Cell-intrinsic lysosomal lipolysis is essential for macrophage alternative activation methods
Aim. Evidence-backed execution summary for Cell-intrinsic lysosomal lipolysis is essential for macrophage alternative activation methods from Cell-intrinsic lysosomal lipolysis is essential for macrophage alternative activation.
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mouse
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
Lipolysis contributes to M2 activation
reagent used in the protocol.
- Use
- Cellular requirements for fatty acids can be met by enzymatically regulated lipolysis of triacylglycerols into diacylglycerols and monoacylglycerols, accompanied by fatty acid release; an endpoint of this process is the release of glycerol from cells in which lipolysis is occurring. We performed global metabolite...
Elevated OXPHOS and optimal M2 activation are dependent on CD36
reagent used in the protocol.
- Use
- The important role for LAL in M2 macrophages suggested that triacylglycerol delivery to lysosomes is a key process in the generation of fatty acids to support FAO and M2 activation. We examined the expression of receptors that mediate the uptake of triacylglycerol-carrying serum lipoproteins in M2 macrophages and ob...
Elevated OXPHOS and optimal M2 activation are dependent on CD36
reagent used in the protocol.
- Use
- Next we tested the ability of monocyte-derived macrophages from a CD36-deficient patient to become alternatively activated in response to IL-4. We found that SRC was substantially diminished in human CD36-deficient M2 macrophages compared to CD36-sufficient human M2 macrophages ( ). Expression of CD206, the mannose...
Elevated OXPHOS and optimal M2 activation are dependent on CD36
reagent used in the protocol.
- Use
- Our data suggested that the lysosomal lipolysis of exogenous triacylglycerols acquired through a CD36-dependent mechanism is important for optimal M2 activation. In this case, we reasoned that depriving macrophages of exogenous triacylglycerols should impact M2 activation. We cultured macrophages in complete medium...
Elevated OXPHOS and optimal M2 activation are dependent on CD36
reagent used in the protocol.
- Use
- These findings raised the question of whether M2 macrophages first incorporate endogenously synthesized fatty acids into triacylglycerols prior to liberating them by lipolysis for FAO, as has been suggested to be the dominant pathway in other cell types. We examined this by first testing the effect of Orlistat on M...
Metabolism assays
reagent used in the protocol.
- Use
- For real-time analysis of extracellular acidification rates (ECAR) and oxygen consumption rates (OCR), macrophages were analyzed using an XF-96 Extracellular Flux Analyzer (EFA, Seahorse Bioscience) as described in detail for bone marrow-derived dendritic cells previously. Three or more consecutive measurements wer...
Isolation of cells from mice
reagent used in the protocol.
- Use
- Peritoneal exudate cells (PECs) were harvested from sacrificed naïve or infected mice by peritoneal lavage with 10 ml of sterile PBS containing 5% FBS (HyClone) per mouse. Total numbers of PECs and peritoneal macrophages were determined by cell counting in combination with multi-color flow cytometry. For analys...
Preparation of macrophages from bone marrow and macrophage activation
reagent used in the protocol.
- Use
- Bone marrow cells were differentiated in the presence of recombinant mouse M-CSF (20 ng/ml; R&D Systems) in complete medium (RPMI 1640 containing 10 mM glucose, 2 mM L-glutamine, 100 U/mL of penicillin/streptomycin and 10% FBS) for 7 days. Day 7 macrophages were washed and variously stimulated with IL-4 (20 ng/ml; P...
Lysosomal acid lipase is responsible for lipolysis upstream of FAO
We noted that, unlike genes encoding many other cellular lipases (including Pnpla2, Lipe, Lipc, Lipg ), expression of Lipa was upregulated in M2 compared to M1 macrophages (, ), suggesting that it could be important for M2 activation. Lipa encodes LAL, a triacylglycerol lipase that is able to breakdown triacylgl...
- Use
- We noted that, unlike genes encoding many other cellular lipases (including Pnpla2, Lipe, Lipc, Lipg ), expression of Lipa was upregulated in M2 compared to M1 macrophages (, ), suggesting that it could be important for M2 activation. Lipa encodes LAL, a triacylglycerol lipase that is able to breakdown triacylgl...
Elevated OXPHOS and optimal M2 activation are dependent on CD36
Next we tested the ability of monocyte-derived macrophages from a CD36-deficient patient to become alternatively activated in response to IL-4. We found that SRC was substantially diminished in human CD36-deficient M2 macrophages compared to CD36-sufficient human M2 macrophages ( ). Expression of CD206, the mannose...
- Use
- Next we tested the ability of monocyte-derived macrophages from a CD36-deficient patient to become alternatively activated in response to IL-4. We found that SRC was substantially diminished in human CD36-deficient M2 macrophages compared to CD36-sufficient human M2 macrophages ( ). Expression of CD206, the mannose...
Metabolism assays
For real-time analysis of extracellular acidification rates (ECAR) and oxygen consumption rates (OCR), macrophages were analyzed using an XF-96 Extracellular Flux Analyzer (EFA, Seahorse Bioscience) as described in detail for bone marrow-derived dendritic cells previously. Three or more consecutive measurements wer...
- Use
- For real-time analysis of extracellular acidification rates (ECAR) and oxygen consumption rates (OCR), macrophages were analyzed using an XF-96 Extracellular Flux Analyzer (EFA, Seahorse Bioscience) as described in detail for bone marrow-derived dendritic cells previously. Three or more consecutive measurements wer...
Methods
C57BL/6J mice, 4get/KN2 mice, B6 Cd36 -/- mice and B6.129P2- Pnpla2tm 1Rze /J ( Pnpla2 -/- ) mice were bred and maintained in specific pathogen free conditions under protocols approved by the institutional animal care committee at Washington University School of Medicine, and were used at 8-1...
- Use
- C57BL/6J mice, 4get/KN2 mice, B6 Cd36 -/- mice and B6.129P2- Pnpla2tm 1Rze /J ( Pnpla2 -/- ) mice were bred and maintained in specific pathogen free conditions under protocols approved by the institutional animal care committee at Washington University School of Medicine, and were used at 8-1...
Isolation of cells from mice
Peritoneal exudate cells (PECs) were harvested from sacrificed naïve or infected mice by peritoneal lavage with 10 ml of sterile PBS containing 5% FBS (HyClone) per mouse. Total numbers of PECs and peritoneal macrophages were determined by cell counting in combination with multi-color flow cytometry. For analys...
- Use
- Peritoneal exudate cells (PECs) were harvested from sacrificed naïve or infected mice by peritoneal lavage with 10 ml of sterile PBS containing 5% FBS (HyClone) per mouse. Total numbers of PECs and peritoneal macrophages were determined by cell counting in combination with multi-color flow cytometry. For analys...
Preparation of macrophages from bone marrow and macrophage activation
Bone marrow cells were differentiated in the presence of recombinant mouse M-CSF (20 ng/ml; R&D Systems) in complete medium (RPMI 1640 containing 10 mM glucose, 2 mM L-glutamine, 100 U/mL of penicillin/streptomycin and 10% FBS) for 7 days. Day 7 macrophages were washed and variously stimulated with IL-4 (20 ng/ml; P...
- Use
- Bone marrow cells were differentiated in the presence of recombinant mouse M-CSF (20 ng/ml; R&D Systems) in complete medium (RPMI 1640 containing 10 mM glucose, 2 mM L-glutamine, 100 U/mL of penicillin/streptomycin and 10% FBS) for 7 days. Day 7 macrophages were washed and variously stimulated with IL-4 (20 ng/ml; P...
Flow Cytometry
Cells were kept at 4 °C and blocked with 5 µg/ml of anti-CD16/32 (clone 93, eBiosciences) before surface staining with antibodies to CD11b (clone M1/70, eBiosciences), F4/80 (clone BM8, eBiosciences), CD206 (clone C068C2, Biolegend), CD301 (clone ER-MP23, AbD Serotec), PD-L2 (clone TY25, eBiosciences), CD6...
- Use
- Cells were kept at 4 °C and blocked with 5 µg/ml of anti-CD16/32 (clone 93, eBiosciences) before surface staining with antibodies to CD11b (clone M1/70, eBiosciences), F4/80 (clone BM8, eBiosciences), CD206 (clone C068C2, Biolegend), CD301 (clone ER-MP23, AbD Serotec), PD-L2 (clone TY25, eBiosciences), CD6...
RNA-sequencing
mRNA was extracted from lysates of cells that had been stimulated for 24 h, using oligodT beads (Invitrogen). cDNA synthesis, sequencing and sequence analysis were performed as described. Raw and processed sequencing data were deposited to Pubmed.
- Use
- mRNA was extracted from lysates of cells that had been stimulated for 24 h, using oligodT beads (Invitrogen). cDNA synthesis, sequencing and sequence analysis were performed as described. Raw and processed sequencing data were deposited to Pubmed.
Statistical analysis
Software used for acquisition, scoring, statistics, or reporting.
- Use
- Data were analyzed using Graphpad Prism (v5). Comparisons for two groups were calculated using One-way ANOVA and, where indicated, unpaired two-tailed Student's t- tests. Differences were considered significant when P -values were below 0.05. No randomization or exclusion of data points was used. Pilot in vivo studi...
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Methods
C57BL/6J mice, 4get/KN2 mice, B6 Cd36 -/- mice and B6.129P2- Pnpla2tm 1Rze /J ( Pnpla2 -/- ) mice were bred and maintained in specific pathogen free conditions under protocols approved by the institutional animal care committee at Washington University School of Medicine, and were used at 8-12 weeks of age. Lipa +/+ and Lipa -/- mice were maintained at Indiana State University under SPF conditions. In some experiments, mice were injected i.p. with tetrahydrolipistatin (Orlistat; 200 mg/kg, Cayman Chemical) or with 300 µl of 3% thioglycollate (Sigma) immediately prior to IL-4 complexed to mAb anti-IL-4 (IL-4c; containing 5 µg of IL-4, PeproTech, and 25 µg of anti-IL-4 clone 11B11, BioXcell). For infection with H. polygyrus bakeri, we followed protocols described in detail previously. Mice were infected orally by gavage with 200 infe...
Isolation of cells from mice
Peritoneal exudate cells (PECs) were harvested from sacrificed naïve or infected mice by peritoneal lavage with 10 ml of sterile PBS containing 5% FBS (HyClone) per mouse. Total numbers of PECs and peritoneal macrophages were determined by cell counting in combination with multi-color flow cytometry. For analysis of lymphocyte activation by flow cytometry, single cell suspensions were prepared from lymphoid organs by forcing them through 70 µm strainers, and washing with RPMI containing 5% FBS (Corning Inc). Red blood cells were lysed with ACK lysis buffer (0.15 M NH 4 Cl, 1 mM KHCO 3, 0.1 mM EDTA). Bone marrow was flushed from the femur and tibia using RPMI containing 5% FBS and dissociated into a single cell suspension by repeated pipetting. Cells were maintained on ice until use or analysis.
Preparation of macrophages from bone marrow and macrophage activation
Bone marrow cells were differentiated in the presence of recombinant mouse M-CSF (20 ng/ml; R&D Systems) in complete medium (RPMI 1640 containing 10 mM glucose, 2 mM L-glutamine, 100 U/mL of penicillin/streptomycin and 10% FBS) for 7 days. Day 7 macrophages were washed and variously stimulated with IL-4 (20 ng/ml; PeproTech) or lipopolysaccharide (LPS, 20 ng/ml; Sigma) plus IFN-γ (50 ng/ml; R&D Systems) in the presence or absence of 200 µM etomoxir (ETO; Sigma), 20 µM sulfo-n-succinimidyl oleate (SSO), 30 µM chloroquine (CLQ; Sigma), 0.1 µM bafilomycin A1 (Baf; Tocris Bioscience), 100 µM Orlistat (Cayman), 20 µM 5-(Tetradecyloxy)-2-furoic Acid (TOFA, Sigma), 5 or 50 µg/ml of LDL (Kalen Biomedical, LLC) and VLDL (Kalen Biomedical, LLC) for 24 h. Macrophages were then harvested and analyzed by flow cytometry for expression of markers of M1 or M2 a...
Isolation of mononuclear cells from human peripheral blood and human macrophage activation
Monocytes were purified from PBMCs using human CD14 MicroBeads (MACS) and then differentiated into macrophages in complete RPMI medium containing 20 ng/ml recombinant human M-CSF (R&D Systems) for 8 days. Human macrophages were harvested on day 8, and stimulated with 20 ng/ml human IL-4 (R&D Systems) or 50 ng/ml human IFN-γ (R&D Systems) plus 20 ng/ml LPS for 24 h.
Retroviral transduction
Retroviral transduction of macrophages was accomplished using a protocol that we have used previously to transduce bone marrow derived dendritic cells. Sequences for luciferase and for Lipa short hairpin RNAs (shRNAs) were obtained from Open Biosystems and cloned into the MSCV-LTRmir30-PI8 retroviral vector, encoding huCD8 as a reporter. Two independent sequences were used to target Lipa: TCAAGTCCAGCATTCACTG, and TGTGCTTCAGAGACCAGGT. Recombinant retroviruses were used for spin infection (800 x g, 2 h) of day 3 bone marrow macrophage cultures. After 7 days in culture with M-CSF, macrophages were harvested and transduced macrophages were gated based on huCD8 expression. Both shRNAs were used for experiments, with similar results, but only data from experiments that utilized TGTGCTTCAGAGACCAGGT are shown. For the bone marrow chimera, bone marrow cells from donor mice were collected and...
Transmission electron microscopy
For ultrastructural analysis, cells were fixed in 2% paraformaldehyde, 2.5% glutaraldehyde (Polysciences Inc.), 0.05% malachite green (Sigma) in 100 mM sodium cacodylate buffer, pH 7.2 for 1 h at 22°C. The malachite green was incorporated into the fixative to stabilize lipid constituents soluble in aqueous glutaraldehyde. Samples were washed in cacodylate buffer and post-fixed in 1% osmium tetroxide (Polysciences Inc.) for 1 h. Samples were then rinsed extensively in distilled H 2 O prior to en bloc staining with 1% aqueous uranyl acetate (Ted Pella Inc.) for 1 h. Following several rinses in dH 2 O, samples were dehydrated in a graded series of ethanol and embedded in Eponate 12 resin (Ted Pella Inc.). Sections of 95 nm were cut with a Leica Ultracut UC7 ultramicrotome (Leica Microsystems Inc.), stained with uranyl acetate and lead citrate, and viewed on a JEOL 1200 EX transmissi...
Measurement outputs
What raw and processed outputs should exist?
Cellular requirements for fatty acids can be met by enzymatically regulated lipolysis of triacylglycerols into diacylglycerols and monoacylglycerols, accompanied by fatty acid r...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Our data implicated lipolysis as a critical mechanism for the generation of fatty acids for FAO and M2 activation in macrophages stimulated with IL-4. In other cells fatty acids...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
We noted that, unlike genes encoding many other cellular lipases (including Pnpla2, Lipe, Lipc, Lipg ), expression of Lipa was upregulated in M2 compared to M1 macrophages (...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
The important role for LAL in M2 macrophages suggested that triacylglycerol delivery to lysosomes is a key process in the generation of fatty acids to support FAO and M2 activat...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Analysis plan
How should the outputs become interpretable results?
Acquisition
Collect raw experimental outputs with enough metadata to preserve sample identity, condition, and timing.
inferred from protocolPreprocessing / cleaning
Cellular requirements for fatty acids can be met by enzymatically regulated lipolysis of triacylglycerols into diacylglycerols and monoacylglycerols, accompanied by fatty acid release; an endpoint of this process is the release of glycerol from cells in which lipolysis is occ...
from paperScoring or quantification
Quantify the primary readouts for this experiment: Cellular requirements for fatty acids can be met by enzymatically regulated lipolysis of triacylglycerols into diacylglycerols and monoacylglycerols, accompanied by fatty acid r...; Our data implicated lipolysis as a critical mechanism for the generation of fatty acids for FAO and M2 activation in macrophages stimulated with IL-4. In other cells fatty acids...; We noted that, unlike genes encoding many other cellular lipases (including Pnpla2, Lipe, Lipc, Lipg ), expression of Lipa was upregulated in M2 compared to M1 macrophages (...; The important role for LAL in M2 macrophages suggested that triacylglycerol delivery to lysosomes is a key process in the generation of fatty acids to support FAO and M2 activat....
from paperStatistical comparison
Cellular requirements for fatty acids can be met by enzymatically regulated lipolysis of triacylglycerols into diacylglycerols and monoacylglycerols, accompanied by fatty acid r...; Next we tested the ability of monocyte-derived macrophages from a CD36-deficient patient to become alternatively activated in response to IL-4. We found that SRC was substantial...; Data were analyzed using Graphpad Prism (v5). Comparisons for two groups were calculated using One-way ANOVA and, where indicated, unpaired two-tailed Student's t- tests. Differ...
from paperReporting output
Report representative outputs alongside summary comparisons for Cellular requirements for fatty acids can be met by enzymatically regulated lipolysis of triacylglycerols into diacylglycerols and monoacylglycerols, accompanied by fatty acid r..., Our data implicated lipolysis as a critical mechanism for the generation of fatty acids for FAO and M2 activation in macrophages stimulated with IL-4. In other cells fatty acids..., We noted that, unlike genes encoding many other cellular lipases (including Pnpla2, Lipe, Lipc, Lipg ), expression of Lipa was upregulated in M2 compared to M1 macrophages (..., The important role for LAL in M2 macrophages suggested that triacylglycerol delivery to lysosomes is a key process in the generation of fatty acids to support FAO and M2 activat....
inferred from protocolStructured statistical methods
Cellular requirements for fatty acids can be met by enzymatically regulated lipolysis of triacylglycerols into diacylglycerols and monoacylglycerols, accompanied by fatty acid r...; Next we tested the ability of monocyte-derived macrophages from a CD36-deficient patient to become alternatively activated in response to IL-4. We found that SRC was substantial...; Data were analyzed using Graphpad Prism (v5). Comparisons for two groups were calculated using One-way ANOVA and, where indicated, unpaired two-tailed Student's t- tests. Differ...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (6)
C57BL/6J mice, 4get/KN2 mice, B6 Cd36 -/- mice and B6.129P2- Pnpla2tm 1Rze /J ( Pnpla2 -/- ) mice were bred and maintained in specific pathogen free conditions under protocols approved by the institutional animal care committee at Washington University School of Medicine, and were used at 8-12 weeks of age. Lipa +/+ and Lipa -/- mice were maintained at Indiana State University under SPF conditions. In some experiments, mice were injected i.p. with tetrahydrolipistatin (Orlistat; 200 mg/kg, Cayman Chemical) or with 300 µl of 3% thioglycollate (Sigma) immediately prior to IL-4 complexed to mAb anti-IL-4 (IL-4c; containing 5 µg of IL-4, PeproTech, and 25 µg of anti-IL-4 clone 11B11, BioXcell). For infection with H. polygyrus bakeri, we followed protocols described in detail previously. Mice were infected orally by gavage with 200 infective L3 stage larvae. For secondary infection experiments, adult H. polygyrus were eliminated from infected mice by oral administration of pyrantel pamoate (1 mg/mouse; Columbia Laboratory) at day 14 post-primary infection, and > 5 weeks later the mice were challenge-infected with 200 L3 stage larv...
Peritoneal exudate cells (PECs) were harvested from sacrificed naïve or infected mice by peritoneal lavage with 10 ml of sterile PBS containing 5% FBS (HyClone) per mouse. Total numbers of PECs and peritoneal macrophages were determined by cell counting in combination with multi-color flow cytometry. For analysis of lymphocyte activation by flow cytometry, single cell suspensions were prepared from lymphoid organs by forcing them through 70 µm strainers, and washing with RPMI containing 5% FBS (Corning Inc). Red blood cells were lysed with ACK lysis buffer (0.15 M NH 4 Cl, 1 mM KHCO 3, 0.1 mM EDTA). Bone marrow was flushed from the femur and tibia using RPMI containing 5% FBS and dissociated into a single cell suspension by repeated pipetting. Cells were maintained on ice until use or analysis.
Bone marrow cells were differentiated in the presence of recombinant mouse M-CSF (20 ng/ml; R&D Systems) in complete medium (RPMI 1640 containing 10 mM glucose, 2 mM L-glutamine, 100 U/mL of penicillin/streptomycin and 10% FBS) for 7 days. Day 7 macrophages were washed and variously stimulated with IL-4 (20 ng/ml; PeproTech) or lipopolysaccharide (LPS, 20 ng/ml; Sigma) plus IFN-γ (50 ng/ml; R&D Systems) in the presence or absence of 200 µM etomoxir (ETO; Sigma), 20 µM sulfo-n-succinimidyl oleate (SSO), 30 µM chloroquine (CLQ; Sigma), 0.1 µM bafilomycin A1 (Baf; Tocris Bioscience), 100 µM Orlistat (Cayman), 20 µM 5-(Tetradecyloxy)-2-furoic Acid (TOFA, Sigma), 5 or 50 µg/ml of LDL (Kalen Biomedical, LLC) and VLDL (Kalen Biomedical, LLC) for 24 h. Macrophages were then harvested and analyzed by flow cytometry for expression of markers of M1 or M2 activation. In some experiments the survival of macrophages was monitored following M2 or M1 activation. For these experiments, bone marrow-derived macrophages (0.5 × 10 6 cells per well of a 48-well plate) were cultured in complete medium with mouse M-CSF (20 ng/ml) and stimulated with IL-4 or...
Monocytes were purified from PBMCs using human CD14 MicroBeads (MACS) and then differentiated into macrophages in complete RPMI medium containing 20 ng/ml recombinant human M-CSF (R&D Systems) for 8 days. Human macrophages were harvested on day 8, and stimulated with 20 ng/ml human IL-4 (R&D Systems) or 50 ng/ml human IFN-γ (R&D Systems) plus 20 ng/ml LPS for 24 h.
Retroviral transduction of macrophages was accomplished using a protocol that we have used previously to transduce bone marrow derived dendritic cells. Sequences for luciferase and for Lipa short hairpin RNAs (shRNAs) were obtained from Open Biosystems and cloned into the MSCV-LTRmir30-PI8 retroviral vector, encoding huCD8 as a reporter. Two independent sequences were used to target Lipa: TCAAGTCCAGCATTCACTG, and TGTGCTTCAGAGACCAGGT. Recombinant retroviruses were used for spin infection (800 x g, 2 h) of day 3 bone marrow macrophage cultures. After 7 days in culture with M-CSF, macrophages were harvested and transduced macrophages were gated based on huCD8 expression. Both shRNAs were used for experiments, with similar results, but only data from experiments that utilized TGTGCTTCAGAGACCAGGT are shown. For the bone marrow chimera, bone marrow cells from donor mice were collected and cultured in complete RPMI medium containing 10 ng/ml IL-3, 10 ng/ml IL-6 and 50 ng/ml murine c-kit ligand for 24 h. Cells were then transduced with retroviral shRNAs targeting Lipa or luciferase, and 5 × 10 5 transduced cells were intravenously injected into irradiated recipients. Recipients w...
For ultrastructural analysis, cells were fixed in 2% paraformaldehyde, 2.5% glutaraldehyde (Polysciences Inc.), 0.05% malachite green (Sigma) in 100 mM sodium cacodylate buffer, pH 7.2 for 1 h at 22°C. The malachite green was incorporated into the fixative to stabilize lipid constituents soluble in aqueous glutaraldehyde. Samples were washed in cacodylate buffer and post-fixed in 1% osmium tetroxide (Polysciences Inc.) for 1 h. Samples were then rinsed extensively in distilled H 2 O prior to en bloc staining with 1% aqueous uranyl acetate (Ted Pella Inc.) for 1 h. Following several rinses in dH 2 O, samples were dehydrated in a graded series of ethanol and embedded in Eponate 12 resin (Ted Pella Inc.). Sections of 95 nm were cut with a Leica Ultracut UC7 ultramicrotome (Leica Microsystems Inc.), stained with uranyl acetate and lead citrate, and viewed on a JEOL 1200 EX transmission electron microscope (JEOL USA Inc.) equipped with an AMT 8 megapixel digital camera (Advanced Microscopy Techniques).
Machine-readable layer
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"name": "Cell-intrinsic lysosomal lipolysis is essential for macrophage alternative activation methods",
"description": "Evidence-backed execution summary for Cell-intrinsic lysosomal lipolysis is essential for macrophage alternative activation methods from Cell-intrinsic lysosomal lipolysis is essential for macrophage alternative activation.",
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"step": [
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"position": 1,
"name": "Methods",
"text": "C57BL/6J mice, 4get/KN2 mice, B6 Cd36 -/- mice and B6.129P2- Pnpla2tm 1Rze /J ( Pnpla2 -/- ) mice were bred and maintained in specific pathogen free conditions under protocols approved by the institutional animal care committee at Washington University School of Medicine, and were used at 8-12 weeks of age. Lipa +/+ and Lipa -/- mice were maintained at Indiana State University under SPF conditions. In some experiments, mice were injected i.p. with tetrahydrolipistatin (Orlistat; 200 mg/kg, Cayman Chemical) or with 300 µl of 3% thioglycollate (Sigma) immediately prior to IL-4 complexed to mAb anti-IL-4 (IL-4c; containing 5 µg of IL-4, PeproTech, and 25 µg of anti-IL-4 clone 11B11, BioXcell). For infection with H. polygyrus bakeri, we followed protocols described in detail previously. Mice were infected orally by gavage with 200 infe..."
},
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"@type": "HowToStep",
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"name": "Isolation of cells from mice",
"text": "Peritoneal exudate cells (PECs) were harvested from sacrificed naïve or infected mice by peritoneal lavage with 10 ml of sterile PBS containing 5% FBS (HyClone) per mouse. Total numbers of PECs and peritoneal macrophages were determined by cell counting in combination with multi-color flow cytometry. For analysis of lymphocyte activation by flow cytometry, single cell suspensions were prepared from lymphoid organs by forcing them through 70 µm strainers, and washing with RPMI containing 5% FBS (Corning Inc). Red blood cells were lysed with ACK lysis buffer (0.15 M NH 4 Cl, 1 mM KHCO 3, 0.1 mM EDTA). Bone marrow was flushed from the femur and tibia using RPMI containing 5% FBS and dissociated into a single cell suspension by repeated pipetting. Cells were maintained on ice until use or analysis."
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"name": "Preparation of macrophages from bone marrow and macrophage activation",
"text": "Bone marrow cells were differentiated in the presence of recombinant mouse M-CSF (20 ng/ml; R&D Systems) in complete medium (RPMI 1640 containing 10 mM glucose, 2 mM L-glutamine, 100 U/mL of penicillin/streptomycin and 10% FBS) for 7 days. Day 7 macrophages were washed and variously stimulated with IL-4 (20 ng/ml; PeproTech) or lipopolysaccharide (LPS, 20 ng/ml; Sigma) plus IFN-γ (50 ng/ml; R&D Systems) in the presence or absence of 200 µM etomoxir (ETO; Sigma), 20 µM sulfo-n-succinimidyl oleate (SSO), 30 µM chloroquine (CLQ; Sigma), 0.1 µM bafilomycin A1 (Baf; Tocris Bioscience), 100 µM Orlistat (Cayman), 20 µM 5-(Tetradecyloxy)-2-furoic Acid (TOFA, Sigma), 5 or 50 µg/ml of LDL (Kalen Biomedical, LLC) and VLDL (Kalen Biomedical, LLC) for 24 h. Macrophages were then harvested and analyzed by flow cytometry for expression of markers of M1 or M2 a..."
},
{
"@type": "HowToStep",
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"name": "Isolation of mononuclear cells from human peripheral blood and human macrophage activation",
"text": "Monocytes were purified from PBMCs using human CD14 MicroBeads (MACS) and then differentiated into macrophages in complete RPMI medium containing 20 ng/ml recombinant human M-CSF (R&D Systems) for 8 days. Human macrophages were harvested on day 8, and stimulated with 20 ng/ml human IL-4 (R&D Systems) or 50 ng/ml human IFN-γ (R&D Systems) plus 20 ng/ml LPS for 24 h."
},
{
"@type": "HowToStep",
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"name": "Retroviral transduction",
"text": "Retroviral transduction of macrophages was accomplished using a protocol that we have used previously to transduce bone marrow derived dendritic cells. Sequences for luciferase and for Lipa short hairpin RNAs (shRNAs) were obtained from Open Biosystems and cloned into the MSCV-LTRmir30-PI8 retroviral vector, encoding huCD8 as a reporter. Two independent sequences were used to target Lipa: TCAAGTCCAGCATTCACTG, and TGTGCTTCAGAGACCAGGT. Recombinant retroviruses were used for spin infection (800 x g, 2 h) of day 3 bone marrow macrophage cultures. After 7 days in culture with M-CSF, macrophages were harvested and transduced macrophages were gated based on huCD8 expression. Both shRNAs were used for experiments, with similar results, but only data from experiments that utilized TGTGCTTCAGAGACCAGGT are shown. For the bone marrow chimera, bone marrow cells from donor mice were collected and..."
},
{
"@type": "HowToStep",
"position": 6,
"name": "Transmission electron microscopy",
"text": "For ultrastructural analysis, cells were fixed in 2% paraformaldehyde, 2.5% glutaraldehyde (Polysciences Inc.), 0.05% malachite green (Sigma) in 100 mM sodium cacodylate buffer, pH 7.2 for 1 h at 22°C. The malachite green was incorporated into the fixative to stabilize lipid constituents soluble in aqueous glutaraldehyde. Samples were washed in cacodylate buffer and post-fixed in 1% osmium tetroxide (Polysciences Inc.) for 1 h. Samples were then rinsed extensively in distilled H 2 O prior to en bloc staining with 1% aqueous uranyl acetate (Ted Pella Inc.) for 1 h. Following several rinses in dH 2 O, samples were dehydrated in a graded series of ethanol and embedded in Eponate 12 resin (Ted Pella Inc.). Sections of 95 nm were cut with a Leica Ultracut UC7 ultramicrotome (Leica Microsystems Inc.), stained with uranyl acetate and lead citrate, and viewed on a JEOL 1200 EX transmissi..."
}
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"tool": [
{
"@type": "HowToTool",
"name": "Lysosomal acid lipase is responsible for lipolysis upstream of FAO"
},
{
"@type": "HowToTool",
"name": "Elevated OXPHOS and optimal M2 activation are dependent on CD36"
},
{
"@type": "HowToTool",
"name": "Metabolism assays"
},
{
"@type": "HowToTool",
"name": "Methods"
},
{
"@type": "HowToTool",
"name": "Isolation of cells from mice"
},
{
"@type": "HowToTool",
"name": "Preparation of macrophages from bone marrow and macrophage activation"
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