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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

Knockdown of C/EBPδ using siRNA.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 4–48 hours. Representative immunocytochemistry images of C/EBPδ with treatments are shown (a) and the percentage of cells positive for nuclear C/EBPδ was quantified (b). RNA was extracted following a six hour treatment with IL-1β and C/EBPδ transcript expression was determined via RT-qPCR (c). Protein was extracted following a 24 hour treatment with IL-1β and C/EBPδ expression determined by western blotting (d,e). Blots are cropped to improve clarity. Full-length blots are presented in Supplementary Figure S2. *** = p < 0.001. Scale bar = 100 μm.
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f5: Knockdown of C/EBPδ using siRNA.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 4–48 hours. Representative immunocytochemistry images of C/EBPδ with treatments are shown (a) and the percentage of cells positive for nuclear C/EBPδ was quantified (b). RNA was extracted following a six hour treatment with IL-1β and C/EBPδ transcript expression was determined via RT-qPCR (c). Protein was extracted following a 24 hour treatment with IL-1β and C/EBPδ expression determined by western blotting (d,e). Blots are cropped to improve clarity. Full-length blots are presented in Supplementary Figure S2. *** = p < 0.001. Scale bar = 100 μm.

Mentions: C/EBPδ has been widely reported to modify inflammatory gene expression in various cell types. To investigate its effects on human brain pericyte inflammatory responses, a C/EBPδ siRNA construct was employed. Transfection of 50 nM C/EBPδ siRNA significantly reduced IL-1β induced C/EBPδ expression at four hours (control siRNA 62.70 ± 1.18%, C/EBPδ siRNA 18.17 ± 3.07%; p < 0.001), 24 hours (control siRNA 49.63 ± 1.51%, C/EBPδ siRNA 16.14 ± 3.05%; p < 0.001) and 48 hours (control siRNA 29.26 ± 1.01%, C/EBPδ siRNA 9.71 ± 1.89%; p < 0.001), however, had no effect on basal levels (control siRNA 3.89 ± 0.98%, C/EBPδ siRNA 4.99 ± 0.99%; p > 0.05) as determined by immunocytochemistry (Fig. 5a,b). Using qRT-PCR a reduction in both basal (0.22 ± 0.02 fold; p < 0.001) and IL-1β induced gene expression (control siRNA 4.86 ± 0.62 fold, C/EBPδ siRNA 1.18 ± 0.17; p < 0.001; Fig. 5c) was observed with C/EBPδ siRNA. Western blotting analysis revealed no basal change in C/EBPδ with siRNA treatment (0.05 ± 0.03 fold; p > 0.05), however a significant attenuation of IL-1β induced expression was observed (control siRNA 8.80 ± 1.85 fold, C/EBPδ siRNA 0.37 ± 0.23; p < 0.001; Fig. 5d,e).


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)

Knockdown of C/EBPδ using siRNA.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 4–48 hours. Representative immunocytochemistry images of C/EBPδ with treatments are shown (a) and the percentage of cells positive for nuclear C/EBPδ was quantified (b). RNA was extracted following a six hour treatment with IL-1β and C/EBPδ transcript expression was determined via RT-qPCR (c). Protein was extracted following a 24 hour treatment with IL-1β and C/EBPδ expression determined by western blotting (d,e). Blots are cropped to improve clarity. Full-length blots are presented in Supplementary Figure S2. *** = p < 0.001. Scale bar = 100 μm.
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f5: Knockdown of C/EBPδ using siRNA.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 4–48 hours. Representative immunocytochemistry images of C/EBPδ with treatments are shown (a) and the percentage of cells positive for nuclear C/EBPδ was quantified (b). RNA was extracted following a six hour treatment with IL-1β and C/EBPδ transcript expression was determined via RT-qPCR (c). Protein was extracted following a 24 hour treatment with IL-1β and C/EBPδ expression determined by western blotting (d,e). Blots are cropped to improve clarity. Full-length blots are presented in Supplementary Figure S2. *** = p < 0.001. Scale bar = 100 μm.
Mentions: C/EBPδ has been widely reported to modify inflammatory gene expression in various cell types. To investigate its effects on human brain pericyte inflammatory responses, a C/EBPδ siRNA construct was employed. Transfection of 50 nM C/EBPδ siRNA significantly reduced IL-1β induced C/EBPδ expression at four hours (control siRNA 62.70 ± 1.18%, C/EBPδ siRNA 18.17 ± 3.07%; p < 0.001), 24 hours (control siRNA 49.63 ± 1.51%, C/EBPδ siRNA 16.14 ± 3.05%; p < 0.001) and 48 hours (control siRNA 29.26 ± 1.01%, C/EBPδ siRNA 9.71 ± 1.89%; p < 0.001), however, had no effect on basal levels (control siRNA 3.89 ± 0.98%, C/EBPδ siRNA 4.99 ± 0.99%; p > 0.05) as determined by immunocytochemistry (Fig. 5a,b). Using qRT-PCR a reduction in both basal (0.22 ± 0.02 fold; p < 0.001) and IL-1β induced gene expression (control siRNA 4.86 ± 0.62 fold, C/EBPδ siRNA 1.18 ± 0.17; p < 0.001; Fig. 5c) was observed with C/EBPδ siRNA. Western blotting analysis revealed no basal change in C/EBPδ with siRNA treatment (0.05 ± 0.03 fold; p > 0.05), however a significant attenuation of IL-1β induced expression was observed (control siRNA 8.80 ± 1.85 fold, C/EBPδ siRNA 0.37 ± 0.23; p < 0.001; Fig. 5d,e).

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