Detection of Inflammasome Activation and Pyroptotic Cell Death in Murine Bone Marrow-derived Macrophages

Summary

We describe the detection of NLRP3 inflammasome activation on cellular basis using fluorescence microscopy and staining for active caspase-1 and the adaptor, ASC. A lactate dehydrogenase release assay is presented to detect pyroptotic lysis on a population basis. These techniques can be adapted to study many aspects of inflammasome biology.

Abstract

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote inflammatory and autoinflammatory diseases. Inflammasomes are activated by cytosolic pattern recognition receptors, including members of the NOD-like receptor (NLR) family. These receptors oligomerize upon the detection of microbial or damage-associated stimuli. Subsequent recruitment of the adaptor protein ASC forms a microscopically visible inflammasome complex, which activates caspase-1 through proximity-induced auto-activation. Following the activation, caspase-1 cleaves pro-IL-1β and pro-IL-18, leading to the activation and secretion of these pro-inflammatory cytokines. Caspase-1 also mediates the inflammatory form of cell death termed pyroptosis, which features the loss of membrane integrity and cell lysis. Caspase-1 cleaves gasdermin D, releasing the N-terminal fragment which forms plasma membrane pores, leading to osmotic lysis.

In vitro, the activation of caspase-1 can be determined by labeling bone marrow-derived macrophages with the caspase-1 activity probe FAM-YVAD-FMK and by labeling the cells with antibodies against the adaptor protein ASC. This technique allows the identification of inflammasome formation and caspase-1 activation in individual cells using fluorescence microscopy. Pyroptotic cell death can be detected by measuring the release of cytosolic lactate dehydrogenase into the medium. This procedure is simple, cost effective and performed in a 96-well plate format, allowing adaptation for screening. In this manuscript, we show that activation of the NLRP3 inflammasome by nigericin leads to the co-localization of the adaptor protein ASC and active caspase-1, leading to pyroptosis.

Introduction

Inflammasome-mediated inflammation is a critical component of the defense against pathogenic organisms¹, but also underlies the etiology of many diseases². The inflammatory response to a wide range of infections is triggered by cytosolic detection of pathogen associated molecular patterns (PAMPs) or damage associated molecular patterns (DAMPs). Pattern recognition receptors (PRR), including members of the NOD-like receptor (NLR) family, oligomerize upon the detection of these PAMPs and DAMPs. This triggers the formation of a multi-protein complex termed the inflammasome, which contains the PRR, the adaptor protein Apopto....

Protocol

All animal procedures were approved and conducted under University of Washington Institutional Animal Care and Use Committee guidelines.

1. Harvest of Bone Marrow

1. Aseptically remove the femur and tibia from a mouse and clean the bones using sterile instruments. Place the cleaned bones in Dulbecco Modified Eagle Medium (DMEM) + 5 mM HEPES + 0.2 mg/mL L-glutamine + 0.05 mM 2-mercaptoethanol + 50 mg/mL gentamicin sulfate + 10,000 U/mL penicillin/streptomycin + 10% fetal bovine serum (FBS, DMEM-10 complete) and incubate the tube on ice for 15 min.
2. Remove the proximal and distal ball joints using scissors. Insert a 25 G nee....

Results

To detect caspase-1 activation following the exposure to nigericin, the cells were processed as described in the protocol section. We combined both FAM-YVAD-FMK and ASC antibody labeling in order to show that ASC and active caspase-1 co-localize following NLRP3-mediated inflammasome activation. Figure 1A shows that wild type macrophages exposed to 5 μM nigericin have formation of an ASC focus in the perinuclear region. These foci also contain active caspase-1.......

Discussion

In this manuscript, we have presented two techniques to examine inflammasome activation and the consequence of caspase-1 activation following NLRP3 stimulation in murine bone marrow-derived macrophages. The first technique allows the researcher to determine the level of caspase-1 activation on a cellular basis using the fluorescent reporter FAM-YVAD-FMK. This reporter is highly specific for binding the caspase-1 family of enzymes, as seen by the absence of staining in caspase-1/11 deficient macrophages. Specificity was a.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
E-MEM	ATCC	30-2003	For growing L929 cells
NCRC clone 929 (L929)	ATCC	CCL-1
Fixation/Permeabilization Kit	BD Biosciences	554714	Includes fixation and permeabilization solution, and wash buffer. Proprietary formulations. Contains 4.2% formaldehyde
Glycine	Biorad	161-0718
Cytotox96 Nonradioactive cytotoxicity assay	Fisher Scientific	PR-G1780	Includes Substrate Mix and Assay Buffer; Proprietary formulations. Stop solution is 1 M Acetic acid
FAM-YVAD-FMK	Immunocytochemistry Technologies	98
DMEM, high glucose, no glutamine	Invitrogen	11960044
DMEM, high glucose, no glutamine, no phenol red	Invitrogen	31053028
Dulbecco's PBS, no calcium, no magnesium	Invitrogen	14190144
Fetal Bovine Serum, qualified, US origin	Invitrogen	26140079	Heat inactivated at 55°C for 50 min
Gentamicin	Invitrogen	15750060
ProLong Gold Mounting Medium	Invitrogen	p36934	Proprietary formulation. Curing mounting medium. Hardens to refractory index of 1.46
goat anti-mouse ALEXA555	Invitrogen	A-21422
HEPES (Ultra Pure)	Invitrogen	11344041
L-Glutamine	Invitrogen	21051024
Penicillin-Streptomycin	Invitrogen	15140122
TO-PRO-3 Iodide	Invitrogen	T3605	far-red fluorescent nucleic acid stain
C57BL/6J mouse	Jackson Laboratoy	000664
caspase-1/11-/- mouse	Jackson Laboratory	016621
Leica SP8X Confocal Microscope	Leica
Ultrapure LPS from Salmonella minnesota R595 (Re)	List Biologicals	434
anti-ASC clone 2EI-7	Millipore-Sigma	04-417
beta-mercapto-ethanol	Millipore-Sigma	M6250-10ML
DMSO	Millipore-Sigma	D2650-5X10ML
EDTA	Millipore-Sigma	E5391
Spectramax M3 plate reader	Molecular Devices	5000414