Proteomic Analysis of Human Macrophage Polarization Under a Low Oxygen Environment

Summary

We present a protocol to obtain proteomic signatures of human macrophages and apply this to determination of the impact of a low oxygen environment on macrophage polarization.

Abstract

Macrophages are innate immune cells involved in a number of physiological functions ranging from responses to infectious pathogens to tissue homeostasis. The various functions of these cells are related to their activation states, which is also called polarization. The precise molecular description of these various polarizations is a priority in the field of macrophage biology. It is currently acknowledged that a multidimensional approach is necessary to describe how polarization is controlled by environmental signals. In this report, we describe a protocol designed to obtain the proteomic signature of various polarizations in human macrophages. This protocol is based on a label-free quantification of macrophage protein expression obtained from in-gel fractionated and Lys C/trypsin-digested cellular lysis content. We also provide a protocol based on in-solution digestion and isoelectric focusing fractionation to use as an alternative. Because oxygen concentration is a relevant environmental parameter in tissues, we use this protocol to explore how atmospheric composition or a low oxygen environment affects the classification of macrophage polarization.

Introduction

Macrophages are innate immune cells involved in a number of physiological functions ranging from responses to infectious pathogens to tissue homeostasis, including removal of apoptotic cells and remodelling of the extracellular matrix¹. These cells are characterized by a strong phenotypic plasticity² that translates into a many possible activation states, which are also called polarizations. The precise molecular description of these various polarizations is a priority in the field of macrophage biology³. It has been proposed to classify these polarizations using the so-called M1/M2 dichotomy, in ....

Protocol

Human blood samples (LRSC) from healthy, de-identified donors were obtained from EFS (French National Blood Service) as part of an authorized protocol (CODECOH DC-2018–3114). Donors gave signed consent for the use of blood.

1. Media and Buffer Preparation

1. Prepare the macrophage medium [RPMI glutamax + 10 mM HEPES + 1x non-essential amino acids (NEAA)] and warm it to 37 °C.
2. Prepare the macrophage medium + 10% human serum from AB plasma (SAB), filter it (0.22 µm filter), then warm it to 37 °C (referred to as macrophage medium + 10% SAB hereafter).
3. Prepare the sorting buffer [1x phosphate bu....

Results

Starting from peripheral blood mononuclear cells (PBMCs) obtained by differential centrifugation, the protocol permits the obtaining of a population of CD14⁺ monocytes with an assessed purity of more than 98% by flow cytometry (Figure 1). These monocytes are secondarily differentiated toward various polarizations (Figure 2). When a fractionation on gel is chosen, the migration on SDS-page gels is adapted to obtain the .......

Discussion

Because proteomics is a powerful tool to study the expression of different proteins from a whole cell or subcellular compartments, optimization of the cell lysis protocol and digestion of proteins has been addressed by a number of studies. There are three main classes of methods, which include in-gel digestion (digestion of proteins in polyacrylamide gel matrix)¹⁷, digestion in solution¹⁸ and filter-aided sample preparation¹⁹. This last method, at fi.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Hypoxia Working Station	Oxford Optronix	Hypoxylab
C6 Flow cytometer	BD	Accuri C6
Urea	Agilent Technologies	5188-6435
Formic acid (FA)	ARISTAR	450122M
R-250 Coomassie blue	Biorad	1,610,436
Lipopolysaccharide, E.Coli (LPS)	Calbiochem	437627
2D clean-up kit	GE Healthcare	80-6484-51
RPMI 1640 medium, glutamax supplement	Gibco	61870044
HEPES 1 M	Gibco	15630-080
MEM Non-Essential Amino Acids (NEAA) Solution 100X	Gibco	11140-035
Phosphate Buffered Saline (PBS) 1X	Gibco	14190-094
Harvard Apparatus column Reverse C18 micro spin column	Harvard Apparatus	74-4601
EDTA 0.5 M, pH 8.0	Invitrogen	AM9260G
NuPAGE Bis-Tris 4-12%	Life Technologies SAS	NP0321 BOX
CD14 Microbeads human	Miltenyi Biotec	130-050-201
MACS separation column LS	Miltenyi Biotec	130-042-401
Macrophage colony-stimulating factor (M-CSF)	Miltenyi Biotec	130-096-485
Interleukin 4 (IL4)	Miltenyi Biotec	130-093-917
Interleukin 13 (IL13)	Miltenyi Biotec	130-112-410
Interferon gamma (INFγ)	Miltenyi Biotec	130-096-482
CD14-FITC (clone TÜK4)	Miltenyi Biotec	130-080-701
MACSmix Tube Rotator	Miltenyi Biotec	130-090-753
Trifluoroacetic Acid (TFA)	Pierce	28904
Trypsin/Lys-C Mix	PROMEGA	V5073
Complete Mini, EDTA-free Protease Inhibitor cocktail	Roche	11836170001
Density Gradient Solution (Histopaque 1077)	Sigma Aldrich	10771-100ML
Accumax	Sigma Aldrich	A7089-100ML
Human Serum from human male AB plasma (SAB)	Sigma Aldrich	H4522-100ML
Bovine Serum Albumin (BSA) solution 30%	Sigma Aldrich	A9576-50ML
Trisma-base	Sigma Aldrich	T1503
Glycerol	Sigma Aldrich	49767
β-Mercaptoethanol	Sigma Aldrich	M3148
Bromophenol blue	Sigma Aldrich	114405
Sodium Dodecyl Sulfate (SDS) 20%	Sigma Aldrich	5030
Ammonium bicarbonate	Sigma Aldrich	9830
Acetonitrile	Sigma Aldrich	34888
Dithiothreitol	Sigma Aldrich	43819
Iodoacetamide	Sigma Aldrich	57670
Thiourea	Sigma Aldrich	T8656
CHAPS	Sigma Aldrich	C9426
Micro BCA Assay Kit	ThermoFisher	23235
5 mL sterile plastic pipette	VWR	612-1685
Thermomixer C Eppendorf	VWR	460-0223
Sep-Pak tC18 reverse phase cartridges, 100 mg	Waters	WAT036820