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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

Time-course and concentration-dependant induction of C/EBPδ expression.Human brain pericytes were treated with vehicle or 0.01–10 ng/mL IL-1β for 24 hours. Representative immunocytochemistry images of C/EBPδ are shown (a). The percentage of cells positive for nuclear C/EBPδ was determined by immunocytochemistry (b) and C/EBPδ intensity was analysed by western blotting (c,d). Blots are cropped to improve clarity. Full-length blots are presented in Supplementary Figure S2. Human brain pericytes were treated with 10 ng/mL IL-1β for 0–48 hours. The percentage of C/EBPδ positive cells was determined by immunocytochemistry (e) and C/EBPδ intensity was analysed by western blotting (f,g). Data is displayed as mean ± SEM from three independent experiments. * = p < 0.05 compared to vehicle control, ** = p < 0.01 compared to vehicle control, *** = p < 0.001 compared to vehicle control. Scale bar = 100 μm.
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f4: Time-course and concentration-dependant induction of C/EBPδ expression.Human brain pericytes were treated with vehicle or 0.01–10 ng/mL IL-1β for 24 hours. Representative immunocytochemistry images of C/EBPδ are shown (a). The percentage of cells positive for nuclear C/EBPδ was determined by immunocytochemistry (b) and C/EBPδ intensity was analysed by western blotting (c,d). Blots are cropped to improve clarity. Full-length blots are presented in Supplementary Figure S2. Human brain pericytes were treated with 10 ng/mL IL-1β for 0–48 hours. The percentage of C/EBPδ positive cells was determined by immunocytochemistry (e) and C/EBPδ intensity was analysed by western blotting (f,g). Data is displayed as mean ± SEM from three independent experiments. * = p < 0.05 compared to vehicle control, ** = p < 0.01 compared to vehicle control, *** = p < 0.001 compared to vehicle control. Scale bar = 100 μm.

Mentions: As IL-1β treatment resulted in the greatest induction of C/EBPδ all subsequent experiments were performed using this inflammatory cytokine. To further understand the profile of C/EBPδ expression in human brain pericytes, a concentration-response curve of IL-1β induced expression was performed. Using immunocytochemistry, C/EBPδ was found to be induced by IL-1β in a concentration-dependant manner (Fig. 4a,b). A significant induction was observed by concentrations as low as 0.1 ng/mL IL-1β (vehicle 13.07 ± 1.20%, IL-1β 26.18 ± 1.16%; p < 0.001) with maximal induction by 10 ng/mL (53.73 ± 2.49%; p < 0.001). Western blotting revealed a significant induction of C/EBPδ using 1 ng/mL (10.12 ± 3.06 fold; p < 0.05) and 10 ng/mL (8.57 ± 0.99 fold; p < 0.05) IL-1β (Fig. 4c,d). In order to investigate the temporal profile of C/EBPδ induction a time-course was performed with 10 ng/mL IL-1β. By immunocytochemistry C/EBPδ was induced as early as two hours after IL-1β stimulation (vehicle 13.95 ± 0.85%, IL-1β 23.03 ± 1.71%; p < 0.05), maximally induced four hours after treatment (72.83 ± 3.11%; p < 0.001) and remained elevated 48 hours later (34.43% ± 1.45%; p < 0.001; Fig. 4e). Western blotting analysis revealed a similar trend with significant induction of C/EBPδ at two (23.42 ± 5.67 fold; p < 0.05), four (27.32 ± 10.55 fold; p < 0.05) and 24 hour treatments (12.45 ± 3.24 fold; p < 0.05) with IL-1β. However, it was not significantly elevated at 48 hours (9.43 + 1.85 fold; p > 0.05; Fig. 4f,g).


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)

Time-course and concentration-dependant induction of C/EBPδ expression.Human brain pericytes were treated with vehicle or 0.01–10 ng/mL IL-1β for 24 hours. Representative immunocytochemistry images of C/EBPδ are shown (a). The percentage of cells positive for nuclear C/EBPδ was determined by immunocytochemistry (b) and C/EBPδ intensity was analysed by western blotting (c,d). Blots are cropped to improve clarity. Full-length blots are presented in Supplementary Figure S2. Human brain pericytes were treated with 10 ng/mL IL-1β for 0–48 hours. The percentage of C/EBPδ positive cells was determined by immunocytochemistry (e) and C/EBPδ intensity was analysed by western blotting (f,g). Data is displayed as mean ± SEM from three independent experiments. * = p < 0.05 compared to vehicle control, ** = p < 0.01 compared to vehicle control, *** = p < 0.001 compared to vehicle control. Scale bar = 100 μm.
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f4: Time-course and concentration-dependant induction of C/EBPδ expression.Human brain pericytes were treated with vehicle or 0.01–10 ng/mL IL-1β for 24 hours. Representative immunocytochemistry images of C/EBPδ are shown (a). The percentage of cells positive for nuclear C/EBPδ was determined by immunocytochemistry (b) and C/EBPδ intensity was analysed by western blotting (c,d). Blots are cropped to improve clarity. Full-length blots are presented in Supplementary Figure S2. Human brain pericytes were treated with 10 ng/mL IL-1β for 0–48 hours. The percentage of C/EBPδ positive cells was determined by immunocytochemistry (e) and C/EBPδ intensity was analysed by western blotting (f,g). Data is displayed as mean ± SEM from three independent experiments. * = p < 0.05 compared to vehicle control, ** = p < 0.01 compared to vehicle control, *** = p < 0.001 compared to vehicle control. Scale bar = 100 μm.
Mentions: As IL-1β treatment resulted in the greatest induction of C/EBPδ all subsequent experiments were performed using this inflammatory cytokine. To further understand the profile of C/EBPδ expression in human brain pericytes, a concentration-response curve of IL-1β induced expression was performed. Using immunocytochemistry, C/EBPδ was found to be induced by IL-1β in a concentration-dependant manner (Fig. 4a,b). A significant induction was observed by concentrations as low as 0.1 ng/mL IL-1β (vehicle 13.07 ± 1.20%, IL-1β 26.18 ± 1.16%; p < 0.001) with maximal induction by 10 ng/mL (53.73 ± 2.49%; p < 0.001). Western blotting revealed a significant induction of C/EBPδ using 1 ng/mL (10.12 ± 3.06 fold; p < 0.05) and 10 ng/mL (8.57 ± 0.99 fold; p < 0.05) IL-1β (Fig. 4c,d). In order to investigate the temporal profile of C/EBPδ induction a time-course was performed with 10 ng/mL IL-1β. By immunocytochemistry C/EBPδ was induced as early as two hours after IL-1β stimulation (vehicle 13.95 ± 0.85%, IL-1β 23.03 ± 1.71%; p < 0.05), maximally induced four hours after treatment (72.83 ± 3.11%; p < 0.001) and remained elevated 48 hours later (34.43% ± 1.45%; p < 0.001; Fig. 4e). Western blotting analysis revealed a similar trend with significant induction of C/EBPδ at two (23.42 ± 5.67 fold; p < 0.05), four (27.32 ± 10.55 fold; p < 0.05) and 24 hour treatments (12.45 ± 3.24 fold; p < 0.05) with IL-1β. However, it was not significantly elevated at 48 hours (9.43 + 1.85 fold; p > 0.05; Fig. 4f,g).

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