Data from: Crystalline silica-induced proinflammatory eicosanoid storm in novel alveolar macrophage model quelled by docosahexaenoic acid

# 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 >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 < 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 > 3 and Quality > 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 < 3 and/or Quality < 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.