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An anti-inflammatory role for C/EBPδ in human brain pericytes.

Rustenhoven J, Scotter EL, Jansson D, Kho DT, Oldfield RL, Bergin PS, Mee EW, Faull RL, Curtis MA, Graham SE, Park TI, Dragunow M - Sci Rep (2015)

Bottom Line: Our results reveal that C/EBPδ is induced in a concentration- and time-dependent fashion in human brain pericytes by interleukin-1β (IL-1β).C/EBPδ knockdown enhanced IL-1β-induced production of intracellular adhesion molecule-1 (ICAM-1), interleukin-8, monocyte chemoattractant protein-1 (MCP-1) and IL-1β, whilst attenuating cyclooxygenase-2 and superoxide dismutase-2 gene expression.Altered ICAM-1 and MCP-1 protein expression were confirmed by cytometric bead array and immunocytochemistry.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Pharmacology and Clinical Pharmacology [2] Centre for Brain Research.

ABSTRACT
Neuroinflammation contributes to the pathogenesis of several neurological disorders and pericytes are implicated in brain inflammatory processes. Cellular inflammatory responses are orchestrated by transcription factors but information on transcriptional control in pericytes is lacking. Because the transcription factor CCAAT/enhancer binding protein delta (C/EBPδ) is induced in a number of inflammatory brain disorders, we sought to investigate its role in regulating pericyte immune responses. Our results reveal that C/EBPδ is induced in a concentration- and time-dependent fashion in human brain pericytes by interleukin-1β (IL-1β). To investigate the function of the induced C/EBPδ in pericytes we used siRNA to knockdown IL-1β-induced C/EBPδ expression. C/EBPδ knockdown enhanced IL-1β-induced production of intracellular adhesion molecule-1 (ICAM-1), interleukin-8, monocyte chemoattractant protein-1 (MCP-1) and IL-1β, whilst attenuating cyclooxygenase-2 and superoxide dismutase-2 gene expression. Altered ICAM-1 and MCP-1 protein expression were confirmed by cytometric bead array and immunocytochemistry. Our results show that knock-down of C/EBPδ expression in pericytes following immune stimulation increased chemokine and adhesion molecule expression, thus modifying the human brain pericyte inflammatory response. The induction of C/EBPδ following immune stimulation may act to limit infiltration of peripheral immune cells, thereby preventing further inflammatory responses in the brain.

No MeSH data available.


Related in: MedlinePlus

C/EBPδ knockdown enhances IL-1β induced MCP-1 and ICAM-1 protein expression.Human brain pericytes were transfected with 50 nM of control or C/EBPδ siRNA for 48 hours. Following transfection, cells were treated with vehicle or 10 ng/mL IL-1β for 2–24 hours and cells fixed and immunostained for ICAM-1 and MCP-1. Representative images of ICAM-1 and MCP-1 immunostaining respectively with vehicle and control siRNA (a,f), vehicle with C/EBPδ siRNA (b,g), 24 hours IL-1β with control siRNA (c,h) and 24 hours IL-1β with C/EBPδ siRNA are shown (d,i). Intensity of ICAM-1 staining (e) and the percentage of MCP-1 positive cells (j) were determined. Data is displayed as mean ± SEM from three independent experiments. ** = p < 0.01, *** = p < 0.001. Scale bar = 100 μm.
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f7: C/EBPδ knockdown enhances IL-1β induced MCP-1 and ICAM-1 protein expression.Human brain pericytes were transfected with 50 nM of control or C/EBPδ siRNA for 48 hours. Following transfection, cells were treated with vehicle or 10 ng/mL IL-1β for 2–24 hours and cells fixed and immunostained for ICAM-1 and MCP-1. Representative images of ICAM-1 and MCP-1 immunostaining respectively with vehicle and control siRNA (a,f), vehicle with C/EBPδ siRNA (b,g), 24 hours IL-1β with control siRNA (c,h) and 24 hours IL-1β with C/EBPδ siRNA are shown (d,i). Intensity of ICAM-1 staining (e) and the percentage of MCP-1 positive cells (j) were determined. Data is displayed as mean ± SEM from three independent experiments. ** = p < 0.01, *** = p < 0.001. Scale bar = 100 μm.

Mentions: In order to determine whether changes at the RNA level correlated with altered protein expression, immunocytochemistry for ICAM-1 and MCP-1 was performed. Unstimulated pericytes showed low basal expression of ICAM-1 and this was unaffected by C/EBPδ siRNA (1.00 ± 0.10 AU and 1.82 ± 0.30 AU respectively; Fig. 7a,b,e). A significant induction of ICAM-1 expression was observed with IL-1β treatment at four (104.61 ± 7.98AU; p < 0.001; Fig. 7e), 24 (518.92 ± 53.80AU; p < 0.001; Fig. 7c,e) and 48 hours (245.31 ± 15.61AU; p < 0.001; Fig. 7e). Compared to the control siRNA, C/EBPδ siRNA further increased IL-1β stimulated ICAM-1 expression at four (230.93 ± 14.85AU; p < 0.01; Fig. 7e), 24 (843.56 ± 128.70AU; p < 0.001; Fig. 7c, e) and 48 hours (394.52 ± 27.55AU; p < 0.01; Fig. 7e). Immunocytochemical analysis of MCP-1 demonstrated low basal levels which were also unaffected by C/EBPδ siRNA (12.77 ± 0.89% and 13.77 ± 1.03% respectively; Fig. 7f,g,j). The percentage of MCP-1 positive cells was significantly increased following IL-1β treatment at two (42.73 ± 0.81%; p < 0.001; Fig. 7j), four (63.47 ± 1.62%; Fig. 7j), 24 (52.51 ± 1.61%; Fig. 7h,j) and 48 hours(49.56 ± 2.97%; Fig. 7j). Compared to control siRNA, C/EBPδ siRNA further increased IL-1β stimulated MCP-1 expression at each of the measured time point (2 hours, 59.83 ± 1.13%; four hours 80.67 ± 0.83%; 24 hours 73.51 ± 2.25%; 48 hours 70.39 ± 4.15%; p < 0.001; Fig. 7i,j).


An anti-inflammatory role for C/EBPδ in human brain pericytes.

Rustenhoven J, Scotter EL, Jansson D, Kho DT, Oldfield RL, Bergin PS, Mee EW, Faull RL, Curtis MA, Graham SE, Park TI, Dragunow M - Sci Rep (2015)

C/EBPδ knockdown enhances IL-1β induced MCP-1 and ICAM-1 protein expression.Human brain pericytes were transfected with 50 nM of control or C/EBPδ siRNA for 48 hours. Following transfection, cells were treated with vehicle or 10 ng/mL IL-1β for 2–24 hours and cells fixed and immunostained for ICAM-1 and MCP-1. Representative images of ICAM-1 and MCP-1 immunostaining respectively with vehicle and control siRNA (a,f), vehicle with C/EBPδ siRNA (b,g), 24 hours IL-1β with control siRNA (c,h) and 24 hours IL-1β with C/EBPδ siRNA are shown (d,i). Intensity of ICAM-1 staining (e) and the percentage of MCP-1 positive cells (j) were determined. Data is displayed as mean ± SEM from three independent experiments. ** = p < 0.01, *** = p < 0.001. Scale bar = 100 μm.
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Related In: Results  -  Collection

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f7: C/EBPδ knockdown enhances IL-1β induced MCP-1 and ICAM-1 protein expression.Human brain pericytes were transfected with 50 nM of control or C/EBPδ siRNA for 48 hours. Following transfection, cells were treated with vehicle or 10 ng/mL IL-1β for 2–24 hours and cells fixed and immunostained for ICAM-1 and MCP-1. Representative images of ICAM-1 and MCP-1 immunostaining respectively with vehicle and control siRNA (a,f), vehicle with C/EBPδ siRNA (b,g), 24 hours IL-1β with control siRNA (c,h) and 24 hours IL-1β with C/EBPδ siRNA are shown (d,i). Intensity of ICAM-1 staining (e) and the percentage of MCP-1 positive cells (j) were determined. Data is displayed as mean ± SEM from three independent experiments. ** = p < 0.01, *** = p < 0.001. Scale bar = 100 μm.
Mentions: In order to determine whether changes at the RNA level correlated with altered protein expression, immunocytochemistry for ICAM-1 and MCP-1 was performed. Unstimulated pericytes showed low basal expression of ICAM-1 and this was unaffected by C/EBPδ siRNA (1.00 ± 0.10 AU and 1.82 ± 0.30 AU respectively; Fig. 7a,b,e). A significant induction of ICAM-1 expression was observed with IL-1β treatment at four (104.61 ± 7.98AU; p < 0.001; Fig. 7e), 24 (518.92 ± 53.80AU; p < 0.001; Fig. 7c,e) and 48 hours (245.31 ± 15.61AU; p < 0.001; Fig. 7e). Compared to the control siRNA, C/EBPδ siRNA further increased IL-1β stimulated ICAM-1 expression at four (230.93 ± 14.85AU; p < 0.01; Fig. 7e), 24 (843.56 ± 128.70AU; p < 0.001; Fig. 7c, e) and 48 hours (394.52 ± 27.55AU; p < 0.01; Fig. 7e). Immunocytochemical analysis of MCP-1 demonstrated low basal levels which were also unaffected by C/EBPδ siRNA (12.77 ± 0.89% and 13.77 ± 1.03% respectively; Fig. 7f,g,j). The percentage of MCP-1 positive cells was significantly increased following IL-1β treatment at two (42.73 ± 0.81%; p < 0.001; Fig. 7j), four (63.47 ± 1.62%; Fig. 7j), 24 (52.51 ± 1.61%; Fig. 7h,j) and 48 hours(49.56 ± 2.97%; Fig. 7j). Compared to control siRNA, C/EBPδ siRNA further increased IL-1β stimulated MCP-1 expression at each of the measured time point (2 hours, 59.83 ± 1.13%; four hours 80.67 ± 0.83%; 24 hours 73.51 ± 2.25%; 48 hours 70.39 ± 4.15%; p < 0.001; Fig. 7i,j).

Bottom Line: Our results reveal that C/EBPδ is induced in a concentration- and time-dependent fashion in human brain pericytes by interleukin-1β (IL-1β).C/EBPδ knockdown enhanced IL-1β-induced production of intracellular adhesion molecule-1 (ICAM-1), interleukin-8, monocyte chemoattractant protein-1 (MCP-1) and IL-1β, whilst attenuating cyclooxygenase-2 and superoxide dismutase-2 gene expression.Altered ICAM-1 and MCP-1 protein expression were confirmed by cytometric bead array and immunocytochemistry.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Pharmacology and Clinical Pharmacology [2] Centre for Brain Research.

ABSTRACT
Neuroinflammation contributes to the pathogenesis of several neurological disorders and pericytes are implicated in brain inflammatory processes. Cellular inflammatory responses are orchestrated by transcription factors but information on transcriptional control in pericytes is lacking. Because the transcription factor CCAAT/enhancer binding protein delta (C/EBPδ) is induced in a number of inflammatory brain disorders, we sought to investigate its role in regulating pericyte immune responses. Our results reveal that C/EBPδ is induced in a concentration- and time-dependent fashion in human brain pericytes by interleukin-1β (IL-1β). To investigate the function of the induced C/EBPδ in pericytes we used siRNA to knockdown IL-1β-induced C/EBPδ expression. C/EBPδ knockdown enhanced IL-1β-induced production of intracellular adhesion molecule-1 (ICAM-1), interleukin-8, monocyte chemoattractant protein-1 (MCP-1) and IL-1β, whilst attenuating cyclooxygenase-2 and superoxide dismutase-2 gene expression. Altered ICAM-1 and MCP-1 protein expression were confirmed by cytometric bead array and immunocytochemistry. Our results show that knock-down of C/EBPδ expression in pericytes following immune stimulation increased chemokine and adhesion molecule expression, thus modifying the human brain pericyte inflammatory response. The induction of C/EBPδ following immune stimulation may act to limit infiltration of peripheral immune cells, thereby preventing further inflammatory responses in the brain.

No MeSH data available.


Related in: MedlinePlus