title: Ligand engagement of Toll-like receptors regulates their expression in cortical microglia and astrocytes.
abstract: 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.
authors.affiliation: Department of Pharmaceutical and Pharmacological Sciences, University of Padua, Largo "E. Meneghetti" 2, 35131, Padua, Italy. carla.marinelli.1@studenti.unipd.it.
authors.forename: Carla
authors.initials: C
authors.lastname: Marinelli
authors.affiliation: Department of Pharmaceutical and Pharmacological Sciences, University of Padua, Largo "E. Meneghetti" 2, 35131, Padua, Italy. rosa.diliddo@unipd.it.
authors.forename: Rosa
authors.initials: R
authors.lastname: Di Liddo
authors.affiliation: Department of Pharmaceutical and Pharmacological Sciences, University of Padua, Largo "E. Meneghetti" 2, 35131, Padua, Italy. laura.facci@unipd.it.
authors.forename: Laura
authors.initials: L
authors.lastname: Facci
authors.affiliation: Department of Pharmaceutical and Pharmacological Sciences, University of Padua, Largo "E. Meneghetti" 2, 35131, Padua, Italy. thomas.bertalot@unipd.it.
authors.forename: Thomas
authors.initials: T
authors.lastname: Bertalot
authors.affiliation: Department of Pharmaceutical and Pharmacological Sciences, University of Padua, Largo "E. Meneghetti" 2, 35131, Padua, Italy. mariateresa.conconi@unipd.it.
authors.forename: Maria Teresa
authors.initials: MT
authors.lastname: Conconi
authors.affiliation: Department of Pharmaceutical and Pharmacological Sciences, University of Padua, Largo "E. Meneghetti" 2, 35131, Padua, Italy. morena.zusso@unipd.it.
authors.forename: Morena
authors.initials: M
authors.lastname: Zusso
authors.affiliation: Department of Pharmaceutical and Pharmacological Sciences, University of Padua, Largo "E. Meneghetti" 2, 35131, Padua, Italy. stephen.skaper@unipd.it.
authors.forename: Stephen D
authors.initials: SD
authors.lastname: Skaper
authors.affiliation: Department of Pharmaceutical and Pharmacological Sciences, University of Padua, Largo "E. Meneghetti" 2, 35131, Padua, Italy. pietro.giusti@unipd.it.
authors.forename: Pietro
authors.initials: P
authors.lastname: Giusti
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D001253:Astrocytes / Q000187:drug effects / Q000378:metabolism*; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000187:drug effects / Q000378:metabolism*; D018920:Coculture Techniques; D005786:Gene Expression Regulation; D008024:Ligands; D017628:Microglia / Q000187:drug effects / Q000378:metabolism*; D011070:Poly I-C / Q000378:metabolism / Q000494:pharmacology; D051381:Rats; D051193:Toll-Like Receptors / Q000819:agonists / Q000096:biosynthesis*; D015054:Zymosan / Q000378:metabolism / Q000494:pharmacology
chemical_list: D008024:Ligands; D051193:Toll-Like Receptors; D015054:Zymosan; D011070:Poly I-C