Monitoring NLRP3 Inflammasome Activation and Exhaustion in Clinical Samples: A Refined Flow Cytometry Protocol for ASC Speck Formation Measurement Directly in Whole Blood after Ex Vivo Stimulation

Coudereau, Rémy; Gossez, Morgane; Py, Bénédicte F.; Henry, Thomas; Lukaszewicz, Anne‐Claire; Monneret, Guillaume; Venet, Fabienne

doi:10.3390/cells11203306

Cited by 3 publications

(3 citation statements)

References 21 publications

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“…Consequently, for ex vivo analyses of inflammasome signaling in human neutrophils it is critical to include additional immunomagnetic selection steps with neutrophil preparations isolated from peripheral human blood by standard gradient centrifugation protocols. Additionally, immunofluorescence (IF) assays at the single cell level or FACS analyses can further buttress support for engagement of particular inflammasome‐related signaling reactions in human neutrophils; published examples include: (i) FACS analyses of ASC speck accumulation 124–127 ; (ii) FACS analyses of P2X7 receptor expression as signal for NLRP3 inflammasome activation 33 ; and (iii) IF analysis of the trafficking of cleaved N‐GSDMD subunits to azurophilic granules rather than the plasma membrane during NLRP3 inflammasome signaling 57 …”

Section: Inflammasomes and Gasdermins As Critical Regulators Of Pyrop...mentioning

confidence: 99%

Pyroptosis in neutrophils: Multimodal integration of inflammasome and regulated cell death signaling pathways

Dubyak

Miller

Pearlman

2023

Immunological Reviews

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Pyroptosis is a proinflammatory mode of lytic cell death mediated by accumulation of plasma membrane (PM) macropores composed of gasdermin-family (GSDM) proteins.It facilitates two major functions in innate immunity: (i) elimination of intracellular replicative niches for pathogenic bacteria; and (ii) non-classical secretion of IL-1 family cytokines that amplify host-beneficial inflammatory responses to microbial infection or tissue damage. Physiological roles for gasdermin D (GSDMD) in pyroptosis and IL-1β release during inflammasome signaling have been extensively characterized in macrophages. This involves cleavage of GSDMD by caspase-1 to generate GSDMD macropores that mediate IL-1β efflux and progression to pyroptotic lysis. Neutrophils, which rapidly accumulate in large numbers at sites of tissue infection or damage, become the predominant local source of IL-1β in coordination with their potent microbiocidal capacity. Similar to macrophages, neutrophils express GSDMD and utilize the same spectrum of diverse inflammasome platforms for caspase-1-mediated cleavage of GSDMD. Distinct from macrophages, neutrophils possess a remarkable capacity to resist progression to GSDMD-dependent pyroptotic lysis to preserve their viability for efficient microbial killing while maintaining GSDMD-dependent mechanisms for export of bioactive IL-1β. Rather, neutrophils employ cell-specific mechanisms to conditionally engage GSDMD-mediated pyroptosis in response to bacterial pathogens that use neutrophils as replicative niches. GSDMD and pyroptosis have also been mechanistically linked to induction of NETosis, a signature neutrophil pathway that expels decondensed nuclear DNA into extracellular compartments for immobilization and killing of microbial pathogens. This review summarizes a rapidly growing number of recent studies that have produced new insights, unexpected mechanistic nuances, and some controversies regarding the regulation of, and roles for, neutrophil inflammasomes, pyroptosis, and GSDMs in diverse innate immune responses.

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Section: Inflammasomes and Gasdermins As Critical Regulators Of Pyrop...mentioning

confidence: 99%

Pyroptosis in neutrophils: Multimodal integration of inflammasome and regulated cell death signaling pathways

Dubyak

Miller

Pearlman

2023

Immunological Reviews

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“…ASC specks were detected on heparinised whole blood as previously described [19]. Briefly, 100 µL of whole heparin blood was first diluted in 100 µL of lysis solution (Versalyse) for 10 min to remove red blood cells.…”

Section: Monocytes Asc Staining In Whole Bloodmentioning

confidence: 99%

“…For this purpose, after doublets exclusion, monocytes were identified among whole blood cells based on CD45 and CD14 co-expressions. Monocytes exhibiting ASC speck formation were identified through the specific signal observed on the bi-parametric ASC-area (ASC-A)/ASC-width (ASC-W) histogram (blue cells, Figure 1A) [19]. While the percentage of ASC+ monocytes was very low at the basal state in unstimulated cells from HD, a large increase in the proportion of ASC+ monocytes was detected after nigericin stimulation (Figure 1B).…”

Section: Detection Of Asc Specks In Monocytes and Activated Caspase-1...mentioning

confidence: 99%

Altered Ex Vivo NLRP3 Inflammasome Activation Is Associated with 28-Day Mortality in Septic Patients

Coudereau,

Monneret,

Lukaszewicz

et al. 2023

Viruses

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Sepsis is a life-threatening organ dysfunction caused by a dysregulated response to infection. In this context, the aberrant activation of the NLRP3 inflammasome has been documented mostly through the measurement of increased plasmatic concentrations of IL-1β and IL-18. At the cellular level, contradictory results have been published. However, no study has comprehensively monitored NLRP3 inflammasome activation at the basal level and after ex vivo reactivation of whole blood monocytes and neutrophils focusing on ICU patients with bacterial and viral sepsis, including a longitudinal analysis. Thus, we conducted a prospective longitudinal study, examining NLRP3 inflammasome functionality in COVID-19 ICU patients (n = 15) and bacterial septic shock patients (n = 17) during the first week of ICU hospitalization, compared with healthy donors. Using two whole-blood flow cytometry assays, we detected ASC speck-positive monocytes (i.e., monocytes presenting the polymerization of ASC proteins) and activated caspase-1 in polymorphonuclear cells as read-outs, both at baseline and following nigericin stimulation, a drug that forms pores and activates the NLRP3 inflammasome. Our findings showed that, at baseline and regardless of the type of infection, patients exhibited reduced ASC speck-positive monocytes and decreased activated caspase-1 in PMN compared to healthy volunteers. This decrease was prominent at day 0. Following nigericin stimulation, this reduction was also observed and persisted throughout the first week of hospitalization, irrespective of the cellular population or parameter being considered. Notably, at day 0, this diminished activation and response to stimulation of NLRP3 was associated with a higher 28-day mortality rate. Consequently, our observations highlighted a concurrent decline in both basal expression and ex vivo activation of the NLRP3 inflammasome in circulating myeloid cells from patients with bacterial and viral sepsis in association with increased mortality.

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The double sides of NLRP3 inflammasome activation in sepsis

Vigneron

Monneret

et al. 2023

Clinical Science

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Sepsis is defined as a life-threatening organ dysfunction induced by a dysregulated host immune response to infection. Immune response induced by sepsis is complex and dynamic. It is schematically described as an early dysregulated systemic inflammatory response leading to organ failures and early deaths, followed by the development of persistent immune alterations affecting both the innate and adaptive immune responses associated with increased risk of secondary infections, viral reactivations, and late mortality. In this review, we will focus on the role of NACHT, leucin-rich repeat and pyrin-containing protein 3 (NLRP3) inflammasome in the pathophysiology of sepsis. NLRP3 inflammasome is a multiproteic intracellular complex activated by infectious pathogens through a two-step process resulting in the release of the pro-inflammatory cytokines IL-1β and IL-18 and the formation of membrane pores by gasdermin D, inducing a pro-inflammatory form of cell death called pyroptosis. The role of NLRP3 inflammasome in the pathophysiology of sepsis can be ambivalent. Indeed, although it might protect against sepsis when moderately activated after initial infection, excessive NLRP3 inflammasome activation can induce dysregulated inflammation leading to multiple organ failure and death during the acute phase of the disease. Moreover, this activation might become exhausted and contribute to post-septic immunosuppression, driving impaired functions of innate and adaptive immune cells. Targeting the NLRP3 inflammasome could thus be an attractive option in sepsis either through IL-1β and IL-18 antagonists or through inhibition of NLRP3 inflammasome pathway downstream components. Available treatments and results of first clinical trials will be discussed.

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Monitoring NLRP3 Inflammasome Activation and Exhaustion in Clinical Samples: A Refined Flow Cytometry Protocol for ASC Speck Formation Measurement Directly in Whole Blood after Ex Vivo Stimulation

Cited by 3 publications

References 21 publications

Pyroptosis in neutrophils: Multimodal integration of inflammasome and regulated cell death signaling pathways

Pyroptosis in neutrophils: Multimodal integration of inflammasome and regulated cell death signaling pathways

Altered Ex Vivo NLRP3 Inflammasome Activation Is Associated with 28-Day Mortality in Septic Patients

The double sides of NLRP3 inflammasome activation in sepsis

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