Assessment of ASC Oligomerization by Flow Cytometry

Hurtado-Navarro, Laura; Baroja‐Mazo, Alberto; Martínez-Banaclocha, Helios; Pelegrı́n, Pablo

doi:10.1007/978-1-0716-2144-8_1

Cited by 4 publications

(6 citation statements)

References 10 publications

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“…We next analyzed the presence of an ASC oligomer or speck in monocytes as a direct readout of active inflammasome formation by flow cytometry. 24 , 25 Surprisingly, our findings revealed that most monocytes from the pre-anakinra sample from the index patient presented ASC speck under resting conditions, and stimulation of the NLRP3 or pyrin inflammasomes was not able to further induce changes ( Figure 2 B). As expected, in healthy donors, ASC oligomerization occurred only after NLRP3 inflammasome activation with LPS+ATP or pyrin inflammasome activation with LPS+TcdB ( Figure 2 B).…”

Section: Resultsmentioning

confidence: 69%

NLRP3 inflammasome activation and symptom burden in KRAS-mutated CMML patients is reverted by IL-1 blocking therapy

Hurtado-Navarro,

Cuenca-Zamora,

Zamora

et al. 2023

Cell Reports Medicine

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Section: Resultsmentioning

confidence: 69%

NLRP3 inflammasome activation and symptom burden in KRAS-mutated CMML patients is reverted by IL-1 blocking therapy

Hurtado-Navarro,

Cuenca-Zamora,

Zamora

et al. 2023

Cell Reports Medicine

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“…Intracellular ASC-speck formation in human monocytes was evaluated by seeding 50 µl of individuals’ whole blood samples in polystyrene flow cytometry tubes (Falcon) with RPMI 1640 medium (Lonza) containing 10% FCS and 2 mM Glutamax. Following treatments with inhibitor or triggers, cells were stained for the detection of ASC specks by Time-of-Flight Inflammasome Evaluation (TOFIE) 65 , 66 using the PE conjugated mouse monoclonal anti-ASC antibody (clone HASC-71, catalog 653903, Biolegend, 1:500). Monocytes were gated using the FITC conjugated mouse monoclonal anti-CD14 antibody (clone M5E2, catalog 557153, BD Biosciences, 1:10) and using the PE-Cy7 conjugated mouse monoclonal anti-CD16 antibody (clone 3G8, catalog 557744, BD Biosciences 1:10).…”

Section: Methodsmentioning

confidence: 99%

Pathogenic NLRP3 mutants form constitutively active inflammasomes resulting in immune-metabolic limitation of IL-1β production

Molina-López,

Hurtado-Navarro,

García

et al. 2024

Nat Commun

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Cryopyrin-associated periodic syndrome (CAPS) is an autoinflammatory condition resulting from monoallelic NLRP3 variants that facilitate IL-1β production. Although these are gain-of-function variants characterized by hypersensitivity to cell priming, patients with CAPS and animal models of the disease may present inflammatory flares without identifiable external triggers. Here we find that CAPS-associated NLRP3 variants are forming constitutively active inflammasome, which induce increased basal cleavage of gasdermin D, IL-18 release and pyroptosis, with a concurrent basal pro-inflammatory gene expression signature, including the induction of nuclear receptors 4 A. The constitutively active NLRP3-inflammasome of CAPS is responsive to the selective NLRP3 inhibitor MCC950 and its activation is regulated by deubiquitination. Despite their preactivated state, the CAPS inflammasomes are responsive to activation of the NF-κB pathway. NLRP3-inflammasomes with CAPS-associated variants affect the immunometabolism of the myeloid compartment, leading to disruptions in lipids and amino acid pathways and impaired glycolysis, limiting IL-1β production. In summary, NLRP3 variants causing CAPS form a constitutively active inflammasome inducing pyroptosis and IL-18 release without cell priming, which enables the host’s innate defence against pathogens while also limiting IL-1β–dependent inflammatory episodes through immunometabolism modulation.

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“…PBMCs from responders and nonresponders were analyzed at baseline before treatment and after 6 months of fingolimod treatment. After PBMC stimulation, cells were stained for the detection of an ASC oligomer by time-of-flight inflammasome evaluation 14 , 15 using the phycoerythrin-conjugated mouse monoclonal anti-ASC antibody (clone HASC-71, catalog 653903, BioLegend, 1:500). Monocytes were gated using the fluorescein isothiocyanate-conjugated mouse monoclonal anti-CD14 antibody (clone M5E2, catalog 557153, BD Biosciences, 1:10) and using the phycoerythrin-Cy7–conjugated mouse monoclonal anti-CD16 antibody (clone 3G8, catalog 557744, BD Biosciences, 1:10).…”

Section: Methodsmentioning

confidence: 99%

Increased NLRP3 Inflammasome Activation and Pyroptosis in Patients With Multiple Sclerosis With Fingolimod Treatment Failure

Malhotra

Hurtado-Navarro

Pappolla

et al. 2023

Neurol Neuroimmunol Neuroinflamm

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Background and ObjectivesInflammasomes are involved in the pathogenesis of different neuroimmune and neurodegenerative diseases, including multiple sclerosis (MS). In a previous study by our group, the nucleotide-binding oligomerization domain, leucine-rich repeat receptor and pyrin-domain–containing 3 (NLRP3) inflammasome was reported to be associated with the response to interferon-beta in MS. Based on recent data showing the potential for the oral therapy fingolimod to inhibit NLRP3 inflammasome activation, here we investigated whether fingolimod could also be implicated in the response to this therapy in patients with MS.MethodsNLRP3gene expression levels were measured by real-time PCR in peripheral blood mononuclear cells at baseline and after 3, 6, and 12 months in a cohort of patients with MS treated with fingolimod (N = 23), dimethyl fumarate (N = 21), and teriflunomide (N = 21) and classified into responders and nonresponders to the treatment according to clinical and radiologic criteria. In a subgroup of fingolimod responders and nonresponders, the percentage of monocytes with an oligomer of ASC was determined by flow cytometry, and the levels of interleukin (IL)-1β, IL-18, IL-6, tumor necrosis factor (TNF)α, and galectin-3 were quantified by ELISA.ResultsNLPR3expression levels were significantly increased in fingolimod nonresponders after 3 (p= 0.03) and 6 months (p= 0.008) of treatment compared with the baseline but remained similar in responders at all time points. These changes were not observed in nonresponders to the other oral therapies tested. The formation of an oligomer of ASC in monocytes after lipopolysaccharide and adenosine 5′-triphosphate stimulation was significantly decreased in responders (p= 0.006) but increased in nonresponders (p= 0.0003) after 6 months of fingolimod treatment compared with the baseline. Proinflammatory cytokine release from stimulated peripheral blood mononuclear cells was comparable between responders and nonresponders, but galectin-3 levels on cell supernatants, as a marker of cell damage, were significantly increased in fingolimod nonresponders (p= 0.02).DiscussionThe differential effect of fingolimod on the formation of an inflammasome-triggered ASC oligomer in monocytes between responders and nonresponders could be used as a response biomarker after 6 months of fingolimod treatment and suggests that fingolimod may exert their beneficial effects by reducing inflammasome signaling in a subset of patients with MS.

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Assessment of ASC Oligomerization by Flow Cytometry

Cited by 4 publications

References 10 publications

NLRP3 inflammasome activation and symptom burden in KRAS-mutated CMML patients is reverted by IL-1 blocking therapy

NLRP3 inflammasome activation and symptom burden in KRAS-mutated CMML patients is reverted by IL-1 blocking therapy

Pathogenic NLRP3 mutants form constitutively active inflammasomes resulting in immune-metabolic limitation of IL-1β production

Increased NLRP3 Inflammasome Activation and Pyroptosis in Patients With Multiple Sclerosis With Fingolimod Treatment Failure

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