Time‐dependent dual effect of NLRP3 inflammasome in brain ischemia

AbstractBackground and purposePost-ischemic inflammation contributes to worsening of ischemic brain injury and in this process, the inflammasomes play a key role. Inflammasomes are cytosolic multiprotein complexes which upon assembly activate the maturation and secretion of the inflammatory cytokines IL-1β and IL-18. However, participation of the NLRP3 inflammasome in ischemic stroke remains controversial. Our aims were to determine the role of NLRP3 in ischemia and to explore the mechanism involved in the potential protective effect of the neurovascular unit.MethodsWT and NLRP3 knock-out mice were subjected to ischemia by middle cerebral artery occlusion (60 minutes) with or without treatment with MCC950 at different time points post-stroke. Brain injury was measured histologically with 2,3,5-triphenyltetrazolium chloride (TTC) staining.ResultsWe identified a time-dependent dual effect of NLRP3. While neither the pre-treatment with MCC950 nor the genetic approach (NLRP3 KO) proved to be neuroprotective, post-reperfusion treatment with MCC950 significantly reduced the infarct volume in a dose-dependent manner. Importantly, MCC950 improved the neuro-motor function and reduced the expression of different pro-inflammatory cytokines (IL-1β, TNF-α), NLRP3 inflammasome components (NLRP3, pro-caspase-1), protease expression (MMP9) and endothelial adhesion molecules (ICAM, VCAM). We observed a marked protection of the blood-brain barrier (BBB), which was also reflected in the recovery of the tight junctions proteins (ZO-1, Claudin-5). Additionally, MCC950 produced a reduction of the CCL2 chemokine in blood serum and in brain tissue, which lead to a reduction in the immune cell infiltration.ConclusionsThese findings suggest that post-reperfusion NLRP3 inhibition may be an effective acute therapy for protecting the blood-brain barrier in cerebral ischemia with potential clinical translation.

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Time-dependent dual effect of NLRP3 inflammasome in brain ischemia

10.22541/au.160943650.03302626/v1 ◽

2020 ◽

Author(s):

Alejandra Palomino-Antolin ◽

Paloma Narros-Fernández ◽

Victor Farré-Alins ◽

Javier Sevilla-Montero ◽

Celine Decouty-Pérez ◽

...

Keyword(s):

Brain Injury ◽

Blood Brain Barrier ◽

Inflammatory Cytokines ◽

Nlrp3 Inflammasome ◽

Neurovascular Unit ◽

Time Dependent ◽

Brain Barrier ◽

Dependent Manner ◽

Dual Effect ◽

Blood Brain

Background: Post-ischemic inflammation contributes to worsening of ischemic brain injury and in this process, the inflammasomes play a key role. Inflammasomes are cytosolic multiprotein complexes which upon assembly activate the maturation and secretion of the inflammatory cytokines IL-1β and IL-18. However, participation of the NLRP3 inflammasome in ischemic stroke remains controversial. Our aims were to determine the role of NLRP3 in ischemia and to explore the mechanism involved in the potential protective effect of the neurovascular unit. Methods: WT and NLRP3 knock-out mice were subjected to ischemia by middle cerebral artery occlusion (60 minutes) with or without treatment with MCC950 at different time points post-stroke. Brain injury was measured histologically with 2,3,5-triphenyltetrazolium chloride (TTC) staining. Results: We identified a time-dependent dual effect of NLRP3. While neither the pre-treatment with MCC950 nor the genetic approach (NLRP3 KO) proved to be neuroprotective, post-reperfusion treatment with MCC950 significantly reduced the infarct volume in a dose-dependent manner. Importantly, MCC950 improved the neuro-motor function and reduced the expression of different pro-inflammatory cytokines (IL-1β, TNF-α), NLRP3 inflammasome components (NLRP3, pro-caspase-1), protease expression (MMP9) and endothelial adhesion molecules (ICAM, VCAM). We observed a marked protection of the blood-brain barrier (BBB), which was also reflected in the recovery of the tight junctions proteins (ZO-1, Claudin-5). Additionally, MCC950 produced a reduction of the CCL2 chemokine in blood serum and in brain tissue, which lead to a reduction in the immune cell infiltration. Conclusions: These findings suggest that post-reperfusion NLRP3 inhibition may be an effective acute therapy for protecting the blood-brain barrier in cerebral ischemia with potential clinical translation.

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Aggregated Tau-PHF6 (VQIVYK) Potentiates NLRP3 Inflammasome Expression and Autophagy in Human Microglial Cells

Cells ◽

10.3390/cells10071652 ◽

2021 ◽

Vol 10 (7) ◽

pp. 1652

Author(s):

Chinmaya Panda ◽

Clara Voelz ◽

Pardes Habib ◽

Christian Mevissen ◽

Thomas Pufe ◽

...

Keyword(s):

Tau Protein ◽

Nlrp3 Inflammasome ◽

Time Dependent ◽

Microglial Cells ◽

Inflammasome Activation ◽

Dependent Manner ◽

Progressive Dementia ◽

Microglial Polarization ◽

Protein Levels ◽

Pyrin Domain

Intra-neuronal misfolding of monomeric tau protein to toxic β-sheet rich neurofibrillary tangles is a hallmark of Alzheimer’s disease (AD). Tau pathology correlates not only with progressive dementia but also with microglia-mediated inflammation in AD. Amyloid-beta (Aβ), another pathogenic peptide involved in AD, has been shown to activate NLRP3 inflammasome (NOD-like receptor family, pyrin domain containing 3), triggering the secretion of proinflammatory interleukin-1β (IL1β) and interleukin-18 (IL18). However, the effect of tau protein on microglia concerning inflammasome activation, microglial polarization, and autophagy is poorly understood. In this study, human microglial cells (HMC3) were stimulated with the unaggregated and aggregated forms of the tau-derived PHF6 peptide (VQIVYK). Modulation of NLRP3 inflammasome was examined by qRT-PCR, immunocytochemistry, and Western blot. We demonstrate that fibrillar aggregates of VQIVYK upregulated the NLRP3 expression at both mRNA and protein levels in a dose- and time-dependent manner, leading to increased expression of IL1β and IL18 in HMC3 cells. Aggregated PHF6-peptide also activated other related inflammation and microglial polarization markers. Furthermore, we also report a time-dependent effect of the aggregated PHF6 on BECN1 (Beclin-1) expression and autophagy. Overall, the PHF6 model system-based study may help to better understand the complex interconnections between Alzheimer’s PHF6 peptide aggregation and microglial inflammation, polarization, and autophagy.

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Time dependent dual effect of anti-inflammatory treatments on sarin-induced brain inflammation: Suggested role of prostaglandins

NeuroToxicology ◽

10.1016/j.neuro.2019.05.006 ◽

2019 ◽

Vol 74 ◽

pp. 19-27

Author(s):

Shira Chapman ◽

Ettie Grauer ◽

Rellie Gez ◽

Inbal Egoz ◽

Shlomi Lazar

Keyword(s):

Brain Inflammation ◽

Time Dependent ◽

Dual Effect ◽

Anti Inflammatory

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Retention of the NLRP3 Inflammasome-primed Neutrophils in the Bone Marrow is Essential for Myocardial Infarction-induced Granulopoiesis

Circulation ◽

10.1161/circulationaha.121.056019 ◽

2021 ◽

Author(s):

Gopalkrishna Sreejit ◽

Sunil K. Nooti ◽

Robert M. Jaggers ◽

Baskaran Athmanathan ◽

Ki Ho Park ◽

...

Keyword(s):

Myocardial Infarction ◽

Bone Marrow ◽

Cardiac Function ◽

Nlrp3 Inflammasome ◽

Molecular Mechanisms ◽

Elevated Serum ◽

Serum Levels ◽

Time Dependent ◽

New Paradigm ◽

Sites Of Action

Background: Acute myocardial infarction (MI) results in overzealous production and infiltration of neutrophils to the ischemic heart. This is mediated in-part by granulopoiesis induced by the S100A8/A9-NLRP3-IL-1β signaling axis in injury-exposed neutrophils. Despite the transcriptional upregulation of the NLRP3 inflammasome and associated signaling components in neutrophils, the serum levels of IL-1β, the effector molecule in granulopoiesis was not impacted by MI suggesting that IL-1β is not released systemically. We hypothesize that IL-1β is released locally within the bone marrow (BM) by inflammasome-primed and reverse-migrating neutrophils. Methods: Using a combination of time-dependent parabiosis and flow cytometry techniques, we first characterized the migration patterns of different blood cell types across the parabiotic barrier. We next induced MI in parabiotic mice by permanent ligation of the LAD artery, and examined the ability of injury-exposed neutrophils to permeate the parabiotic barrier and induce granulopoiesis in non-infarcted parabionts. Finally, utilizing multiple neutrophil adoptive and BM transplant studies, we studied the molecular mechanisms that govern reverse migration and retention of the primed neutrophils, IL-1β secretion and granulopoiesis. Cardiac function was assessed by echocardiography. Results: MI promoted greater accumulation of the inflammasome-primed neutrophils in the BM. Introducing a time-dependent parabiotic barrier to the free movement of neutrophils inhibited their ability to stimulate granulopoiesis in the non-infarcted parabionts. Prior priming of the NLRP3 inflammasome is not a prerequisite, but the presence of a functional CXCR4 (C-X-C-motif chemokine receptor 4) on the primed neutrophils and elevated serum S100A8/A9 levels are necessary for homing and retention of the reverse-migrating neutrophils. In the BM, the primed neutrophils secrete IL-1β through formation of gasdermin D pores and, promote granulopoiesis. Pharmacological and/ or genetic strategies aimed at inhibition of neutrophil homing or release of IL-1β in the BM markedly suppressed MI-induced granulopoiesis and, improved cardiac function. Conclusions: Our data reveal a new paradigm of how circulatory cells establish a direct communication between organs by delivering signaling molecules (e.g., IL-1β) directly at the sites of action rather through systemic release. We suggest that this pathway may exist to limit the off-target effects of systemic IL-1β release.

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Time-Dependent Entropy Estimation of EEG Rhythm Changes Following Brain Ischemia

Annals of Biomedical Engineering ◽

10.1114/1.1541013 ◽

2003 ◽

Vol 31 (2) ◽

pp. 221-232 ◽

Cited By ~ 85

Author(s):

A. Bezerianos ◽

S. Tong ◽

N. Thakor

Keyword(s):

Brain Ischemia ◽

Time Dependent ◽

Entropy Estimation ◽

Eeg Rhythm

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The Differences in the Cellular and Plasma Antioxidative Capacity Between Transient and Defined Focal Brain Ischemia: Does it Suggest Supporting Time-Dependent Neuroprotection Therapy?

Cellular and Molecular Neurobiology ◽

10.1007/s10571-015-0262-y ◽

2015 ◽

Vol 36 (5) ◽

pp. 789-800 ◽

Cited By ~ 2

Author(s):

Srdjan Ljubisavljevic ◽

Tatjana Cvetkovic ◽

Lilika Zvezdanovic ◽

Svetlana Stojanovic ◽

Ivana Stojanovic ◽

...

Keyword(s):

Brain Ischemia ◽

Time Dependent ◽

Antioxidative Capacity ◽

Focal Brain Ischemia

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P4145Endothelial NLRP3-Inflammasome impairs vascular function via microparticles

European Heart Journal ◽

10.1093/eurheartj/ehz745.0717 ◽

2019 ◽

Vol 40 (Supplement_1) ◽

Author(s):

P Pfeifer ◽

A Ackerschott ◽

S Ebert ◽

J Jehle ◽

E Latz ◽

...

Keyword(s):

Endothelial Cells ◽

Nlrp3 Inflammasome ◽

Time Dependent ◽

Target Cells ◽

Vascular Cells ◽

Endothelial Microparticles ◽

Scratch Assay ◽

Microscopic Imaging ◽

Caspase 1 ◽

Viability Assay

Abstract Background In recent years inflammation has emerged to the centre of attention of cardiovascular research. One of its key figures is the NLRP3-inflammasome a multimeric protein complex that stimulates inflammatory responses in atherogenesis through proinflammatory cytokines like caspases IL-1β and -18. It is activated by danger signals such as cholesterol crystals, oxidized LDL, ATP or uric acids. Microparticles (MP) are extracellular vesicles that are released by activated or apoptotic cells. They are known as a vector for the intercellular transfer of biological information. The body of evidence indicates that endothelial microparticles contribute to the development and complications in atherosclerosis. With this study we sought to elucidate the effects microparticles, that are discharged by inflammasome activated endothelial cells, exert on arterial vascular cells. Methods and results RTPCR experiments showed that activation of human coronary artery endothelial cells (HCAEC) with LPS and Nigericin leads to NLRP3-inflammasome-specific upregulation of NLRP3 and IL1β. Analysis of the supernatant of aforementioned cells via westernblot revealed release of cleaved caspase-1 while donorcells undergo pyroptosis. FACS and electronmicroscopy experiments revealed time dependent release of endothelial microparticles (EMP) by inflammasome activated HCAEC, while western blot demonstrated that EMP enclose active caspase-1. Fluorescence microscopic imaging illustrated time dependent incorporation of EMP by HCAEC. Stimulation of HCAEC with EMP revealed detrimental biological effects on recipient cells as viability assay and scratch assay showed decreased viability and proliferation/ migration, cytotoxity assay showed increased cytotoxity and RT-PCR experiments showed increased expression of NALP3, IL-1b, VCAM and ICAM. The fact that treatment of recipient cells with the NLRP3-Inhibitor isoliquiritigenin (ILG), heat-inactivation of EMP and rupturing the EMP-membrane by freezing is able to diminish harmful effects EMP exert on recipient cells shown by viability assay, scratch assay and microscopic imaging underlines detrimental effects being exerted by EMP-encapsuled inflamasome-components. Conclusion Our findings verify that MP released from inflammasome-activated endothelial cells are incorporated by vascular cells which in turn sustain a reduction of cell viability, migration and proliferation. EMP effectuate activation of the NLRP3-inflammasme in their target cells. The cytotoxic effects of EMP are suppressed by inhibitors of the NLRP3-inflammasome and affection of EMP-membrane. Our results emphasize the immunological role of endothelial cells and indicate that inflammasome activation is transferable through microparticle-associated communication. This in turn facilitates cell death and possibly initiates a vicious cycle of inflammation suggesting a role in the advancement of atherosclerosis. Acknowledgement/Funding Else Kröner Scholarship

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Inflammageing in the cardiovascular system: mechanisms, emerging targets, and novel therapeutic strategies

Clinical Science ◽

10.1042/cs20191213 ◽

2020 ◽

Vol 134 (17) ◽

pp. 2243-2262

Author(s):

Danlin Liu ◽

Gavin Richardson ◽

Fehmi M. Benli ◽

Catherine Park ◽

João V. de Souza ◽

...

Keyword(s):

Cardiovascular Diseases ◽

Cardiovascular System ◽

Nlrp3 Inflammasome ◽

Molecular Mechanisms ◽

Molecular Targets ◽

Therapeutic Interventions ◽

Low Grade ◽

Cardiovascular Research ◽

Inflammatory Processes ◽

Ach Receptors

Abstract In the elderly population, pathological inflammation has been associated with ageing-associated diseases. The term ‘inflammageing’, which was used for the first time by Franceschi and co-workers in 2000, is associated with the chronic, low-grade, subclinical inflammatory processes coupled to biological ageing. The source of these inflammatory processes is debated. The senescence-associated secretory phenotype (SASP) has been proposed as the main origin of inflammageing. The SASP is characterised by the release of inflammatory cytokines, elevated activation of the NLRP3 inflammasome, altered regulation of acetylcholine (ACh) nicotinic receptors, and abnormal NAD+ metabolism. Therefore, SASP may be ‘druggable’ by small molecule therapeutics targeting those emerging molecular targets. It has been shown that inflammageing is a hallmark of various cardiovascular diseases, including atherosclerosis, hypertension, and adverse cardiac remodelling. Therefore, the pathomechanism involving SASP activation via the NLRP3 inflammasome; modulation of NLRP3 via α7 nicotinic ACh receptors; and modulation by senolytics targeting other proteins have gained a lot of interest within cardiovascular research and drug development communities. In this review, which offers a unique view from both clinical and preclinical target-based drug discovery perspectives, we have focused on cardiovascular inflammageing and its molecular mechanisms. We have outlined the mechanistic links between inflammageing, SASP, interleukin (IL)-1β, NLRP3 inflammasome, nicotinic ACh receptors, and molecular targets of senolytic drugs in the context of cardiovascular diseases. We have addressed the ‘druggability’ of NLRP3 and nicotinic α7 receptors by small molecules, as these proteins represent novel and exciting targets for therapeutic interventions targeting inflammageing in the cardiovascular system and beyond.

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