While glial cells are recognized for their roles in maintaining neuronal function, there is growing appreciation of the ability of resident brain cells to initiate and/or regulate inflammation following trauma or infection in the central nervous system (CNS). Published work from our laboratory has shed light on the possible mechanisms that underlie glial activation with the demonstration that these cells functionally express NOD2, a member of the nucleotide-binding oligomerization domain (NOD) family of proteins that serves as an intracellular pattern recognition receptor for a discrete bacterial peptidoglycan motif. In the present R03 pilot study we are assessing the role of NOD2 proteins in the initiation and/or maintenance of inflammatory immune responses of murine glia in vitro and in vivo to clinically relevant bacterial pathogens of the CNS. We have made great strides in these studies and recently published our findings. We demonstrated 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 showed that NOD2 is an important component in the in vitro inflammatory responses of resident glia to bacterial antigens. Finally, we established that NOD2 is required, at least in part, for the astrogliosis, demyelination, behavioral changes, and elevated inflammatory cytokine levels observed following in vivo bacterial infection. In this competitive R03 revision application, we propose to extend our findings to human cells. We will employ commercially available primary human astrocytes and microglia to determine whether these cells express NOD2 constitutively or following bacterial challenge. Furthermore, we will employ siRNA techniques to knock down expression of this novel intracellular pattern recognition receptor to establish the relative importance of NOD2 in the inflammatory immune responses of these key glial cell types. Results from these studies are anticipated to reveal that the expression of NOD2 is conserved in human astrocytes and microglia and that this molecule represents an important mechanism underlying the initiation and/or maintenance of glial immune responses following exposure to clinically relevant bacterial pathogens. Importantly, these additional R03 studies will provide a solid rationale for more comprehensive investigations into the regulation and role of this novel microbial pattern recognition receptor during CNS infection for which R01 mechanism funding will be sought in the near future.
In the present R03 pilot study we are assessing the role of NOD2 proteins in the initiation and/or maintenance of inflammatory immune responses of murine glia in vitro and in vivo to clinically relevant bacterial pathogens of the CNS. In this competitive R03 revision application, we propose to extend our studies to primary human astrocytes and microglia to establish the relative importance of NOD2 in the inflammatory immune responses of these key glial cell types.
