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 which M1 represents pro-inflammatory and M2 represents anti-inflammatory macrophages. This model fits well in various pathological situations like acute infections, allergy, and obesity⁴. However, in chronically inflamed tissues and cancer, it has been demonstrated that this classification is unable to grasp the broad phenotypic repertoire that macrophages present in certain cellular environments^5,6,7. The current consensus is that macrophage polarization is better described using a multidimensional model to integrate specific microenvironmental signals⁸. This conclusion has been confirmed through transcriptomic analysis of human macrophages showing that the M1/M2 model is inefficient in describing the obtained polarizations⁹.

The study presented aims to provide a protocol to obtain proteomic signatures of various polarizations in human macrophages. We describe how to differentiate human macrophages in environments of various oxygen levels and obtain peptides from the whole macrophage proteome to perform a label-free quantification. This quantification allows the comparison of expression levels of various proteins. As research on stem cells has revealed the importance of oxygen as an environmental key parameter¹⁰, we seek to understand how this tissue parameter can influence macrophage polarization in humans. The partial pressure of oxygen has been found to range from 3 to 20% (of total atmospheric pressure) in the human body, where 20% corresponds roughly to what is commonly used in a cell culture incubator (the exact value is around 18.6% while taking the presence of water into account).

Previous work has shown that alveolar differ from interstitial macrophages from functional and morphological point of views¹¹ and that these differences are probably partially due to the different oxygen levels to which they are exposed¹². Furthermore, bone marrow-derived macrophages show an increased ability to phagocytize bacteria when exposed to a low oxygen environment¹². The opposite effect has been found for THP1-differentiated human macrophages¹³, but these results support the idea that oxygen is a regulator of macrophage biology and that it is necessary to clarify this role at the molecular level in human macrophages. In a previous study, we have applied a proteomics approach to address these issues. By measuring expression levels for thousands of proteins simultaneously, we highlighted the impact of oxygen on polarization and provided a list of new molecular markers. We were also able to relate these findings to some macrophages functions. Notably, we found that the rate of phagocytosis of apoptotic cells was increased in IL4/IL13-polarized macrophages, which was linked to the upregulation of ALOX15 as revealed by the proteomic analysis¹⁴. In the present study, we describe how to perform such an analysis.

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 buffered saline (PBS) + 0.5% bovine serum albumin (BSA) + 2 mM ethylenediaminetetraacetic acid (EDTA)], filter it (0.22 µm filter), and maintain it at 4 °C.

2. Isolation of Peripheral Blood Mononuclear Cells (PBMCs) from Leukoreduction System Chamber (LRSC)

1. Put 15 mL of density gradient cell separation solution (see Table of Materials) in a 50 mL centrifugation tube so it can warm to room temperature (RT) before receiving the LRSC.
   NOTE: Density depends on temperature. As this product is stored at 4 °C, this step must be done in advance so it can equilibrate to RT.
2. Empty the LRSC into a 50 mL centrifugation tube, add up to 50 mL of 1x PBS, and mix. Very slowly, add 25 mL of the mix prepared during step 2.2 on top of 15 mL of density gradient solution warmed up during step 2.1.
   NOTE: Be careful not to mix the phases during this step. The blood must be added on the density gradient solution without any disturbance of this phase.
3. Centrifuge both centrifugation tubes for 25 min at 700 x g without breaks.
   NOTE: At the end of the density gradient centrifugation, the layers from bottom to top are: the erythrocytes and granulocytes forming the pellet, density gradient solution phase, layer of PBMCs, and plasma.
4. With a pipet, pass through the plasma phase without aspirating it and collect the PBMC layer into a new 50 mL centrifugation tube. Add up to 50 mL of 1x PBS to the PBMCs as a washing step and centrifuge for 10 min at 300 x g.
5. Aspirate the supernatant and resuspend the pellet in 40 mL of macrophage medium.

3. Magnetic Labeling and Isolation of CD14⁺ Cells (Monocytes)

1. Count the PBMCs in a Malassez chamber. Withdraw the amount of PBMCs necessary for conducting the experiment (typically 100 to 300 x 10⁶ cells), place them in a centrifugation tube, and centrifuge for 10 min at 300 x g.
2. Aspirate the supernatant and resuspend the pellet in 80 µL of the sorting buffer prepared during step 1.3 per 10⁷ PBMCs. Add 20 µL of CD14 microbeads per 10⁷ PBMCs. Mix well and incubate for 15 min at 4 °C under constant agitation.
3. Add 1 mL of sorting buffer per 10⁷ PBMCs as a washing step and centrifuge for 10 min at 300 x g. Aspirate the supernatant and resuspend the pellet in 500 µL of sorting buffer per 10⁸ PBMCs.
4. Place a column in the magnetic field of the separator. Prepare the column by rinsing it with 3 mL of sorting buffer.
5. Apply the cell suspension onto the column. The column depends on the number of cells to be isolated (here, LS columns for up to 10⁹ PBMCs are used). Collect flow-through containing unlabeled cells.
   NOTE: Starting at this step, all the tubes (negative and positive selections) are kept for later checking of the different steps by flow cytometry.
6. Wash the column with 3 x 3 mL of sorting buffer. Collect unlabeled cells passing through the same tube from step 3.12. Perform washing steps by adding sorting buffer, be careful not to dry your column. Place a collection tube under the column and remove it from the separator.
7. Pipette 5 mL of sorting buffer into the column. Immediately flush out the magnetically labeled cells by firmly pushing the plunger into the column. To increase purity of the CD14⁺ cells, the eluted fraction is enriched over a second column.
8. Repeat steps 3.4 to 3.7 with a new column.

4. Plating of Monocytes

1. Count the monocytes in a Malassez chamber. Check the purity of the CD14⁺ cells by flow cytometry. Withdraw the amount of monocytes necessary for the experiment and place them in a centrifugation tube.
2. Centrifuge for 10 min at 300 x g. Aspirate the supernatant and resuspend the monocyte pellet in macrophage medium. Plate the cells and let them settle for 50 min to 1 h. Aspirate the medium and replace it with macrophage medium + 10% SAB + 25 ng/mL macrophage colony stimulating factor (M-CSF) to induce differentiation.

5. Polarization of Macrophages at Day 6

1. Aspirate the medium. Replace it with macrophage medium + 10% SAB with various stimuli. For example, add 10 ng/mL interferon gamma (INFγ) + 1 ng/mL lipopolysaccharide (LPS) to obtain M1 polarization, or 20 ng/mL interleukin 4 (IL4) + 20 ng/mL interleukin 13 (IL13) for M2 polarization.
   NOTE: The stimulation can be performed between 24 and 48 h before proceeding to other tests.
2. Harvest cells using a detaching solution or a cell scraper.

6. Cell Culture Under Low Oxygen Conditions

1. Starting from step 4, maintain the monocytes and then macrophages in an oxygen-controlled environment to perform hypoxic condition analysis. Use a hypoxia working station in order to maintain cells under the desired oxygen partial pressure during the experiment.
   NOTE: When working under low oxygen pressure, it is important to prepare all media and washing buffers under the station and wait sufficiently to obtain the correct partial pressure in the liquid. For example, 10 mL of PBS in a 60 mm Petri dish requires roughly 2 h to reach 25 mmHg for O₂ partial pressure starting from atmospheric pressures (as we have measured it using a fiber-optic oxygen sensor). In many hypoxic stations or incubators, the oxygen pressure is set as a percentage of the atmospheric pressure. If precise measurements are necessary, it is better to use a station authorizing to directly set the oxygen pressure in mmHg.

7. Lysis and In-Gel Digestion (Protocol 1)

NOTE: In this and the following sections, two protocols used to obtain peptides and perform LC-MS/MS analysis are described. Protocol 1 describes cell lysis and in-gel fractionation and digestion, and protocol 2 describes in-solution cell lysis followed by in-solution digestion and fractionation using an isoelectric focusing method.

1. Perform cell lysis in Laemmli buffer [234 mM Tris-HCL (pH 6.8), 7.5% SDS, 37% glycerol, 33.3% (v/v) β-mercaptoethanol, bromophenol blue 0.2% w/v]. Load the protein equivalent of 300,000 cells for each sample on 4-12% bis-Tris acrylamide gels.
2. Control the duration of the electrophoretic migration to allow each protein sample to be split into 6 gel bands as exemplified in Figure 3.
3. Fix the gel with a fixing solution (30% ethanol + 7.5% acetic acid for 20 min), then add the staining solution (R-250 Coomassie blue for 45 min). Add the destaining solution (30% ethanol + 7.5% acetic until bands appear) before excising the protein bands with a clean scalpel.
4. Dice each excised band before introduction in 500 µL tubes. A clean glass surface is warranted to avoid contamination with keratins (5% SDS solution in deionized water can be used to clean surfaces).
5. Wash the gel slices 3 times in 200 µL of 25 mM ammonium bicarbonate for 20 min at 37 °C, followed by one wash in 25 mM ammonium bicarbonate and acetonitrile (50% v/v). Dehydrate the gel pieces with 200 µL of 100% acetonitrile for 10 min.
6. Incubate each gel piece with 10 mM DTT (dithiothreitol) in 25 mM ammonium bicarbonate for 45 min at 56 °C (200 µL), followed by 55 mM iodoacetamide in 25 mM ammonium bicarbonate (200 µL) for 35 min in the dark at RT.
7. To stop alkylation, incubate each gel piece with 200 µL of 10 mM DTT in 25 mM ammonium bicarbonate for 10 min at RT. Wash the gel pieces in 200 µL of 25 mM ammonium bicarbonate, then dehydrate with 200 µL of 100% acetonitrile for 10 min.
8. Digest the proteins overnight at 37 °C with Trypsin/Lys-C mix according to the manufacturer’s instructions.
9. Extract the resulting peptides from gel pieces by adding 50 μL of 50% acetonitrile for 15 min, then 50 μL of 5% formic acid for 15 min, and finally, 50 μL of 100% acetonitrile for 15 min. Pool and dry each fraction in low-absorption tubes to limit adsorption of peptides and sample loss. Store the samples at -80 °C until further analysis.

8. Protein Extraction and In-Solution Digestion (Protocol 2)

1. Perform cell lysis (2 x 10⁶ cells) with 150 µL of the following lysis buffer:
   1. 7 M urea, 2 M thiourea, 40 mM Tris, and 4% CHAPS, supplemented with protease inhibitors (complete mini, EDTA-free protease inhibitor cocktail).
2. Homogenize the solutions for 30 min at RT with a thermoshaker. Centrifuge at 13,800 x g for 20 min RT and keep the supernatant.
3. Remove contaminants with a 2D clean-up kit:
   1. The kit contains precipitant solution, co-precipitant solution, wash buffer, and wash additive.
   2. Add 300 µL of precipitant solution and mix well. Incubate on ice for 15 min. Add 300 µL of co-precipitant solution. Centrifuge the tubes (at least) at 12,000 x g for 5 min. A small pellet should be visible. Proceed rapidly to the next step to avoid resuspension or dispersion of the pellet. Remove the supernatant without disturbing the pellet.
   3. Centrifuge the tubes again with the cap-hinge and pellet facing outward to bring any remaining liquid to the bottom of the tube. A brief pulse is sufficient. There should be no visible liquid remaining in the tubes.
   4. Without disturbing the pellet, add 40 µL of co-precipitant solution. Let the tube sit on ice for 5 min. Centrifuge for 5 min, then remove and discard the wash. Add 25 µL of de-ionized water. Vortex each tube for 5-10 s. The pellet should disperse but not dissolve in the water.
   5. Add 1 mL of wash buffer (pre-chilled for at least 1 h at -20°C) and 5 µL of wash additive. Vortex until the pellet is fully dispersed. Incubate the tubes at -20°C for at least 30 min. Vortex for 20-30 s every 10 min
      NOTE: The tubes can be stored at -20°C for up to 1 week with minimal protein degradation or modification.
   6. Centrifuge the tubes (at least) at 12,000 × g for 5 min. Carefully remove and discard the supernatant. A white pellet should be visible. Allow the pellet to airdry for no more than 5 min (if the pellet is too dry, it will be difficult to resuspend).
4. Resuspend the protein pellet in 300 µL of 8 M urea and 0.1 M ammonium bicarbonate. Vortex strongly for 1 min. Determine the protein concentration using a colorimetric assay.

9. In-Solution Digestion (Protocol 2)

1. Reduce disulfide bridges by adding 5.1 µL of a 700 mM DTT aqueous solution (final concentration 12.5 mM) to the resuspended proteins from step 8.4 and incubate at 37 °C for 30 min with a thermoshaker. Alkylate cysteine residues by adding 20.3 µL of a 700 mM iodoacetamide aqueous solution (final concentration 40 mM) and incubating at 25°C for 30 min in the dark with a thermoshaker.
2. Add 990 µL of 0.1 M ammonium bicarbonate to the sample. Add a corresponding volume of Trypsin/Lys-C mix (enzyme:substrate ratio 1:100 w/w). Incubate at 37°C overnight with a thermoshaker.

10. Clean-up Cartridge (Protocol 2)

1. Wet a cartridge with 1 column-volume (1 mL) of methanol. Clean the cartridge with 1 column-volume (1 mL) of 80% acetonitrile/HPLC-grade water and discard the flow-through. Equilibrate the cartridge with 4 column-volumes (4 mL) of 0.1% formic acid/HPLC-grade water and discard the flow-through.
2. Acidify samples with 90 µL of 10% formic acid or water to pH 2-3 (check the pH with a pH indicator). Load the acidified samples and collect the flow-through. Reload the flow-through (containing the not-retained peptides). Wash the cartridge with 6 column-volume (6 mL) of 0.1% formic acid/HPLC-grade water.
3. Elute peptides from the cartridge with 1 column-volumes (1 mL) of 0.1% formic acid/50% acetonitrile/HPLC-grade water. Transfer to a 1.5 mL microcentrifuge tube. Concentrate the sample using a vacuum concentrator (150 x g, vacuum at 160 mBar).

11. Fractionation by Isoelectric Focusing (Protocol 2)

NOTE: Peptides are separated according to their isoelectric points using an off-gel fractionator on a 13 cm strip covering a pH range from 3 to 10. We used the following protocol provided by the supplier (summarized below):

1. Prepare the following solutions: solution A (600 µL of glycerol solution, 60 µL of OFFGEL buffer, 4.34 mL of ultrapure water) and solution B (1.776 mL of solution A and 444 µL of ultrapure water).
2. Assemble the IPG strips, frames, and electrodes according to the manufacturer’s instructions.
3. Resuspend the sample with 1.8 mL of solution B. Add 40 µL of solution B into each well. Load 150 µL of sample into each well.
4. Select the default method for peptides: OG12PE00 (OFFGEL default method for peptides for use with a 3100 OFFGEL Low Res Kit, pH 3-10, 12-well frames. Wait until this method has been completed (~20 h). Collect the fractions in properly labeled tubes.

12. Clean-up Harvard Apparatus Column Reverse C18 Post-IEF (Protocol 2)

1. Progressively add a few μL at a time of 1% TFA in de-ionized water to each fraction to acidify the sample. Check using pH paper that the pH is about 3 or below.
2. Prepare the following solutions: solution 1 (5 mL of acetonitrile, 10 µL of formic acid, 4.99 mL of ultrapure water) and solution 2 (0.5 mL of acetonitrile, 10 µL of formic acid, 9.49 mL of ultrapure water).
3. Pre-wet the spin column with 150 µL of solution 1. Centrifuge for 90 s at 750 x g and discard the flow-through. Wash the spin column with 150 μL of solution 2. Centrifuge for 90 s at 750 x g and discard the flow-through.
4. Pass the fraction through the column. Centrifuge for 90 s at 750 x g and discard the flow-through. Wash with 150 μL of solution 2. Centrifuge for 90 s at 750 x g and discard the flow-through.
5. Elute the fraction with 50 μL of solution 1. Centrifuge for 90 s at 750 x g. Repeat these steps once more.
6. Dry-fraction using a vacuum concentrator (150 x g, vacuum 160 mBar) and store at -80°C

13. Analysis of Proteomic Data and Bioinformatics¹⁸

1. Analyze data obtained by a nano-LC MS/MS mass spectrometer using quantification software such as MaxQuant (version 1.5.2.8) and the Andromeda search engine.
2. Set false discovery rate (FDR) to 1% for both proteins and peptides and a minimum length of 7 amino acids. Set enzyme specificity as C-terminal to Arg and Lys. Allow 2 missed cleavages at proline bonds. Select carbamidomethylation of cysteine as a fixed modification and N-terminal protein acetylation and methionine oxidation as variable modifications.
3. Further analyze the data with statistical analysis software. Perform a functional enrichment analysis using FunRich software (www.funrich.org/). Perform a gene ontology enrichment analysis using DAVID software (https://david.ncifcrf.gov/).

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