Chlamydia Infection’s Role in Neurological Diseases

Abstract

Chlamydia infections are common infections that are transmitted through sexual C. pneumonia is a pathogen that causes different acute and chronic infections. Due to the increase in biological knowledge and the use of more sensitive and specific techniques in the detection of the pathogen in recent years, it is thought that C. pneumonia has a role in various cardiovascular and central nervous system (CNS) diseases. There is increasing evidence that C. pneumonia may have a role in various chronic neurologic diseases, especially Alzheimer’s disease (AD) and multiple sclerosis (MS). C. pneumonia crosses the blood-brain barrier via monocytes and triggers neuroinflammation in the central nervous system. Various diagnostic methods (molecular, histopathologic, and culture) have shown the presence of C. pneumonia in patients with late-onset AD dementia. It is thought that C. pneumonia may be a cofactor in the development of MS disease by causing chronic permanent brain infection in MS patients. There are also reports of C. pneumonia causing other CNS diseases such as Guillaine Barre syndrome, encephalitis/meningoencephalitis, and cerebellar ataxia. In this section, the relationship between Chlamydia infections and neurological diseases will be discussed based on scientific research.

1. Introduction

Chlamydiae have been identified as viruses because they have a life cycle within the host cell and are smaller than bacteria. However, they were later classified as bacteria because they can also live outside the cell. With these conditions, they were named as “obligate intracellular bacteria” [1].

There are four species in the genus Chlamydia: C. pecorum, C. psittaci, C. trachomatis and C. pneumoniae. These species are classified according to their disease, antigenic structures, and intracellular inclusions. C. pecorum does not cause disease in humans. Others may cause disease in humans.

C. trachomatis is the most frequently sexually transmitted bacterium today [2]. It most commonly causes urethritis in men and cervicitis most commonly in women. If left untreated, it can progress to pelvic inflammatory disease in women. C. trachomatis is the most common cause of nongonococcal urethritis [3]. As a significant proportion of patients are asymptomatic, they continue to be contagious and act as vector [4]. This complicates the management of the disease and results in a serious socioeconomic burden, even in developed countries [5]. The three main clinical manifestations of urethritis—urethral discharge, itching, and dysuria—are mild or absent in some cases [6]. Therefore, diagnosing C. trachomatis infection is important. Although conventional diagnostic methods have low efficiency, PCR tests have high sensitivity and specificity and are currently the gold standard in the diagnosis of C. trachomatis infection. C. trachomatis can also cause ocular trachoma, lymphogranuloma venerum, and neonatal infections [7].

Chlamydia Psittaci; it causes a systemic disease that often progresses with pneumonia, being more common in occupational groups that come into contact with birds.

C. pneumoniae; it causes respiratory tract (such as pneumonia, bronchitis, sinusitis, and pharyngitis) infections and is also associated with atherosclerosis and cardiovascular diseases [8].

Chlamydia pneumonia is transmitted from person to person by direct respiratory route. The infection spreads slowly [9, 10]. It has a longer incubation period than many pathogens that cause respiratory tract disease, and this period is about a few weeks [11]. If it is within the family, the infection spreads in a shorter time [12].

Chlamydiae are obligate intracellular parasites and have a biphasic life cycle. They can grow and multiply using the host cell’s ATP. Chlamydia are in the bacteria class because they are sensitive to antibiotics, reproduce by division, contain both DNA and RNA, and have cell membranes similar to gram-negative bacteria [13, 14].

Chlamydia reproduces by forming inclusion bodies in the cytoplasm of host cells. Chlamydia pneumonia has two forms called “Elementary body” (EB) and “Reticulate body” (RB). These two forms are functionally and morphologically different from each other and undergo regular change. The EB form is the metabolically inactive, extracellular form and causes contamination. It attaches to the mucosal surfaces of the respiratory tract by inhalation and enters the host cell by endocytosis, where it transforms into the RB form.

The RB form is metabolically active and utilizes the host cell’s metabolism. It multiplies in the host cell, breaks up this cell, spreads around as newly formed elementary body bodies and continues to be transmitted. In its RB form, it is protected from the host cell’s endocytic lysosomal digestive tract, where it can be stored for years. In this way, it causes a chronic inflammatory process in the body [15, 16].

Being located intracellularly provides the ability of this bacterium to transform into a resistant form [8, 17].

C. pneumonia lives in the host cell as a non-degradable inclusion separate from the cytoplasm of the cell. For this reason, it is protected from the host cell’s defense systems by arranging the signal pathways used by the host cell for defense. In this way, C. pneumoniae cannot be eliminated because the host defense mechanisms are insufficient and may lead to persistent infection [18, 19, 20]. While C. pneumoniae lives in mononuclear cells, it multiplies from time to time and creates a chronic infection. In this chronic infection, heat shock protein ((HsP) and proinflammatory cytokine production takes place, and this process can initiate an autoimmunity process over time. As a result of chronic infection, C. pneumonia increases the expression of its own HsP60 proteins. The immune response of the host to microbial HsP60 is over time to human HsP60. This may contribute to the development of chronic diseases such as asthma, atherosclerosis, and coronary artery disease [11, 21, 22].

In recent years, there has been a great deal of information about the physiological effects of chlamydia infection on the host cell [23]. Although it is not known exactly how C. pneumonia may cause chronic infection in the central nervous system by affecting apoptotic pathways [24, 25].

In recent years, it has been determined that C. pneumonia may have a role in other chronic diseases in addition to respiratory tract diseases, as more powerful tests have been developed to show the presence of C. pneumonia [26].

It has also been shown to cause progressive diseases with chronic inflammation processes such as lung cancer, Alzheimer’s disease, multiple sclerosis, arthritis, and atherosclerosis [18, 27, 28, 29, 30, 31].

The demonstration that various human cells (smooth muscle, monocytes, lymphocytes, macrophages, endothelium, and epithelium) are infected by C. pneumonia after a respiratory infection supports the systemic spread after respiratory tract infection [32]. Thus, C. pneumonia spreads everywhere through the circulatory system. Chlamydia, which invades the arterial wall as a result of endothelial damage, participates in the atherosclerosis process [33].

Chlamydia that invades the arterial wall as a result of endothelial dysfunction contributes to the atherosclerosis process [33]. However, C. pneumoniae infection has also been shown to promote monocytic migration via human brain endothelial cells, which is thought to be a mechanism by which the organism is able to enter the CNS. This mechanism may explain how the organism enters the CNS and causes chronic damage [34].

This chapter examines the potential role of chlamydial infections in different neurological diseases and the underlying mechanisms in light of the literature.

2. Chlamydial infection and cerebrovascular disease

At present, cerebrovascular diseases constitute an important public health problem because of the associated mortality and functional losses in the acute and chronic period. According to studies conducted in the USA, 500,000 new or recurrent stroke cases are observed every year [35]. Approximately 80–85% of these are cases of ischemic stroke [36, 37].

Although studies have identified many etiological factors for stroke, none of these factors can be identified in approximately 40% of cases. The number of patients with no detectable risk factors is increasing, especially in the patient population under 45 years of age [38]. The research, identification, and (if possible) treatment of potential risk factors have become a priority in order to reduce the incidence and consequences of the disease [35, 39, 40].

Many researchers have suggested that viruses play a role in the development of atherosclerosis and have shown that these pathogens are closely linked to the rapidly progressive CAD that develops in heart transplant recipients [41, 42, 43, 44, 45].

The main infectious agents implicated are C. pneumoniae, cytomegalovirus, Helicobacter pylori, Streptococcus mutans, Porphyromonas gingivalis, Actinobacillus, and Prevotella intermedia. Although there are a few publications in the literature on the role of C. pneumoniae in ischemic cerebrovascular diseases, the number of studies on this subject is steadily increasing [46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57].

Çalık et al. investigated the presence of C. pneumoniae antibodies in patients and healthy controls and did not detect IgM positivity, an indicator of acute C. pneumoniae infection, in either group. Both IgA and IgG antibodies were more common in the patient group, with IgG antibodies detected in 37 (74%) of patients and 28 (56%) of controls, and IgA antibodies detected in 31 (62%) of patients and 16 (32%) of controls. However, the difference was statistically significant only for IgA antibodies (P < 0.01). Chronic persistent C. pneumoniae infection was not present in any of the controls but was detected in eight patients (16%). Findings of atherosclerosis in the carotid and vertebral arteries were statistically more frequent in the patient group. When they compared patients with and without atherosclerosis on Doppler ultrasound, those with atherosclerosis showed higher rates of C. pneumoniae IgG and IgA positivity, higher mean titers of these antibodies, and more frequent chronic persistent infection. However, the differences were not statistically significant [41].

Studies have generally shown that IgA antibodies are associated with ischemic cerebrovascular disease while IgG antibodies are not [49, 50, 53].

In a study by Cook et al., acute C. pneumoniae infection or reinfection was found to be associated with ischemic cerebrovascular disease [55].

IgG positivity is an indicator of a previous infection and can remain positive for years [11]. However, IgA antibodies have a very short half-life of 5–6 days on average. Therefore, IgA antibodies are useful in determining persistent and active carrier status. Based on observations, anti-Chlamydia IgA antibodies become positive (titer of 1/16 or higher) early in primary chlamydial infections, and the titer increases 2–4 times within 20–40 days, even if the patient is treated. This elevated titer rapidly decreases to former values after treatment [58]. However, infection status can be ascertained by simultaneously determining IgG and IgM antibody status. The presence of IgM antibodies is a definitive indicator of acute infection. In chronic or re-infection, IgM antibodies do not rise at all or are positive at very low titers. Thus, IgM negativity and IgA positivity (≥1/32) accompanied by IgG positivity (≥1/128) indicate chronic or re-infection [11, 53, 56, 59].

Acute C. pneumoniae infection was not detected in any of the patients included in the study by Çalık et al. [41]. Similarly, Elkind et al. reported not detecting IgM antibodies in any of the patients in their ischemic stroke study [53].

Although criteria have been established for the diagnosis of chronic persistent infection, a positive IgA titer is an indirect indicator that the causative pathogen is present in the body [60].

Studies have emphasized the relationship between C. pneumoniae infections and atherosclerosis, focusing mostly on the coronary and carotid arteries [17, 46, 51, 61].

Schmidt et al. reported that C. pneumoniae seropositivity was associated with an increase in intima media thickness in the main carotid arteries [51]. Grayston et al. demonstrated the presence of C. pneumoniae in endarterectomy specimens by PCR and immunocytochemistry and concluded that C. pneumoniae infections may cause atherosclerosis or play a role in its pathogenesis [62]. The findings support that C. pneumoniae infections may be a strong risk factor for atherosclerosis and related vascular diseases [41]. However, the mechanism underlying this relationship has not been elucidated. Different mechanisms have been described in relation to the contribution of infections in the formation or emergence of atherosclerotic diseases. Infections have both direct and indirect effects on the vasculature. The main direct effects are endothelial cell destruction or dysfunction, smooth muscle cell proliferation, and local inflammation. Indirect effects include chronic systemic inflammation, cross-immunoreactivity of antibodies against the pathogen with host tissues, and the impact of the host response to the pathogen on known atherosclerotic risk factors.

C. pneumoniae can infect various types of cells. Among the cells it infects are endothelial cells, vascular smooth muscle cells, and macrophages, all of which have important roles in the pathogenesis of atherosclerosis. Changes in the function and structure of these cells are an expected result of direct invasion by the pathogen [41]. Indeed, many studies have demonstrated the presence of C. pneumoniae in vascular endothelium affected by atherosclerosis [62, 63]. In addition to local vessel wall invasion and destruction, C. pneumoniae induces a procoagulant state by causing tumor necrosis factor (TNF) and interleukin (IL)-2 release and affecting lipoprotein levels and tissue factors because of its lipopolysaccharide cell wall components [50, 62]. Hsp60, one of the antigens in the protein structure of C. pneumoniae, can trigger atherogenesis indirectly through certain immunological mechanisms [64]. The effects of chronic C. pneumoniae infection on blood clotting factors have also been demonstrated. Toss et al. determined that chronic persistent C. pneumoniae infection was associated with increased serum fibrinogen levels but were unable to explain the mechanism behind this increase [65].

C. pneumoniae infections were also reported to have a number of effects on the blood lipid profile, which has an important role in the development of atherosclerosis [64]. In their study including 1053 patients, Laurila et al. showed that individuals with C. pneumoniae IgG antibody titer values of 1/128 and above had higher serum triglyceride levels and lower HDL/total cholesterol ratio compared to individuals with lower antibody titers [66]. The host response to the lipopolysaccharides in the structure of C. pneumoniae found in the atherosclerotic region alters the synthesis of certain cytokines. These cytokines cause a complex set of changes in the serum lipid profile [64].

In summary, evidence pointing to the relationship between C. pneumoniae and atherosclerosis is as follows: (a) sero-epidemiological studies have demonstrated its association with atherosclerotic diseases (CAD, ischemic cardiovascular disease, and carotid atherosclerosis); (b) the pathogen has been demonstrated in atherosclerotic lesions; and (c) several small clinical studies have shown the benefits of using anti-inflammatory antibiotics in the secondary prevention in patients with CAD [52]. If the possible relationship between infections and vascular occlusive diseases such as ischemic cardiovascular disease and CAD is confirmed, adding antibiotics to treatment protocols for stroke and coronary events may help prevent ischemic events [50].

3. C. pneumoniae and neurodegenerative and demyelinating diseases

Information regarding the role of C. pneumoniae in chronic neurological diseases has been increasing recently. This was supported by the detection of C. pneumoniae genomic material in the cerebrospinal fluid of MS and AD patients [11, 18, 19, 20, 34].

3.1 C. pneumoniae and multiple sclerosis

MS is a chronic autoimmune and demyelinating disease that affects the CNS, usually in young adults [67]. MS was first described in 1838. During the six decades following its identification, German and French physicians determined the clinical and pathological features of the disease. Previously documented only as cases, MS became one of the most common diseases in neurology at the turn of the twentieth century. The disease is characterized by damage to the myelin sheaths, oligodendrocytes, and to a lesser extent, axons and neurons. There are currently 2.5 million people with MS worldwide, and their treatment and care cost billions of dollars [68]. MS is a highly heterogeneous disease and can present with widely variable clinical signs and symptoms including motor, sensory, autonomic, and cognitive disorders, depending on the part of the CNS affected [69].

MS develops in genetically predisposed individuals as a result of a combination of environmental factors, viral or bacterial pathogens, cytokines secreted in inflammatory and autoimmune response, and other yet unidentified etiological agents. Various lesions can be seen in patients with MS, such as CNS inflammatory infiltrates, astrogliosis, demyelination, and early axonal damage [70, 71, 72].

In MS, CD4+ T helper (Th)-1 and Th17 cells perceive myelin sheath components as foreign antigens and develop an autoreaction against myelin [67].

3.1.1 Axonal and neuronal damage in MS

Inflammatory CNS damage in MS has frequently been associated with axonal damage. Although MS is classically defined as a condition primarily characterized by axonal myelin loss, axonal damage has also been described in the early pathological findings of MS lesions. Modern techniques have yielded definitive findings showing axonal damage. Antibodies to amyloid precursor proteins (APP) reveal damaged axons in the active areas of MS lesions [72].

The distinctive pathological feature of MS is demyelinating plaques, which represent areas of demyelination and gliosis around blood vessels [73]. Acute lesions show macrophage infiltration and phagocytosis of myelin membranes, as well as perivascular lymphocytes and plasma cells. The continuous destruction and regeneration of myelin has been demonstrated within progressive MS plaques [74]. Toll-like receptors (TLR) are strongly associated with many neurodegenerative and demyelinating disorders, including MS, with a significant increase in TLR expression in MS lesions. PCR studies have shown that TLR1–8 s are expressed in microglial cells obtained from MS patients [75]. In addition, healthy white matter from MS patients does not contain TLR, whereas active lesions are associated with increased TLR3 and TLR4 expression by microglia and astrocytes. Late active lesions also contain astrocytes with surface expression of TLR3 and TLR4 [75]. This indicates that early lesions are characterized by microglial infiltration, while astrocytes are also active in late active lesions. However, the exact role of TLR3 and TLR4 activation in these lesions remains unclear. TLRs have been shown to recognize highly conserved sites (pathogen-associated molecular patterns) in various microorganisms, including C. pneumoniae, thereby stimulating a strong inflammatory response that contributes to pathogen clearance [76]. In one study, overexpression of TLR-2 and TLR-4 messenger RNA (mRNA) was detected in the peripheral blood, but not in the CSF of MS patients with the relapsing-remitting form, and the combined activation of these TLR has been reported to play a significant role in activating and modulating cellular immune response during chronic C. pneumoniae infection [77].

Based on epidemiological observations, it has been suggested that along with genetic predisposition, exposure to an environmental factor such as an infectious agent may play a role in the pathogenesis of MS [78].

The risk of MS is increased by the presence of specific genes in the human MHC, or human leukocyte antigen (HLA) complex, on chromosome 6. In particular, HLA-DR and HLA-DQ genes, which are involved in antigen presentation, are strongly associated with the development of disease. However, although the risk of disease is higher in monozygotic than in dizygotic twins (approximately 30% and 5%, respectively), the low concurrence rate among identical twins suggests that nongenetic factors may contribute to the etiology of MS. In this regard, the etiopathogenesis of MS is complex and remains a subject of debate.

To date, about 20 microorganisms, including viruses, have been associated with MS [79]. The screening techniques used in these studies varied from serology to PCR, and the quality and number of controls examined varied greatly. The most recent pathogen associated with MS is C. pneumoniae [26].

Sriram et al. reported the first evidence pointing to the potential role of C. pneumoniae in the pathogenesis of MS [80]. A year later, a larger study from the same group strongly confirmed that the frequency of C. pneumoniae in the CSF of individuals with MS was significantly higher than in control patients with other neurological diseases (OND) [18]. Specifically, C. pneumoniae was isolated in culture in 24/37 (65%) of MS patients and 3/27 (11%) of OND patients; CSF PCR for major outer membrane protein (MOMP) was positive in 36/37 (97%) of MS patients and 5/27 positive (18%) of OND patients, and CSF anti-C. pneumoniae IgG test by enzyme-linked immunosorbent assay (ELISA) was positive in 32/37 (86%) of MS patients and none of the OND patients. After this groundbreaking report, a number of studies suggested that C. pneumoniae infection may be associated with MS, while no association could be established in others [81, 82].

In recent years, the possible role of C. pneumoniae in the development of MS has been supported by sero-epidemiological, cultural, molecular, immunological, and therapeutic studies. However, the fact is that there are not many studies supporting the role of the organism in MS. Firstly, some reports have shown that C. pneumoniae seropositivity is associated with the risk of developing progressive forms of MS but only moderately associated with the developing MS [83], while others found no association between serum anti-Chlamydia antibody titers and MS risk [84]. Secondly, the microorganism was detected in the throat with increasing serological titers during MS relapses [85]. Thirdly, MS relapses have long been known to follow respiratory tract infections, including sore throats or pneumonia, a typical clinical pattern of respiratory tract infection caused by C. pneumoniae. However, studies to isolate the pathogen in cultures of CSF and brain tissue have repeatedly failed in MS patients or shown culture positivity in only a small proportion of MS patients [86, 87, 88, 89, 90].

Dong-Si et al. reported gene transcription of C. pneumoniae mRNA in the CSF of MS patients, a finding that supports active infection with this pathogen [91].

In another study, active transcription of DNA of the organism indicating in a persistent and metabolically active state was detected in cultured CSF and PBMCs from MS patients but not controls [92]. Other investigators were able to culture C. pneumoniae in buffy coat samples from a healthy blood donor population and demonstrated 24.6% carriage of Chlamydia in the circulating white blood cells. Because of the difficulties of isolating C. pneumoniae in culture, PCR-based nucleic acid amplification methods have become the preferred method for the detection of this microorganism. However, PCR procedures also often differ in several aspects that may affect their sensitivity, reproducibility, and specificity [26]. In this context, collaborative studies involving different laboratories examining the presence of C. pneumoniae in blinded CSF samples further highlighted the lack of an accepted standardized PCR protocol [93, 94]. A series of PCR studies failed to provide evidence of C. pneumoniae DNA in the CSF of MS patients. Most of these studies were conducted using single or nested PCR targeting the MOMP or 16 s ribosomal (rRNA) chlamydial genes [26].

In contrast, a substantial number of studies from around the world have provided clear evidence that C. pneumoniae has a role in MS. In this setting, most studies found positive PCR results, with rates of DNA or mRNA positivity ranging from 2.9–69% [26]. Some reports also showed that C. pneumoniae DNA was more common in the CSF of MS patients with gadolinium-enhancing lesions on MRI [95, 96]. Furthermore, detection of 16 s rRNA and Hsp60 mRNA by reverse transcriptase (RT)-PCR of CSF was more frequent in MS patients than controls, indicating the presence of high gene transcription and thus a more active C. pneumoniae metabolism in MS [91]. In 2004, a new amplification program for the MOMP gene was developed by analyzing CSF samples from patients with MS, other inflammatory neurological disorders (OIND), and non-inflammatory neurological disorders (NIND), and using three gene targets in parallel (MOMP, 16 s rRNA, and Hsp70) to achieve high sensitivity and specificity [97]. PCR positivity for MOMP and 16 s rRNA in CSF was present in a small proportion of MS patients (37%), OIND (28%), and NIND (37%), and there was no difference between MS and controls. Also, PCR positivity for MOMP and 16 s rRNA in CSF was more frequent in relapsing-remitting MS than in its progressive forms, as well as clinically and MRI stable MS compared to clinically and MRI active MS. In contrast, CSF PCR positivity for HsP70 was observed in only three patients with active relapsing-remitting MS. Therefore, the presence of C. pneumoniae, especially in a specific subgroup of active relapsing-remitting MS patients, cannot be disregarded. Early in the disease course, activated infected blood-derived monocytes cross the blood-brain barrier via transendothelial migration, resulting in inflammatory immune activation in the CNS. Alternatively, the presence of C. pneumoniae DNA in the CSF at high rates in this subset of MS patients may reflect selective infiltration of monocytes to the brain only after activation, thus suggesting that C. pneumoniae plays a role only as a silent passenger. In a PCR study targeting multiple genes in both CSF and PBMCs, 64% of active relapsing-remitting MS patients were positive for C. pneumoniae DNA and mRNA, while only three control patients were found to be positive for Chlamydia, showing that C. pneumoniae may exist in a persistent and metabolically active state at both the peripheral and intrathecal levels in MS patients but not in controls [92]. C. pneumoniae DNA was found in PBMCs, which can cross the blood-brain barrier into the intrathecal area and induce a chronic persistent brain infection that may be a cofactor in the development of the disease. Recently, they found that intrathecal synthesis of anti-C. pneumoniae IgG evaluated by antibody specific index was more common in MS (16.9%) and OND (21.6%) than in NIND (1.9%) patients, as well as in progressive forms of MS than in relapsing-remitting MS [98]. In addition, in patients with intrathecally produced anti-C. pneumoniae IgG, it was shown that CSF C. pneumoniae-specific high affinity antibodies are more common in the subgroup of patients with progressive forms of MS than in patients with OND and were not found at all in patients with remitting-relapsing MS and NIND. To further examine a possible relationship between C. pneumoniae infection and MS, Sriram et al. published a study examining autopsy samples of brain tissue and CSF using immunohistochemical staining with anti-C. pneumoniae monoclonal antibodies in addition to molecular and ultrastructural methods [99]. These techniques provided evidence for the presence of C. pneumoniae, which was more common in MS patients (90, 62, and 55%, respectively) than in control patients. The authors first demonstrated the presence of chlamydial EBs on the ependymal surfaces and periventricular regions by electron microscopy in 40% of patients with MS but not in the control group. Collectively, MS patients are more likely to have detectable levels of C. pneumoniae compared with patients with OND. However, a review of 26 studies including 1332 MS patients and 1464 controls reported that the findings were insufficient to establish an etiological relationship between C. pneumoniae and MS after controlling for the confounding effects of gender differences [88].

Treatment targeting the inflammatory process is only partly effective on the course of MS. In relapsing-remitting MS, this type of therapy slows the progression of disability, while the same therapy has been shown to have little or no effect on the progression of disability in primary progressive MS. Reports regarding antimicrobial therapy in MS have also yielded conflicting results. In one trial, the antibiotic minocycline resulted in a reduction in the number of gadolinium-enhancing lesions detected by MRI [100]. Another study showed that anti-chlamydial therapy reduced brain atrophy, but showed no beneficial effect on the number of gadolinium-enhancing lesions on MRI [101].

From the data presented, there is evidence that Chlamydia does not have a causal role in MS disease. Therefore, the findings on this topic are still confusing. While some studies have stated that the presence of Chlamydia is merely an epiphenomenon of ongoing inflammation in MS, others have reported that it plays a role as a cofactor in the development and progression of the disease by enhancing a pre-existing immune response in a subgroup of MS patients, as supported by recent immunological and molecular findings [81, 92].

There are also studies showing a possible association between MS and Parachlamydia-like organisms, which are thought to act alone or in conjunction with C. pneumoniae as a cofactor in the development and progression of MS [102].

Finally, we cannot rule out the possibility that other pathogens could be involved in the development of MS. Viruses are often considered potential candidates because they are known to cause demyelinating disease in experimental animals and humans and are generally known to cause diseases that have prolonged latent periods and manifest clinically with relapsing and remitting symptoms [103]. However, research conducted to date has not identified any single virus that plays an important role in MS. Among the viruses proposed as MS cofactors are ubiquitous members of the Herpesviridae family, human herpesvirus 6, and Epstein-Barr virus [26]. The MS-related human retrovirus of the endogenous retrovirus family has also been identified as a potential pathogen in MS [104].

3.2 C. pneumoniae and Alzheimer’s disease

AD is among the most severe dementias and is increasing as the population ages. AD is associated with neuronal atrophy/death in certain areas of the brain and occurs in two main forms: an early-onset form that is primarily genetically determined, and late-onset AD, which is a non-familial, progressive neurodegenerative disease that is currently the most common and severe form of dementia in older adults. The descriptive neuropathology of both familial and sporadic AD includes neuritic senile plaques (NSPs), consisting mainly of amyloid-β protein, and neurofibrillary tangles (NFTs), the major component of which is modified tau protein, which affect nerve synapses and nerve-nerve cell communication. Genetic, biochemical, and immunological analyses have provided relatively detailed information about these entities [31]. The disease usually initially presents as a gradual loss in short-term memory and later progresses to major cognitive dysfunction. Later it can manifest with various behavioral disorders, disorientation, language difficulties, and impairment in activities of daily living [105]. Estimates of the gross incidence of AD range from 7.03 to 23.8 per 1000 person-years [106, 107]. The incidence of AD increases with age in both sexes. Women have approximately 33% higher incidence and prevalence than men [105, 106, 107, 108, 109]. Although AD was discovered by Alois Alzheimer in 1907, the cause of this pathology and neurodegeneration is unknown. CNS infections have been shown to stimulate inflammatory responses that may result in neurodegeneration [110]. Several groups have investigated the relationship between various infectious agents and AD, but none of these pathogens were confirmed as etiological factors of disease development or worsening of neuropathology. Interesting insights came from a study that identified herpes simplex virus 1 infection as a risk factor for AD development in subjects expressing the apolipoprotein-E (APOE)-4 allele [110, 111].

Viruses such as the measles virus, adenovirus, lentiviruses, and others were initially evaluated but later ruled out [112, 113]. Bacterial pathogens, including C. trachomatis, Coxiella burnetii, Mycoplasma species, and spirochetes, have also been investigated for involvement in the neuropathogenesis of AD and were rejected [114, 115].

Prions were also considered but later excluded [116].

The first article reporting an association between C. pneumoniae infection and late-onset AD was published by Balin et al. [31]. In this study, highly sensitive and specific PCR tests for C. pneumoniae were performed on different brain regions (hippocampus, cerebellum, temporal cortex, and prefrontal cortex) showing varying degrees of AD pathology and were positive in 90% of the brains [117]. Electron microscope studies revealed C. pneumoniae-like particles containing EBs and RBs in brain tissue, and immunohistochemical analysis showed strong labeling in the parts of the brain most affected by AD, while no labeling was observed in controls. Furthermore, C. pneumoniae were detected and visualized in some CNS cells associated with plaques and tangles, RNA transcripts of C. pneumoniae showing metabolically active organisms were demonstrated by RT-PCR of frozen tissue samples, and the organisms were subsequently isolated and grown in cell culture. As demonstrated in reactive arthritis, Balin et al. reported a strong correlation between the APOE-4 genotype and C. pneumoniae infection in 58% (11/19) of patients with AD, suggesting that the APOE-4 gene may support some aspects of C. pneumoniae pathobiology in AD [31]. This report attracted great interest from the public and science, and attempts to replicate their findings were made at reputable laboratories worldwide. Two independent research teams (Ossewaarde et al., 2000 and Mahony et al., 2000, unpublished data) found C. pneumoniae in the brains of patients with AD using PCR and immunohistochemistry, confirming the results of Balin et al. However, subsequent studies conducted by different authors using the same procedures but different protocols in paraffin-embedded brain tissues yielded conflicting results [26]. The contradictory results obtained in these studies may be related to differences in diagnostic criteria or demographic differences such as the patients’ geographic location, season of death, and history.

AD patients included in the Balin study may have recently been exposed to C. pneumoniae and therefore may have been at high risk of systemic dissemination from the respiratory tract to sites within the CNS where advanced AD pathology was already present [118]. As an extension of these findings, 2 years later Gerard demonstrated the presence of C. pneumoniae in 80% of AD specimens and 11.1% of controls using a technique targeting two Chlamydia genes [1046, 0695] genes. Although the AD patients (mean age: 79.3 years) and controls (65.9 years) were as well matched for age and sex as possible, the controls were younger and only 22.7% were male [119]. Cultures of brain specimens showed that the organism was viable in the AD brain, and further reverse-transcriptase PCR analyses identified primary rRNA gene transcripts from C. pneumoniae, demonstrating metabolic activity of the organism in these tissues. Interestingly, immunohistochemical analyses have also shown that astrocytes, microglia, and neurons all serve as host cells for C. pneumoniae. These infected cells were found in the AD brain close to both NSPs and NFTs.

Recent studies in cultured astrocytes and microglia have shown that C. pneumoniae exhibits an active rather than persistent growth phenotype, indicating possible simultaneous destruction with the rupture of some host cell components at the end of this cycle [120].

In the years immediately following Balin et al.’s study, some experimental discoveries provided insight into the pathogenetic mechanisms of AD. First, there is a relationship between carriage of the APOE-4 allele and the pathobiology of C. pneumoniae, and the C. pneumoniae load in the AD brain varies by ApoE genotype [121, 122]. Second, infection of human microvascular endothelial cells cultured with C. pneumoniae results in an increase in the expression of proteins involved in the organism’s CNS access, including N-cadherin and b-catenin [123]. Third, the expression of occluding, a protein associated with tight connections was attenuated in C. pneumoniae-infected cells. Fourth, infection with C. pneumoniae via the olfactory pathways in nontransgenic young female BALB/c mice, which generally do not develop AD, was shown to promote the production of extracellular amyloid-like plaques [124]. Because C. pneumoniae resides in the respiratory tract and has a tendency to infect epithelial cells, the olfactory epithelium in the nasal passages is a possible target of infection. After entry into these epithelia, potential damage and/or cell death may occur in the main olfactory bulb and olfactory cortex, opening the way for further retrograde neuronal damage [117, 125].

In a recent study by Chacko et al., it was shown that C. pneumoniae rapidly infects both the olfactory and trigeminal nerves that nasal epithelial injury exacerbated peripheral nerve infection but reduced brain infection and that C. pneumoniae inclusions in the olfactory nerve and bulb were associated with amyloid-β deposits. It was also reported that C. pneumoniae replication occurred in the glial cells of the olfactory/trigeminal nerves and the brain and that C. pneumoniae infection may lead to differential regulation of the genes associated with AD. Thus, C. pneumoniae can spread very quickly from the periphery to the CNS via the nerves connecting the nasal cavity and the brain, without infecting the bloodstream. This study demonstrated in vivo that there is a rapid accumulation of amyloid-β in response to C. pneumoniae infection of the primary olfactory nervous system. C. pneumoniae inclusions were detected in the olfactory bulb and nerve fiber layer/glomerular layer. The detection of inclusion bodies in the olfactory piriform cortex as well suggested that C. pneumoniae progressed deeper into the olfactory bulb, as previously reported.

The ability to infect glia is considered the key to invading the CNS via cranial neural pathways. The study by Chacko et al. demonstrated that C. pneumoniae could infect, survive, and replicate (form inclusions) within the glia of the peripheral nervous system (olfactory ensheathing cells and trigeminal Schwann cells) and the CNS (astrocytes and microglia). C. pneumoniae antigens have been detected in both astrocytes and microglia in human brains post-mortem. Olfactory ensheathing cells, Schwann cells, and astrocytes are all innate immune cells that can respond to and phagocytose bacteria, and microglia (macrophages of the CNS) are well-characterized professional phagocytes. The ability of C. pneumoniae to form inclusions in these cells suggests that the bacteria may overcome phagocytic destruction, at least to some extent, which may be an important mechanism that allows this bacterium to invade and establish long-term infection of the CNS.

In addition, their study also revealed localized amyloid-β accumulation adjacent to C. pneumoniae inclusion bodies and in the olfactory bulb 7 days and 28 days after inoculation. Diffuse/scattered amyloid-β immunoreactivity was also present in these tissues in control mice, but the common localization of amyloid-β deposits and C. pneumoniae inclusions in vaccinated mice was clear and pronounced. Their findings and those of previous studies suggest that amyloid-β secretion occurs in response to infection. One reason for this may be that amyloid-β is secreted as an antimicrobial agent, but alternatively, it may be secreted in response to infection due to pathway activation for subsequent processing of APP into secreted amyloid-β. Future studies may clarify the secretion and role of amyloid-β in this context.

Thus, the secretion of amyloid-β may be a normal immune response to any microbe that might invade the nervous system, and if the infection is cleared, the accumulated amyloid-β can be cleared by phagocytic glia. However, if the bacteria are not cleared and instead become persistent or latent in neural cells, continuous amyloid-β deposition may occur, which contributes to late-onset dementia and/or accelerates amyloid-β accumulation in familial AD. In the case of C. pneumoniae, one study in wild-type mice showed that amyloid-β deposits from infection were subsequently cleared, while another study showed that deposits did not disappear for several months [125].

C. pneumoniae infection also leads to the upregulation of key pathways involved in the pathogenesis of AD. The pathological features of AD, such as the production of activated microglia, inflammatory mediators, and reactive oxygen species, were highly regulated in infected brain tissue after inoculation. These neuroinflammatory responses are considered an important driving factor in patients with neurodegeneration and AD pathology, which begins in the early stages of the disease, before the formation of amyloid-β plaques in the brain. After infection, activated microglia and astrocytes (which were shown to be hosts for Chlamydia) have been shown to secrete pro-inflammatory cytokines including IL-1β, TNFα, and IL-6. These cytokines are neurotoxic and can directly increase amyloid-β production through activation of β-secretase, which cleaves APP and initiates the amyloid cascade. Microglial activation reduces amyloid-β accumulation in the brain by increasing phagocytosis, clearance, and degradation. However, neuroinflammation associated with AD may be a double-edged sword, as persistent microglia activation stimulated by microglia binding to amyloid-β may increase the production of inflammatory mediators and reactive oxygen species, further strengthening the neuroinflammatory response [125].

In addition to considering key pathways, it is also useful to consider changes in individual gene expression. Long-term C. pneumoniae infection triggered downregulation of many other key genes involved in the pathogenesis of AD. Most importantly, the downregulation of the protective Hsp (Hspa1b or Hsp70–2), which was associated with increased oxidative stress and the onset of AD pathology, and of Bag2, a B-cell lymphoma-2-associated co-chaperone gene that controls Hsp70 functionality, which led to further failure of the system to protect cells from oxidative damage. Persistent infection was also reported to be associated with AD by leading to mitochondrial dysfunction, gene modulations, increased unfolded protein response, and oxidative stress. In fact, long-term infection has also been associated with low expression of CD2-associated protein, which was previously associated with AD pathology exacerbated by increased amyloid-β deposition and tau-induced neurotoxicity. In this study, it was concluded that nerves extending between the nasal cavity and brain constitute invasion pathways by which C. pneumoniae was able to rapidly invade the CNS, triggering genetic and molecular changes in the longer term and contributing to the initiation of AD pathogenesis [125].

Because chlamydial chronic infections are characterized by a “chlamydial persistent state” inaccessible to traditional antichlamydial agents, there have been several clinical studies determining the efficacy of antibiotic therapy against C. pneumoniae in AD. In a first randomized, placebo-controlled, multicenter clinical trial to determine whether a 3-month course of doxycycline and rifampin reduced the decline in cognitive function in AD patients, the antibiotic group exhibited significantly less cognitive decline at 6 months and less dysfunctional behavior at 3 months compared to controls [126]. Although these observations did not show a causal relationship between C. pneumoniae and CNS infection, they paved the way for further research on the eradication of chronic C. pneumoniae infection and AD neuropathogenesis. In this context, animal modeling will be required to describe in detail how chlamydial infection can lead to AD-related pathological changes in CNS and to provide a better understanding of infection parameters. In vitro and mouse model studies have shown that metal protein attenuating compounds support the dissolution and clearing of extracellular senile plaques composed of amyloid-β. The antiprotozoal metal chelator clioquinol, which has been reported to reduce amyloid-β plaques, possibly through chelation associated with copper and zinc, is currently in clinical trials as a potential treatment for AD [127, 128].

Scientific knowledge about AD and C. pneumoniae infection is still growing. Standardization of diagnostic techniques will certainly allow for better comparability of studies. However, other systemic infections should also be considered as potential contributors to AD pathogenesis.

3.3 C. pneumoniae and AIDS dementia

Many authors have investigated the possibility that C. pneumoniae is involved in neurodegenerative disorders other than AD. However, the available data are few and not significant. One study investigated the possible link between AIDS-dementia complex and C. pneumoniae [129]. AIDS-dementia complex is an HIV-induced neuropathological disease characterized by infection of macrophages and microglial cells and release of proinflammatory cytokines into the parenchyma [130]. In this report, C. pneumoniae was identified in the CNS by PCR. Four (17.4%) of 23 HIV-infected patients with stage 3 AIDS-dementia complex diagnosed according to the AIDS-dementia complex scheme and confirmed by autopsy were found to have C. pneumoniae in their CNS by PCR for C. pneumoniae MOMP and 16 s rRNA gene. Sequence analysis revealed important homologies with C. pneumoniae when compared with C. trachomatis and C. psittaci. In addition, ELISA demonstrated high mean levels of CSF specific anti-C. pneumoniae antibodies and significantly elevated C. pneumoniae antibody-specific index values in these patients. These findings suggest that although the low rate of isolation does not represent the incidence of C. pneumoniae, an increase in the “trafficking” of monocytes containing C. pneumoniae to the brain in the late stages of HIV infection may carry this organism to regions that are major reservoirs of productive HIV replication and contribute to neuronal damage in HIV-infected patients [129]. Furthermore, the possibility cannot be ruled out that in a subset of patients this organism exists is not an “innocent bystander,” as in atherosclerosis and other chlamydial diseases, but is able to survive and reproduce in CNS macrophages [26].

4. C. pneumoniae and other neurological complications

A number of reports have focused on the involvement of C. pneumoniae in other CNS disorders, particularly encephalitis or meningoencephalitis. Reported cases have not been not very frequent [26]. Most patients were young patients presenting with different neurological symptoms and/or neuro-radiological changes on computed tomography or MRI. In most cases, there were also accompanying well-defined respiratory symptoms, although in some cases these occurred prior to the onset of neurological records. Three patients had cerebellar ataxia, acute demyelinating encephalitis, and Guillain-Barrè syndrome. Chlamydia was almost always detected serologically using microimmunofluorescence test (four-fold increase in IgG titer) and ELISA techniques based on the detection of specific anti-C. pneumoniae antibodies. One study found the presence of IgA-type antibodies, suggesting re-infection [131]. One note reported the use of PCR in a tracheal swab and increased Chlamydia IgM antibody titers [132]. These cases and a review of the literature have shown that C. pneumoniae infection, in addition to other Chlamydia species, can present with significant neurological symptoms. Therefore, the differential diagnosis of respiratory tract infections with neurological presentation should include chlamydial infections as well as Mycoplasma and Legionella infections.

5. Conclusions

Thanks to the deep knowledge of Chlamydia biology of and the use of more advanced techniques than those traditionally used, the presence of C. pneumoniae genomic material has been demonstrated in a large number of people suffering from different acute and chronic diseases. Over the past 10 years, an increasing number of reports have indicated a possible link between C. pneumoniae and atherosclerosis and CNS diseases including various neurobehavioral disorders, MS, and AD. The main obstacle to determining the exact role of C. pneumoniae in chronic diseases is the lack of any method to safely and reliably diagnose chronic infection. The causal role of C. pneumoniae infection in cardiovascular disease has not been definitively established. Despite molecular and genetic studies of the role of C. pneumoniae in the progression of atherosclerosis, some important questions urgently need answers, such as whether C. pneumoniae is an innocent passenger or whether it is actively involved in the onset or progression of atherosclerotic disease. In particular, C. pneumoniae Hsp60 should be further investigated as a potential culprit and therapeutic target [86]. Efforts should be made to find a truly effective treatment targeting chronic C. pneumoniae. At the same time, the development of an effective vaccine should continue [133].

Although astrocytes, microglia, and neurons have been shown to be host cells for C. pneumoniae in the brain of patients with AD, and infected cells can be found near both NSPs and NFTs, most studies have been conducted with different diagnostic methods, none of which have yet been standardized. This has led to wide variation in interlaboratory test performance even when using the same test and the same criteria. Therefore, the actual involvement of C. pneumoniae in AD remains a subject of debate and requires further understanding through standardized cultural and molecular protocols.

Recent molecular, ultrastructural, and cultural developments have provided evidence that C. pneumoniae is viable and metabolically active in different biological compartments such as CSF and PBMCs in MS patients compared to controls, suggesting a relationship between this pathogen and the disease. The role of Chlamydia has been demonstrated in a subgroup of relapsing-remitting MS patients with clinical and MRI disease activity who experience the early inflammatory phase representing the development of the disease [81, 82, 92, 96]. However, growing evidence suggests that C. pneumoniae is not just an innocent bystander epiphenomenon due to ongoing MS inflammation, but is a cofactor in the development and progression of the disease by strengthening a pre-existing autoimmune response in a subset of MS patients [81, 92, 134].

For both AD and MS, there is an urgent need for further well-designed studies to determine the importance of C. pneumoniae involvement in the disease and the usefulness of antibiotic treatment.

Abbreviations

C	Chlamydia
CNS	Central nervous system
MS	Multiple sclerosis
AD	Alzheimer’s disease
TWAR	Chlamydia pneumoniae
USA	United States of America
EB	Elementary body
RB	Reticulate body
Hsp	Heat shock protein
PCR	Polymerase chain reaction
CAD	Coronary artery disease
TNF	Tumor necrosis factor
IL	Interleukin
CSF	Cerebrospinal fluid
APC	Antigen-presenting cell
FLAIR	Fluid-attenuated inversion recovery
MHC	Major histocompatibility complex
APP	Amyloid precursor protein
TLR	Toll-like receptor
mRNA	Messenger RNA
HLA	Human leukocyte antigen
OND	Other neurologic diseases
MOMP	Major outer membrane protein
ELISA	Enzyme-linked immunosorbent assay
PBMC	Peripheral blood mononuclear cell
rRNA	Ribosomal RNA
OIND	Other inflammatory neurological disease
NIND	Non-inflammatory neurological disease
NSP	Neuritic senile plaques
NFT	Neurofibrillary tangles
APOE	Apolipoprotein-E