Lymphocytic Choriomeningitis Virus (LCMV) infection of CNS glial cells results in TLR2-MyD88/Mal-dependent inflammatory responses

https://doi.org/10.1016/j.jneuroim.2007.11.018Get rights and content

Abstract

In response to invading pathogens, Toll-like receptors (TLR) play a critical role in the initiation of the innate immune response, which can be either beneficial or detrimental to the host. In the present study, we demonstrated that central nervous system (CNS) glial cells are activated by Lymphocytic Choriomeningitis Virus (LCMV) in a TLR2-MyD88/Mal-dependent manner. Specifically, in response to LCMV, both astrocytes and microglial cells isolated from wild-type (WT) mice produced chemokines, such as MCP-1, RANTES and TNF-α. Similar responses occurred in TLR3 KO and TLR4 KO glial cells. In striking contrast, both astrocytes and microglial cells isolated from mice deficient in TLR2, MyD88, and Mal did not produce any of these chemokines. In addition, LCMV infection of glial cells induced up-regulation of TLR2, MHC class-I and II, CD40, CD86 in a MyD88-dependent manner. These results define a functional role for TLR signaling in viral infection-induced activation of CNS glial cells as well as for the immunopathology in the CNS.

Introduction

Toll-like receptors (TLRs) are important components of innate immunity (Finberg and Kurt-Jones, 2004, Medzhitov, 2001, Takeda and Akira, 2005). More than 10 TLRs have been identified so far (Kawai and Akira, 2007). All TLRs, with the exception of TLR3, use Myeloid Differentiation factor 88 (MyD88) as the adaptor molecule to activate the downstream signaling pathways. TLR3 ligand activates a Toll/IL-1 receptor (TIR) domain-containing adaptor protein inducing IFN-β (TRIF)-dependent signaling pathway. In addition, another TIR domain-containing adaptor, TIR domain-containing adaptor protein (Mal), is essential for both TLR2 and TLR4 activated MyD88-dependent signaling pathways. Following engagement with TLR ligands, TLR-dependent signals lead to the activation of the transcriptional factors nuclear factor κB (NF-κB), activator protein-1 (AP-1), or Interferon Regulatory Factors (IRFs), thus coordinately regulating host defense against microbial infections. There is increasing evidence that TLR/MyD88 mediated signals play a central role in the activation of the innate and adaptive immune response to microbial pathogens (Palliser et al., 2004, Pasare and Medzhitov, 2004, Schnare et al., 2001, Zhou et al., 2005). However, the exact mechanisms by which TLRs regulate viral pathogenesis are poorly defined (Lang et al., 2006). Astrocytes and microglial cells are the predominant non-neuronal parenchymal cells in the brain and are essential for maintaining CNS homeostasis (Hertz et al., 1990). They are crucial for the induction of innate immune responses, including secreting chemokines within the brain to protect neuronal cells from invading pathogens (Dong and Benveniste, 2001, Frei et al., 1994, Olson and Miller, 2004). On the other hand, glial cell-mediated inflammation in the brain can also contribute to damage of the CNS (Lehnardt et al., 2006). Chemokines are involved in the development of viral infection-mediated inflammatory diseases of the CNS and peripheral tissues (Allan et al., 1987, de Jong et al., 2006, Kamperschroer and Quinn, 2002). The production of chemokines in the brain parenchyma acts as a signal to attract pathogen-specific CD4+and CD8+T cells to the brain and therefore contributes to the control of infection but also to immune-mediated diseases (Kamperschroer and Quinn, 2002). Recent studies have demonstrated that both CNS astrocytes and microglial cells express TLRs (Bowman et al., 2003, Farina et al., 2005, McKimmie and Fazakerley, 2005). The role of TLRs in both innate and adaptive immunity suggests that TLRs could be involved in the initiation of CNS inflammation in response to viral pathogens.

LCMV is a non-cytolytic virus and most of the diseases associated with LCMV infection in mice are mediated by innate and acquired immune responses rather than direct cytopathic effects of the virus (Allan et al., 1987, Oldstone, 2002, Zinkernagel et al., 1985). LCMV is used as a model to evaluate viral infection-induced protective immune responses and immune-mediated pathogenesis. It has been found that intracranial LCMV infection leads to the up-regulation of several chemokine genes in the brain, including MCP-1, TNF-α, and RANTES (Asensio and Campbell, 1997, Campbell et al., 1994). Recent studies have demonstrated that the production of these chemokines occurs in a TLR-dependent manner (Takeda and Akira, 2005), suggesting that TLRs may also be involved in LCMV-induced chemokine responses within the brain. Although it has been known for years that intracranial LCMV infection in immunocompetent mice induces a robust chemokine gene expression and a MHC class-I-restricted CD8 T cell response, which is related to LCMV-induced death, the mechanisms associated with the immunopathological damage in the brain and death are not fully understood (Allan et al., 1987, Andersen et al., 1991, Wacher et al., 2007). The aim of this study was to explore the role of the innate immune system in the activation of CNS glial cells in response to LCMV. Our studies demonstrate that the TLR2-MyD88/Mal pathway is required for the activation and induction of chemokines in mouse CNS glial cells.

Section snippets

Virus

The Armstrong strain of LCMV (LCMV-Arm) was originally obtained from Dr. Liisa K. Selin (University of Massachusetts Medical School, Worcester, MA). LCMV-Arm was propagated on BHK-21 cells (ATCC) at low MOI (0.01). Viral titer was determined with an immunological focus assay (Battegay et al., 1991). Rat anti-LCMV NP antibody was kindly provided by Dr. Demetrius Moskophidis (Medical College of Georgia, Augusta, GA).

Mice

TLR2, TLR3, TLR4, MyD88, and Mal knockout (KO) mice were a gift of Dr. S Akira

LCMV-induced chemokine responses in mouse CNS astrocytes and microglial cells are TLR2/MyD88/Mal-dependent

We have previously shown that the TLR2-MyD88 pathway is essential for LCMV-induced chemokine and cytokine responses ex vivo in mouse peritoneal macrophages and in vivo in intravenously (i.v.) LCMV-infected mice (Zhou et al., 2005). To determine whether the TLR2-MyD88 signaling pathway is also important for cytokine production in brain glial cells, we established primary brain mixed glial cell cultures from WT mice as well as mice deficient in TLR2 or the TLR2 adaptor molecules MyD88 and Mal. As

Discussion

The main findings of the current study are as follows: 1) TLR2-MyD88/Mal signaling pathways play a central role in LCMV-induced chemokine (cytokine) responses in CNS glial cells, 2) TLR2-MyD88-dependent signaling is essential for the LCMV-induced activation of both astrocytes and microglial cells, and 3) Microglial cells and astrocytes differ in the type of chemokines (cytokines) they produce in response to viral infection (microglial cells produce primarily TNF-α and astrocytes produce

Acknowledgments

We thank Junko Kato for excellent secretarial assistance. This work was supported by NIAID Regional Center of Excellence Grant AI 057159, NIH grants R01 AI 49309 and P01 AI 0577484 (RWF), NIH grant AI 51415 (EAKJ) and the German Academic Exchange Program (AH).

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