Assessment of Inflammasome Formation by Flow Cytometry

Sester, David P.; Zamoshnikova, Alina; Thygesen, Sara J.; Vajjhala, Parimala R.; Cridland, Simon O.; Schroder, Kate; Stacey, Katryn J.

doi:10.1002/cpim.13

Cited by 30 publications

(35 citation statements)

References 20 publications

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“…By contrast, treatment with the known inflammasome‐mediated pyroptosis inducer nigericin (Gustin et al, ), produced a significant increase in LDH release (Figure e), demonstrating that microglia are capable of undergoing significant pyroptosis through inflammasome activation, but this is not detectable when caspase‐1 is activated in response to SOD1 G93A . Another key event in the activation of many inflammasomes is the formation of ASC specks (Sester et al, ). We, therefore, performed immunocytochemistry experiments to identify ASC speck formation in response to SOD1 G93A protein.…”

Section: Resultsmentioning

confidence: 99%

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The microglial NLRP3 inflammasome is activated by amyotrophic lateral sclerosis proteins

Deora

Lee

Albornoz

et al. 2019

Glia

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150

118

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Microglial NLRP3 inflammasome activation is emerging as a key contributor to neuroinflammation during neurodegeneration. Pathogenic protein aggregates such as β‐amyloid and α‐synuclein trigger microglial NLRP3 activation, leading to caspase‐1 activation and IL‐1β secretion. Both caspase‐1 and IL‐1β contribute to disease progression in the mouse SOD1G93A model of amyotrophic lateral sclerosis (ALS), suggesting a role for microglial NLRP3. Prior studies, however, suggested SOD1G93A mice microglia do not express NLRP3, and SOD1G93A protein generated IL‐1β in microglia independent to NLRP3. Here, we demonstrate using Nlrp3‐GFP gene knock‐in mice that microglia express NLRP3 in SOD1G93A mice. We show that both aggregated and soluble SOD1G93A activates inflammasome in primary mouse microglia leading caspase‐1 and IL‐1β cleavage, ASC speck formation, and the secretion of IL‐1β in a dose‐ and time‐dependent manner. Importantly, SOD1G93A was unable to induce IL‐1β secretion from microglia deficient for Nlrp3, or pretreated with the specific NLRP3 inhibitor MCC950, confirming NLRP3 as the key inflammasome complex mediating SOD1‐induced microglial IL‐1β secretion. Microglial NLRP3 upregulation was also observed in the TDP‐43Q331K ALS mouse model, and TDP‐43 wild‐type and mutant proteins could also activate microglial inflammasomes in a NLRP3‐dependent manner. Mechanistically, we identified the generation of reactive oxygen species and ATP as key events required for SOD1G93A‐mediated NLRP3 activation. Taken together, our data demonstrate that ALS microglia express NLRP3, and that pathological ALS proteins activate the microglial NLRP3 inflammasome. NLRP3 inhibition may therefore be a potential therapeutic approach to arrest microglial neuroinflammation and ALS disease progression.

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Section: Resultsmentioning

confidence: 99%

“…Another key event in the activation of many inflammasomes is the formation of ASC specks (Sester et al, 2016). We, therefore, performed immunocytochemistry experiments to identify ASC speck formation in response to SOD1 G93A protein.…”

Section: Reactive Oxygen Species (Ros) Assaymentioning

confidence: 99%

The microglial NLRP3 inflammasome is activated by amyotrophic lateral sclerosis proteins

Deora

Lee

Albornoz

et al. 2019

Glia

Self Cite

150

118

View full text Add to dashboard Cite

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“…Sester and colleagues have employed FCM to monitor ASC redistribution in primary macrophages and monocytes, detecting changes in fluorescence peak height and width of immunostained or fluorescently tagged ASC as detailed in ref. .…”

Section: Introductionmentioning

confidence: 99%

Guidelines for the use of flow cytometry and cell sorting in immunological studies (second edition)

et al. 2019

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These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer‐reviewed by leading experts in the field, making this an essential research companion.

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“…7,8 Then, in addition to the release of IL-1b, the activation of the inflammasome was further assessed using the flow cytometry technique time-of-flight inflammasome evaluation. 9 After NLRP3 activation by LPS and ATP stimulation, the CD33 + cells presented oligomeric ASC specks, whereas the CD33 À population did not …”

Section: Isolated Human Peritoneal Macrophages Are Functionalmentioning

confidence: 98%

Isolation of functional mature peritoneal macrophages from healthy humans

Ruiz–Alcaraz

Martínez-Banaclocha²,

Marín-Sánchez

et al. 2019

Immunol Cell Biol

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a Equal contributors. b These authors jointly supervised this work. AbstractMacrophages play an important role in the inflammatory response. Their various biological functions are induced by different membrane receptors, including Toll-like receptors, which trigger several intracellular signaling cascades and activate the inflammasomes, which in turn elicit the release of inflammatory mediators such as cytokines. In this study, we present a novel method for the isolation of human mature peritoneal macrophages. This method can be easily implemented by gynecologists who routinely perform laparoscopy for sterilization by tubal ligation or surgically intervene in benign gynecological pathologies. Our method confirms that macrophages are the main peritoneal leukocyte subpopulation isolated from the human peritoneum in homeostasis. We showed that primary human peritoneal macrophages present phagocytic and oxidative activities, and respond to activation of the main proinflammatory pathways such as Toll-like receptors and inflammasomes, resulting in the secretion of different proinflammatory cytokines. Therefore, this method provides a useful tool for characterizing primary human macrophages as control cells for studies of molecular inflammatory pathways in steady-state conditions and for comparing them with those obtained from pathologies involving the peritoneal cavity. Furthermore, it will facilitate advances in the screening of anti-inflammatory compounds in the human system.

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Assessment of Inflammasome Formation by Flow Cytometry

Cited by 30 publications

References 20 publications

The microglial NLRP3 inflammasome is activated by amyotrophic lateral sclerosis proteins

The microglial NLRP3 inflammasome is activated by amyotrophic lateral sclerosis proteins

Guidelines for the use of flow cytometry and cell sorting in immunological studies (second edition)

Isolation of functional mature peritoneal macrophages from healthy humans

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