Toll-like receptors as transducer of inflammatory signals in glia:the astrocyte-microglia connection

In physiological conditions glia in the central nervous system (CNS) can produce and release protective factors such as anti-oxidant molecules and neurotrophic factors (Sofroniew et al., 2010). Events that impinge on CNS homeostatic balance can induce local inflammatory responses (Carson et al., 2006). Reactive glia can participate producing pro-inflammatory mediators such as chemokines, cytokines, purines and free radicals. Toll-like receptors (TLRs) are involved in injury responses of nervous system tissue and in neuropathic pain. Here we have investigated the cross-talk mechanisms between glial cells in the CNS making use of an in vitro cellular model, evaluating how glia respond to TLR agonists based on cytokine synthesis and release as well as TLR mRNA/protein expression as readouts. In order to analyze specific molecular parameters involved in the genesis and maintenance of inflammation, purified microglia and astrocyte-enriched cultures were generated from cerebral cortex of 1-2 day-old rat pups. For some experiments the enriched astrocyte cultures were purified by treatment with L-leucyl-L- leucine methyl ester (L-LME), which selectively depletes cultures of microglia. Activation of microglia and astrocytes (± L-LME) was achieved by treatment with lipopolysaccharide (LPS, TLR4 agonist); zymosan (TLR2 agonist) and poly(I:C) (TLR3 agonist) for 6 and 24 hours. Gene expression analysis (Real Time-polymerase chain reaction) revealed the ability of microglia to induce mRNA coding for interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). In contrast, purified (nominally microglia-free) astrocyte cultures were not responsive to TLR agonists – unlike their astrocyte-enriched counterpart. Mediator production and release into the culture medium (analysed by ELISA) confirmed that microglia themselves respond to pathogenic stimuli. Utilizing flow-cytometric analysis we evaluated the expression of TLR receptors on the cell surface (TLR2/4) or in endosomal membranes (TLR3) after 1, 6 or 24 hours of stimulation with TLR agonists. Non-neuronal cell responsiveness to pathogenic stimuli is almost always linked to the production of inflammatory mediators. In this context we asked if the apparent inability of purified astrocytes to express a pro-inflammatory phenotype was dependent on the absence of the relevant TLR. Using confocal microscopy, stimulation with LPS conjugated with a fluorochrome showed the presence of TLR4 on the astrocyte cell surface. and Western blot analysis revealed the presence of the co-receptors MD2 and CD14. As consequence, purified astrocytes have been studied in flow cytometry to evaluate alteration in TLR protein expression. Moreover, we reconstituted the inflammatory profile in astrocyte cell cultures by adding fixed numbers of purified microglia (10% of contaminating cells final). Although the latter 'co-cultures' express pro-inflammatory cytokines after TLR agonist stimulation the absolute levels are inferior to those measured in enriched astrocytes (<5% of contaminating microglia. To further address the issue of whether microglial cell activation in the presence of astrocytes results from either physical interaction between cell membranes or chemical induction mediated by the release of mediator(s) into the culture medium, a “Transwell insert” system was used. The astrocyte/microglia co-culture paradigm described here may provide a useful starting point to elucidate the molecular mechanisms underlying astrocyte- and microglia-specific responses pertaining to, although not limited to, CNS inflammation, especially where TLR activation plays a role.