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 agonists up-regulate pro-inflammatory cytokine gene expression at 6 and 24 h in purified rat cortical microglia. Cells were challenged with a LPS (100 ng/ml); b zymosan (10 μg/ml); c poly(I:C) (50 μg/ml) and processed after 6 and 24 h for IL-1β, IL-6 and TNF-α mRNA expression by RT-PCR. Data are means ± SEM (triplicate culture wells) normalized to GAPDH levels and are representative of three experiments. **p < 0.01 and ***p < 0.001 vs control (‘Ctr’)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig2: TLR agonists up-regulate pro-inflammatory cytokine gene expression at 6 and 24 h in purified rat cortical microglia. Cells were challenged with a LPS (100 ng/ml); b zymosan (10 μg/ml); c poly(I:C) (50 μg/ml) and processed after 6 and 24 h for IL-1β, IL-6 and TNF-α mRNA expression by RT-PCR. Data are means ± SEM (triplicate culture wells) normalized to GAPDH levels and are representative of three experiments. **p < 0.01 and ***p < 0.001 vs control (‘Ctr’)

Mentions: Rat cortical microglia responded to TLR2, TLR3 and TLR4 ligands (LPS, zymosan and (poly(I:C)), respectively) after 6 h with a robust up-regulation of mRNA for the pro-inflammatory cytokines IL-1β, IL-6 and TNF-α (Fig. 2a–c, left columns). These mRNA levels remained elevated at 24 h, albeit reduced compared to 6 h (Fig. 2a–c, right columns). Changes in gene expression were accompanied by the corresponding cytokine product in the culture medium at both 6 and 24 h (Tables 2, 3 and 4). The results confirm cellular functionality (see also [7, 18]). Zymosan depleted is a Saccharomyces cerevisiae cell wall preparation treated with hot alkali to remove all its TLR-stimulating properties (InvivoGen). Zymosan depleted activates Dectin-1 (which is also expressed in microglia) but not TLR2. Zymosan depleted, up to a concentration of 100 μg/ml, failed to elicit cytokine gene changes or protein release from microglia (data not shown).Fig. 2


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 agonists up-regulate pro-inflammatory cytokine gene expression at 6 and 24 h in purified rat cortical microglia. Cells were challenged with a LPS (100 ng/ml); b zymosan (10 μg/ml); c poly(I:C) (50 μg/ml) and processed after 6 and 24 h for IL-1β, IL-6 and TNF-α mRNA expression by RT-PCR. Data are means ± SEM (triplicate culture wells) normalized to GAPDH levels and are representative of three experiments. **p < 0.01 and ***p < 0.001 vs control (‘Ctr’)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig2: TLR agonists up-regulate pro-inflammatory cytokine gene expression at 6 and 24 h in purified rat cortical microglia. Cells were challenged with a LPS (100 ng/ml); b zymosan (10 μg/ml); c poly(I:C) (50 μg/ml) and processed after 6 and 24 h for IL-1β, IL-6 and TNF-α mRNA expression by RT-PCR. Data are means ± SEM (triplicate culture wells) normalized to GAPDH levels and are representative of three experiments. **p < 0.01 and ***p < 0.001 vs control (‘Ctr’)
Mentions: Rat cortical microglia responded to TLR2, TLR3 and TLR4 ligands (LPS, zymosan and (poly(I:C)), respectively) after 6 h with a robust up-regulation of mRNA for the pro-inflammatory cytokines IL-1β, IL-6 and TNF-α (Fig. 2a–c, left columns). These mRNA levels remained elevated at 24 h, albeit reduced compared to 6 h (Fig. 2a–c, right columns). Changes in gene expression were accompanied by the corresponding cytokine product in the culture medium at both 6 and 24 h (Tables 2, 3 and 4). The results confirm cellular functionality (see also [7, 18]). Zymosan depleted is a Saccharomyces cerevisiae cell wall preparation treated with hot alkali to remove all its TLR-stimulating properties (InvivoGen). Zymosan depleted activates Dectin-1 (which is also expressed in microglia) but not TLR2. Zymosan depleted, up to a concentration of 100 μg/ml, failed to elicit cytokine gene changes or protein release from microglia (data not shown).Fig. 2

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