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 language | English |
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Title of host publication | Neuroscience Meeting Planner |
Subtitle of host publication | Society for Neuroscience |
Publication status | Published - 2015 |
Event | Society for Neuroscience 2015 - Chicago, USA United States Duration: 17 Oct 2015 → 21 Oct 2015 |
Conference
Conference | Society for Neuroscience 2015 |
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Country/Territory | USA United States |
Period | 17/10/15 → 21/10/15 |
Keywords
- Depression
- Neuroinflammation
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Amanda Mackenzie
- Department of Life Sciences - Deputy Head of Department
- Centre for Bioengineering & Biomedical Technologies (CBio)
Person: Research & Teaching, Affiliate staff