Viral CNS infections: role of glial pattern recognition receptors in neuroinflammation.

Viruses are the major causative agents of central nervous system (CNS) infection worldwide. RNA and DNA viruses trigger broad activation of glial cells including microglia and astrocytes, eliciting the release of an array of mediators that can promote innate and adaptive immune responses. Such responses can limit viral replication and dissemination leading to infection resolution. However, a defining feature of viral CNS infection is the rapid onset of severe neuroinflammation and overzealous glial responses are associated with significant neurological damage or even death. The mechanisms by which microglia and astrocytes perceive neurotropic RNA and DNA viruses are only now becoming apparent with the discovery of a variety of cell surface and cytosolic molecules that serve as sensors for viral components. In this review we discuss the role played by members of the Toll-like family of pattern recognition receptors (PRRs) in the inflammatory responses of glial cells to the principle causative agents of viral encephalitis. Importantly, we also describe the evidence for the involvement of a number of newly described intracellular PRRs, including retinoic acid-inducible gene I and DNA-dependent activator of IFN regulatory factors, that are thought to function as intracellular sensors of RNA and DNA viruses, respectively. Finally, we explore the possibility that cross-talk exists between these disparate viral sensors and their signaling pathways, and describe how glial cytosolic and cell surface/endosomal PRRs could act in a cooperative manner to promote the fulminant inflammation associated with acute neurotropic viral infection.

A role for DNA-dependent activator of interferon regulatory factor in the recognition of herpes simplex virus type 1 by glial cells.

BACKGROUND: The rapid onset of potentially lethal neuroinflammation is a defining feature of viral encephalitis. Microglia and astrocytes are likely to play a significant role in viral encephalitis pathophysiology as they are ideally positioned to respond to invading central nervous system (CNS) pathogens by producing key inflammatory mediators. Recently, DNA-dependent activator of IFN regulatory factor (DAI) has been reported to function as an intracellular sensor for DNA viruses. To date, the expression and functional role of DAI in the inflammatory responses of resident CNS cells to neurotropic DNA viruses has not been reported. METHODS: Expression of DAI and its downstream effector molecules was determined in C57BL/6-derived microglia and astrocytes, either at rest or following exposure to herpes simplex virus type 1 (HSV-1) and/or murine gammaherpesvirus-68 (MHV-68), by immunoblot analysis. In addition, such expression was studied in ex vivo microglia/macrophages and astrocytes from uninfected animals or mice infected with HSV-1. Inflammatory cytokine production by glial cultures following transfection with a DAI specific ligand (B-DNA), or following HSV-1 challenge in the absence or presence of siRNA directed against DAI, was assessed by specific capture ELISA. The production of soluble neurotoxic mediators by HSV-1 infected glia following DAI knockdown was assessed by analysis of the susceptibility of neuron-like cells to conditioned glial media. RESULTS: We show that isolated microglia and astrocytes constitutively express DAI and its effector molecules, and show that such expression is upregulated following DNA virus challenge. We demonstrate that these resident CNS cells express DAI in situ, and show that its expression is similarly elevated in a murine model of HSV-1 encephalitis. Importantly, we show B-DNA transfection can elicit inflammatory cytokine production by isolated glial cells and DAI knockdown can significantly reduce microglial and astrocyte responses to HSV-1. Finally, we demonstrate that HSV-1 challenged microglia and astrocytes release neurotoxic mediators and show that such production is significantly attenuated following DAI knockdown. CONCLUSIONS: The functional expression of DAI by microglia and astrocytes may represent an important innate immune mechanism underlying the rapid and potentially lethal inflammation associated with neurotropic DNA virus infection.

RIG-I mediates nonsegmented negative-sense RNA virus-induced inflammatory immune responses of primary human astrocytes.

While astrocytes produce key inflammatory mediators following exposure to neurotropic nonsegmented negative-sense RNA viruses such as rabies virus and measles virus, the mechanisms by which resident central nervous system (CNS) cells perceive such viral challenges have not been defined. Recently, several cytosolic DExD/H box RNA helicases including retinoic acid-inducible gene I (RIG-I) have been described that function as intracellular sensors of replicative RNA viruses. Here, we demonstrate that primary human astrocytes constitutively express RIG-I and show that such expression is elevated following exposure to a model neurotropic RNA virus, vesicular stomatitis virus (VSV). Evidence for the functional nature of RIG-I expression in these cells comes from the observation that this molecule associates with its downstream effector molecule, interferon promoter stimulator-1, following VSV infection and from the finding that a specific ligand for RIG-I elicits astrocyte immune responses. Importantly, RIG-I knockdown significantly reduces inflammatory cytokine production by VSV-infected astrocytes and inhibits the production of soluble neurotoxic mediators by these cells. These findings directly implicate RIG-I in the initiation of inflammatory immune responses by human glial cells and provide a potential mechanism underlying the neuronal cell death associated with acute viral CNS infections.

Vesicular stomatitis virus infects resident cells of the central nervous system and induces replication-dependent inflammatory responses.

Vesicular stomatitis virus (VSV) infection of mice via intranasal administration results in a severe encephalitis with rapid activation and proliferation of microglia and astrocytes. We have recently shown that these glial cells express RIG-I and MDA5, cytosolic pattern recognition receptors for viral RNA. However, it is unclear whether VSV can replicate in glial cells or if such replication is required for their inflammatory responses. Here we demonstrate that primary microglia and astrocytes are permissive for VSV infection and limited productive replication. Importantly, we show that viral replication is required for robust inflammatory mediator production by these cells. Finally, we have confirmed that in vivo VSV administration can result in viral infection of glial cells in situ. These results suggest that viral replication within resident glial cells might play an important role in CNS inflammation following infection with VSV and possibly other neurotropic nonsegmented negative-strand RNA viruses.

NOD2 plays an important role in the inflammatory responses of microglia and astrocytes to bacterial CNS pathogens.

While glial cells are recognized for their roles in maintaining neuronal function, there is growing appreciation that resident central nervous system (CNS) cells initiate and/or augment inflammation following trauma or infection. We have recently demonstrated that microglia and astrocytes constitutively express nucleotide-binding oligomerization domain-2 (NOD2), a member of the novel nucleotide-binding domain leucine-rich repeat region containing a family of proteins (NLR) that functions as an intracellular receptor for a minimal motif present in all bacterial peptidoglycans. In this study, we have confirmed the functional nature of NOD2 expression in astrocytes and microglia and begun to determine the relative contribution that this NLR makes in inflammatory CNS responses to clinically relevant bacterial pathogens. We demonstrate the increased association of NOD2 with its downstream effector molecule, Rip2 kinase, in primary cultures of murine microglia and astrocytes following exposure to bacterial antigens. We show that this cytosolic receptor underlies the ability of muramyl dipeptide to augment the production of inflammatory cytokines by glia following exposure to specific ligands for disparate Toll-like receptor homologues. In addition, we demonstrate that NOD2 is an important component in the in vitro inflammatory responses of resident glia to N. meningitidis and B. burgdorferi antigens. Finally, we have established that NOD2 is required, at least in part, for the astrogliosis, demyelination, behavioral changes, and elevated inflammatory cytokine levels observed following in vivo infection with these pathogens. As such, we have identified NOD2 as an important component in the generation of damaging CNS inflammation following bacterial infection.

Characterization of retinoic acid-inducible gene-I expression in primary murine glia following exposure to vesicular stomatitis virus.

Vesicular stomatitis virus (VSV) is a negative-sense single-stranded RNA virus that closely resembles its deadly cousin, rabies virus. In mice, VSV elicits a rapid and severe T cell-independent encephalitis, indicating that resident glial cells play an important role in the initiation of central nervous system (CNS) inflammation. Recently, retinoic acid-inducible gene I (RIG-I)-like helicases have been shown to function as intracellular pattern recognition receptors for replicative viral RNA motifs. In the present study, we demonstrate that the expression of two members of this RIG-I-like receptor family (RLR), RIG-I and melanoma differentiation-associated antigen 5 (MDA5), are elevated in mouse brain tissue following intranasal administration of VSV. Using isolated cultures of primary murine glial cells, we demonstrate that microglia and astrocytes constitutively express both RIG-I and MDA5 transcripts and protein. Importantly, we show that such expression is elevated following challenge with VSV or another negative-sense RNA virus, Sendai virus. The authors provide evidence that such induction is indirect and secondary to the production of soluble mediators by infected cells. Circumstantial evidence for the functional nature of RLR expression in glial cells comes from the observation that microglia express the RLR downstream effector molecule, interferon promoter stimulator-1, and demonstrate diminished levels of the negative RLR regulator, laboratory of genetics and physiology 2, following viral challenge. These findings raise the exciting possibility that RLR molecules play important roles in the detection of viral CNS pathogens and the initiation of protective immune responses or, alternatively, the progression of damaging inflammation within the brain.