Different Sensitivity of Macrophages to Phospholipidosis Induction by Amphiphilic Cationic Drugs
Abstract
:1. Introduction
2. Results
2.1. Characterization of Macrophages (Cell Area, Lysosome Area, Lysosome Number/Cell, Cytotoxicity to CADs)
2.2. Identification of Optimal Staining
2.3. Effect of CADs on Cell Area
2.4. Detection of Phospholipidosis and Neutral Lipids
2.5. Effect of Amiodarone and Chloroquine on Number, Area and Staining Intensity of Lysosomes
3. Discussion
3.1. Establishment of Macrophage Models and Staining
3.2. Action of Amiodarone and Chloroquine on Lysosomes
4. Materials and Methods
4.1. Cell Culture
4.2. Differentiation to THP-1 Macrophages
Characterization of THP-1 Macrophages
4.3. Generation of Monocyte-Derived Macrophages (MDMs)
4.3.1. Differentiation to M1 and M2 State
4.3.2. Cytotoxicity Screening Using Measurement of Dehydrogenase Activity
4.3.3. Identification of Lysosomes by LysoSensor Staining
4.3.4. Measurement of Membrane Potential by Acridine Orange Staining
4.3.5. Lipid Accumulation by Nile Red Staining
4.3.6. Neutral Lipid and Phospholipid Accumulation by HCS LipidTOX Dyes
4.4. Lysosome Morphology by LysoID Staining
4.5. Statistics
Supplementary Materials
Author Contributions
Funding
Conflicts of Interest
Abbreviations
Amio | Amiodarone |
AD | Distilled water |
CAD | Cationic amphiphilic drug |
Chloro | Chloroquine |
EDTA | Ethylenediaminetetraacetic acid |
DIILD | Drug induced interstitial lung disease |
DMEM | Dulbecco’s Modified Eagle Medium |
DMSO | Dimethylsulfoxide |
DPBS | Dulbecco’s PBS |
DPPC | Dipalmitoylphosphatidylcholine |
FBS | Fetal bovine serum |
GM-CSF | Granulocyte-macrophage colony stimulating factor |
GM-MDM | M1 macrophages |
HCS | High content screening |
ILD | Interstitial lung disease |
LDL | Low density lipoprotein |
LPS | Lipopolysaccharide |
LRS | Leukoreduction system |
M-CSF | Macrophage colony stimulating factor |
M-MDM | M2 macrophages |
MDM | Monocyte-derived macrophage |
NEAA | non-essential amino acids |
PLD | Phospholipidosis |
NBD-PE | N-(7-nitrobenz-2-oxa-1,3 diazol-4-yl)1,2 dihexadecanoyl-sn-glycero-3-phosphoethanolamine |
PBMC | Peripheral blood mononuclear cells |
PMA | Phorbol 12-myristate 13-acetate |
P/S | Penicillin/Streptomycin |
RPMI | Roswell Park Memorial Institute |
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Macrophages | Cell Area (DMSO, µm2) | Cell Area (AD, µm2) | Lysosome Number (DMSO) | Lysosome Number (AD) | Lysosome Area (DMSO, µm2) | Lysosome Area (AD, µm2) |
---|---|---|---|---|---|---|
DMBM-2 | 253 ± 6 * | 229 ± 2.4 | 30 ± 0.5 | 30 ± 0.4 | 3.5 ± 0.014 | 3.3 ± 0.009 |
J774A.1 | 183 ± 1.8 * | 172 ± 1.6 | 24 ± 0.4 | 23 ± 0.4 | 1.5 ± 0.008 | 1.4 ± 0.006 |
RAW264.7 | 131 ± 1.6 * | 120 ± 1.7 | 13 ± 0.2 | 14 ± 0.2 | 1.1 ± 0.003 | 1.1 ± 0.004 |
THP-1 | 208 ± 3.8 | 215 ± 3.3 | 13 ± 0.4 | 14 ± 0.5 | 1.5 ± 0.01 | 1.5 ± 0.01 |
GM-MDM | 241 ± 4.5 | 238 ± 4.0 | 11 ± 0.2 | 10 ± 0.2 | 2.1 ± 0.01 | 2.2 ± 0.01 |
Treatment | DMBM-2 | J774 | RAW264.7 | THP-1 | GM-MDM | M-MDM |
---|---|---|---|---|---|---|
DMSO | 100 ± 5 | 100 ± 2 | 100 ± 3 | 100 ± 5 | 100 ± 9 | 100 ± 2 |
Amio2.5 | 98 ± 3 | 100 ± 1 | 103 ± 4 | 86 ± 3 | 92 ± 7 | 101 ± 9 |
Amio5.0 | 107 ± 3 | 105 ± 3 | 111 ± 2 | 89 ± 3 | 89 ± 7 | 123 ± 11 |
Amio7.5 | 104 ± 2 | 108 ± 2 | 107 ± 3 | 93 ± 6 | 97 ± 8 | 137 ± 2 |
Amio10.0 | 90 ± 4 | 111 ± 2 | 115 ± 2 | 105 ± 3 | 96 ± 9 | 150 ± 3 |
AD | 100 ± 5 | 100 ± 1 | 100 ± 2 | 100 ± 4 | 100 ± 5 | 100 ± 12 |
Chloro2.5 | 97 ± 6 | 99 ± 1 | 102 ± 2 | 97 ± 10 | 97 ± 8 | 99 ± 9 |
Chloro5.0 | 99 ± 3 | 107 ± 2 | 104 ± 5 | 92 ± 5 | 96 ± 7 | 95 ± 11 |
Chloro7.5 | 99 ± 7 | 108 ± 3 | 102 ± 4 | 91 ± 5 | 97 ± 7 | 108 ± 6 |
Chloro10.0 | 98 ± 5 | 111 ± 4 | 107 ± 4 | 97 ± 6 | 99 ± 4 | 131 ± 5 |
Amiodarone | Chloroquine | |||
---|---|---|---|---|
Macrophage | Phospholipid Staining | Neutral Lipid Staining | Phospholipid Staining | Neutral Lipid Staining |
DMBM-2 | - | - | Area (2.5, 5 µM) | Area (≥2.5 µM) |
J774 | Intensity (≥7.5 µM) Area (≥2.5 µM) | - | Intensity (≥5 µM) Area (≥5 µM) | Intensity (10 µM) |
RAW264.7 | Intensity (≥5 µM) Area (≥5 µM) | Area (5, 7.5 µM) | Intensity (≥5 µM) Area (2.5 µM) | Intensity (≥7.5 µM) |
THP-1 | Intensity (2.5 µM) | Intensity (≥2.5 µM) | Intensity (2.5 µM) | Intensity (7.5 µM) |
GM-MDM | Intensity (≥2.5 µM) Area (≥2.5 µM) | - | Area (5 µM) | - |
M-MDM | Intensity (≥2.5 µM) | - | Intensity (10 µM) | Intensity (≥7.5 µM) Area (≥7.5 µM) |
Amiodarone | Chloroquine | |||||
---|---|---|---|---|---|---|
Macrophage | Number | Area | Intensity | Number | Area | Intensity |
DMBM-2 | 2.5, 5 µM | 2.5–7.5 µM | ≥2.5 µM | 10 µM | ≥2.5 µM | ≥2.5 µM |
J774 | ≥2.5 µM | ≥2.5 µM | ≥2.5 µM | 2.5, 10 µM | 2.5–7.5 µM | 2.5–7.5 µM |
RAW264.7 | ≥2.5 µM | ≥2.5 µM | ≥2.5 µM | ≥2.5 µM | ≥2.5 µM | ≥2.5 µM |
THP-1 | ≥2.5 µM | ≥5 µM | ≥2.5 µM | ≥2.5 µM | 10 µM | ≥2.5 µM |
GM-MDM | ≥2.5 µM | 2.5, 5, 10 µM | ≥2.5 µM | 10 µM | 10 µM | ≥2.5 µM |
Medium | DMSO | AD | Amio | Chloro | |
---|---|---|---|---|---|
DMBM-2 | 0.42 | 0.37 | 0.41 | 0.35 | 0.25 |
RAW264.7 | 0.48 | 0.35 | 0.48 | 0.47 | 0.49 |
J774 | 0.63 | 0.63 | 0.63 | 0.47 | 0.44 |
GM-MDM | 0.56 | 0.63 | 0.53 | 0.57 | 0.55 |
THP-1 | 0.67 | 0.65 | 0.53 | 0.66 | 0.63 |
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Öhlinger, K.; Absenger-Novak, M.; Meindl, C.; Ober, J.; Fröhlich, E. Different Sensitivity of Macrophages to Phospholipidosis Induction by Amphiphilic Cationic Drugs. Int. J. Mol. Sci. 2020, 21, 8391. https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21218391
Öhlinger K, Absenger-Novak M, Meindl C, Ober J, Fröhlich E. Different Sensitivity of Macrophages to Phospholipidosis Induction by Amphiphilic Cationic Drugs. International Journal of Molecular Sciences. 2020; 21(21):8391. https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21218391
Chicago/Turabian StyleÖhlinger, Kristin, Markus Absenger-Novak, Claudia Meindl, Jennifer Ober, and Eleonore Fröhlich. 2020. "Different Sensitivity of Macrophages to Phospholipidosis Induction by Amphiphilic Cationic Drugs" International Journal of Molecular Sciences 21, no. 21: 8391. https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21218391