In response to pathogens, glial cells dynamically and differentially regulate Toll-like receptor gene expression

https://doi.org/10.1016/j.jneuroim.2005.08.006Get rights and content

Abstract

The mechanisms that mediate innate immune recognition of CNS infections are unknown. This study provides a comparison of Toll-like receptor (TLR) gene expression in resting and virus infected CNS cells. N2a neuroblastoma cells expressed TLR 3 but demonstrated no change in TLR gene expression in response to either LPS or virus infection. N9 microglia and differentiated primary astrocytes expressed most TLR genes. TLR 2 expression was highest in N9 microglia and TLR 7 in astrocytes. In both glial cell types, LPS stimulation upregulated pro-inflammatory cytokines, TLR 2 and TLR 3 gene expression but down-regulated other TLR genes. RNA virus infection substantially increased levels of type-I interferon (IFN) and TLR 3 transcripts and to a lesser extent TLR 9 transcripts. Microglia and astrocytes thus have the ability to discriminate between pathogens and elicit an appropriate response.

Introduction

The mechanisms that mediate innate recognition of infections in the immunospecialised environment of the central nervous system (CNS) have not been characterized. Over recent years it has become clear that many pathogens are at first recognized by the broad specificity of pattern recognition receptors. Prominent amongst these are the Toll-like receptors (TLR). A growing consensus suggests that these are key to the instigation of innate immune responses (Akira and Takeda, 2004). TLR are principally expressed on cell types likely to be the first to encounter antigen such as phagocytic cells. TLR 1 to 9 are expressed in both mice and humans. TLR 10 is present in mice only as a degenerate pseudogene. TLR 11, 12 and 13 are expressed in mice but may lack human orthologs (Chuang and Ulevitch, 2001, Chuang and Ulevitch, 2000, Du et al., 2000, Medzhitov et al., 1997, Rock et al., 1998, Tabeta et al., 2004, Takeuchi et al., 1999b). All TLR share a similar structure with extracellular leucine rich repeat (LRR) motifs and an intracellular Toll/IL-1 receptor domain. The repeating patterns on the structural components of bacterial pathogens are principally recognized by TLR 1, 2, 4, 5 and 6. TLR 2 is known to be involved in the recognition of bacterial peptidoglycan, fungal components and viral glycoproteins (Bieback et al., 2002, Takeuchi et al., 1999a, Underhill et al., 1999). TLR 4 is involved in the recognition of lipopolysaccharide (LPS) and viral envelope proteins (Poltorak et al., 1998, Rassa et al., 2002), whilst TLR 5 can recognize bacterial flagellin (Hayashi et al., 2001). TLR 1, 2 and 6 act together to discriminate between bacterial lipoproteins (Takeuchi et al., 2001, Wyllie et al., 2000). The nucleic acid of pathogens is also recognized by several TLR. TLR 3 can recognize virus dsRNA (Alexopoulou et al., 2001), TLR 7 and 8 can recognize virus ssRNA (Diebold et al., 2004, Heil et al., 2004) and TLR 9 is involved in the recognition of CpG motifs present in bacterial and viral DNA (Hemmi et al., 2000, Tabeta et al., 2004). In contrast to TLR that recognize structural components these TLR have also been observed in intracellular compartments (Matsumoto et al., 2003).

In the mature CNS, the majority of neurons are post-mitotic and cannot be replaced if lost or damaged and the CNS environment is therefore carefully regulated to avoid neuronal damage. The absence of immune cells and plasma components is one consequence of this highly specialized environment. However, substantial inflammatory responses can be initiated in the CNS. Glial cells, in particular microglia and astrocytes, have been reported to be capable of initiating or perpetuating immune responses. In response to a variety of pathogens these cells can undergo hypertrophy and hyperplasia and produce cytokines. Astrocytes can initiate the production of complement proteins and microglia can become active phagocytes, capable of producing many of the same anti-microbial molecules as activated macrophages and capable of activating T cells (Carpentier et al., 2005, Mack et al., 2003, Olson and Miller, 2004). It remains unclear how this glial cell response is initiated; signaling via TLR may be one mechanism. To date, there are few and limited studies on TLR expression profiles in glial cells.

Determination of TLR gene expression and signaling in neural cells is likely to be relevant in understanding the neuropathology of a multitude of diseases from nervous system infections to neurodegeneration. Many viruses cause encephalitis from DNA viruses such as Herpes simplex virus type-I to RNA viruses such as rabies virus or measles virus. Glial cell activation and inflammatory infiltrates, composed predominantly of mononuclear cells, are common features of encephalitis. Glial cell activation is also observed in the absence of inflammatory infiltrates, in neurodegenerative diseases such as Alzheimer's and the transmissible spongiform encephalopathies. How glial cells become activated in these diseases remains unclear. Many studies of glial cells utilize cell lines, which while different in many respects to cells in vivo, are useful tools in research. Here we investigate TLR gene expression in specific pure neural cell populations in vitro. In vitro cultures are devoid of the complexity of inflammatory mediators produced by infiltrating leukocytes and therefore provide a system to study basal TLR gene expression and changes in expression in response to specific stimuli. In this study, two stimuli were used, lipopolysaccharide (LPS) and virus infection.

Section snippets

Cell culture and reagents

Primary astrocyte cultures were grown from neonatal mouse brain by a modification of an established protocol to generate mature cells, analogous to their in vivo counterparts in the adult brain (Juurlink and Hertz, 1985). Briefly, post-natal day 2 mouse brains (CBA crossed with C57Bl/6) were harvested and meninges removed. The cortex and cerebellum were cut into small pieces, the cells were dissociated by enzymatic digestion in papain and DNAse I for 1 h at 37 Ā°C, and then triturated through 19

Results

TLR gene transcript levels in resting, LPS activated or virus infected cells were determined by quantitative (Q)PCR in RNA isolated from cultures of N9 microglia, astrocyte and N2a neuroblastoma cells. N9 microglia and N2a neuroblastoma cells are well-characterized examples of neural cells which have been extensively used in many other studies of glial cell biology (Bate et al., 2004, Enari et al., 2001, Ferrari et al., 1996, Meda et al., 1995, Olmsted et al., 1970, Righi et al., 1989, Shea and

Discussion

This paper provides a systematic comparison of TLR gene expression in cultured neural cells. The finding that cells of the CNS have the ability via TLR expression to detect infection and discern its type provides an important contribution to understanding pathological processes in this tissue. Parenchymal microglia populate the CNS during embryogenesis and in the adult are distinct from tissue macrophages derived from circulating monocytes (Ford et al., 1995, Hickey and Kimura, 1988, Streit,

Acknowledgments

We thank Claire Cotterill for assistance with astrocyte culture and GFAP staining. C.S. McKimmie was funded by the Wellcome Trust. Funding from the EU SFVECTORS programme contributed to this research.

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