Rho proteins are negative regulators of TLR2, TLR3, and TLR4 signaling in astrocytes

Abstract

The family of Toll-like receptors (TLRs) expressed by innate immune cells recognizes a spectrum of microbial components as well as molecules released from injured tissues. TLR ligation activates intracellular signaling cascades that culminate in the up-regulation of proinflammatory genes. We have recently demonstrated that the up-regulation of inflammatory cytokines mediated by TLR4 in astrocytes is negatively controlled by the monomeric GTPases of Rho subfamily. The present study was undertaken to examine further the involvement of Rho proteins in the inflammatory response of astrocytes elicited by the ligation of three TLRs that use divergent signaling pathways. Astrocyte cultures established from newborn rat brains were challenged with ligands of TLR2, TLR3, and TLR4. The expression of genes encoding interleukin (IL)-1beta, IL-6, tumor necrosis factor-alpha (TNFalpha), interferon-beta (IFNbeta), and inducible nitric oxide synthase (NOS2) was up-regulated 24 hr after the challenge as determined by real-time RT-PCR. Pretreatment of the cells with toxin B, which specifically inactivates Rho proteins, enhanced the up-regulation of gene expression. The extent of this enhancement was both receptor and gene dependent. The enhancing effect of Rho protein inactivation was also evident at the protein level of IL-6 and NOS2 as revealed by ELISA and immunoblot analyses, respectively. These results suggest that Rho proteins control TLR-mediated up-regulation of inflammatory genes in astrocytes by interfering with multiple events along the signaling pathways.