DOI: 10.5061/dryad.w3r2280wn
title: Data from: Crystalline silica-induced proinflammatory eicosanoid storm in novel alveolar macrophage model quelled by docosahexaenoic acid

description: Introduction: Workplace exposure to respirable crystalline silica (cSiO2)
 is associated with chronic inflammatory and autoimmune diseases. At the
 mechanistic level, cSiO2 particles are quickly phagocytosed by resident
 alveolar macrophages (AMs) in the lung, causing a robust cycle of
 proinflammatory cytokine release, lysosomal rupture, mitochondrial
 toxicity, and immunogenic cell death if the particle is not efficiently
 cleared by the lung. We and others have demonstrated in bone
 marrow-derived and transformed macrophage models that supplementation with
 the ω-3 polyunsaturated fatty acid (PUFA) docosahexaenoic acid (DHA)
 contributes to increased membrane phospholipid content of DHA and
 subsequent suppression of cSiO2-triggered inflammatory responses. However,
 mechanistic exploration of ω-3 PUFA effects in AMs is challenging due to
 reliance on short-lived primary AMs derived from lung lavage fluid.
 Methods: To address these limitations, we have employed a recently
 developed novel self-renewing AM model from C57BL/6 mice, fetal
 liver-derived alveolar-like macrophages (FLAMs), that is phenotypically
 representative of primary lung AM populations. We found that incubation of
 FLAMs with 25 µM  DHA as ethanolic suspensions or as complexes
 with bovine serum albumin were equally effective at increasing ω-3 PUFA
 content of phospholipids at the expense of the ω-6 PUFA arachidonic acid
 (ARA) and the ω-9 monounsaturated fatty acid oleic acid. Based on these
 findings, FLAMs were treated with 25 µM DHA in EtOH or EtOH vehicle (VEH)
 for 24 h, with or without LPS for 2 h, and with or without cSiO2 for 1.5
 or 4 h then proinflammatory cytokine release, lysosomal membrane
 permeabilization, and mitochondrial depolarization assessed. In addition,
 oxylipin metabolites were measured using a targeted LC-MS lipidomics panel
 of 156 metabolites. Results: Regardless of whether FLAMs were LPS-primed,
 cSiO2-triggered lysosomal permeability, mitochondrial toxicity, and cell
 death were not impacted by DHA. LPS+cSiO2 elicited marked IL-1α, IL-1β,
 and TNF-α release after 1.5 and 4 h of cSiO2 exposure, which was
 significantly inhibited by DHA. In VEH-treated cells, cSiO2 alone and
 LPS+cSiO2 induced synthesis of ARA-derived proinflammatory oxylipins
 including prostaglandins, leukotrienes, and thromboxanes that was
 suppressed by DHA. In addition, DHA promoted synthesis of pro-resolving
 DHA-derived oxylipins at the expense of ARA-derived oxylipins.
 Discussion:  FLAMs were amenable to lipidome modulation by DHA,
 which suppressed cSiO2-triggered proinflammatory cytokine responses and
 ARA-derived oxylipins that potentially contribute to the particle’s
 toxicity in the lung. FLAMs are a promising in vitro alternative to
 primary AMs for investigating interventions against toxicant-triggered
 inflammation and autoimmunity in the lung.
description: Experimental design Fetal liver-derived
 alveolar-like macrophages (FLAMs) were seeded in 6-well plates at a
 density of 4.50×10<sup>5</sup> cells/well in complete FLAM
 medium (RPMI 1640, 10% fetal bovine serum, 1% penicillin-streptomycin, 30
 ng/ml GM-CSF, 20 ng/ml TGF-β). Cells were incubated overnight to achieve
 70-90% confluency before beginning treatments. The next day, cells washed
 once with sterile PBS, fresh complete FLAM medium containing either 25 µM
 ethanolic DHA or ethanol vehicle (VEH) added, then cells incubated for 24
 h. Following DHA or VEH treatment, cells were washed once with sterile
 PBS, treated with either 20 ng/ml lipopolysaccharide (LPS) or VEH in
 DPBS+/+ (DPBS containing calcium and magnesium) for 2 h, then exposed to
 12.5 µg/cm<sup>2</sup> crystalline silica
 (cSiO<sub>2</sub>) or VEH for 1.5 or 4 h. Cell culture
 supernatants and cells were pooled together and collected at t = 2 h
 (after 2 h LPS priming), 3.5 h (after 1.5 h cSiO<sub>2</sub>
 exposure), and 6 h (after 4 h cSiO<sub>2 </sub>exposure). LPS
 and cSiO<sub>2</sub> exposures were done in DPBS+/+ to
 minimize interference from fatty acids present in cell culture medium
 during oxylipin analyses. Measurement of intracellular
 and extracellular oxylipins Sample
 preparation Following treatments, ice-cold methanol
 was added to each well, resulting in a final sample volume of 3 ml (1 ml
 cell culture supernatant + 2 ml methanol). To each sample, 60 µl of
 antioxidant cocktail (0.2 mg/ml butylated hydroxytoluene, 0.2 mg/ml
 triphenylphosphine, 0.6 mg/ml EDTA) was added to achieve a total cocktail
 concentration of 5% (v/v). Cells and supernatants within each well were
 pooled together, then samples were frozen at -80 °C until liquid
 chromatography-mass spectrometry (LC-MS) analysis.
 Liquid chromatography-mass spectrometry (LC-MS)
 analysis Targeted LC-MS lipidomics for 156 lipid
 metabolites was conducted at the Lipidomics Core Facility at Wayne State
 University. Briefly, 100 µl aliquots of cellular samples were thawed and
 spiked with a cocktail of deuterated internal standards (5 ng each of
 PGE1-d<sub>4</sub>, RvD2-d<sub>5</sub>,
 LTB4-d<sub>4</sub>, and 15[S]-HETE-d<sub>8</sub>;
 Cayman Chemical, Ann Arbor, MI) for quantification of oxylipins and
 recovery. Then, fatty acyl lipid metabolites were extracted by using C18
 extraction columns that were washed with 15% (v/v) methanol and
 subsequently hexane, dried in a vacuum, eluted with methanol containing
 0.1% (v/v) formic acid, dried under nitrogen gas, and dissolved in a 1:1
 mixture of methanol:25 mM aqueous ammonium acetate. Extracted fatty acyl
 metabolites were subjected to high-performance liquid chromatography
 (HPLC) using a Luna C18 (3 µm, 2.1×150 mm) column connected to a
 Prominence XR system (Shimadzu, Somerset, NJ) then analyzed with a
 QTrap5500 mass spectrometer (AB Sciex, Singapore) set to negative ion
 mode. Analyst 1.6 software (AB Sciex) and MultiQuant software (AB Sciex)
 were used to collect and quantify the data in units of ng,
 respectively. Data analysis and statistics
 For oxylipin data, MetaboAnalyst Version 5.0 (Xia Lab, Quebec,
 Canada, www.metaboanalyst.ca/) were used to conduct statistical analyses.
 First, raw ng values were converted to corresponding pmol values in
 Microsoft Excel. Then, in MetaboAnalyst, the one factor statistical
 analysis module was chosen, and data were uploaded as a comma separated
 values (.csv) file with samples in unpaired columns and features (i.e.,
 metabolites) in rows. Features with &gt;70% missing data were removed
 from the dataset, and remaining missing values were estimated by replacing
 with the corresponding limits of detection (LODs; 1/5 of the minimum
 positive value of each variable). After the data was cleaned, the data was
 normalized by auto scaling only, then the data editor option was used to
 select experimental groups of interest to compare. For comparisons between
 experimental groups, one-way analysis of variance (ANOVA) (FDR = 0.05)
 followed by Tukey’s honestly significant difference (HSD) post-hoc test
 was used, with FDR q &lt; 0.05 considered statistically
 significant.
description: Applications needed: Microsoft Excel, MetaboAnalyst Version 5.0
 (Xia Lab, Quebec, Canada, www.metaboanalyst.ca/).
description: # General Information Title of Dataset: Data from: Omega-3 docosahexaenoic
 acid suppresses silica-induced proinflammatory cytokine release and
 oxylipin mediator production in novel fetal liver-derived alveolar-like
 macrophages Author/Principal Investigator Information Name: Dr. Olivia
 Favor ORCID: 0000-0001-9164-7077 Institution: Michigan State University
 Address: 567 Wilson Rd, Office 4183, East Lansing, MI 48824 Email:
 Author/Associate or Co-Investigator Information
 Name: Dr. James Pestka ORCID: 0000-0003-4689-2756 Institution: Michigan
 State University Address: 567 Wilson Rd, Office 4176, East Lansing, MI
 48824 Email: Author/Alternate Contact Information
 Name: Dr. Kathryn Wierenga ORCID: 0000-0003-1725-4616 Institution: Utrecht
 University Address: Heidelberglaan 8, 3584 CS Utrecht, The Netherlands
 Email: Author/Alternate Contact
 Information Name: Dr. Lichchavi Rajasinghe ORCID: 0000-0002-5136-7422
 Institution: AstraZeneca Address: 1800 Concord Pike, Wilmington, DE 19803
 Email: Author/Alternate
 Contact Information Name: Dr. Krishna Maddipati ORCID: 0000-0003-1445-791X
 Institution: Wayne State University Address: 435 Chemistry Bldg., 5101
 Cass Ave, Detroit, MI 48202 Email: 
 Author/Associate or Co-Investigator Information Name: Dr. Kin Sing Stephen
 Lee ORCID: 0000-0003-4541-3063 Institution: Michigan State University
 Address: Address: 1355 Bogue St, Room B330A, East Lansing, MI 48824 Email:
 Author/Alternate Contact Information Name: Dr. Andrew
 Olive ORCID: 0000-0003-3441-3113 Institution: Michigan State University
 Address: 567 Wilson Rd, Office 5198, East Lansing, MI 48824 Email:
 Date of data collection: 2021 Geographic location
 of data collection: Wayne State University Information about funding
 sources that supported the collection of the data: National Institute of
 Environmental Health Sciences, ES027353; National Institutes of
 Health/National Center for Research Resources, S10RR027926. --- #
 Description of the Data and File structure File List: File 1 - Oxylipin
 Profiling Data: contains area ratios, signal-to-noise (SN) ratios,
 chromatographic peak quality values, calculated quantities with
 validation, and final quantities for 143 unique oxylipins analyzed by
 LC-MS. Zip File 1 - MS Spectra for Representative Oxylipins: contains mass
 spectrometry spectra for representative omega-6 eicosanoids (i.e., PGE2,
 LTB4, TXB2), hydroxy-fatty acids (i.e., 5-HETE, 5-HEPE, 4-HDoHE),
 epoxy-fatty acids (i.e., 14,15-EpETrE, 19,20-EpDPE), dihydroxy-fatty acids
 (i.e., 14,15-DiHETrE, 19,20-DiHDoPE), and specialized pro-resolving
 mediators (i.e., RvD6, MaR1[n-3 DPA]) in PDF format for Windows OS and Mac
 OS. Relationship between files, if important: Additional related data
 collected that was not included in the current data package: Are there
 multiple versions of the dataset? No If yes, name of file(s) that was
 updated: Why was the file updated? When was the file updated? #
 Methodological Information MEASUREMENT OF INTRACELLULAR AND EXTRACELLULAR
 OXYLIPINS SAMPLE PREPARATION Following treatments, ice-cold methanol was
 added to each well, resulting in a final sample volume of 3 ml (1 ml cell
 culture supernatant + 2 ml methanol). To each sample, 60 l of antioxidant
 cocktail (0.2 mg/ml butylated hydroxytoluene, 0.2 mg/ml
 triphenylphosphine, 0.6 mg/ml EDTA) was added to achieve a total cocktail
 concentration of 5% (v/v). Cells and supernatants within each well were
 pooled together, then samples were frozen at -80 C until liquid
 chromatography-mass spectrometry (LC-MS) analysis. LIQUID
 CHROMATOGRAPHY-MASS SPECTROMETRY (LC-MS) ANALYSIS Targeted LC-MS
 lipidomics for 156 lipid metabolites was conducted at the Lipidomics Core
 Facility at Wayne State University. 100 l aliquots of cellular samples
 were thawed and spiked with a cocktail of deuterated internal standards (5
 ng each of PGE1-d4, RvD2-d5, LTB4-d4, and 15[S]-HETE-d8; Cayman Chemical,
 Ann Arbor, MI) for quantification of oxylipins and recovery. Then, fatty
 acyl lipid metabolites were extracted by using C18 extraction columns that
 were washed with 15% (v/v) methanol and subsequently hexane, dried in a
 vacuum, eluted with methanol containing 0.1% (v/v) formic acid, dried
 under nitrogen gas, and dissolved in a 1:1 mixture of methanol:25 mM
 aqueous ammonium acetate. Extracted fatty acyl metabolites were subjected
 to high-performance liquid chromatography (HPLC) using a Luna C18 (3 m,
 2.1150 mm) column connected to a Prominence XR system (Shimadzu, Somerset,
 NJ) then analyzed with a QTrap5500 mass spectrometer (AB Sciex, Singapore)
 set to negative ion mode. Analyst 1.6 software (AB Sciex) and MultiQuant
 software (AB Sciex) were used to collect and quantify the data in units of
 ng, respectively. DATA ANALYSIS AND STATISTICS MetaboAnalyst Version 5.0
 (Xia Lab, Quebec, Canada,
 [www.metaboanalyst.ca/](http://www.metaboanalyst.ca/)) was used to conduct
 statistical analyses. First, raw ng values were converted to corresponding
 pmol values in Microsoft Excel. Then, in MetaboAnalyst, the one factor
 statistical analysis module was chosen, and data were uploaded as a comma
 separated values (.csv) file with samples in unpaired columns and features
 (i.e., metabolites) in rows. Features with &gt;70% missing data were
 removed from the dataset, and remaining missing values were estimated by
 replacing with the corresponding limits of detection (LODs; 1/5 of the
 minimum positive value of each variable). After the data was cleaned, the
 data was normalized by auto scaling only, then the data editor option was
 used to select experimental groups of interest to compare. For comparisons
 between experimental groups, one-way analysis of variance (ANOVA) (FDR =
 0.05) followed by Tukeys honestly significant difference (HSD) post-hoc
 test was used, with FDR q &lt; 0.05 considered statistically significant.
 Instrument- or software-specific information needed to interpret the data:
 Microsoft Excel, MetaboAnalyst Version 5.0 (Xia Lab, Quebec, Canada,
 [www.metaboanalyst.ca/](http://www.metaboanalyst.ca/))
 Environmental/experimental conditions: Fetal liver-derived alveolar-like
 macrophages (FLAMs) were seeded in 6-well plates at a density of 4.5010^5
 cells/well in complete FLAM medium (RPMI 1640, 10% fetal bovine serum, 1%
 penicillin-streptomycin, 30 ng/ml GM-CSF, 20 ng/ml TGF-beta). Cells were
 incubated overnight to achieve 70-90% confluency before beginning
 treatments. The next day, cells washed once with sterile PBS, fresh
 complete FLAM medium containing either 25 uM ethanolic DHA or ethanol
 vehicle (VEH) added, then cells incubated for 24 h. Following DHA or VEH
 treatment, cells were washed once with sterile PBS, treated with either 20
 ng/ml lipopolysaccharide (LPS) or VEH in DPBS+/+ (DPBS containing calcium
 and magnesium) for 2 h, then exposed to 12.5 ug/cm2 crystalline silica
 (cSiO2) or VEH for 1.5 or 4 h. Cell culture supernatants and cells were
 pooled together and collected at t = 2 h (after 2 h LPS priming), 3.5 h
 (after 1.5 h cSiO2 exposure), and 6 h (after 4 h cSiO2 exposure). LPS and
 cSiO2 exposures were done in DPBS+/+ to minimize interference from fatty
 acids present in cell culture medium during oxylipin analyses. Describe
 any quality-assurance procedures performed on the data: People involved
 with sample collection, processing, analysis, and/or submission: Olivia
 Favor, Dr. Lichchavi Rajasinghe, Dr. Krishna Maddipati, Dr. James Pestka #
 DATA-SPECIFIC INFORMATION FOR: Oxylipin Profiling Data Number of
 variables: Three variables - 1) exposure (VEH, LPS, cSiO2, LPS+cSiO2), 2)
 treatment (VEH, DHA), and 3) timepoint (2, 3.5, 6) Variable list: VEH -
 PBS vehicle LPS - lipopolysaccharide, 20 ng/ml cSiO2 - crystalline silica,
 12.5 ug/cm^2 DHA - docosahexaenoic acid, 25 uM ## SHEET 1 - Key
 Description: Contains Sample ID numbers and corresponding
 exposure/treatment groups with timepoints Number of cases/rows: 61 Missing
 data codes: None Specialized formats of other abbreviations used: VEH -
 PBS vehicle LPS - lipopolysaccharide, 20 ng/ml cSiO2 - crystalline silica,
 12.5 ug/cm^2 LPS+cSiO2 - simultaneous LPS (20 ng/ml) and cSiO2 (12.5
 ug/cm^2) exposure DHA - docosahexaenoic acid, 25 uM ## SHEET 2 - Area
 Ratio Description: Ratio of the chromatographic peak area to that of the
 Internal Standard added to the sample at the start of sample processing
 for analysis. Number of cases/rows: 147 Missing data codes: N.D. - not
 determined Specialized formats of other abbreviations used: VEH - PBS
 vehicle LPS - lipopolysaccharide, 20 ng/ml cSiO2 - crystalline silica,
 12.5 ug/cm^2 LPS+cSiO2 - simultaneous LPS (20 ng/ml) and cSiO2 (12.5
 ug/cm^2) exposure DHA - docosahexaenoic acid, 25 uM ## SHEET 3 - SN
 Description: Signal to noise ratio of the detected peak in the
 chromatogram. Minimum acceptable for quantitation: 3. Number of
 cases/rows: 147 Missing data codes: N.D. - not determined Specialized
 formats of other abbreviations used: VEH - PBS vehicle LPS -
 lipopolysaccharide, 20 ng/ml cSiO2 - crystalline silica, 12.5 ug/cm^2
 LPS+cSiO2 - simultaneous LPS (20 ng/ml) and cSiO2 (12.5 ug/cm^2) exposure
 DHA - docosahexaenoic acid, 25 uM ## SHEET 4 - Quality Description:
 Chromatographic peak quality based on symmetry, noise, data points, etc.
 Range 0 1. Minimum accepted for quantitation 0.2. Number of cases/rows:
 147 Missing data codes: N.D. - not determined Specialized formats of other
 abbreviations used: VEH - PBS vehicle LPS - lipopolysaccharide, 20 ng/ml
 cSiO2 - crystalline silica, 12.5 ug/cm^2 LPS+cSiO2 - simultaneous LPS (20
 ng/ml) and cSiO2 (12.5 ug/cm^2) exposure DHA - docosahexaenoic acid, 25 uM
 ## SHEET 5 - Validity\&Calc Description: Calculated data of each
 analyte after validating that the SN &gt; 3 and Quality &gt; 0.2. The
 calculation includes multiplication of the Area Ratio with the quantity of
 internal standard used and adjustment to any dilutions. Number of
 cases/rows: 147 Missing data codes: #VALUE! - value that fails validation
 (SN &lt; 3 and/or Quality &lt; 0.2) Specialized formats of other
 abbreviations used: VEH - PBS vehicle LPS - lipopolysaccharide, 20 ng/ml
 cSiO2 - crystalline silica, 12.5 ug/cm^2 LPS+cSiO2 - simultaneous LPS (20
 ng/ml) and cSiO2 (12.5 ug/cm^2) exposure DHA - docosahexaenoic acid, 25 uM
 ## SHEET 6 - ng per sample Description: Provides quantities of oxylipins
 in each sample in units of ng/sample. This worksheet is essentially the
 same as Validity&Calc data but without the formulas and other
 qualification information. Number of cases/rows: 147 Missing data codes:
 N.D. - not determined Specialized formats of other abbreviations used: VEH
 - PBS vehicle LPS - lipopolysaccharide, 20 ng/ml cSiO2 - crystalline
 silica, 12.5 ug/cm^2 LPS+cSiO2 - simultaneous LPS (20 ng/ml) and cSiO2
 (12.5 ug/cm^2) exposure DHA - docosahexaenoic acid, 25 uM # DATA-SPECIFIC
 INFORMATION FOR: MS Spectra for Representative Oxylipins Description: Zip
 file provides mass spectrometry (MS) spectra for representative omega-6
 eicosanoids (i.e., PGE2, LTB4, TXB2), hydroxy-fatty acids (i.e., 5-HETE,
 5-HEPE, 4-HDoHE), epoxy-fatty acids (i.e., 14,15-EpETrE, 19,20-EpDPE),
 dihydroxy-fatty acids (i.e., 14,15-DiHETrE, 19,20-DiHDoPE), and
 specialized pro-resolving mediators (i.e., RvD6, MaR1[n-3 DPA]). Each MS
 spectrum contains an abbreviation for a representative oxylipin (e.g.,
 4-HDoHE), blue peaks labeled with mass-to-charge (m/z) ratios (given in
 Daltons, Da) for the most characteristic molecular ion fragments, negative
 enhanced product ion (-EPI) scan with m/z ratio for specific molecular ion
 fragment analyzed, charge, collision energy (CE), collision energy spread
 (CES), fourier transform (FT), and counts per second (cps). Number of
 cases/rows: 12 oxylipins: 4-HDoHE, 5-HEPE, 5-HETE, 14(15)-EpETrE,
 14-15-DiHETrE, 19(20)-EpDPE, 19-20-DiHDoPE, LTB4, MaR1(n-3-DPA), PGE2,
 RvD6, TxB2 Missing data codes: N/A Specialized formats of other
 abbreviations used: 4-HDoHE: 4-hydroxydocosahexaenoic acid 5-HEPE:
 5-hydroxyeicosapentaenoic acid 5-HETE: 5-hydroxyeicosatetraenoic acid
 14(15)-EpETrE: 14,15-epoxyeicosatrienoic acid 14-15-DiHETrE:
 14,15-dihydroxyeicosatrienoic acid 19(20)-EpDPE:
 19,20-epoxydocosapentaenoic acid 19-20-DiHDoPE:
 19,20-dihydroxydocosapentaenoic acid LTB4: leukotriene B4 MaR1(n-3-DPA):
 maresin-1, derived from omega-3 DPA PGE2: prostaglandin E2 RvD6: resolvin
 D6 TxB2: thromboxane B2 m/z: mass-to-charge ratio, given in Daltons (Da)
 Rel. Int.: relative intensity or abundance of molecular ion fragments,
 given in percentage (%) -EPI: negative enhanced product ion scan CE:
 collision energy CES: collision energy spread FT: fourier transform cps:
 counts per second # Sharing/access Information Licenses/restrictions
 placed on the data: None Links to publications that cite or use the data:
 https://doi.org/10.3389/fimmu.2023.1274147 Links to other publicly
 accessible locations of the data: None Links/relationships to ancillary
 data sets: None Was data derived from another source? No If yes, list
 source(s): Recommended citation for this dataset: doi:
 10.3389/fimmu.2023.1274147 CC0 license waiver: The uploaded supplemental
 files are compatible with the CC0 license waiver required by Dryad for
 publication and are not included within the related manuscript.