Visualization of Inflammatory Caspases Induced Proximity in Human Monocyte-Derived Macrophages

Summary

This protocol describes the workflow to obtain monocytes-derived macrophages (MDM) from human blood samples, a simple method to efficiently introduce inflammatory caspase Bimolecular Fluorescence Complementation (BiFC) reporters into human MDM without compromising cell viability and behavior, and an imaging-based approach to measure inflammatory caspase activation in living cells.

Abstract

Inflammatory caspases include caspase-1, -4, -5, -11, and -12 and belong to the subgroup of initiator caspases. Caspase-1 is required to ensure correct regulation of inflammatory signaling and is activated by proximity-induced dimerization following recruitment to inflammasomes. Caspase-1 is abundant in the monocytic cell lineage and induces maturation of the pro-inflammatory cytokines interleukin (IL)-1β and IL-18 to active secreted molecules. The other inflammatory caspases, caspase-4 and -5 (and their murine homolog caspase-11) promote IL-1β release by inducing pyroptosis. Caspase Bimolecular Fluorescence Complementation (BiFC) is a tool used to measure inflammatory caspase induced proximity as a readout of caspase activation. The caspase-1, -4, or -5 prodomain, which contains the region that binds to the inflammasome, is fused to non-fluorescent fragments of the yellow fluorescent protein Venus (Venus-N [VN] or Venus-C [VC]) that associate to reform the fluorescent Venus complex when the caspases undergo induced proximity. This protocol describes how to introduce these reporters into primary human monocyte-derived macrophages (MDM) using nucleofection, treat the cells to induce inflammatory caspase activation, and measure caspase activation using fluorescence and confocal microscopy. The advantage of this approach is that it can be used to identify the components, requirements, and localization of the inflammatory caspase activation complex in living cells. However, careful controls need to be considered to avoid compromising cell viability and behavior. This technique is a powerful tool for the analysis of dynamic caspase interactions at the inflammasome level as well as for the interrogation of the inflammatory signaling cascades in living MDM and monocytes derived from human blood samples.

Introduction

The caspases are a family of cysteine aspartate proteases that can be grouped into initiator caspases and executioner caspases. Executioner caspases comprise caspase-3, -6 and -7. They are naturally found in cells as dimers and are cleaved by the initiator caspases to execute apoptosis¹. Initiator caspases include human caspase-1, -2, -4, -5, -8, -9, -10 and -12. They are found as inactive zymogens (pro-caspases) that are activated by proximity-induced dimerization and stabilized by auto-proteolytic cleavage^2,3. The inflammatory caspases are a subset of the initiator caspases

Protocol

This protocol follows the guidelines of Baylor College of Medicine's human research ethics committee for the manipulation of human samples. Blood samples are handled following the institutional safety guidelines for human samples. Blood samples are obtained at a regional blood bank, where they are collected with citrate phosphate dextrose (CPD) solution. However, blood collected with other anticoagulants like sodium heparin, lithium heparin, or EDTA can also be used for this protocol^28,29.

1. Isolation of human monocytes and differentiation into macrophages

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Results

The scheme shown in Figure 2 gives an overview of how to obtain, transfect, and image human MDM. After incubation of the selected CD14+ monocytes with GM-CSF for 7 days, the cell morphology changes over the course of the differentiation period (Figure 3A), going from spherical suspension cells to spindly and fully attached (days 3 and 4), and lastly to more spread cells when fully differentiated (day 7). Fully differentiated cells are then detached from the plat.......

Discussion

This protocol describes the workflow to obtain macrophages from monocytes isolated from human blood samples and a method to efficiently introduce the inflammatory caspase BiFC reporters into human MDM without compromising cell viability and behavior.

This protocol takes advantage of the BiFC technique³⁵ to tag the inflammatory caspases at the caspase recruitment domain (CARD) with non-fluorescent fragments of the split fluorescent protein Venus. Inflammatory caspases en.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
48 well tissue culture2:34 plates	Genesee Scientific	25-108
10 cm Tissue Culture Dishes	VWR	25382-166
2 Mercaptoethanol 1000x	Thermo Fisher Scientific	21985023
8 well chambered coverglass with 1.5 HP coverglass	Cellvis	c8-1.5H-N
AutoMACS columns	Miltenyi (Biotec)	130-021-101	For automated separation using AutoMACS Pro Separator only
AutoMACS Pro Separator	Miltenyi (Biotec)	130-092-545	For automated separation using AutoMACS Pro Separator only
AutoMACS Pro Washing Solution	Miltenyi (Biotec)	130-092-987	For automated separation using AutoMACS Pro Separator only
AutoMACS Rinsing Solution	Miltenyi (Biotec)	130-091-222	For automated separation using AutoMACS Pro Separator only
AutoMacs running buffer	Miltenyi (Biotec)	130-091-221	For manual or automated separation using QuadroMACS or AutoMACS pro Separator
Axio Observer Z1 motorized inverted microscope equipped with a CSU-X1A 5000 spinning disk unit	Zeiss		Any confocal microscope equipped with a laser module fitted with laser lines of 568 nm (RFP) and 488 or 512 nm (GFP or YFP) wavelengths can be used
AxioObserver A1, Research Grade Inverted Microscope	Zeiss		Any epifluorescence microscope with fluorescence filters capable of exciting 568 nm (RFP) and 488 or 512 nm (GFP or YFP) wavelengths can be used
CD14+ MICROBEADS	Miltenyi (Biotec)	130-050-201	For manual or automated separation using QuadroMACS or AutoMACS pro Separator
DPBS without calcium chloride and magnesium chloride	Sigma	D8537-6x500ML
DsRed mito plasmid	Clontech	632421	Similar plasmids that can be used as fluorescent reporters can be found on Addgene
Fetal Bovine Serum	Thermo Fisher Scientific	10437028
Ficoll-Paque PLUS 6 x 100 mL	Sigma	GE17-1440-02
GlutaMAX Supplement (100x)	Thermo Fisher Scientific	35050079
GM-CSF	Thermo Fisher Scientific	PHC2011
Hemin BioXtra, from Porcine, ≥96.0% (HPLC)	Sigma	51280-1G
HEPES	Thermo Fisher Scientific	15630106
Inflammatory caspase BiFC plasmids			Available by request from LBH lab
LPS-EB Ultrapure	Invivogen	TLRL-3PELPS
LS Columns	Miltenyi (Biotec)	130-042-401	For manual separation using QuadroMACS Separator only
MACS 15 mL Tube Rack	Miltenyi (Biotec)	130-091-052	For manual separation using QuadroMACS Separator only
MACS MultiStand	Miltenyi (Biotec)	130-042-303	For manual separation using QuadroMACS Separator only
mCherry plasmid	Yungpeng Wang Lab		Similar plasmids that can be used as fluorescent reporters can be found on Addgene
Neon Transfection System	Thermo Fisher Scientific	MPK5000	Includes Neon electroporation device, pipette and pipette station
Neon Transfection System 10 µL Kit	Thermo Fisher Scientific	MPK1096	Includes resuspension buffer R, resuspension buffer T, electrolytic buffer E, 96 x 10 µL Neon tips and Neon electroporation tubes
Nigericin sodium salt, ready made solution	Sigma	SML1779-1ML
Penicillin-Streptomycin (10,000 U/mL)	Thermo Fisher Scientific	15140122
Poly-D-Lysine Hydrobromide	Sigma	P7280-5mg
QuadroMACS Separator	Miltenyi (Biotec)	130-090-976	For manual separation using QuadroMACS Separator only
qVD-OPh	Fisher (ApexBio)	50-101-3172
RPMI 1640 Medium	Thermo Fisher Scientific	11875119
Trypsin-EDTA (0.25%), phenol red	Thermo Fisher Scientific	25200072
UltraPure 0.5 M EDTA, pH 8.0	Thermo Fisher Scientific	15575020
Zeiss Zen 2.6 (blue edition) software	Zeiss		Any software used to operate the confocal microscope of choice