In Vitro Stimulation and Visualization of Extracellular Trap Release in Differentiated Human Monocyte-derived Macrophages

Summary

Presented here is a protocol to detect macrophage extracellular trap (MET) production in live cell culture using microscopy and fluorescence staining. This protocol can be further extended to examine specific MET protein markers by immunofluorescence staining.

Abstract

The release of extracellular traps (ETs) by neutrophils has been identified as a contributing factor to the development of diseases related to chronic inflammation. Neutrophil ETs (NETs) consist of a mesh of DNA, histone proteins, and various granule proteins (i.e., myeloperoxidase, elastase, and cathepsin G). Other immune cells, including macrophages, can also produce ETs; however, to what extent this occurs in vivo and whether macrophage extracellular traps (METs) play a role in pathological mechanisms has not been examined in detail. To better understand the role of METs in inflammatory pathologies, a protocol was developed for visualizing MET release from primary human macrophages in vitro, which can also be exploited in immunofluorescence experiments. This allows further characterization of these structures and their comparison to ETs released from neutrophils. Human monocyte-derived macrophages (HMDM) produce METs upon exposure to different inflammatory stimuli following differentiation to the M1 pro-inflammatory phenotype. The release of METs can be visualized by microscopy using a green fluorescent nucleic acid stain that is impermeant to live cells (e.g., SYTOX green). Use of freshly isolated primary macrophages, such as HMDM, is advantageous in modeling in vivo inflammatory events that are relevant to potential clinical applications. This protocol can also be used to study MET release from human monocyte cell lines (e.g., THP-1) following differentiation into macrophages with phorbol myristate acetate or other macrophage cell lines (e.g., the murine macrophage-like J774A.1 cells).

Introduction

The release of ETs from neutrophils was first identified as an innate immune response triggered by bacterial infection¹. They consist of a DNA backbone to which various granule proteins with anti-bacterial properties are bound, including neutrophil elastase and myeloperoxidase². The primary role of neutrophil ETs (NETs) is to capture pathogens and facilitate their elimination³. However, in addition to the protective role of ETs in immune defense, an increasing number of studies have also discovered a role in disease pathogenesis, particularly during the development of inflammation-driven diseases ....

Protocol

The HMDM were isolated from human buffy coat preparations supplied by the blood bank with ethics approval from the Sydney Local Health District.

1. HMDM Culture

1. Isolate the monocytes from buffy coat preparations prepared from the peripheral blood of healthy human donors using a commercially available preparation to isolate lymphocytes, followed by countercurrent centrifugal elutriation^19,20.
2. Confirm the presence of monocytes by cytospinning and staining with modified Giemsa stain for monocyte characterization¹⁹.
3. Under sterile c....

Results

Brightfield images showing the morphological changes of HMDM in response to stimuli for cell differentiation are shown in Figure 1. M1 polarized macrophages from experiments with HMDM exposed to IFNγ and LPS showed an elongated and spindle-like cell shape, as indicated by the black arrows in Figure 1 (middle panel). For comparison, the morphology of the M2 polarized macrophages after exposure of HMDM to IL-4 for 48 h were typically round and flat, as indicated b.......

Discussion

The generation and visualization of MET formation using M1 differentiated HMDMs represents a new in vitro model that may be useful for investigating the potential pathological role of these macrophage structures, particularly under chronic inflammatory conditions. It provides a robust protocol for the stimulation of primary human macrophages to release METs, which can also be utilized in related studies with human monocyte or murine macrophage cell lines. The successful implementation of this protocol for the stimulation.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
120Q broad spectrum fluorescent light source	EXFO Photonic Solutions, Toronto, Canada		x-cite series
Corning CellBIND Multiple Well Plate (12 wells)	Sigma-Aldrich	CLS3336	For cell culture
Differential Quik Stain Kit (Modified Giemsa)	Polysciences Inc.	24606	Characterisation of monocytes
Hanks balanced salt solution (HBSS)	Thermo-Fisher	14025050	For washing steps and HOCl treatment
Hypochlorous acid (HOCl)	Sigma-Aldrich	320331	For MET stimulation
Interferon gamma	Thermo-Fisher	PMC4031	For M1 priming
Interleukin 4	Integrated Sciences	rhil-4	For M2 priming
Interleukin 8	Miltenyl Biotec	130-093-943	For MET stimulation
L-Glutamine	Sigma-Aldrich	59202C	Added to culture media
Lipopolysaccharide	Integrated Sciences	tlrl-eblps	For M1 priming
Lymphoprep	Axis-Shield PoC AS	1114544	For isolation of monocytes
Olympus IX71 inverted microscope	Olympus, Tokyo, Japan
Phorbol 12- myristate 13-acetate (PMA)	Sigma-Aldrich	P8139	For MET stimulation
Phosphate buffered saline (PBS)	Sigma-Aldrich	D5652	For washing steps
RPMI-1640 media	Sigma-Aldrich	R8758	For cell culture
SYTOX green	Life Technologies	S7020	For MET visulaization
TH4-200 brightfield light source	Olympus, Tokyo, Japan		x-cite series
Tumor necrosis factor alpha	Lonza	300-01A-50	For MET stimulation