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

Marinelli C, Di Liddo R, Facci L, Bertalot T, Conconi MT, Zusso M, Skaper SD, Giusti P - J Neuroinflammation (2015)

Bottom Line: 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.L-LME treatment effectively removed microglia from the latter (real-time polymerase chain reaction).The effects of LPS on TLR2 mRNA in both cell populations were antagonized by a nuclear factor-κB inhibitor.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical and Pharmacological Sciences, University of Padua, Largo "E. Meneghetti" 2, 35131, Padua, Italy. carla.marinelli.1@studenti.unipd.it.

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.

No MeSH data available.


Related in: MedlinePlus

TLR ligands influence expression of their receptors in purified astrocytes. Purified (L-LME-treated) astrocytes were challenged with LPS (100 ng/ml), zymosan (10 μg/ml) or poly(I:C) (50 μg/ml) for 1, 6 and 24 h then processed for flow cytometric analysis, as described in ‘Methods’. Based on MFI values of each TLR and corresponding II AB-matched negative control detected in resting (a) and primed cells, we compared the relative MFI values [MFI TLR expression/MFI II AB-matched sample] to define ratio values [relative MFI (primed cells)/relative MFI (resting cells)] (b) as indicators of TLR expression change induced by treatment. Data are mean ± SD of three independent experiments conducted in duplicate. **p < 0.01 vs control
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4696218&req=5

Fig10: TLR ligands influence expression of their receptors in purified astrocytes. Purified (L-LME-treated) astrocytes were challenged with LPS (100 ng/ml), zymosan (10 μg/ml) or poly(I:C) (50 μg/ml) for 1, 6 and 24 h then processed for flow cytometric analysis, as described in ‘Methods’. Based on MFI values of each TLR and corresponding II AB-matched negative control detected in resting (a) and primed cells, we compared the relative MFI values [MFI TLR expression/MFI II AB-matched sample] to define ratio values [relative MFI (primed cells)/relative MFI (resting cells)] (b) as indicators of TLR expression change induced by treatment. Data are mean ± SD of three independent experiments conducted in duplicate. **p < 0.01 vs control

Mentions: After treatment of purified microglia and astrocyte cultures with TLR agonists, the change in TLR expression level was defined by FCM using untreated cultures (Ctr 10 % FBS) as reference (Figs. 9 and 10). Based on MFI values of each TLR and corresponding II AB-matched negative control detected in resting (Figs. 9a and 10a) and primed (data not shown) cells, we compared each pair of relative MFI values to define ratio values (Figs. 9b and 10b) as indicators of TLR expression change induced by treatment. A complex pattern of regulation was observed in purified cortical microglia: LPS treatment significantly down-regulated surface expression of TLR2, TLR4 at all time points (1, 6, 24 h) examined (Fig. 9b). Zymosan caused a transient (1 h) increase in TLR2 and TLR4; the former was significantly lower at 24 h. Much like LPS, poly(I:C) treatment led to a significant reduction in all three TLRs at both 6 and 24 h, with a transient rise only in intracellular TLR3.Fig. 9


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

Marinelli C, Di Liddo R, Facci L, Bertalot T, Conconi MT, Zusso M, Skaper SD, Giusti P - J Neuroinflammation (2015)

TLR ligands influence expression of their receptors in purified astrocytes. Purified (L-LME-treated) astrocytes were challenged with LPS (100 ng/ml), zymosan (10 μg/ml) or poly(I:C) (50 μg/ml) for 1, 6 and 24 h then processed for flow cytometric analysis, as described in ‘Methods’. Based on MFI values of each TLR and corresponding II AB-matched negative control detected in resting (a) and primed cells, we compared the relative MFI values [MFI TLR expression/MFI II AB-matched sample] to define ratio values [relative MFI (primed cells)/relative MFI (resting cells)] (b) as indicators of TLR expression change induced by treatment. Data are mean ± SD of three independent experiments conducted in duplicate. **p < 0.01 vs control
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4696218&req=5

Fig10: TLR ligands influence expression of their receptors in purified astrocytes. Purified (L-LME-treated) astrocytes were challenged with LPS (100 ng/ml), zymosan (10 μg/ml) or poly(I:C) (50 μg/ml) for 1, 6 and 24 h then processed for flow cytometric analysis, as described in ‘Methods’. Based on MFI values of each TLR and corresponding II AB-matched negative control detected in resting (a) and primed cells, we compared the relative MFI values [MFI TLR expression/MFI II AB-matched sample] to define ratio values [relative MFI (primed cells)/relative MFI (resting cells)] (b) as indicators of TLR expression change induced by treatment. Data are mean ± SD of three independent experiments conducted in duplicate. **p < 0.01 vs control
Mentions: After treatment of purified microglia and astrocyte cultures with TLR agonists, the change in TLR expression level was defined by FCM using untreated cultures (Ctr 10 % FBS) as reference (Figs. 9 and 10). Based on MFI values of each TLR and corresponding II AB-matched negative control detected in resting (Figs. 9a and 10a) and primed (data not shown) cells, we compared each pair of relative MFI values to define ratio values (Figs. 9b and 10b) as indicators of TLR expression change induced by treatment. A complex pattern of regulation was observed in purified cortical microglia: LPS treatment significantly down-regulated surface expression of TLR2, TLR4 at all time points (1, 6, 24 h) examined (Fig. 9b). Zymosan caused a transient (1 h) increase in TLR2 and TLR4; the former was significantly lower at 24 h. Much like LPS, poly(I:C) treatment led to a significant reduction in all three TLRs at both 6 and 24 h, with a transient rise only in intracellular TLR3.Fig. 9

Bottom Line: 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.L-LME treatment effectively removed microglia from the latter (real-time polymerase chain reaction).The effects of LPS on TLR2 mRNA in both cell populations were antagonized by a nuclear factor-κB inhibitor.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical and Pharmacological Sciences, University of Padua, Largo "E. Meneghetti" 2, 35131, Padua, Italy. carla.marinelli.1@studenti.unipd.it.

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.

No MeSH data available.


Related in: MedlinePlus