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

Zymosan regulates TLR2, TLR3 and TLR4 mRNA expression in rat cortical microglia. Cells were incubated with 10 μg/ml zymosan for 6 or 24 h and then processed for RT-PCR analysis. Data are means ± SEM (n = 3) normalized to GAPDH levels and are representative of three experiments. *p < 0.05, **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

Fig5: Zymosan regulates TLR2, TLR3 and TLR4 mRNA expression in rat cortical microglia. Cells were incubated with 10 μg/ml zymosan for 6 or 24 h and then processed for RT-PCR analysis. Data are means ± SEM (n = 3) normalized to GAPDH levels and are representative of three experiments. *p < 0.05, **p < 0.01 and ***p < 0.001 vs control (‘Ctr’)

Mentions: The ability of TLR ligation to influence TLR subtype expression in CNS glia remains to be explored. To address this question, we incubated purified cortical microglia with LPS, zymosan or poly(I:C) and evaluated TLR mRNA expression by RT-PCR after 6 or 24 h. LPS treatment significantly decreased or increased, respectively, TLR4 and TLR2 gene levels at both time points (Fig. 4). In contrast, TLR3 gene expression was unchanged at 6 h and significantly reduced at 24 h. Zymosan treatment of microglia likewise produced significant decreases and increases in TLR4 and TLR2 mRNA levels, respectively, at 6 and 24 h (Fig. 5), while also significantly lowering levels of TLR3 mRNA at 6 and 24 h. Engagement of TLR3 with poly(I:C) produced somewhat different results: TLR4 mRNA was decreased at 6 h only, TLR2 significantly elevated at 6 and 24 h, and no change in TLR3 gene at either time point (Fig. 6).Fig. 4


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)

Zymosan regulates TLR2, TLR3 and TLR4 mRNA expression in rat cortical microglia. Cells were incubated with 10 μg/ml zymosan for 6 or 24 h and then processed for RT-PCR analysis. Data are means ± SEM (n = 3) normalized to GAPDH levels and are representative of three experiments. *p < 0.05, **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

Fig5: Zymosan regulates TLR2, TLR3 and TLR4 mRNA expression in rat cortical microglia. Cells were incubated with 10 μg/ml zymosan for 6 or 24 h and then processed for RT-PCR analysis. Data are means ± SEM (n = 3) normalized to GAPDH levels and are representative of three experiments. *p < 0.05, **p < 0.01 and ***p < 0.001 vs control (‘Ctr’)
Mentions: The ability of TLR ligation to influence TLR subtype expression in CNS glia remains to be explored. To address this question, we incubated purified cortical microglia with LPS, zymosan or poly(I:C) and evaluated TLR mRNA expression by RT-PCR after 6 or 24 h. LPS treatment significantly decreased or increased, respectively, TLR4 and TLR2 gene levels at both time points (Fig. 4). In contrast, TLR3 gene expression was unchanged at 6 h and significantly reduced at 24 h. Zymosan treatment of microglia likewise produced significant decreases and increases in TLR4 and TLR2 mRNA levels, respectively, at 6 and 24 h (Fig. 5), while also significantly lowering levels of TLR3 mRNA at 6 and 24 h. Engagement of TLR3 with poly(I:C) produced somewhat different results: TLR4 mRNA was decreased at 6 h only, TLR2 significantly elevated at 6 and 24 h, and no change in TLR3 gene at either time point (Fig. 6).Fig. 4

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