Ligand engagement of Toll-like receptors regulates their expression in cortical microglia and astrocytes

BACKGROUND: Toll-like receptor (TLR) activation on microglia and astrocytes are key elements in neuroinflammation which accompanies a number of neurological disorders. While TLR activation on glia is well-established to up-regulate pro-inflammatory mediator expression, much less is known about how ligand engagement of one TLR may affect expression of other TLRs on microglia and astrocytes. METHODS: In the present study, we evaluated the effects of agonists for TLR2 (zymosan), TLR3 (polyinosinic-polycytidylic acid (poly(I:C)), a synthetic analogue of double-stranded RNA) and TLR4 (lipopolysaccaride (LPS)) in influencing expression of their cognate receptor as well as that of the other TLRs in cultures of rat cortical purified microglia (>99.5 %) and nominally microglia-free astrocytes. Elimination of residual microglia (a common contaminant of astrocyte cultures) was achieved by incubation with the lysosomotropic agent L-leucyl-L-leucine methyl ester (L-LME). RESULTS: Flow cytometric analysis confirmed the purity (essentially 100 %) of the obtained microglia, and up to 5 % microglia contamination of astrocytes. L-LME treatment effectively removed microglia from the latter (real-time polymerase chain reaction). The three TLR ligands robustly up-regulated gene expression for pro-inflammatory markers (interleukin-1 and interleukin-6, tumor necrosis factor) in microglia and enriched, but not purified, astrocytes, confirming cellular functionality. LPS, zymosan and poly(I:C) all down-regulated TLR4 messenger RNA (mRNA) and up-regulated TLR2 mRNA at 6 and 24 h. In spite of their inability to elaborate pro-inflammatory mediator output, the nominally microglia-free astrocytes (>99 % purity) also showed similar behaviours to those of microglia, as well as changes in TLR3 gene expression. LPS interaction with TLR4 activates downstream mitogen-activated protein kinase and nuclear factor-κB signalling pathways and subsequently causes inflammatory mediator production. The effects of LPS on TLR2 mRNA in both cell populations were antagonized by a nuclear factor-κB inhibitor. CONCLUSIONS: TLR2 and TLR4 activation in particular, in concert with microglia and astrocytes, comprise key elements in the initiation and maintenance of neuropathic pain. The finding that both homologous (zymosan) and heterologous (LPS, poly(I:C)) TLR ligands are capable of regulating TLR2 gene expression, in particular, may have important implications in understanding the relative contributions of different TLRs in neurological disorders associated with neuroinflammation

Varying constants, Gravitation and Cosmology

Fundamental constants are a cornerstone of our physical laws. Any constant varying in space and/or time would reflect the existence of an almost massless field that couples to matter. This will induce a violation of the universality of free fall. It is thus of utmost importance for our understanding of gravity and of the domain of validity of general relativity to test for their constancy. We thus detail the relations between the constants, the tests of the local position invariance and of the universality of free fall. We then review the main experimental and observational constraints that have been obtained from atomic clocks, the Oklo phenomenon, Solar system observations, meteorites dating, quasar absorption spectra, stellar physics, pulsar timing, the cosmic microwave background and big bang nucleosynthesis. At each step we describe the basics of each system, its dependence with respect to the constants, the known systematic effects and the most recent constraints that have been obtained. We then describe the main theoretical frameworks in which the low-energy constants may actually be varying and we focus on the unification mechanisms and the relations between the variation of different constants. To finish, we discuss the more speculative possibility of understanding their numerical values and the apparent fine-tuning that they confront us with.Comment: 145 pages, 10 figures, Review for Living Reviews in Relativit