Abstract
Background:
Microglia promote neuroinflammation following haemorrhagic stroke, when activated in their proinflammatory states. Thrombin activates microglia after haemorrhagic stroke, inducing pro-inflammatory signalling pathways and cytokines production (e.g. IL-1b). Thrombin activates the NLRP3 inflammasome, while inflammasome-dependent release of HMGB1 following thrombin stimulation has not been studied.
Aims:
To investigate the effects of thrombin on the induction of pro-inflammatory phenotypes in microglia and the expression of NLRP3 and HMGB1 in microglia.
Method:
Spontaneously immortalised microglia (SIM-A9) were exposed to thrombin (10, 20 and 40 U/ml), LPS (1mg/ ml, positive control), or vehicle controls for 24 hours. Pro-inflammatory populations, NLRP3 and HMGB1 expression, MAPK and NF-jB activation, as well as IL-1b released were then examined using flow cytometry, RTqPCR, immunoblotting and ELISA, respectively.
Results/Conclusions:
Increased pro-inflammatory populations were recorded in all groups, including the untreated ones (Figure 1), while expression of NLRP3 and HMGB1 was statistically similar before and after thrombin stimulation (n ¼ 3, p>0.05). Preliminary results detected activated MAPK signalling in both thrombintreated and untreated groups (n ¼ 1), and no detectable IL-1b levels. These preliminary results showed that thrombin does not induce pro-inflammatory microglial polarisation or NLRP3 and HMGB1 expression versus vehicle controls. We recommend that pre-existing pro-inflammatory states of SIM-A9 cells are investigated for future studies using SIM-A9s for assessing microglia as protagonists for neuroinflammation.
Microglia promote neuroinflammation following haemorrhagic stroke, when activated in their proinflammatory states. Thrombin activates microglia after haemorrhagic stroke, inducing pro-inflammatory signalling pathways and cytokines production (e.g. IL-1b). Thrombin activates the NLRP3 inflammasome, while inflammasome-dependent release of HMGB1 following thrombin stimulation has not been studied.
Aims:
To investigate the effects of thrombin on the induction of pro-inflammatory phenotypes in microglia and the expression of NLRP3 and HMGB1 in microglia.
Method:
Spontaneously immortalised microglia (SIM-A9) were exposed to thrombin (10, 20 and 40 U/ml), LPS (1mg/ ml, positive control), or vehicle controls for 24 hours. Pro-inflammatory populations, NLRP3 and HMGB1 expression, MAPK and NF-jB activation, as well as IL-1b released were then examined using flow cytometry, RTqPCR, immunoblotting and ELISA, respectively.
Results/Conclusions:
Increased pro-inflammatory populations were recorded in all groups, including the untreated ones (Figure 1), while expression of NLRP3 and HMGB1 was statistically similar before and after thrombin stimulation (n ¼ 3, p>0.05). Preliminary results detected activated MAPK signalling in both thrombintreated and untreated groups (n ¼ 1), and no detectable IL-1b levels. These preliminary results showed that thrombin does not induce pro-inflammatory microglial polarisation or NLRP3 and HMGB1 expression versus vehicle controls. We recommend that pre-existing pro-inflammatory states of SIM-A9 cells are investigated for future studies using SIM-A9s for assessing microglia as protagonists for neuroinflammation.
| Original language | English |
|---|---|
| Article number | 323 |
| Pages (from-to) | 194 |
| Number of pages | 1 |
| Journal | Journal of Cerebral Blood Flow and Metabolism |
| Volume | 42 |
| Issue number | 1S |
| Early online date | 29 May 2022 |
| DOIs | |
| Publication status | Published - 30 Jun 2022 |
Keywords
- microglia
- thrombin
- haemorrhagic stroke