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.

Neurogenic exacerbation of microglial and astrocyte responses to Neisseria meningitidis and Borrelia burgdorferi.

Although glial cells are recognized for their roles in maintaining neuronal function, there is growing appreciation of the ability of resident CNS cells to initiate and/or augment inflammation following trauma or infection. The tachykinin, substance P (SP), is well known to augment inflammatory responses at peripheral sites and its presence throughout the CNS raises the possibility that this neuropeptide might serve a similar function within the brain. In support of this hypothesis, we have recently demonstrated the expression of high affinity receptors for SP (Neurokinin-1 (NK-1) receptors) on microglia and shown that this tachykinin can significantly elevate bacterially induced inflammatory prostanoid production by isolated cultures of these cells. In the present study, we demonstrate that endogenous SP/NK-1R interactions are an essential component in the initiation and/or progression of CNS inflammation in vivo following exposure to two clinically relevant bacterial CNS pathogens, Neisseria meningitidis and Borrelia burgdorferi. We show that in vivo elevations in inflammatory cytokine production and decreases in the production of an immunosuppressive cytokine are markedly attenuated in mice genetically deficient in the expression of the NK-1R or in mice treated with a specific NK-1R antagonist. Furthermore, we have used isolated cultures of microglia and astrocytes to demonstrate that SP can augment inflammatory cytokine production by these resident CNS cell types following exposure to either of these bacterial pathogens. Taken together, these studies indicate a potentially important role for neurogenic exacerbation of resident glial immune responses in CNS inflammatory diseases, such as bacterial meningitis.

Characterization of nucleotide-binding oligomerization domain (NOD) protein expression in primary murine microglia.

We demonstrate that primary microglia express nucleotide-binding oligomerization domain (NOD) proteins that are thought to serve as novel pattern recognition receptors for bacterial peptidoglycan motifs. NOD2 is constitutively present in microglia and is upregulated following exposure to Borrelia burgdorferi or Neisseria meningitidis. Its expression is also elevated following exposure to Toll-like receptor (TLR) ligands and muramyl dipeptide (MDP), a putative ligand for NOD2. Microglia express essential downstream effector molecules for NOD2-mediated cell responses, and MDP augments TLR ligand-induced inflammatory cytokine production. Together these data suggest that NOD2 may contribute to microglial immune responses to bacterial pathogens.

Functional expression of NOD2, a novel pattern recognition receptor for bacterial motifs, in primary murine astrocytes.

There is growing appreciation that resident brain cells can initiate and/or regulate inflammation after trauma or infection in the central nervous system (CNS). Recent studies from our laboratory have begun to shed light on the mechanisms by which astrocytes perceive bacterial challenges by demonstrating the functional expression of Toll-like receptors (TLR) in this cell type. In the present study, we demonstrate that astrocytes also express members of the novel nucleotide-binding oligomerization domain (NOD) family of proteins that can serve as cytosolic pattern recognition receptors. We show that isolated cultures of murine astrocytes constitutively express robust levels of NOD2, a molecule that can recognize a minimal peptidoglycan motif. Expression of NOD2 is significantly upregulated after exposure to two disparate and clinically relevant bacterial pathogens of the CNS, Borrelia burgdorferi and Neisseria meningitidis. Similarly, NOD2 protein expression is elevated after exposure to specific bacterial ligands for TLRs. Importantly, we show that astrocytes express Rip2 kinase, an essential downstream effector molecule for NOD-mediated cell responses, and demonstrate that this expression is upregulated after bacterial challenge. Furthermore, we confirm the functional nature of NOD2 in astrocytes by demonstrating that a specific ligand for this receptor induces significant inflammatory cytokine production and augments immune responses induced by TLR ligation. Taken together, the present demonstration that astrocytes express functional NOD2 proteins may represent a potentially important mechanism by which this glial cell type initiates either protective host responses within the brain or the progression of damaging CNS inflammation.