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 regulate TLR2, TLR3 and TLR4 gene expression in rat cortical astrocytes. Enriched or purified (L-LME-treated) astrocytes were challenged with LPS (100 ng/ml), zymosan (10 μg/ml) or poly(I:C) (50 μg/ml) and processed after 6 h for TLR2, TLR3 and TLR4 mRNA expression by RT-PCR. 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’). §§p < 0.01 and §§§p < 0.001 vs L-LME-treated cells. Qualitatively similar results were obtained after 24 h, although relative mRNA levels were lower
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig8: TLR agonists regulate TLR2, TLR3 and TLR4 gene expression in rat cortical astrocytes. Enriched or purified (L-LME-treated) astrocytes were challenged with LPS (100 ng/ml), zymosan (10 μg/ml) or poly(I:C) (50 μg/ml) and processed after 6 h for TLR2, TLR3 and TLR4 mRNA expression by RT-PCR. 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’). §§p < 0.01 and §§§p < 0.001 vs L-LME-treated cells. Qualitatively similar results were obtained after 24 h, although relative mRNA levels were lower

Mentions: Given the general lack of responsiveness of purified astrocytes to TLR ligands in terms of pro-inflammatory cytokine induction, we then asked whether or not these cells possessed the capability to react to TLR ligation at the level of TLR expression. Enriched and L-LME-purified astrocytes were challenged with LPS, zymosan or poly(I:C) and processed after 6 h for RT-PCR analysis of TLR gene expression. LPS significantly diminished TLR4 mRNA in both astrocyte populations, while increasing expression levels of TLR2 and TLR3 (Fig. 8, left column). Likewise, zymosan and poly(I:C) each reduced the TLR4 mRNA level, while raising TLR2 and TLR3 mRNA (Fig. 8, middle and right columns, respectively). Qualitatively similar trends were observed at 24 h (data not shown). In all cases, mRNA levels were lower in purified astrocytes, possibly indicative of removal of the residual microglia subset. However, this explanation alone appears insufficient to fully account for these responses as, for example, the increase in TLR3 mRNA induced by all three TLR ligands is at variance with the lack of change in LPS- and poly(I:C)-treated microglia (Figs. 4, 5 and 6). These last findings suggest that either astrocytes are more responsive in the presence of microglia or the small numbers of contaminating microglia exhibit altered behaviours in the presence of astrocytes [7, 8, 25, 29].Fig. 8


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 regulate TLR2, TLR3 and TLR4 gene expression in rat cortical astrocytes. Enriched or purified (L-LME-treated) astrocytes were challenged with LPS (100 ng/ml), zymosan (10 μg/ml) or poly(I:C) (50 μg/ml) and processed after 6 h for TLR2, TLR3 and TLR4 mRNA expression by RT-PCR. 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’). §§p < 0.01 and §§§p < 0.001 vs L-LME-treated cells. Qualitatively similar results were obtained after 24 h, although relative mRNA levels were lower
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig8: TLR agonists regulate TLR2, TLR3 and TLR4 gene expression in rat cortical astrocytes. Enriched or purified (L-LME-treated) astrocytes were challenged with LPS (100 ng/ml), zymosan (10 μg/ml) or poly(I:C) (50 μg/ml) and processed after 6 h for TLR2, TLR3 and TLR4 mRNA expression by RT-PCR. 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’). §§p < 0.01 and §§§p < 0.001 vs L-LME-treated cells. Qualitatively similar results were obtained after 24 h, although relative mRNA levels were lower
Mentions: Given the general lack of responsiveness of purified astrocytes to TLR ligands in terms of pro-inflammatory cytokine induction, we then asked whether or not these cells possessed the capability to react to TLR ligation at the level of TLR expression. Enriched and L-LME-purified astrocytes were challenged with LPS, zymosan or poly(I:C) and processed after 6 h for RT-PCR analysis of TLR gene expression. LPS significantly diminished TLR4 mRNA in both astrocyte populations, while increasing expression levels of TLR2 and TLR3 (Fig. 8, left column). Likewise, zymosan and poly(I:C) each reduced the TLR4 mRNA level, while raising TLR2 and TLR3 mRNA (Fig. 8, middle and right columns, respectively). Qualitatively similar trends were observed at 24 h (data not shown). In all cases, mRNA levels were lower in purified astrocytes, possibly indicative of removal of the residual microglia subset. However, this explanation alone appears insufficient to fully account for these responses as, for example, the increase in TLR3 mRNA induced by all three TLR ligands is at variance with the lack of change in LPS- and poly(I:C)-treated microglia (Figs. 4, 5 and 6). These last findings suggest that either astrocytes are more responsive in the presence of microglia or the small numbers of contaminating microglia exhibit altered behaviours in the presence of astrocytes [7, 8, 25, 29].Fig. 8

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