Saturated Fatty Acids Induce Ceramide-associated Macrophage Cell Death

Summary

We illustrate a straight-forward method to derive murine primary macrophages from bone marrow cells and a simple method to prepare BSA-fatty acid conjugates. Then we demonstrate that saturated fatty acids can induce macrophage cell death, and such cell death is positively associated with cellular accumulation of ceramide levels.

Abstract

Macrophages highly express epidermal fatty acid-binding protein and adipose fatty acid-binding protein. They actively uptake saturated and unsaturated fatty acids, which might play a critical role in regulating their immune functions. Numerous studies have shown that various fatty acids, saturated or unsaturated, may possess different impacts on cell growth and function. However, the approaches used for fatty acid preparation vary, which may lead to non-physiological results. Serum albumin, a natural carrier for fatty acids in mammalian peripheral blood, is recommended for forming a conjugate complex with the sodium salt of fatty acids to study fatty acid function in mammalian cells, thus minimizing the toxicity of fatty acid soap. Thus, a simple, relatively quick heating and sonicating method is developed and presented here for BSA-fatty acid conjugate formation. We describe a protocol using saturated fatty acids, especially stearic acids to induce severe cell death in mouse bone-marrow derived macrophages. We further demonstrate that saturated fatty acid-induced cell death is positively associated with accumulated cellular ceramide levels. This method can be extended for studies of the impact of fatty acid on other mammalian cells.

Introduction

Fatty acids play a critical role in energy metabolism and in the synthesis of membrane phospholipids in different kinds of cells. Fatty acids have a low aqueous solubility. Appropriate preparation of fatty acid is of critical importance for studying the biological functions of fatty acids in mammalian cells. When fatty acids are prepared with ethanol, many fatty acids may show their toxic soap (detergent) effect on the cell membrane, even at relatively low concentrations¹. As a natural, major transporter for free fatty acids in the serum, serum albumin is considered a good carrier for fatty acid delivery in vitro for fatty acid function assays^2,3,4. However, the details of the preparation of fatty acid and serum albumin conjugate are usually not available even though many research papers using fatty acids have been published.

Macrophages highly express epidermal fatty acid-binding protein and adipose fatty acid-binding protein^5,6,7,8. They actively uptake saturated and unsaturated fatty acids which may regulate their immune functions. To study the impact of fatty acids on macrophages and other cells, different methods of fatty acid preparation were applied^1,7,9. Using appropriately prepared fatty acid/serum albumin conjugates to investigate the impact of fatty acids on macrophage function is of critical importance in obtaining biologically meaningful data. Studies on the impact of fatty acids on macrophage function can provide basic knowledge and potential therapeutic targets relating to fatty acid metabolism in macrophage-involved diseases.

Protocol

The protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of University of Louisville.

1. Mouse Bone Marrow Derived Macrophages (BMDMs)

1. Euthanize a 6- to 8-week-old, healthy wild type mouse using CO₂. Pin it down to a foam board and spray it with 70% ethanol until it is soaked. Remove the tibia/femur bones with forceps and scissors. Put them in a petri dish with 5 mL 1x phosphate-buffered saline (PBS). Perform all procedures under sterile conditions.
2. Cut open both ends of the tibia/femur bone with a scissor in a sterile tissue culture hood. Flush the bone with a 25 G needle with PBS + 2% fetal bovine serum (FBS) into a 15 mL tube.
3. Centrifuge the bone marrow cells at 500 x g, 4 °C for 5 min.
4. Resuspend the cell pellet in 1 mL red blood cell lysis buffer to lyse red blood cells (RBC) for less than 1 min. Dilute immediately with 9 mL 1x PBS. Centrifuge again as in step 1.3. Decant the supernatants.
5. Resuspend the cell pellet in 10 mL 1x PBS, filter the cell suspension through a sterile 40 μm nylon mesh into another 15 mL tube to remove cell debris/clumps. Centrifuge again as in 1.3. Decant the supernatants.
6. Resuspend the cell pellet in 15 mL Roswell Park Memorial Institute medium (RPMI) 1640 with 5% FBS and 10 μg/mL gentamicin, plate the cells in a 100-mm tissue culture dish at 37 ^oC incubator for 60 min. Gently swirl the dish, take out the floating cells, and count them.
7. Plate 6x10⁶ cells in a 100-mm dish with 12 mL macrophage differentiating media (RPMI 1640 with 5% FBS and 10 μg/mL gentamicin, mixed with 30% L929 conditioned media¹⁰) with 10 ng/mL recombinant mouse macrophage colony-stimulating factor (M-CSF) for 2 days.
8. After culturing for 2 days in a 37 °C incubator with 5% CO₂, feed each dish with 6 mL fresh macrophage differentiating media with 10 ng/mL M-CSF.
9. On day 5, take 8 mL of the old media out without disturbing the cells and add 10 mL fresh macrophage differentiating media with 10 ng/mL M-CSF.
10. On day 7, harvest the bone marrow-derived macrophages (BMDMs) using a cell lifter.
    NOTE: Attached macrophages can also be dissociated with 5 mL 1 mM EDTA in 1x PBS for 5 to 10 min after removing the float cells and washing plate twice with 5 mL 1x PBS. Centrifuge the cells as in step 1.3., resuspend them in fresh macrophage differentiating media, and count them with a hemocytometer.
    1. Confirm the BMDMs' phenotype by flow cytometry as described⁵. Prepare the derived mouse macrophages in a certain concentration for the following studies.

2. BSA-fatty Acid Conjugate Preparation

1. Prepare 2 mM BSA in sterile PBS. For example, weigh 6.65 g BSA, add 30 mL 1x PBS to dissolve, then add more PBS until 50 mL, scaling components to make 2 mM BSA in 1x PBS.
2. Prepare 5 mM individual sodium salt of fatty acids in 2 mM BSA. Heat to 37 °C or higher, if necessary, and sonicate the mixture of 2 mM BSA and sodium salt of fatty acids until a clear solution is obtained.
   NOTE: Compared to unsaturated fatty acids, saturated fatty acids need a higher temperature and more sonication time for dissolving.
3. Filter the fatty-acid-BSA solution through a 0.22 μm filter, aliquot it into 1.5 mL sterile microcentrifuge tubes, and store them at 4 °C for short term use or at -20 °C for long term storage. No precipitation should be observed after storage at 4 °C in the refrigerator.

3. Saturated Fatty Acids Induce Cell Death of Mouse Bone Marrow Derived Macrophages

1. Plate the BMDMs in a 24-well tissue culture plate with 0.4 x 10⁶/mL in macrophage differentiating media.
2. Treat the cells with the 0.4 mM palmitic acid and stearic acid for 16 to 24 h at a 37 °C incubator with 5% CO₂. Use BSA as the negative control.
3. Harvest the cells with a cell lifter after treatment and spin down the cells at 500 x g, 4 °C for 5 min.
4. Stain the cells with Annexin V-Alexa 488 and 7-aminoactinomycin D (7-AAD) in 100 μL annexin V binding buffer for 15 min at room temperature.
5. Dilute the sample with 200 µL of annexin-binding buffer, mix gently, then keep the samples on ice after the incubation period.
6. Analyze the stained cells as soon as possible by flow cytometry⁵. Annexin V-Alexa 488 is detected in FITC channel and 7-AAD is detected in PerCP/PerCP-Cy5.5 channel.

4. Intracellular Ceramide Staining

1. After fatty acid treatment, harvest the cells as in step 3.3. Then fix the cells in 4% paraformaldehyde for 30 min.
2. Wash the sample with 1 mL 1x PBS, spin down as step in 3.3, and decant the supernatant.
3. Stain the cells with anti-ceramide primary antibody (1:200 dilution) in 100 μL 1x permeabilization buffer for 30 min.
4. Wash and spin down the cells again as in step 4.2.
5. Stain the cells with anti-mouse IgM secondary antibody (1:500 dilution) in 100 μL 1x permeabilization buffer for 30 min.
6. Wash and spin down the cells again as in step 4.2. Add 250 μL 1x PBS and analyze the stained cells by flow cytometry⁵.
   NOTE: See the detailed reagent list in the Table of equipment and reagents.

Results

Obesity increases free fatty acid concentrations in serum. As professional phagocytes, macrophages actively take up fatty acids to maintain host homeostasis. During these processes, overloaded lipids may induce macrophage cell death. To this end, we cultured BMDMs in vitro with obese levels of dietary fatty acids and measured macrophage cell death using flow cytometric staining. Compared to the BSA control, saturated fatty acids, in particular stearic acids, induced significant c...

Discussion

The proper preparation of fatty acid solution is of critical importance to study the biological function of fatty acids. The neutralization of fatty acid increases its solubility in aqueous solution. However, sodium salts of fatty acids, especially saturated fatty acids, are still of low solubility in water or PBS as we observed. One method is to use 95 - 100% ethanol to help dissolve fatty acid¹. Using this method, higher toxicity to cells may be observed even at lower concentrations for less tox...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
CPX Ultrasonic Bath	Bransonic	Model 2800
Sodium palmitate (PA)	Nu-Chek Prep, Inc.	S-1109	M.W. 278
Sodium stearate (SA)	Nu-Chek Prep, Inc.	S-1111	M.W. 306
Bovine serum albumin (BSA), fatty acid-free	Fisher Scientific	9048-46-8
Mouse macrophage colony stimulating factor (mM-CSF)	Cell Signaling Technology,  Inc.	5228
RPMI 1640	VWR International	71002-878
Annexin V, Alexa Fluor 488 conjugate	Fisher Scientific	A13201
7-AAD	BD Biosciences	559925
Monoclonal anti-ceramide antibody (mouse IgM)	Sigma	C8104-50TST	Clone: MID 15B4
Goat Anti-Mouse IgM Antibody, µ chain, FITC conjugate	Sigma	AP128F
Fixation buffer	Biolegend	420801
Permeabilization buffer	Ebioscience	4307693
Red Blood Cell Lysis Buffer	Sigma	11814389001
Annexin V Binding Buffer	BD Biosciences	556454
L929 cells	ATCC	CCL-1
Corning Cell Lifter	Fisher Scientific	07-200-364
Note: M.W. is for molecular weight.