous plaques

Although most studies conducted in different centers in many parts of the world were able to demonstrate the presence of Chlamydia in atherosclerotic lesions, they could not be detected in other studies. This was thought to be related to differences or technical incompatibilities between the imaging methods, leading to debate regarding which diagnostic methods are most appropriate. As a result, polymerase chain reaction (PCR), immunohistochemistry (microimmunofluorescence), and electron microscopy imaging are the most widely accepted techniques. These studies focus on the bacteria’s DNA signature or directly demonstrate bacterial presence instead of utilizing indirect methods.

PCR is a diagnostic method for detecting chlamydial DNA or RNA and focuses on the bacteria’s genetic material [37]. It is a sensitive and specific test based on the degradation of genetic material in atheromatous plaques by electrophoresis [38, 39]. Immunogold labeling is based on the direct observation of Chlamydia bacteria with an electron microscope. The detection of monoclonal antibodies clearly demonstrates the presence of Chlamydia. Immunocytochemistry (ICC) is based on the demonstration of anti-chlamydial immunoglobulins adhering to the Chlamydia bacteria using immunofluorescent microscopy. This method has lower specificity and sensitivity than PCR. Serologic studies are based on the measurement of the host response to chlamydial invasion. Results are obtained by demonstrating host immunoglobulins. It has low specificity and sensitivity.

In most studies using ICC, PCR, and culture methods for C. pneumoniae, detection rates were higher in atherosclerotic vessels than in those without atherosclerosis [40, 41].

3. Atherosclerosis

Atherosclerosis can be defined as a disease that causes progressive arterial stenosis and obstruction due to intimal plaques containing lipids, fibroblasts, macrophages, smooth muscle cells, and extracellular substances in varying proportions, leading to loss of the elasticity and antithrombotic properties of the arterial walls. Atherosclerosis is a multifactorial, morbid and mortal systemic disease that affects not only the coronary vessels but all arterial structures. The coronary arteries, internal carotid arteries, and abdominal aorta are vessels most commonly affected [42].

Atherosclerotic lesions appear as focal thickenings in the intima and subintimal space of arteries. When the content of the thickened region is examined, it is seen to contain vascular endothelial cells, smooth muscle cells, connective tissue, and lipid deposits, as well as inflammatory and immune cells from the blood. Historically, with our nascent understanding of atherosclerosis, treatment focused on cholesterol-lowering drugs and a low-cholesterol diet. However, research on therapeutic processes started to diversify after the role of inflammation in atherosclerosis came to light.

3.1 Atherosclerosis and inflammation

Atherosclerotic vascular disease is a typical environment-gene interaction. Environmental risk factors trigger a proinflammatory response in people with genetic predisposition. Epidemiological studies have demonstrated the role of risk factors such as cigarette exposure, cholesterol, hypertension, and diabetes mellitus in the development of atherosclerosis. Experimental studies have shown that these risk factors induce a general inflammatory response, causing a widespread reaction in the body. In response to risk factors, systemic acute phase reactants are activated and there is the onset of signal traffic from the endothelium. In light of this information, atherosclerosis is defined as a multifactorial disease that is associated with the inflammatory process and in which chronic inflammation plays a role in every stage, from onset to progression [43, 44, 45].

At the onset of atherosclerosis, leukocytes and macrophages attach to the endothelium and cross into the subendothelial space. This occurs because of adhesion molecules [46, 47]. The best-known adhesion molecules are endothelial leukocyte adhesion molecule 1 (ELAM-1), membrane-bound vascular cell adhesion molecule 1 (VCAM-1), and intracellular adhesion molecule 1 (ICAM-1) [48, 49].

Cytokines are also an integral factor in the inflammatory process. The best-known proinflammatory cytokines are tumor necrosis factor alpha (TNF-ɑ), interleukin (IL)-1, and IL-6. TNF-ɑ is released from macrophages, vascular smooth muscle cells, and endothelial cells. These cytokines trigger the production of other cytokines in the inflammatory cycle [50].

Another marker is plasma fibrinogen values, which are an indicator of both the inflammatory response and the thrombotic response. Increased fibrinogen values have been shown to significantly increase coronary event risk. In addition, elevated fibrinogen levels have been detected in healthy individuals with a family history of atherosclerosis [51, 52].

C-reactive protein (CRP) is a good indicator of inflammation because its values are stable over time [53, 54]. It does not increase due to anything other than inflammation. It can be measured with a highly sensitive and inexpensive test. Studies have shown that CRP levels have an additive effect on other risk markers. The cholesterol/high-density lipoprotein (HDL) ratio is a strong indicator of cardiovascular risk [55]. However, the risk predictivity increases when CRP values are added. The PROVE-IT study showed that highest risk group is those with both high total cholesterol/HDL ratio and high CRP levels [56].

Elevated CRP levels, increased leukocyte counts in peripheral blood counts, and high serum fibrinogen levels are strong predictors of coronary artery disease and atherosclerotic diseases [57, 58].

The normal arterial structure consists of three main layers, the intima, media, and adventitia from innermost to outermost. The intima layer is covered with a single cell layer endothelium. The intact endothelial surface is resistant to thrombus formation because it secretes nitric oxide (NO) and prostacyclin (PGI2) and is covered with heparin sulfate.

3.2 Endothelial inflammation

The endothelium is the first vascular structure affected by risk factors [59, 60]. Normally shiny, slippery, and antithrombotic, risk factors cause the endothelium to lose its slipperiness and become sticky and prothrombotic. Endothelial cells exposed to risk factors from an early age start producing adhesion molecules (VCAM-1, ICAM), growth factors (platelet-derived growth factor [PDGF], basic fibroblast growth factor [FGF], transforming growth factor beta [TGF-β], IL-1, TNF-α), and cytokines (macrophage colony-stimulating factor [M-CSF], granulocyte-macrophage colony stimulating factor [GM-CSF]). VCAM-1 binds both monocytes and T lymphocytes. Atherosclerosis-related leukocyte adhesion molecule, or athero-ELAM, is released from endothelial cells, triggering mononuclear cell migration [61]. This initiates the chemotactic process on monocytes, macrophages, and lymphocytes and triggers the inflammation process [62]. The result is a vicious cycle in which inflammation stimulates cytokine release and cytokines increase inflammation. These proteins, which are expressed due to the vascular inflammatory response, are the main cause of early atherosclerotic lesions [62].

On the one hand, there is an inflammatory response in the endothelium, while on the other hand, there is subclinical systemic inflammation. Proinflammatory risk factors such as oxidized low-density lipoprotein (LDL) activate IL-1 and TNF-α, which are called primary proinflammatory cytokines [53, 63]. These primary proinflammatory cytokines activate IL-6, resulting in the release of acute phase reactants. The presence of subclinical systemic inflammation can be understood by measuring some acute phase reactants such as CRP, fibrinogen, factor 7, plasminogen activator inhibitor-1 (PAI-1), tissue plasminogen activator (tPA), and lipoprotein (a) in the blood or by measuring endothelium-derived peripheral markers [53, 64, 65].

3.3 Medial inflammation

In the atherosclerosis process, smooth muscle cells migrate from the media to the intima and there is a reduction in the contractile protein content and an increase in the number of synthetic organelles. Smooth muscle cells migrating to intima change from the contractile phenotype to the synthetic phenotype and contribute to proliferation. Smooth muscle cells in the media respond to vasoconstrictors such as endothelin, catecholamine, angiotensin II, and vasodilators such as NO and PGI2, while those in the intima respond to mitogens such as PDGF. In addition, the balance shifts from vasodilation to vasoconstriction, from antithrombotic to prothrombotic, and from antiproliferative to proliferative properties. Adhesion molecules, cytokines (IL-1, TNF-α), chemokines (monocyte chemoattractant protein-1 [MCP-1], IL-8), and growth factors (PDGF, FGF) are released from dysfunctional endothelial cells. IL-8 triggers the inflammatory cascade by binding to chemokine receptor 2 on leukocytes [66]. MCP-1 mediates selective directed migration of monocytes to the subendothelial space. Transgenic experimental animals unable to express MCP-1 were found to have nearly absent subendothelial lipid accumulation [67] All these processes allow defense cells to migrate to the inflammation site, leading to the onset of volumetric thickening of the vessel wall.

3.4 Lipid deposition and atherosclerosis

Lipids are a key cell component that serves as one of the main building blocks of cell membranes and organelles, as well as having nutrient and energy functions. Fatty acids, the simplest lipid form, are divided into different classes depending on the length of their structure, the number of carbon atoms, and whether the bonds are saturated or unsaturated. Phosphoglycerides are the main class of lipids comprising cell membranes. Cholesterols are also part of a large group of fats called sterols and are another important component of cellular membranes. LDL particles in the blood are made of lipids and protein, including cholesterol esters, triglycerides, phospholipids, and apoB-100 protein.

Many studies indicate that atherosclerosis begins with endothelial damage. However, histopathological studies have revealed atherosclerotic plaque formations with an intact endothelial structure [68, 69]. This raises the question of how lipid and cell passage into the subendothelial space occurs without endothelial damage.

It has been observed that eating even a single meal of excessively fatty food disrupts endothelial function, raises CRP levels, and increases adhesion molecules [70]. Animal experiments have shown that in subjects fed a high-cholesterol diet, the endothelium becomes sticky and begins expressing adhesion molecules within a few weeks [71, 72, 73]. The first alteration in the arterial endothelium of experimental animals fed a cholesterol-rich diet was shown to be leukocyte adhesion [74].

The main damage caused by cholesterol particles occurs through LDL. LDL particles are believed to penetrate the arterial wall by passing through the endothelial cells and initiate a number of remarkable changes involving various different processes. Subintimal lipid particles have been shown to be ingested by macrophages and smooth muscle cells, where they are degraded in the intracellular lipid oxidation and peroxidation chains. In the sub intimal space, LDL particles are modified with a different phospholipid and fatty ester structure and begin to form lipid clusters [75]. These lipid clusters trigger free radicals produced by chain chemical reactions, induce the inflammatory process, and cause chemotaxis.

3.5 Fibrous plaque–fibrous cap

Although immunohistochemical studies have demonstrated a cascade mechanism that allows inflammatory cells to infiltrate the subintimal layer at this early stage of atherosclerosis, pathological studies show that the endothelium is intact at this stage and there is no physical damage to this layer on microscopic examination [68]. Light microscopy examination of very small early lesions showed that primary damage occurred in the muscle cell component of the intima.

Monocytes accumulated in the subendothelial space transform into macrophages and begin to express scavenger receptors. This enables them to phagocytose the oxidized LDL.

As cholesterol esters accumulate in the macrophages, foam cells are formed. Macrophages accumulate lipids while continuing to release inflammatory mediators. M-CSF released from activated endothelial cells increases macrophage accumulation in the region. M-CSF also stimulates the immune system. A proinflammatory cytokine called CD40 ligand is one of the inflammatory mediators that contribute to progression. T cells accumulate in the subendothelial space due to the effect of different chemokines (e.g., interferon gamma-induced protein 10 [IP-10], monokine induced by interferon gamma [MIG]). Mast cells have recently been shown to accumulate via similar mechanisms. T lymphocytes also accumulate in the intima and continue to release proinflammatory cytokines. Another interesting function of T cells is to activate macrophages to stimulate the release of collagen, matrix metalloproteinases (MMP), and cytokines. Thus, the atheromatous plaque gradually grows. Oxidized LDL and heat shock protein (HSP) increase inflammation by stimulating toll-like receptors [76, 77]. Experimental studies have shown that toll-like receptor blockade can reduce atherosclerosis. Toll-like receptors accelerate atherosclerosis by triggering cytokine release in the inflammation cascade and stimulating the immune response [78].

As the lesions progress, extracellular lipids begin to accumulate. The extracellular lipid pool is largely a result of foam cell apoptosis and the release of their stored cholesterol esters. A very small proportion comprises lipoproteins that pass from the lumen. The “fibrous plaque” that begins to form in the subintimal layer initially appears microscopically as lipid nuclei, large amounts of smooth muscle cells, macrophages, foam cells, T lymphocytes, and extracellular matrix, and macroscopically as white lesions that enlarge mostly towards the artery lumen [3].

Smooth muscle cells in the fibrous plaque continue to produce extracellular matrix, while macrophages degrade the connective tissue. This construction and destruction is mediated by numerous cytokines. Even if fibrous plaques significantly narrow the vessel lumen, they are believed not to cause significant clinical events as long as they remain intact. The structure on the luminal side of this plaque is called the “fibrous cap.” A thicker fibrous cap is associated with greater plaque stability.

Plaques that are rich in lipids and inflammatory cells and have a thin fibrous cap have higher risk of rupture (vulnerable plaque). Metalloproteinases (collagenase, elastase, stromelysin) secreted by macrophages surrounding the lipid nucleus degrade the collagenous matrix of the fibrous cap. In addition, the synergistic effect of IL-1β and TNF-α released from activated macrophages and interferon gamma (IFN-γ) released by T lymphocytes results in smooth muscle cell death and reduced extracellular matrix. As a result of increased destruction and decreased construction, the fibrous cap weakens and eventually ruptures. Procoagulant substances in plaques with a disrupted fibrous cap interact with blood elements and clotting factors, triggering thrombus formation [65].

The ruptured plaque remains unstable for some time, after which the healing process begins. Smooth muscle cells capable of making extracellular matrix act as reparative cells. Smooth muscle cells produce large amounts of matrix proteins, such as glycosaminoglycan, elastin, and collagen, which are needed to repair the vessel and form the fibrous cap over the lipid-rich plaque nucleus. By synthesizing its contents, they enable the plaque capsule to stabilize the atherosclerotic lesion and separate the thrombogenic lipid-rich plaque nucleus from the platelets and coagulation cascade proteins in the blood. Thus, vascular smooth muscle cells have a critical role in ensuring plaque stability and inhibiting fatal thrombogenic outcomes. Some authors have argued that smooth muscle cells migrating into the intima play a constructive and reparative role, rather than a destructive role, in atherosclerosis [45, 79].

3.6 Atherosclerosis and immune response

It has recently become understood that both the natural and adaptive immune systems play important roles in the development of atherosclerosis [80, 81]. The natural immune system is responsible for the initial inflammatory response to a microorganism or pathogen. Immune cells, namely T cells, monocytes, macrophages and mast cells, circulating through various tissues (including the atherosclerotic artery) seeking antigen. When T cells encounter and bind to an antigen, a series of cytokines are released to launch an inflammatory response. Scavenger and toll-like receptors are the main receptors responsible for natural immunity in atherothrombosis [82]. Toll-like receptors are found on fibroblasts and macrophages in the intimal and adventitial layers of coronary atherothrombotic plaques.

The adaptive immune system is more specific than the natural immune system. This system includes an organized immune response leading to the formation of T and B cell receptors and immunoglobulins that recognize foreign antigens. Modified lipoproteins, HSPs, beta₂-glycoprotein I, and infectious agents can stimulate the adaptive immune system [83].

4. Chlamydia pneumoniae and atherosclerosis

The first findings regarding the association of C. pneumoniae and coronary artery disease were presented by Shor et al. [84] in 1992. Their study examined coronary artery atherosclerosis and vascular fatty streaks in seven autopsy studies and demonstrated the presence of TWAR-like C. pneumoniae. In a later, extended autopsy study, Kuo et al. [21] demonstrated the presence of C. pneumoniae in atherosclerotic lesions by PCR and culture studies.

In a 1998 study by Saiku et al. including 40 patients with acute myocardial infarction, 30 patients with coronary artery disease and 41 controls, chlamydial IgA and IgG antibodies were detected in 68% and 50% by micro immunofluorescence, respectively. Both frequencies were significantly higher than in the controls (7.17%). In 68% of patients with acute myocardial infarction, a significant seroconversion was demonstrated in enzyme immunoassay with LPS antigen; this response was absent in patients with coronary heart disease and in all but one of the controls [85].

The presence of C. pneumoniae organisms in atherosclerotic lesions was later demonstrated using immunohistochemistry, PCR, in situ DNA hybridizations, and electron microscopy [21, 86, 87, 88].

Chlamydia has been detected not only in plaques in the coronary arteries, but also in the aortic tissue, aortic aneurysms, and plaques in the carotid and peripheral arteries [22, 24, 89, 90, 91, 92].

4.1 Chlamydia pneumoniae and the immune response

In studies demonstrating the relationship between C. pneumoniae and atherosclerosis, circulating cholesterol-containing immune complexes were shown to be present in 50–70% of patients with acute myocardial infarction [93, 94]. These immune complexes (comprising IgG and apolipoprotein (a)) have a proatherogenic effect [95]. Patients with immunocomplexes containing C. pneumoniae-specific IgG and apolipoprotein (a) were found to have a 3.8 times higher risk of developing acute myocardial infarction than the control group [96].

Several mechanisms have been proposed to explain the formation of immunocomplexes containing C. pneumoniae-specific IgG and apolipoprotein (a). According to one mechanism, structurally similar elements in C. pneumoniae and apolipoprotein (a), which is found in lipoprotein (a), causes anti-C. pneumoniae antibodies to form an immune complex with apolipoprotein (a) [34, 35, 97].

Another mechanism involves the formation of antibodies against apolipoprotein (a) in association with HLA tissue groups, which is facilitated by C. pneumoniae infection. It was reported that HLA class II DR genotypes were more common in patients with high lipoprotein (a) levels and early coronary artery disease compared to the healthy control group. This finding indicates that an immune response to apolipoprotein (a) may occur in connection with the HLA system [95, 98].

Many of the properties of the single-cell-layer endothelium that forms the innermost layer of the arteries are mediated by NO. NO is synthesized from L-arginine by NO synthetase, an endothelial enzyme. NO is a potent inhibitor of platelet aggregation on endothelial cells and a potent vasodilator that acts by reducing vascular tone. In addition, it inhibits atherosclerosis at every stage through its anti-inflammatory properties, which it exerts by preventing the expression of genes that synthesize molecules that cause inflammation, such as ICAM-1, VCAM-1, MCP-1, and P selectin. Conditions known to predispose to atherosclerosis, such as hypertension, diabetes mellitus, smoking, or increased super oxide levels, have been associated with reduced endothelial production or increased destruction of NO [99]. Chlamydial infection in the endothelial layer results in the disruption of these endothelial properties. Stimulation of the release of endothelin 1, which is an especially powerful vasoconstrictor, causes endothelial cells to revert to their proliferative form, initiating the atherosclerosis process [100]. Chlamydial infection also stimulates the release of ELAM-1, VCAM-1, and ICAM-1 from the endothelium. Studies have shown that C. pneumoniae is associated with damage to the endothelium, which forms the intima layer of the arteries, in the early stages of the atherosclerosis process [84].

Host monocytes infected with C. pneumoniae begin secreting the adhesion molecules E-selectin, ICAM-1, and VCAM-1. These molecules allow adhesion of monocytes from the endothelium to the subendothelium. There the monocytes turn into macrophages and start to increase their cytokine production. Macrophages enlarged from the phagocytosis of oxidized LDL rupture, releasing the bacteria within them into the atherosclerotic plaque to infect neighboring cells [92].

In addition, C. pneumoniae proliferating within monocytes and macrophages stimulates the release of IL-6, TNF-ɑ, monocyte chemoattractant protein-1 (MCP-1), and macrophage inflammatory protein 1-a (MIP1-a) [84].

Another mechanism that may explain the relationship between C. pneumoniae infection and atherosclerosis is based on the similarities in structure between the heat shock protein (HSP-60) produced by nearly all bacteria and the HSP produced by humans. Antibodies against the HSP-60 in bacteria may cross-react with human HSP [40]. The immune response to C. pneumoniae and/or human HSP in the vascular wall is thought to activate atherosclerosis. Wong et al. showed that C. pneumoniae and human HSPs coexisted in atherosclerotic lesions, and incubating mouse macrophages with this HSP caused an increase in matrix-degrading metalloproteinases and TNF-α activity. Chlamydia-infected endothelial cells trigger smooth muscle cell proliferation by stimulating the synthesis of endogenous HSP-60 and PDGF [40].

In addition, C. pneumoniae HSP has been shown to increase the secretion of lectin-like oxidized LDL receptor 1 (LOX-1) in hypercholesterolemic rabbit endothelial cells. Increased LOX-1 disrupts the LDL regulation system in the host and induces oxidized-LDL-mediated atherosclerosis. LOX-1 forces macrophages to phagocytose oxidized LDL, thereby turning macrophages into foam cells through the phagocytosis of high amounts of oxidized LDL, and foam cells are known to be one of the main players in the atherosclerotic process [74].

Chlamydia has been shown to stimulate the toll-like receptor system in host tissue. Toll-like receptor stimulation is considered one of the factors that initiates and promotes atherosclerosis by triggering the cytokine and inflammatory cascade. Thus, smooth muscle cell migration into the media layer is stimulated and the macrophage/foam cell diapedesis process that causes subintimal thickening progresses [101].

An experimental study by Justin et al. [102] showed that after contaminating porcine coronary arteries with C. pneumonia in culture medium, Chlamydia proliferating in the arterial wall quickly stimulated smooth muscle cell proliferation in the medial layer of the artery and caused atherosclerosis and significant narrowing in the lumen.

4.2 Chlamydia pneumoniae and lipid metabolism

C. pneumoniae has been shown to adversely affect the regulation of lipid metabolism in host tissue. C. pneumoniae continues to live in immune cells after undergoing phagocytosis and thus can survive even in chronic inflammation environments [103].

The unmodified, natural level of LDL is controlled by LDL receptors and does not normally lead to the formation of macrophage-derived foam cells. However, the oxidation of LDL due to chlamydial infection disrupts this balance. It has been shown that C. pneumoniae-infected macrophages incubated with LDL turn into foam cells within 22 hours [40]. This effect occurs mostly through the induction of LDL oxidation, phagocytosis of oxidated LDL, and induction of lipid accumulation within cells and in the atherosclerotic plaque. Liu et al. [104] reported that both active and inactive Chlamydia trigger lipid accumulation and induce foam cell formation.

A study conducted by Zhao et al. [105] showed that C. pneumoniae negatively affects lipid metabolism by decreasing ATP-binding cassette transporter A1 (ABCA1) level, which has an important role in cholesterol transport in macrophages. In a study conducted by Tumurkhuu et al. [106] on the same system, it was shown that C. pneumoniae infection affected the lipid reuptake system by stimulating extracellular IL-1β and caused intracellular cholesterol accumulation by reducing the synthesis of ABCA1 and G protein-coupled receptor 109A (GPR109a), which are involved in the niacin and ketone receptor system.

In a study on the effects of Chlamydia on lipid metabolism in humans, it was found that chronic inflammation associated with C. pneumoniae infection caused a significant increase in cardiovascular risk in individuals with familial hypercholesterolemia [104].

In experimental studies, rabbits intranasally infected with C. pneumoniae exhibited findings consistent with early atherosclerosis characterized by aortic inflammation when fed a high-fat diet but not in those fed a normal diet [107, 108]. The combination of hyperlipidemia and C. pneumoniae infection has been shown to significantly increase the development of atherosclerosis.

Blessing et al. [109, 110] demonstrated in their study that C. pneumoniae inoculation causes inflammation in the heart and aorta in normolipidemic C57BL/6J mice. In the same model, atherosclerosis was shown to accelerate and become widespread when the animals were fed a high-cholesterol diet.

Lantos et al. [111] showed that hyperlipidemic diet-induced atherosclerosis in ApoB100only/LDLR−/− mice accelerated threefold in the presence of C. pneumoniae infection.

Apolipoprotein E (apoE) is involved in chylomicron and very-low-density lipoprotein (VLDL) metabolism and has a key role in LDL and cholesterol metabolism. ApoE deficiency leads to dyslipidemia and increases susceptibility to atherosclerosis. In mice with apoE enzyme deficiency, even a single dose of Chlamydia inoculation significantly increased atherosclerosis compared to uninfected subjects [112].

New Zealand rabbits do not develop atherosclerosis unless they are fed a hyperlipidemic diet. However, when infected with C. pneumoniae, atherosclerosis was observed in these animals within 2 weeks despite being fed a normal diet [113]. These results suggest that C. pneumoniae also triggers atherosclerosis independently of lipid levels and acts as an independent factor in the development of atherosclerosis.

In their study on C57BL/6J mice fed a high-cholesterol diet, Zafiratos et al. [114] concluded that the coexistence of Chlamydia infection and hyperlipidemia significantly increased levels of TNF receptors 1 and 2 and caused inflammation when compared with hyperlipidemia or Chlamydia infection alone. Similarly, a study conducted on IL-17A-deficient mice showed that a hyperlipidemic diet did not result in a significant difference in inflammation or atherosclerosis compared to the control group. However, after infection with C. pneumoniae, the control group exhibited significant elevation in inflammation markers in the blood (IL-12p40 and IFN-γ) and increased macrophage accumulation in atherosclerotic plaques compared to the IL-17A-deficient group. This showed that IL-17A plays a role in the Chlamydia-induced atherosclerosis process in hyperlipidemic subjects [115].

These studies demonstrate the important role of Chlamydia, both independent of lipid physiology and as a cofactor of hyperlipidemia, in the different stages of initiating and advancing atherosclerosis.

5. Treatment strategies

5.1 Antibiotic studies

The multitude of studies indicating that C. pneumoniae plays a role in the pathogenesis of atherosclerosis led to the investigation of whether this pathogenesis can be treated with antibiotic therapy. Some experimental studies showed that antichlamydial antibiotherapy slowed atherosclerosis to some extent [116].

The ACADEMIC study included 302 coronary artery patients with positive Chlamydia IgG tests. Of these, 152 patients received a placebo and 150 patients received azithromycin for 6 months. At the end of the study, there were significant decreases in CRP, IL-1, IL-6, and TNF levels in the azithromycin group, but the expected significant change in serological tests was not observed. In addition, there was no difference in terms of rates of clinical events related to coronary artery disease [117]. This supports the theory that inflammation can be reduced with antichlamydial antibiotherapy.

However, in a multi-center antichlamydial antibiotic study including more than 15,000 people in total, although there were significant changes in laboratory parameters, it unfortunately did not yield the expected results in terms of reducing rates of clinical symptoms and events [118, 119, 120].

The failure of antibiotic therapy to provide secondary protection against atherosclerosis complications in individuals infected with C. pneumoniae has been attributed to the treatment being too late. It is believed that the atherosclerosis process is already triggered in people infected with C. pneumoniae and that antichlamydial treatment does not protect against infection. Experimental studies have indicated that antibiotic treatment initiated immediately after chlamydial infection may protect against atherosclerosis [121, 122]. Research on this subject is ongoing.

5.2 Vaccine studies

Could the failure to obtain expected results in antibiotic studies be due to late treatment? Taking measures before inflammatory and immune responses are induced might facilitate the management of the process. Antichlamydial vaccine studies are the focus in this regard.

Due to the different strains and pathogenetic mechanisms in the Chlamydia family, vaccines against all chlamydial species have long been a research topic of interest. C. trachomatis is the most common sexually transmitted disease in the world and causes health problems of concern to all of humanity in both men and women, from urinary tract infection to infertility, from pneumonia to blindness. Thus, antichlamydial vaccine development started about 100 years ago with C. trachomatis. Using the inactivated EB form for immunization provided results, but the short duration of immunity was disappointing [123].

Chlamydia contain two main bacteria-specific antigens: HSP-60 and major outer membrane protein (MOMP). MOMP activates both cellular and humoral immunity. Most current antichlamydial vaccine studies are focused on MOMP. HSP-60 is a receptor that can be found in many bacterial species and human cells, and no significant progress has been made to date in vaccine studies targeting this antigen [124].

Li et al. [125] demonstrated that administering a recombinant chlamydial protease-like activity factor (rCPAF) and IL-2 vaccine slowed the atherosclerosis process in their study on mice in which atherosclerosis was induced by a hyperlipidemic diet.

Recombinant protein vaccine studies have also yielded promising results in terms of reducing the immune response occurring after chlamydial contamination [126].

There have been nearly 200 antichlamydial vaccine studies to date, and an average of 10–12 new studies are conducted each year. However, although antichlamydial vaccine studies have investigated numerous specific antigenic targets and achieved partial success in mice, the results from whole-cell vaccine targets have not yet reached the clinical implementation stage [123].

5.3 Treatments targeting inflammation triggers (risk factors)

There is ample evidence regarding the anti-inflammatory effect of lifestyle modification. Regular exercise have been shown to both directly improve endothelial function and reduce inflammatory mediators, as well as mitigate risk factors [54, 55  83]. Adipose tissue is an important source of IL-6, which is known to be elevated and linked to inflammation in people with obesity. Lipid-lowering diet and treatments are effective in reducing inflammation. Statins not only have a strong lipid-lowering effect, but anti-inflammatory activity is known to be one of their pleiotropic effects. Statins reduce the release of adhesion molecules and inflammatory cytokines. In addition, they correct endothelial function, reduce oxidative stress, inhibit platelet aggregation, prevent clustering of T cell antigen receptors due to immune activation, and generally stabilize vulnerable plaques. The anti-inflammatory effects of statins have been demonstrated in patients with rheumatoid arthritis. In clinical studies, statins have been shown to reduce CRP values independently of LDL levels. In the CARE study, subgroup analyses revealed that the patient group with high CRP levels benefited more from statin treatment, which was more effective in reducing coronary events in this group [127, 128]. In the PROVE-IT study, the greatest benefit was seen in the group with aggressively reduced LDL and decreased CRP, which demonstrated that patients with the most inflammation benefited most and supported the use of statins in acute coronary syndromes [129]. In the JUPITER study, statin therapy resulted in significant reduction of cardiovascular events in individuals with high CRP but without high LDL, offering further evidence of the importance of reducing inflammation [130]. That study included 17,802 healthy individuals with normal LDL-cholesterol level and CRP above 2 mg/l who were randomized to receive rosuvastatin 20 mg or a placebo, and was terminated after only 1.9 years because of the significant reduction in cardiovascular events in the rosuvastatin arm. This rapid benefit was thought to be a result of the anti-inflammatory effect of rosuvastatin rather than its lipid-lowering effect [131].

There are studies on treatment methods that target risk factors, as well as treatments that directly target inflammation [73]. Some evidence has indicated that acetyl salicylic acid also has an important anti-inflammatory effect in addition to its inhibition of platelet aggregation [73].

An emerging treatment method that is still mostly in the animal testing phase is cytokine blockade. The newly identified CD40 pathway has been shown to play an important role in inflammation, the increase in adhesion molecules, and thrombosis. Blockade of this pathway with monoclonal antibodies has allowed a reduction in the development of atherosclerotic lesions in animal experiments [132].

Another treatment method based on cytokine blockade is polyclonal IgG injection. With polyclonal IgG injections, it is possible to block receptors in phagocytic cells and inhibit antibody synthesis and cytokine production. In mouse studies, intravenous IgG injection prevented the development of fatty streaks at several sites and reduced the area of atheromatous plaques [133]. The latest method of cytokine blockade are treatments to shrink atheromatous plaques with IL-10 injection, but these are still in the animal trial stage [134].

Anti-atherosclerosis vaccine development studies have intensified in recent years. The basic principle here is to modulate the immune response and protect against the development of atherosclerosis. For example, developing antibodies against oxidized LDL and creating antibodies by activating B lymphocytes have been shown to have a protective effect against atherosclerosis. In brief, inflammation is a fundamental mechanism that is present at every stage of atherosclerosis and must be suppressed. The anti-inflammatory effects of lifestyle modification, statin therapy, and some other treatments have been demonstrated, while the search for new anti-inflammatory therapies continues.

6. Conclusions

The presence of C. pneumoniae in human atherosclerotic plaques is now a generally accepted fact. Numerous studies in various centers have repeatedly confirmed this with many different diagnostic methods, laboratory tests, and imaging techniques.

The association between C. pneumoniae and the atherosclerosis process leads to new questions. Is Chlamydia a cause of atherosclerosis, or is it a risk factor that participates in the process and predisposes to its complications?

The active pathogenic mechanism of the bacteria, which involves the ability to live within the cytoplasm, persist long-term and even multiple within vacuoles, cause cell necrosis and lysis, and infect the surrounding cells, and its detection even in the fatty streaking stage, which is the initial stage of atherosclerosis, can be regarded as evidence of its role as a triggering factor. However, its ability to survive in macrophages and lymphocytes, cause cell necrosis and fibrosis, and exist and reproduce in plaque inclusion bodies can be regarded as evidence that it is a co-factor associated with and contributing to the atherosclerotic process.

As a result, the pathophysiological mechanisms of Chlamydial infection shown to play a role in atherosclerosis can be summarized under the main headings as follows:

• Arterial wall infection resulting from Chlamydia entering the bloodstream via the respiratory system and invading the arterial wall (initiation of atherosclerosis),

• Systemic inflammation induced by inflammatory mediators released into the circulation in response to infection,

• Chronic infection and a chronic inflammation process due to the biphasic life cycle of Chlamydia,

• Triggering of adhesion and inflammation in the arteries,

• Bacterial infection and consequent accumulation of inflammatory cells in the sub-intimal area,

• Chlamydia-associated lipoprotein deposition and fibro-fatty plaque induction (advancement of atherosclerosis)

• Autoimmunity caused by the host immune response to Chlamydia-specific components,

• Proatherogenic effects of specific bacterial toxins produced by Chlamydia.

Considerable research continues on atherosclerosis and related cardiovascular diseases, which are leading causes of mortality and morbidity worldwide. Vascular interventional techniques and bypass surgeries are performed as palliative interventions. However, atherosclerosis is a major disease that has no definitive treatment and is still awaiting a solution.

Studies on the role of C. pneumoniae in the pathogenesis of atherosclerosis, vaccine studies, and advances in immune response regulation remain scientists’ focus of attention for the treatment of atherosclerosis.

Abbreviations

ABCA-1	ATP-binding cassette transporter A1
apoE	apolipoprotein E
EB	elementary body
ELAM-1	endothelial leukocyte adhesion molecule 1
HSP	heat shock protein
FGF	fibroblast growth factor
ICAM-1	intracellular adhesion molecule 1
ICC	immunohistochemistry
IFN-γ	interferon gamma
IL	interleukin
LOX-1	lectin-like oxidized LDL receptor 1
LPS	lipopolysaccharide
MCP-1	monocyte chemoattractant protein-1
M-CSF	macrophage colony stimulating factor
MIP1-a	macrophage inflammatory protein 1-a
MMP	matrix metalloproteinase
NO	nitric oxide
PAI-1	plasminogen activator inhibitor-1
PCR	polymerase chain Rreaction
PDGF	platelet derived growth factor
PGI2	prostocyclin
TNF-ɑ	tumor necrosis factor alpha
tPA	tissue plasminogen activator
RB	reticulate body
VCAM-1	vascular cell adhesion molecule 1