Specific enhancement of TLR2 activity in astrocytes, previously activated by microglia

Abstract

The role of glial cells in neurodegeneration, toxicology and immunity is an expanding area of biomedical research requiring large numbers of animals. Use of the murine microglial cell line BV-2 would accelerate many research programs, and reduce the necessity of continuous cell preparations, provided that the cell line reproduces the situation in primary microglia (PM) with high fidelity. As recently doubt has been raised on their suitability, here, we re-evaluated strengths and potential shortcomings of BV-2. Their inflammatory response was compared to that of PM. Transcriptome analysis after stimulation by lipopolysaccharide (LPS) indicated a reaction pattern of BV-2 with many similarities to that of PM, although the average upregulation of genes was less pronounced. The cells showed a normal regulation of NO production and a functional response to IFNgamma;, which is important for their interaction with T-cells and neurons. BV-2 were also able to stimulate other glial cells. They triggered the translocation of NF-kB, and a subsequent production of IL-6 in astrocytes. Thus, BV-2 appear to be a good substitute for PM in many experimental settings, including complex cell-cell interaction studies.In CNS inflammation, microglia and astrocytes interact with respect to the regulation of local innate immune reactions. However, up to now the knowledge about microgliaastrocyte interaction under repeated inflammatory stress is relatively scarce. Precedent inflammatory episodes may drastically modify the function and reactivity of cells. We investigated whether priming of astrocytes by microglia-derived cytokines may enhance their subsequent reaction to pathogen-associated danger signals not recognized in the quiescent state. Resting primary murine astrocytes expressed little Toll-like receptor 2 (TLR2), and its ligands FSL1 or Pam3Cys failed to trigger IL-6 release, as functional readout, and translocation or phosphorylation of the transcription factors NF-kB or c-Jun, respectively, as easily detectable and sharply regulated upstream indicators of an inflammatory response. Cells, pre-stimulated with IL-1, with a complete cytokine mix (CCM) consisting of TNFalpha, IL-1ß and IFN-gamma, with conditioned medium of BV-2 cells, which have been activated with LPS, but also directly by activated microglia, showed a fulminant response to FSL1 or Pam3Cys, while other pattern recognition receptors were not sensitized. The heterologous sensitization of transcription factor responses to TLR2 ligands was paralleled by initial upregulation of TLR2, displayed a memory window of at least 6 days, and was largely independent of the length of pre-stimulation. The altered signaling was reflected by altered function, as FSL1 or Pam3Cys triggered the release of IL-6 and other mediators in primed cells. These data confirmed the hypothesis that sensitization towards TLR2 ligands represents a memory of earlier cell activations persisting beyond the initial stimulation by inflammatory mediators.