The innate immune response in primary cultures of human astrocytes

Abstract

The innate immune response forms a critical first line of defense against infectious agents, mediated by pattern recognition receptors that recognize microorganism-specific motifs. A major family of these receptors is the toll-like receptor family (TLR). In the central nervous system (CNS) microglia are considered to be the major cell type involved in innate immune responses. However, the intimate association of astrocytes with the vasculature of the CNS, suggests that they might also play a role. In this study, we investigated TLR expression and function in astrocytes, with a particular emphasis on TLR4 and TLR3.;We found that human fetal astrocytes in culture expressed mRNA and protein for TLR4, however, these cells did not respond to activation with LPS, even in the presence of soluble CD14, a co-receptor required for TLR4 signaling. Given the homology between the IL-1 and TLR-4 signal transduction pathways, we next questioned whether IL-1 activation of astrocytes also induced signaling leading to interferon regulatory factor-3 (IRF-3) activation. Data showed that IL-1 activation led to co-localization of tank binding kinase-1 (TBK-1) with IRF-3, phosphorylation of IRF3, and IRF3 nuclear translocation. Furthermore, IRF-3 responsive genes could be inhibited by a dominant negative form of IRF3.;Our data also showed that astrocytes expressed TLR3, a major inducer of the IRF3 pathway. We investigated astrocytic responsiveness to the TLR3 ligand poly(I:C). The data showed that resting astrocytes were highly activated with 10mug/m1 of poly(I:C). In addition, we found that expression of TLR3 was upregulated in the presence of poly(I:C) or IL-1, and that this upregulation translated into increased expression of IRF3 and NF-kappaB responsive genes. Utilizing microarrays, astrocytes stimulated with poly(I:C) showed a strong bias in anti-viral gene expression. To this end, we demonstrated that poly(I:C) activated astrocytes inhibited active replication of pseudotyped HIV-1. Furthermore, poly(I:C) induced expression of the anti-viral protein viperin/cig5 via an IRF3, NF-kB and interferon-beta-dependent (IFNbeta) pathway. Finally, we demonstrated using viperin-targeted siRNA that IFNbeta and viperin contributed to the anti-viral effects of poly(I:C).;Taken together, our data supports the conclusion that astrocytes form a unique component of the innate immune response within the CNS.