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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 is differentially induced by IFNγ, IL-1β and LPS.Human brain pericytes were treated with vehicle or 10 ng/mL IL-1β, IFNγ, or LPS for 24 hours. Representative immunocytochemistry images of C/EBPδ with treatments are shown (a). The percentage of cells positive for nuclear C/EBPδ was determined by immunocytochemistry (b) and the intensity of C/EBPδ expression was analysed by western blotting (c,d). Blots are cropped to improve clarity. Full-length blots are presented in Supplementary Figure S2. Data is displayed as mean ± SEM of 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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f3: C/EBPδ is differentially induced by IFNγ, IL-1β and LPS.Human brain pericytes were treated with vehicle or 10 ng/mL IL-1β, IFNγ, or LPS for 24 hours. Representative immunocytochemistry images of C/EBPδ with treatments are shown (a). The percentage of cells positive for nuclear C/EBPδ was determined by immunocytochemistry (b) and the intensity of C/EBPδ expression was analysed by western blotting (c,d). Blots are cropped to improve clarity. Full-length blots are presented in Supplementary Figure S2. Data is displayed as mean ± SEM of 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: Having observed an induction of C/EBPδ with a combination of IL-1β and IFNγ we sought to investigate how individual inflammatory stimuli affect this response. IL-1β, IFNγ and LPS were investigated based on prior evidence for their involvement in pericyte inflammatory responses. As determined by immunocytochemistry the basal expression of C/EBPδ in pericyte cultures is low (9.85 ± 1.07%; Fig. 3a,b). Enhanced nuclear expression was observed with IL-1β alone (49.85 ± 3.36%; p < 0.001) and a combination of IL-1β and IFNγ (54.15 ± 2.96%; p < 0.001). Neither IFNγ (18.14 ± 1.80%; p > 0.05) or LPS (18.46 ± 2.57%; p > 0.05) were sufficient to significantly induce C/EBPδ expression (Fig. 3a,b). Western blot analysis revealed a similar trend with both IL-1β alone (7.63 ± 2.52 fold; p < 0.05) and in combination with IFNγ (10.82 ± 1.50 fold; p < 0.01) significantly enhancing C/EBPδ expression whilst IFNγ (3.67 ± 0.77 fold; p > 0.05) and LPS (3.32 ± 0.86 fold; p > 0.05) did not (Fig. 3c,d).


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 is differentially induced by IFNγ, IL-1β and LPS.Human brain pericytes were treated with vehicle or 10 ng/mL IL-1β, IFNγ, or LPS for 24 hours. Representative immunocytochemistry images of C/EBPδ with treatments are shown (a). The percentage of cells positive for nuclear C/EBPδ was determined by immunocytochemistry (b) and the intensity of C/EBPδ expression was analysed by western blotting (c,d). Blots are cropped to improve clarity. Full-length blots are presented in Supplementary Figure S2. Data is displayed as mean ± SEM of 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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4499812&req=5

f3: C/EBPδ is differentially induced by IFNγ, IL-1β and LPS.Human brain pericytes were treated with vehicle or 10 ng/mL IL-1β, IFNγ, or LPS for 24 hours. Representative immunocytochemistry images of C/EBPδ with treatments are shown (a). The percentage of cells positive for nuclear C/EBPδ was determined by immunocytochemistry (b) and the intensity of C/EBPδ expression was analysed by western blotting (c,d). Blots are cropped to improve clarity. Full-length blots are presented in Supplementary Figure S2. Data is displayed as mean ± SEM of 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: Having observed an induction of C/EBPδ with a combination of IL-1β and IFNγ we sought to investigate how individual inflammatory stimuli affect this response. IL-1β, IFNγ and LPS were investigated based on prior evidence for their involvement in pericyte inflammatory responses. As determined by immunocytochemistry the basal expression of C/EBPδ in pericyte cultures is low (9.85 ± 1.07%; Fig. 3a,b). Enhanced nuclear expression was observed with IL-1β alone (49.85 ± 3.36%; p < 0.001) and a combination of IL-1β and IFNγ (54.15 ± 2.96%; p < 0.001). Neither IFNγ (18.14 ± 1.80%; p > 0.05) or LPS (18.46 ± 2.57%; p > 0.05) were sufficient to significantly induce C/EBPδ expression (Fig. 3a,b). Western blot analysis revealed a similar trend with both IL-1β alone (7.63 ± 2.52 fold; p < 0.05) and in combination with IFNγ (10.82 ± 1.50 fold; p < 0.01) significantly enhancing C/EBPδ expression whilst IFNγ (3.67 ± 0.77 fold; p > 0.05) and LPS (3.32 ± 0.86 fold; p > 0.05) did not (Fig. 3c,d).

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