Primary microglia isolated from neonatal mice provide a model for the study of NLRP3 inflammasome and the effect of long-term hypoxia

Robin Wickens, Luc Ver Donck, Sarah Bailey, Amanda Mackenzie

Research output: Chapter in Book/Report/Conference proceedingConference contribution

Abstract

Microglia act as the primary immune cells of the brain and have inflammatory properties aimed at clearing infected or damaged tissue. Neuroinflammation is thought to play a key role in the pathology of many diseases, including Alzheimer’s disease, Parkinson’s disease and even psychiatric disorders such as depression (Amor, S. et al. 2010. Immunol. 129(2); 154-169). The NLRP3 inflammasome is a multi-protein complex responsible for caspase-1 activation and the production of inflammatory cytokines IL-1β and IL-18. Furthermore, while most in vitro studies use 20% O2, the O2 availability within the brain is thought to range between 0.5-7% O2 (Ivanovic, Z. 2009. J Cell Physiol. 219(2); 271-275). We have previously reported that NLRP3 inflammasome signalling is sensitive to acute hypoxia (4 hr; 5% O2) in a microglia cell line (Wickens, R. et al. 2013. pA2 Online. 11(3) 058P). Here, we have aimed to establish a primary microglia culture using mild trypsinization to study NLRP3 inflammasome expression in long term cultures under different oxygen conditions. Mixed glial cultures were obtained from neonate (P0.5) C57BL/6 pups and cultured in normoxic tissue culture conditions for 3 weeks (~20% O2/5% CO2). Astrocytes were removed via mild trypsin incubation (0.0625% w/v). Microglia were kept in conditioned media for 24 hours prior to testing. To study protein expression, cells were treated with lipopolysaccharide (E. Coli LPS; 0.1 μg/ml; ≤4hr) before cell lysates were collected for western blot. To study inflammasome activity, cells were primed with LPS (0.1 μg/ml; ≤4hr) prior to treatment with ATP (5 mM; 30 minutes) to assess IL-1β release by ELISA. All cells were CD11b+/GFAP-, confirming the presence of microglia. We observed a time-dependant increase in NLRP3 (4 hr, p=0.06) and proIL-1β (4 hr, p<0.05) expression following LPS stimulation, along with the expression of adaptor protein ASC and P2X7 in stimulated and unstimulated microglia. We then observed an LPS-dependant IL-1β release following cell stimulation with ATP (4 hr LPS, p<0.001). Furthermore, caspase-1 inhibition (Ac-YVAD-cmk; 10 μM) caused a significant reduction in ATP-induced IL-1β release (p<0.05), indicating the process is caspase-1-dependant. Together, these data suggest the presence of a functional NLRP3 inflammasome. These findings confirm the expression and activity of the NLRP3 inflammasome in neonatal primary microglia. The 3-week microglia culture provides a platform to study NLRP3 inflammasome signalling and the effect of chronic hypoxia, which will give a more accurate insight into microglia activation and inflammasome signalling within the brain and during neuroinflammatory disease.
Original languageEnglish
Title of host publicationNeuroscience Meeting Planner
Subtitle of host publicationSociety for Neuroscience
Publication statusPublished - 2015
EventSociety for Neuroscience 2015 - Chicago, USA United States
Duration: 17 Oct 201521 Oct 2015

Conference

ConferenceSociety for Neuroscience 2015
CountryUSA United States
Period17/10/1521/10/15

Fingerprint

Inflammasomes
Microglia
Caspase 1
Adenosine Triphosphate
Brain
Hypoxia
Proteins
Interleukin-18
Conditioned Culture Medium
Neuroglia
Astrocytes
Trypsin
Parkinson Disease
Psychiatry
Lipopolysaccharides
Alzheimer Disease
Western Blotting
Enzyme-Linked Immunosorbent Assay
Depression
Pathology

Keywords

  • Depression
  • Neuroinflammation

Cite this

Primary microglia isolated from neonatal mice provide a model for the study of NLRP3 inflammasome and the effect of long-term hypoxia. / Wickens, Robin; Ver Donck, Luc; Bailey, Sarah; Mackenzie, Amanda.

Neuroscience Meeting Planner: Society for Neuroscience. 2015. 809.29.

Research output: Chapter in Book/Report/Conference proceedingConference contribution

Wickens, R, Ver Donck, L, Bailey, S & Mackenzie, A 2015, Primary microglia isolated from neonatal mice provide a model for the study of NLRP3 inflammasome and the effect of long-term hypoxia. in Neuroscience Meeting Planner: Society for Neuroscience., 809.29, Society for Neuroscience 2015 , USA United States, 17/10/15.
@inproceedings{3268b7f7dab740429bf38114af4f09af,
title = "Primary microglia isolated from neonatal mice provide a model for the study of NLRP3 inflammasome and the effect of long-term hypoxia",
abstract = "Microglia act as the primary immune cells of the brain and have inflammatory properties aimed at clearing infected or damaged tissue. Neuroinflammation is thought to play a key role in the pathology of many diseases, including Alzheimer’s disease, Parkinson’s disease and even psychiatric disorders such as depression (Amor, S. et al. 2010. Immunol. 129(2); 154-169). The NLRP3 inflammasome is a multi-protein complex responsible for caspase-1 activation and the production of inflammatory cytokines IL-1β and IL-18. Furthermore, while most in vitro studies use 20{\%} O2, the O2 availability within the brain is thought to range between 0.5-7{\%} O2 (Ivanovic, Z. 2009. J Cell Physiol. 219(2); 271-275). We have previously reported that NLRP3 inflammasome signalling is sensitive to acute hypoxia (4 hr; 5{\%} O2) in a microglia cell line (Wickens, R. et al. 2013. pA2 Online. 11(3) 058P). Here, we have aimed to establish a primary microglia culture using mild trypsinization to study NLRP3 inflammasome expression in long term cultures under different oxygen conditions. Mixed glial cultures were obtained from neonate (P0.5) C57BL/6 pups and cultured in normoxic tissue culture conditions for 3 weeks (~20{\%} O2/5{\%} CO2). Astrocytes were removed via mild trypsin incubation (0.0625{\%} w/v). Microglia were kept in conditioned media for 24 hours prior to testing. To study protein expression, cells were treated with lipopolysaccharide (E. Coli LPS; 0.1 μg/ml; ≤4hr) before cell lysates were collected for western blot. To study inflammasome activity, cells were primed with LPS (0.1 μg/ml; ≤4hr) prior to treatment with ATP (5 mM; 30 minutes) to assess IL-1β release by ELISA. All cells were CD11b+/GFAP-, confirming the presence of microglia. We observed a time-dependant increase in NLRP3 (4 hr, p=0.06) and proIL-1β (4 hr, p<0.05) expression following LPS stimulation, along with the expression of adaptor protein ASC and P2X7 in stimulated and unstimulated microglia. We then observed an LPS-dependant IL-1β release following cell stimulation with ATP (4 hr LPS, p<0.001). Furthermore, caspase-1 inhibition (Ac-YVAD-cmk; 10 μM) caused a significant reduction in ATP-induced IL-1β release (p<0.05), indicating the process is caspase-1-dependant. Together, these data suggest the presence of a functional NLRP3 inflammasome. These findings confirm the expression and activity of the NLRP3 inflammasome in neonatal primary microglia. The 3-week microglia culture provides a platform to study NLRP3 inflammasome signalling and the effect of chronic hypoxia, which will give a more accurate insight into microglia activation and inflammasome signalling within the brain and during neuroinflammatory disease.",
keywords = "Depression, Neuroinflammation",
author = "Robin Wickens and {Ver Donck}, Luc and Sarah Bailey and Amanda Mackenzie",
year = "2015",
language = "English",
booktitle = "Neuroscience Meeting Planner",

}

TY - GEN

T1 - Primary microglia isolated from neonatal mice provide a model for the study of NLRP3 inflammasome and the effect of long-term hypoxia

AU - Wickens, Robin

AU - Ver Donck, Luc

AU - Bailey, Sarah

AU - Mackenzie, Amanda

PY - 2015

Y1 - 2015

N2 - Microglia act as the primary immune cells of the brain and have inflammatory properties aimed at clearing infected or damaged tissue. Neuroinflammation is thought to play a key role in the pathology of many diseases, including Alzheimer’s disease, Parkinson’s disease and even psychiatric disorders such as depression (Amor, S. et al. 2010. Immunol. 129(2); 154-169). The NLRP3 inflammasome is a multi-protein complex responsible for caspase-1 activation and the production of inflammatory cytokines IL-1β and IL-18. Furthermore, while most in vitro studies use 20% O2, the O2 availability within the brain is thought to range between 0.5-7% O2 (Ivanovic, Z. 2009. J Cell Physiol. 219(2); 271-275). We have previously reported that NLRP3 inflammasome signalling is sensitive to acute hypoxia (4 hr; 5% O2) in a microglia cell line (Wickens, R. et al. 2013. pA2 Online. 11(3) 058P). Here, we have aimed to establish a primary microglia culture using mild trypsinization to study NLRP3 inflammasome expression in long term cultures under different oxygen conditions. Mixed glial cultures were obtained from neonate (P0.5) C57BL/6 pups and cultured in normoxic tissue culture conditions for 3 weeks (~20% O2/5% CO2). Astrocytes were removed via mild trypsin incubation (0.0625% w/v). Microglia were kept in conditioned media for 24 hours prior to testing. To study protein expression, cells were treated with lipopolysaccharide (E. Coli LPS; 0.1 μg/ml; ≤4hr) before cell lysates were collected for western blot. To study inflammasome activity, cells were primed with LPS (0.1 μg/ml; ≤4hr) prior to treatment with ATP (5 mM; 30 minutes) to assess IL-1β release by ELISA. All cells were CD11b+/GFAP-, confirming the presence of microglia. We observed a time-dependant increase in NLRP3 (4 hr, p=0.06) and proIL-1β (4 hr, p<0.05) expression following LPS stimulation, along with the expression of adaptor protein ASC and P2X7 in stimulated and unstimulated microglia. We then observed an LPS-dependant IL-1β release following cell stimulation with ATP (4 hr LPS, p<0.001). Furthermore, caspase-1 inhibition (Ac-YVAD-cmk; 10 μM) caused a significant reduction in ATP-induced IL-1β release (p<0.05), indicating the process is caspase-1-dependant. Together, these data suggest the presence of a functional NLRP3 inflammasome. These findings confirm the expression and activity of the NLRP3 inflammasome in neonatal primary microglia. The 3-week microglia culture provides a platform to study NLRP3 inflammasome signalling and the effect of chronic hypoxia, which will give a more accurate insight into microglia activation and inflammasome signalling within the brain and during neuroinflammatory disease.

AB - Microglia act as the primary immune cells of the brain and have inflammatory properties aimed at clearing infected or damaged tissue. Neuroinflammation is thought to play a key role in the pathology of many diseases, including Alzheimer’s disease, Parkinson’s disease and even psychiatric disorders such as depression (Amor, S. et al. 2010. Immunol. 129(2); 154-169). The NLRP3 inflammasome is a multi-protein complex responsible for caspase-1 activation and the production of inflammatory cytokines IL-1β and IL-18. Furthermore, while most in vitro studies use 20% O2, the O2 availability within the brain is thought to range between 0.5-7% O2 (Ivanovic, Z. 2009. J Cell Physiol. 219(2); 271-275). We have previously reported that NLRP3 inflammasome signalling is sensitive to acute hypoxia (4 hr; 5% O2) in a microglia cell line (Wickens, R. et al. 2013. pA2 Online. 11(3) 058P). Here, we have aimed to establish a primary microglia culture using mild trypsinization to study NLRP3 inflammasome expression in long term cultures under different oxygen conditions. Mixed glial cultures were obtained from neonate (P0.5) C57BL/6 pups and cultured in normoxic tissue culture conditions for 3 weeks (~20% O2/5% CO2). Astrocytes were removed via mild trypsin incubation (0.0625% w/v). Microglia were kept in conditioned media for 24 hours prior to testing. To study protein expression, cells were treated with lipopolysaccharide (E. Coli LPS; 0.1 μg/ml; ≤4hr) before cell lysates were collected for western blot. To study inflammasome activity, cells were primed with LPS (0.1 μg/ml; ≤4hr) prior to treatment with ATP (5 mM; 30 minutes) to assess IL-1β release by ELISA. All cells were CD11b+/GFAP-, confirming the presence of microglia. We observed a time-dependant increase in NLRP3 (4 hr, p=0.06) and proIL-1β (4 hr, p<0.05) expression following LPS stimulation, along with the expression of adaptor protein ASC and P2X7 in stimulated and unstimulated microglia. We then observed an LPS-dependant IL-1β release following cell stimulation with ATP (4 hr LPS, p<0.001). Furthermore, caspase-1 inhibition (Ac-YVAD-cmk; 10 μM) caused a significant reduction in ATP-induced IL-1β release (p<0.05), indicating the process is caspase-1-dependant. Together, these data suggest the presence of a functional NLRP3 inflammasome. These findings confirm the expression and activity of the NLRP3 inflammasome in neonatal primary microglia. The 3-week microglia culture provides a platform to study NLRP3 inflammasome signalling and the effect of chronic hypoxia, which will give a more accurate insight into microglia activation and inflammasome signalling within the brain and during neuroinflammatory disease.

KW - Depression

KW - Neuroinflammation

M3 - Conference contribution

BT - Neuroscience Meeting Planner

ER -