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Early steps of microglial activation are directly affected by neuroprotectant FK506 in both in vitro inflammation and in rat model of stroke

Zawadzka, Malgorzata, Dabrowski, Michal, Gozdz, Agata, Szadujkis, Barbara, Sliwa, Marcin, Lipko, Maciej, Kaminska, Bozena·2012·Journal of Molecular Medicine
DOI 10.1007/s00109-012-0925-9PMC PMC3506835
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Fig. 1 FK506 and CsA attenuate LPS-induced transformation of microglia. a Primary microglial cultures were prepared as described and kept for 48 h after plating to silence microglia. Morphological alterations of microglia untreated or exposed to 100 ng/mL LPS alone or with immunosuppressants (at concentration of 5 μM) for 24 h were visualized by immunofluorescence. Cells were stained with FITC-conjugated isolectin B4 (green) to visualize cell morphology followed by staining with DAPI for nuclei visualization (blue); ×20 objective. Stained cells with a specified range of size and optical density above fixed threshold value were automatically recognized and counted. Cell body area of microglial cells was measured, and the percentage of cells with body area >2,000 μm2 (activated cells) was calculated. Left panel shows the histograms representing the distribution of microglial cells body area (square micrometers) for all analyzed experimental conditions. Data are expressed as means ± SD from ten fields for all treatments in three independent experiments. b FK506 and CsA do not inhibit LPS-induced growth arrest of microglial cells. Microglial proliferation was determined with BrdU incorporation assay 24 h after treatment. The results are means ± SEM of three independent experiments, each in triplicate. c Immunosuppressants affect migration of activated microglia. Confluent microglial cultures were scratched with a plastic 100-μL tip, washed with PBS, and incubated for 5 h under given conditions. Cells migrating to a cell-free area without the treatment, after addition of 100 ng/mL LPS alone or with immunosuppressants (5 μM), were visualized by phase-contrast microscopy (magnification ×4). Left panel shows quantitative evaluation of motility/migration of microglia treated with LPS alone or with immunosupressants at 6 h time point. Cells from five fields between scratch edges along the line were counted. Results represent means ± SD from two independent experiments each in triplicate
Fig. 1 FK506 and CsA attenuate LPS-induced transformation of microglia. a Primary microglial cultures were prepared as described and kept for 48 h after plating to silence microglia. Morphological alterations of microglia untreated or exposed to 100 ng/mL LPS alone or with immunosuppressants (at concentration of 5 μM) for 24 h were visualized by immunofluorescence. Cells were stained with FITC-conjugated isolectin B4 (green) to visualize cell morphology followed by staining with DAPI for nuclei visualization (blue); ×20 objective. Stained cells with a specified range of size and optical density above fixed threshold value were automatically recognized and counted. Cell body area of microglial cells was measured, and the percentage of cells with body area >2,000 μm2 (activated cells) was calculated. Left panel shows the histograms representing the distribution of microglial cells body area (square micrometers) for all analyzed experimental conditions. Data are expressed as means ± SD from ten fields for all treatments in three independent experiments. b FK506 and CsA do not inhibit LPS-induced growth arrest of microglial cells. Microglial proliferation was determined with BrdU incorporation assay 24 h after treatment. The results are means ± SEM of three independent experiments, each in triplicate. c Immunosuppressants affect migration of activated microglia. Confluent microglial cultures were scratched with a plastic 100-μL tip, washed with PBS, and incubated for 5 h under given conditions. Cells migrating to a cell-free area without the treatment, after addition of 100 ng/mL LPS alone or with immunosuppressants (5 μM), were visualized by phase-contrast microscopy (magnification ×4). Left panel shows quantitative evaluation of motility/migration of microglia treated with LPS alone or with immunosupressants at 6 h time point. Cells from five fields between scratch edges along the line were counted. Results represent means ± SD from two independent experiments each in triplicateCC BY · PubMed Central
Fig. 2 FK506 reduces LPS-induced production of IL-1β by microglial cells. a Kinetics of LPS-induced production of IL-1β. Immunoblot shows the levels of pro-IL-1β (31 kDa) and mature IL-1β (17 kDa) protein in extracts from microglial cells at different times after treatment with 100 ng/mL LPS. β-Actin detection ensured an equal protein loading. Similar results were observed on three independently derived microglial cultures. b Attenuation of LPS-induced production of the mature form of IL-1β in cells exposed to 5 and 20 μM FK506 for 12 h. c Effects of immunosuppressants on IL-1β secretion by LPS-stimulated microglial cultures evaluated by ELISA assay at 12 h after treatment. Results are expressed as percentage of the IL-1β release obtained in untreated cells; means ± SEM from three experiments (each in triplicate) are presented
Fig. 2 FK506 reduces LPS-induced production of IL-1β by microglial cells. a Kinetics of LPS-induced production of IL-1β. Immunoblot shows the levels of pro-IL-1β (31 kDa) and mature IL-1β (17 kDa) protein in extracts from microglial cells at different times after treatment with 100 ng/mL LPS. β-Actin detection ensured an equal protein loading. Similar results were observed on three independently derived microglial cultures. b Attenuation of LPS-induced production of the mature form of IL-1β in cells exposed to 5 and 20 μM FK506 for 12 h. c Effects of immunosuppressants on IL-1β secretion by LPS-stimulated microglial cultures evaluated by ELISA assay at 12 h after treatment. Results are expressed as percentage of the IL-1β release obtained in untreated cells; means ± SEM from three experiments (each in triplicate) are presentedCC BY · PubMed Central
Fig.3 Immunosuppressants affect LPS-induced production of inflammatory mediators in rat microglial cultures. a Immunoblot shows that FK506 and CsA reduce the level of iNOS protein in microglial cells exposed for 24 h to 100 ng/mL LPS alone or together with FK506 or CsA (5 or 10 μM). Densitometry of immunoblots from three independent experiments was performed. The level of iNOS expression was normalized to β-actin content. All values are related to untreated controls and presented as the mean values ± SEM. b Nitric oxide production was determined as nitrite concentration in cell culture supernatants and expressed as percentage of the release by untreated cells. Results represent means ± SEM obtained from three independently derived cultures (each in triplicate). c FK506 (but not CsA) reduces LPS-induced expression of Cox-2 protein in microglial cultures. Densitometry of immunoblots from three independent experiments was performed. The level of Cox-2 expression was normalized to β-actin content, related to controls and presented as means ± SEM
Fig.3 Immunosuppressants affect LPS-induced production of inflammatory mediators in rat microglial cultures. a Immunoblot shows that FK506 and CsA reduce the level of iNOS protein in microglial cells exposed for 24 h to 100 ng/mL LPS alone or together with FK506 or CsA (5 or 10 μM). Densitometry of immunoblots from three independent experiments was performed. The level of iNOS expression was normalized to β-actin content. All values are related to untreated controls and presented as the mean values ± SEM. b Nitric oxide production was determined as nitrite concentration in cell culture supernatants and expressed as percentage of the release by untreated cells. Results represent means ± SEM obtained from three independently derived cultures (each in triplicate). c FK506 (but not CsA) reduces LPS-induced expression of Cox-2 protein in microglial cultures. Densitometry of immunoblots from three independent experiments was performed. The level of Cox-2 expression was normalized to β-actin content, related to controls and presented as means ± SEMCC BY · PubMed Central
Fig.4 FK506 interferes with signal transduction triggered by LPS in microglial cultures. Immunoblots show the levels of phosphorylated forms of MAP kinases (a) their substrates, MAPKAPK2 and c-Jun (b) and IκB (c) Western blot analyses were performed with total extracts from microglial cultures treated with LPS alone or with FK506. Similar results were obtained in four independent experiments. The level of phosphorylated MAPK was quantified by densitometry, related to the β-actin level and then to values obtained for untreated microglia. Results are presented as means ± SEM from three independent experiments
Fig.4 FK506 interferes with signal transduction triggered by LPS in microglial cultures. Immunoblots show the levels of phosphorylated forms of MAP kinases (a) their substrates, MAPKAPK2 and c-Jun (b) and IκB (c) Western blot analyses were performed with total extracts from microglial cultures treated with LPS alone or with FK506. Similar results were obtained in four independent experiments. The level of phosphorylated MAPK was quantified by densitometry, related to the β-actin level and then to values obtained for untreated microglia. Results are presented as means ± SEM from three independent experimentsCC BY · PubMed Central
Fig.5 FK506 blocks upregulation of numerous inflammation-related genes in the ischemic cortex. a Gene Ontology “biological process” terms most significantly associated with the changes in gene expression induced by MCAo in the ischemic cortex, with or without FK506 treatment, and by LPS activation of microglia in vitro. Analysis was performed on the remote RankGOStat server, with ranked lists of log2 changes in expression of the genes with the respective t test p value < 0.05 used as the input files, and RGD selected as the target database. For the plot, the FDR-corrected association p values were log10-transformed and signed, i.e., multiplied by +1 or −1 to reflect association with increased or decreased expression, respectively. Numbers of genes in each GO category are given in parentheses. b Comparison of log2 changes in gene expression induced in microglia by LPS and after MCAo for the genes significantly regulated in both systems (top panel). The changes of the same genes as in the top panel are compared to the changes induced by MCAo in the animals that received FK506 (bottom panel). Red dots represent gene assigned to GO: inflammatory response. c. Venn diagram of the genes affected by FK506 treatment assigned to GO categories: “response to external stimulus,” “response to wounding,” “immune system process,” and “inflammatory response.” A newer local version of the GO database was used than by the RankGOStat server, resulting in larger numbers of genes assigned to the same GO terms than in (a). For details of the gene names and the expression data, see ESM Table 1. d Heat maps of expression profiles of the genes in GO catagories: “response to wounding” and “inflammatory response” that were regulated by FK506. The genes were sorted on the change in expression induced by MCAo (as compared to sham). The genes that were also significantly regulated during microglia activation with LPS are marked with an asterisk
Fig.5 FK506 blocks upregulation of numerous inflammation-related genes in the ischemic cortex. a Gene Ontology “biological process” terms most significantly associated with the changes in gene expression induced by MCAo in the ischemic cortex, with or without FK506 treatment, and by LPS activation of microglia in vitro. Analysis was performed on the remote RankGOStat server, with ranked lists of log2 changes in expression of the genes with the respective t test p value < 0.05 used as the input files, and RGD selected as the target database. For the plot, the FDR-corrected association p values were log10-transformed and signed, i.e., multiplied by +1 or −1 to reflect association with increased or decreased expression, respectively. Numbers of genes in each GO category are given in parentheses. b Comparison of log2 changes in gene expression induced in microglia by LPS and after MCAo for the genes significantly regulated in both systems (top panel). The changes of the same genes as in the top panel are compared to the changes induced by MCAo in the animals that received FK506 (bottom panel). Red dots represent gene assigned to GO: inflammatory response. c. Venn diagram of the genes affected by FK506 treatment assigned to GO categories: “response to external stimulus,” “response to wounding,” “immune system process,” and “inflammatory response.” A newer local version of the GO database was used than by the RankGOStat server, resulting in larger numbers of genes assigned to the same GO terms than in (a). For details of the gene names and the expression data, see ESM Table 1. d Heat maps of expression profiles of the genes in GO catagories: “response to wounding” and “inflammatory response” that were regulated by FK506. The genes were sorted on the change in expression induced by MCAo (as compared to sham). The genes that were also significantly regulated during microglia activation with LPS are marked with an asteriskCC BY · PubMed Central

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Research works printed but not linked: 34

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Metadata

Authors
Zawadzka, M., Dabrowski, M., Gozdz, A., Szadujkis, B., Sliwa, M., Lipko, M., & Kaminska, B.
Venue
Journal of Molecular Medicine
Publisher
Springer Science and Business Media LLC
Published
2012-07-18
Volume
90
Issue
12
Pages
1459-1471
ISSN
  • 0946-2716
  • 1432-1440
DOI
10.1007/s00109-012-0925-9
PMC
PMC3506835
Cited by
34
References
1
Language
en

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