Evaluation of Caspase Activation to Assess Innate Immune Cell Death

Summary

This protocol describes a comprehensive method for assessing caspase activation (caspase-1, caspase-3, caspase-7, caspase-8, caspase-9, and caspase-11) in response to both in vitro and in vivo (in mice) models of infection, sterile insults, and cancer to determine the initiation of cell death pathways, such as pyroptosis, apoptosis, necroptosis, and PANoptosis.

Abstract

Innate immunity provides the critical first line of defense in response to pathogens and sterile insults. A key mechanistic component of this response is the initiation of innate immune programmed cell death (PCD) to eliminate infected or damaged cells and propagate immune responses. However, excess PCD is associated with inflammation and pathology. Therefore, understanding the activation and regulation of PCD is a central aspect of characterizing innate immune responses and identifying new therapeutic targets across the disease spectrum.

This protocol provides methods for characterizing innate immune PCD activation by monitoring caspases, a family of cysteine-dependent proteases that are often associated with diverse PCD pathways, including apoptosis, pyroptosis, necroptosis, and PANoptosis. Initial reports characterized caspase-2, caspase-8, caspase-9, and caspase-10 as initiator caspases and caspase-3, caspase-6, and caspase-7 as effector caspases in apoptosis, while later studies found the inflammatory caspases, caspase-1, caspase-4, caspase-5, and caspase-11, drive pyroptosis. It is now known that there is extensive crosstalk between the caspases and other innate immune and cell death molecules across the previously defined PCD pathways, identifying a key knowledge gap in the mechanistic understanding of innate immunity and PCD and leading to the characterization of PANoptosis. PANoptosis is a unique innate immune inflammatory PCD pathway regulated by PANoptosome complexes, which integrate components, including caspases, from other cell death pathways.

Here, methods for assessing the activation of caspases in response to various stimuli are provided. These methods allow for the characterization of PCD pathways both in vitro and in vivo, as activated caspases undergo proteolytic cleavage that can be visualized by western blotting using optimal antibodies and blotting conditions. A protocol and western blotting workflow have been established that allow for the assessment of the activation of multiple caspases from the same cellular population, providing a comprehensive characterization of the PCD processes. This method can be applied across research areas in development, homeostasis, infection, inflammation, and cancer to evaluate PCD pathways throughout cellular processes in health and disease.

Introduction

The innate immune system acts as the first line of defense during infection and in response to sterile stimuli, such as tissue injury and alterations in homeostasis. Innate immune sensors on the cell surface and in the cytoplasm respond to pathogen- or damage-associated molecular patterns (PAMPs or DAMPs, respectively) to trigger inflammatory signaling pathways and cellular responses. One of the key processes of the innate immune response is the induction of cell death to remove infected or damaged cells and drive further innate and adaptive immune responses. Programmed cell death (PCD) is a highly conserved process across species, highlighting its evolutionary import....

Protocol

The animal use and procedures were approved by the St. Jude Children's Research Hospital Committee on the Use and Care of Animals.

1. Preparing the solutions

1. Prepare L929-conditioned media.
   1. Plate 1 × 10⁶ L929 cells (see Table of Materials) in a 182 cm² tissue culture flask containing 50 mL of L929 culture media (see Table 1 for the preparation of the media).
   2. Grow the cells in a .......

Discussion

Monitoring caspase cleavage and activation provides one of the most comprehensive pictures of innate immune PCD activation as part of the innate immune response. The protocol described here demonstrates a strategy to monitor caspase activation in response to IAV, HSV1, and F. novicida infections and the sterile trigger LPS + ATP, but numerous other stimuli can induce PCD and could be used in this method, as has been shown in several publications^44,48

Materials

Name	Company	Catalog Number	Comments
0.45 μm filter	Millipore	SCHVU05RE
10 mL syringe	BD Biosciences	309604
12% polyacrylamide gel with 10 wells	Bio-Rad	4561043
12-well plate	Corning	07-200-82
18 G needle	BD Biosciences	305195
25 G needle	BD Biosciences	305122
50 mL tube	Fisher Scientific	50-809-218
70 μm cell strainer	Corning	431751
150 mm tissue culture dishes	Corning	430597
182-cm2 tissue culture flask	Genesee Scientific	25-211
Accessory white trans tray	Cytiva	29-0834-18
Anti–caspase-1 antibody	AdipoGen	AG-20B-0042-C100
Anti–caspase-11 antibody	Novus Biologicals	NB120-10454
Anti–caspase-3 antibody	Cell Signaling Technology	9662
Anti–caspase-7 antibody	Cell Signaling Technology	9492
Anti–caspase-8 antibody	Cell Signaling Technology	4927
Anti–caspase-9 antibody	Cell Signaling Technology	9504
Anti–cleaved caspase-3 antibody	Cell Signaling Technology	9661
Anti–cleaved caspase-7 antibody	Cell Signaling Technology	9491
Anti–cleaved caspase-8 antibody	Cell Signaling Technology	8592
Anti-mouse HRP-conjugated secondary antibody	Jackson ImmunoResearch Laboratories	315-035-047
Anti-rabbit HRP-conjugated secondary antibody	Jackson ImmunoResearch Laboratories	111-035-047
Anti-rat HRP-conjugated secondary antibody	Jackson ImmunoResearch Laboratories	112-035-003
Anti–β-Actin antibody (C4) HRP	Santa Cruz	sc-47778 HRP
ATP	InvivoGen	tlrl-atpl
BBL Trypticase Soy Broth	BD Biosciences	211768
Bead bath	Chemglass Life Sciences	CLS-4598-009
Biophotometer D30	Eppendorf	6133000010
BME	Sigma	M6250
Bromophenol blue	Sigma	BO126
Cell scrapers	CellTreat Scientific Products	229315
Chemiluminescence imager (Amersham 600)	Cytiva	29083461
CO2 chamber	VetEquip	901703
Cuvettes	Fisher Scientific	14-955-129
Dissecting scissors	Thermo Fisher Scientific	221S
DMEM	Thermo Fisher Scientific	11995-073
DTT	Sigma	43815
Eelectrophoresis apparatus	Bio-Rad	1658004
Ethanol	Pharmco	111000200
Fetal bovine serum	Biowest	S1620
Filter paper	Bio-Rad	1703965
Forceps	Fisher Scientific	22-327379
Francisella novicida (U112 strain)	BEI Resources	NR-13
Gel releaser	Bio-Rad	1653320
Gentamycin	Gibco	15750060
Glycerol	Sigma	G7893
Glycine	Sigma	G8898
HCl	Sigma	H9892
Heat block	Fisher Scientific	23-043-160
Herpes simplex virus 1 (HF strain)	ATCC	VR-260
High glucose DMEM	Sigma	D6171
Human anti–caspase-1 antibody	R&D Systems	MAB6215
Human anti–caspase-8 antibody	Enzo	ALX-804-242
Humidified incubator	Thermo Fisher Scientific	51026282
Image analysis software	ImageJ	v1.53a
IMDM	Thermo Fisher Scientific	12440-053
Influenza A virus (A/Puerto Rico/8/34, H1N1 [PR8])	constructed per Hoffmann et al.
L929 cells	ATCC	CCL-1	cell line for creating L929-conditioned media
L-cysteine	Thermo Fisher Scientific	BP376-100
Luminata Forte Western HRP substrate	Millipore	WBLUF0500	standard-sensitivity HRP substrate
MDCK cells	ATCC	CCL-34	cell line for determining IAV viral titer
Methanol	Sigma	322415
Microcentrifuge	Thermo Fisher Scientific	75002401
Non-essential amino acids	Gibco	11140050
Nonfat dried milk powder	Kroger
NP-40 solution	Sigma	492016
PBS	Thermo Fisher Scientific	10010023
Penicillin and streptomycin	Sigma	P4333
Petri dish	Fisher Scientific	07-202-011
PhosSTOP	Roche	PHOSS-RO
Power source	Bio-Rad	164-5052
Protease inhibitor tablet	Sigma	S8820
PVDF membrane	Millipore	IPVH00010
Rocking shaker	Labnet	S2035-E
SDS	Sigma	L3771
Sodium chloride	Sigma	S9888
Sodium deoxycholate	Sigma	30970
Sodium hydroxide	Sigma	72068
Sodium pyruvate	Gibco	11360-070
Square Petri dish	Fisher Scientific	FB0875711A
Stripping buffer	Thermo Fisher Scientific	21059
Super Signal Femto HRP substrate	Thermo Fisher Scientific	34580	high-sensitivity HRP substrate
Tabletop centrifuge	Thermo Fisher Scientific	75004524
Trans-Blot semi-dry system	Bio-Rad	170-3940
Tris	Sigma	TRIS-RO
Tween 20	Sigma	P1379
Ultrapure lipopolysaccharide (LPS) from E. coli 0111:B4	InvivoGen	tlrl-3pelps
Vero cells	ATCC	CCL-81	cell line for determining HSV1 viral titer