Elsevier

Neurobiology of Aging

Volume 28, Issue 4, April 2007, Pages 524-532
Neurobiology of Aging

Chlamydia pneumoniae infection of brain cells: An in vitro study

https://doi.org/10.1016/j.neurobiolaging.2006.02.014Get rights and content

Abstract

Inspired by the suggested associations between neurological diseases and infections, we determined the susceptibility of brain cells to Chlamydia pneumoniae (Cpn). Murine astrocyte (C8D1A), neuronal (NB41A3) and microglial (BV-2) cell lines were inoculated with Cpn. Infection was established by immunofluorescence and real-time PCR at various time points. Productive infection was assessed by transferring medium of infected cells to a detection layer. Finally, apoptosis and necrosis post-infection was determined. Our data demonstrate that the neuronal cell line is highly sensitive to Cpn, produces viable progeny and is prone to die after infection by necrosis. Cpn tropism was similar in an astrocyte cell line, apart from the higher production of extracellular Cpn and less pronounced necrosis. In contrast, the microglial cell line is highly resistant to Cpn as the immunohistochemical signs almost completely disappeared after 24 h. Nevertheless, significant Cpn DNA amounts could be detected, suggesting Cpn persistence. Low viable progeny and hardly any necrotic microglial cells were observed. Further research is warranted to determine whether these cell types show the same sensitivity to Cpn in an in vivo setting.

Introduction

Previous data provided a strong link between infectious agents, like human immunodeficiency virus (HIV), herpes simplex virus or cytomegalovirus and diseases of the central nervous system [5], [15], [17], [22], [36]. More recent data then suggested that Chlamydia pneumoniae (Cpn) should be added to the candidate list of microbes responsible for several of these diseases [34]. Cpn, an obligate intracellular bacterium, is a widespread respiratory pathogen able to infect a diversity of eukaryotic cell types [20]. Cpn infections mainly result in acute respiratory tract infections, including pneumonia, sinusitis, bronchitis and pharyngitis [10], [11]. Furthermore, Cpn has been associated with various chronic diseases like asthma [39], atherosclerosis [6], [19] and rheumatoid arthritis. Interestingly, previous studies suggested a potential link between Cpn and several disorders of the central nervous system as well [42].

One of the neurological diseases associated with Cpn is Alzheimer's disease (AD). When post-mortem brain samples were analysed, Cpn was found in 17 out of 19 late-onset AD patients using PCR, immunohistochemistry and electron microscopy while Cpn could be detected in only 1 out of 19 controls [2]. Similarly, Cpn could be detected in other areas of neuropathology in the brain [1]. Additional evidence for a role of Cpn in neurological disorders comes from animal experiments as Balb/c mice develop AD-like pathology after infection with Cpn [23]. However, others failed to identify an association between Cpn and AD [4], [9], [26], [30], [37] and discrepancies exist regarding the presence of Cpn inside the brain in neurological diseases. Nonetheless, it is well recognised that Cpn can traffic towards the brain and cause a local inflammatory reaction [34]. Whether this is induced by direct infection of brain cells or rather by an indirect response is still ambiguous. Therefore, it is important to investigate the susceptibility of specific brain cells to Cpn infection. For this purpose three brain cell lines were selected: neurons (NB41A3), microglial cells (BV-2) and astrocytes (C8D1A). Neurons are particularly involved in intracellular communication, while neuronal death is often connected with a variety of brain disorders like AD and Parkinson's disease. Next, astrocytes play a role in maintaining an appropriate chemical content of the extracellular space for neuronal signaling. Finally, microglial cells have been termed the tissue macrophages of the central nervous system; they proliferate following brain injury and presumably help repair neural damage [32].

In this study, it was our aim to examine the susceptibility of these murine brain cells to Cpn infection by immunohistochemical techniques and real-time polymerase chain reaction (RT-PCR). Furthermore, a productive infection was assessed by using a susceptible cell line (epithelial cell line). Finally, we determined the occurrence of cell death and its nature – apoptotic versus necrotic – in these cell lines following Cpn infection.

Section snippets

C. pneumoniae (Cpn)

The Cpn strain, TWAR 2043, was obtained from the American Type Tissue collection (ATCC VR-1355) and continuously propagated on Hep2 cells as described previously [31]. Bacterial titres, expressed as the number of inclusion forming units per millilitre (IFU/ml), were determined by titration in Hep2 cells as described by Ezzahiri [6] and stained with a Chlamydia LPS-specific, FITC-conjugated mononuclear Antibody (RR402, DAKO, Glostrup, Denmark).

Cell cultures

Murine microglial cells (MMC—BV-2) [3] were kindly

Results

Thus, a reference epithelial cell layer and three brain cell lines were infected with C. pneumoniae (MOI 5). The presence of Cpn antigens and DNA in these cell lines was determined, using immunofluorescent techniques and real-time PCR respectively. A productive Cpn infection was also analyzed. Therefore, Cpn fluorescent signal was investigated in a “susceptible cell layer” after transferring medium of infected cells. Finally, a double-staining with Hoechst 33258 and propidium iodide revealed

Discussion

C. pneumoniae (Cpn), an obligate intracellular gram-negative bacterium, has been associated with both acute (meningitis–encephalitis) as well as chronic diseases (multiple sclerosis) of the central nervous system [12], [33], [34], [40]. Also, recent evidence suggests a role for Cpn in AD [2]. Nevertheless, the data published so far do not prove any causative role for Cpn in AD and many questions have to be answered before we can attribute a definite role to Cpn in the pathogenesis of AD. For

Acknowledgement

This research was funded by the Departments of Medical Microbiology and Cellular Neuroscience of the Maastricht University.

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