Melissa Trotman-Lucas, PhD
Lemarchand E, Barrington J, Chenery A, Haley M, Coutts G, Allen JE, et al. Extent of Ischemic Brain Injury After Thrombotic Stroke Is Independent of the NLRP3 (NACHT, LRR and PYD Domains-Containing Protein 3) Inflammasome. Stroke. 2019;50:1232-1239.
Inflammation plays a key role in the fight against infection. However, following ischaemic brain injury, inflammation can play a very different role, exacerbating the severity of damage. Inflammation results in long lasting, ongoing damage from the onset of vessel blockage through to and during reperfusion of the ischaemic brain area. One possible player within the inflammation related post-stroke damage is the NLR family pyrin domain containing 3 (NLRP3) inflammasome. During ischaemic brain injury, NLRP3 senses multiple stroke-induced stimuli leading to the recruitment of the adaptor protein ASC (the apoptosis-associated speck-like pro-caspase-1) resulting in caspase 1 production leading to downstream IL-1β and IL-18 production and release. IL-1β is well-reported to have significant pro-inflammatory and pro-apoptotic effects during acute ischaemic stroke.
A recent study by Lemarchand et al., published in Stroke, sought to determine the importance of NLRP3 to the damage occurring following ischaemic brain damage. Previous studies have reported associations between NLRP3 and an increase in the severity of ischaemic brain injury, leading to the suggestion that targeting NLRP3 could be a potential therapeutic avenue. These previous studies report NLRP3 inhibition to be protective during ischaemia, alongside data showing that mice deficient in NLRP3 show decreased damage when compared to WT counterparts. However, contrary to this, the group responsible for the paper discussed here have previously reported that ischaemic brain injury develops independent of the NLRP3 inflammasome in a rodent model of stroke, suggesting instead that the NLRC4 (NLR family, CARD containing 4) and AIM2 (absent in melanoma 2) inflammasomes contribute to the resulting brain injury, independent of NLRP3. Lemarchand et al. sought to categorically determine the role of NLRP3 in ischaemic stroke damage, using genetic and pharmacological inhibition of NLRP3. Furthermore, to increase the robustness of the data, the group utilized the FeCl3 (ferric chloride induced thrombosis) model of preclinical ischaemic stroke, where FeCl3 soaked strips are applied to the middle cerebral artery causing localized and immediate thrombus formation, a model that may have considerable clinical relevance.
The study reports no reduction in ischaemic brain injury when NLRP3 inflammasome is inhibited with MCC950 or when using NLRP3-/- (heterozygous maintained) knockout mice. NLRP3 expression was increased within the injured tissue at 24 hours post-stroke onset, a result of which is consistent with previous reports. Furthermore, using flow cytometry, the authors report a marked inflammatory response alongside the significant upregulating of the NRP3 system. To assess the role of NLRP3, the potent and selective inhibitor MCC950 was administered 30 minutes following occlusion onset; however, administration did not result in reduced lesion volume, nor did it affect the recruitment of neutrophils or IL-1β expression. This outcome was the same with MCC950 administration peripherally or if administered at the site of action via the lateral ventricle. The authors note that, unexpectedly, MCC950 administration led to increase lesion volume when using NLRP3-/- animals maintained as homozygous. The authors investigated this to discover significant genetic drift within the line. This unexpected finding was highlighted by the group and discussed to caution the scientific community to the pitfalls of homozygote maintained transgenic lines.
In conclusion, the study reports that irrespective of the increased inflammatory response following ischaemic stroke, NLRP3-dependent inflammation is not involved in the exacerbation of injury and is, therefore, not a target for stroke therapeutics. This study highlights the need for further research to fully assess not only the role of the NLRP3 inflammasome following an ischaemic stroke, but also to determine the exact processes involved in the damage occurring. The findings presented here could be further interrogated through the addition of a tPA reperfusion step — more closely representing the clinical situation. Furthermore, the utilization of other species, aged animals and the presence of comorbidities is needed to fully assess the role of NLRP3 in ischaemic stroke at both the acute and chronic phases of inflammation.