DOI: 10.5061/dryad.5mkkwh79g
title: Data from: Dietary docosahexaenoic acid supplementation inhibits acute pulmonary transcriptional and autoantibody responses to a single crystalline silica exposure in lupus-prone mice

description: Short-term repeated intranasal exposure crystalline silica (cSiO2), a
 known human autoimmune trigger, induces uncontrolled inflammation,
 upregulated IFN-stimulated gene expression, diverse autoantibody
 production, and glomerulonephritis in lupus-prone female NZBWF1 mice.
 Dietary supplementation with the omega-3 fatty acid docosahexaenoic acid
 (DHA) prevents subchronic cSiO2 triggering of these lupus hallmarks. To
 understand how this intervention impacts acute effects of cSiO2, we fed
 NZBWF1 mice control (CON) or DHA-containing diet, subjected them to a
 single acute intranasal instillation of 2.5 mg cSiO2, then compared
 pulmonary inflammatory/autoimmune responses and autoimmune-related gene
 expression in experimental cohorts terminated at 7 and 28 d
 post-instillation (PI). Acute cSiO2 exposure of CON-fed mice elicited
 decreased macrophage and increased neutrophil numbers at 7 d PI, whereas
 at 28 d PI, CON-fed mice treated with particle displayed elevated total
 cell, macrophage, neutrophil, and lymphocyte counts. In contrast, DHA-fed
 mice treated with cSiO2 exhibited less macrophage loss at 7 d PI and
 reduced total cell, macrophage, and lymphocyte accumulation at 28 d PI.
 Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) of
 lung sections suggested that cSiO2 induced more robust cell death at 7 d
 PI in CON-fed than DHA-fed mice. Targeted multiplex ELISA of lung extracts
 showed that at 28 d PI, cSiO2 induced higher concentrations of
 inflammation-associated cytokines (IL-1α, IL-6, and GM-CSF) and
 IFN-stimulated chemokines (CCL2, CCL3, CXCL10) in the CON-fed cohort than
 in the DHA-fed cohort. Autoantigen protein microarray of BALF collected at
 28 d PI indicated that cSiO2 induced higher autoantibody responses for
 representative nuclear, ribosomal, mitochondrial, and complement proteins
 in CON-fed mice than DHA-fed mice. Gene expression analyses with
 NanoString Autoimmune Gene Expression assay revealed greater
 cSiO2-triggered upregulation of genes associated with TLR activation, DNA
 signaling, proinflammatory cytokines,  chemokines, type 1 and 2
 IFN response signatures, lymphocyte trafficking, MHC class 1 antigen
 presentation, B and T cell activation at 7 and 28 d PI in CON-fed mice
 than those fed DHA. Ingenuity Pathway Analysis (IPA) further demonstrated
 that DHA supplementation quelled cSiO2-induced responses top upstream
 regulators of proinflammatory and IFN-regulated gene networks to observed
 in CON-fed mice. Altogether, this short-term model illustrated that DHA
 suppression of aberrant acute cSiO2-induced inflammation is linked to
 altered regulation of autoimmune-related gene expression in lupus-prone
 mice.
description: <strong>NanoString autoimmune
 gene profiling </strong> RNA was extracted from day 7 and day 28
 lung tissue (n=4 samples/gp) using Tri Reagent (Sigma Aldrich, St. Louis,
 MO) and TissueLyser II (Qiagen). Extracted RNA was purified using a Zymo
 RNA Clean and Concentrator Kit with DNase according to the
 manufacturer's instructions (Zymo Research, Irvine, CA, cat no-
 R1017). The resulting RNA was dissolved in nuclease-free water and
 quantified using the Qubit (Thermo Fisher Scientific). RNA integrity was
 assessed using the Bioanalyzer (Agilent Technologies) at the MSU Genomics
 Core. Samples with RNA integrity &gt;8 were analyzed using NanoString
 Autoimmune Gene Expression assay (Cat # XT-CSO-MAIPI1-12) at the MSU
 Genomics Core. Assays were performed and quantified on the nCounter MAX
 system, sample preparation station, and digital analyzer (NanoString
 Technologies) according to the manufacturer’s instructions. Data were
 processed using the nSolver 4.0 advance analysis as described previously
 (6) using NanoString’s software nSolver v3.0.22 with the Advanced Analysis
 Module v2.0. Background subtraction was performed using the eight negative
 controls included with the module. Genes with counts below a threshold of
 2σ of the mean background signal were excluded from subsequent analysis.
 Differential gene expression analyses were performed as previously
 described (6). Three pairwise comparisons within each time point (day 7
 and day 28) were determined as follows: cSiO<sub>2</sub>/Con
 vs Veh/Con, cSiO<sub>2</sub>/DHA vs Veh/Con, and
 cSiO<sub>2</sub>/Con vs cSiO<sub>2</sub>/DHA. A
 statistically significant difference in gene expression was defined as
 1.5-fold change in expression (log2 &gt;0.58 or &lt;-0.58) with BH
 q &lt;0.05. BioVenn was used to create Venn diagrams of significant
 differentially expressed genes (7) using log<sub>2</sub>
 transcript count data for DEGs.ClustVis (8) was used to perform
 unsupervised hierarchical cluster analyses (HCC) and principal component
 analyses (PCA) (https://biit.cs.ut.ee/clustvis/). Values in heat maps were
 centered by row; imputation was used to estimate missing values. Euclidean
 distance and Ward linkage were used to cluster rows. The volcano plot was
 created using the R Shiny online tool (https://paolo.shinyapps).
 <strong>High throughput
 autoantibody (AAb) microarray profiling </strong> High-throughput profiling of IgG AAb
 against a broad range of autoantigens (AAgs) was performed at the
 Microarray and Immune Phenotyping Core Facility at The University of Texas
 Southwestern Medical Center using AAg coated protein arrays as described
 previously (5). Briefly, BALF samples (n=8 samples/gp) were first treated
 with DNAse I to remove free-DNA. Then samples were diluted 1:25 and
 hybridized to protein array plates coated with 122 antigens and 6
 controls. The antibodies binding with the antigens on plate were detected
 with Cy3-conjugated anti-mouse IgG(1:2000, Jackson ImmunoResearch
 Laboratories, PA) and fluorescent images captured with a Genepix 4200A
 scanner (Molecular Devices, CA). Fluorescent images were transformed to
 signal intensity values using GenePix 7.0 software and background
 subtracted and normalized to internal controls for IgG. The processed
 signal intensity value for each AAb was reported as antibody score
 (Ab-score), which is expressed based on the normalized signal intensity
 and signal-to-noise ratio (SNR) using the formula: Ab-score =
 log<sub>2</sub>(NSI x SNR + 1). Normalized and unit
 variance-scaled Ab-score values were represented with heat maps.
 <strong>Fatty acid
 analysis</strong> Lung tissues (n=2 samples/gp) were kept at
 -80°C until the fatty acid analysis. As previously described, gas
 chromatography (GC) with flame ionization detection was used for fatty
 acid analysis at Omega Quant, LLC (Sioux Falls, SD). (4). Fatty acids were
 identified by comparison with a standard mixture of fatty acids (GLC 782,
 NuCheck Prep) and an internal standard (C23:0 FAME, NuCheck Prep).
 Di-C23:0 PL was used to calculate recovery efficiency of the assay and
 applied to all fatty acids. The following 24 fatty acids (by class) were
 identified: saturated (14:0, 16:0, 18:0, 20:0, 22:0 24:0); cis
 monounsaturated (16:1, 18:1, 20:1, 24:1); trans (16:1, 18:1, 18:2 ), cis
 n-6 polyunsaturated (18:2, 18:3, 20:2, 20:3, 20:4, 22:4, 22:5); cis n-3
 polyunsaturated (18:3, 20:5, 22:5, 22:6). The composition of fatty acids
 expressed as a percentage of total identified fatty acids.
description: # Data from: Dietary docosahexaenoic acid supplementation inhibits acute
 pulmonary transcriptional and autoantibody responses to a single
 crystalline silica exposure in lupus-prone mice Author/Principal
 Investigator Information Name: Dr. Preeti Chauhan ORCID:
 0000-0001-8806-9552 Institution: Children's Hospital of Philadelphia
 Address: 3401 Civic Center Blvd, Philadelphia, PA 19104 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. Abby Benninghoff ORCID: 0000-0002-7993-0117 Institution: Utah
 State University Address: 4815 Old Main Hill, AGRS 427, Logan, UT
 84322-4815 Email: Author/Alternate
 Contact 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/Alternate Contact Information Name: Dr. James Wagner ORCID: None
 Institution: Michigan State University Address: 1129 Farm Ln, Room 211,
 East Lansing MI 48824 Email: Author/Alternate
 Contact Information Name: Ryan Lewandowski ORCID: None Institution:
 Michigan State University Address: 1129 Farm Ln, Office 218, East Lansing,
 MI 48824 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. Jack Harkema ORCID: 0000-0003-4682-0824
 Institution: Michigan State University Address: 1129 Farm Lane, Room 212,
 East Lansing, MI 48824 Email: Date of data
 collection: 2018-2019 Geographic location of data collection: 1) Michigan
 State University, East Lansing, MI 48824; 2) Utah State University, Logan,
 UT 84322. Information about funding sources that supported the collection
 of the data: National Institute of Environmental Health Sciences,
 ES027353. --- # Description of the Data and File structure File List: File
 1 - Normalized log2 mRNA Counts and log2 ratios: contains normalized log2
 mRNA counts and log2 ratios for 750 genes in the lung File 2 -
 Autoantibody Array Data (IgG): contains antibody scores (Ab-scores) for
 120 IgG autoantibody classes in the bronchoalveolar lavage fluid (BALF)
 File 3 - Lung Lipid Results: contains masses of individual and total fatty
 acids in the lung and composition of fatty acids expressed as percentage
 of total identified fatty acids in the lung 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? Yes If
 yes, name of file(s) that was updated: File 1 - Normalized log2 mRNA
 Counts and log2 Ratios Why was the file updated? This file was updated
 because the previous version only contained normalized log2 mRNA counts.
 The current version not only contains the original normalized log2 mRNA
 counts but also log2 ratios comparing all groups to a control group. When
 was the file updated? 11/1/2023 # Methodological Information NANOSTRING
 AUTOIMMUNE GENE PROFILING RNA was extracted from day 7 and day 28 lung
 tissue (n=4 samples/gp) using Tri Reagent (Sigma Aldrich, St. Louis, MO)
 and TissueLyser II (Qiagen). Extracted RNA was purified using a Zymo RNA
 Clean and Concentrator Kit with DNase according to the manufacturer's
 instructions (Zymo Research, Irvine, CA, cat no- R1017). The resulting RNA
 was dissolved in nuclease-free water and quantified using the Qubit
 (Thermo Fisher Scientific). RNA integrity was assessed using the
 Bioanalyzer (Agilent Technologies) at the MSU Genomics Core. Samples with
 RNA integrity &gt;8 were analyzed using NanoString Autoimmune Gene
 Expression assay (Cat # XT-CSO-MAIPI1-12) at the MSU Genomics Core. Assays
 were performed and quantified on the nCounter MAX system, sample
 preparation station, and digital analyzer (NanoString Technologies)
 according to the manufacturers instructions. Data were processed using the
 nSolver 4.0 advance analysis as described previously (6) using NanoStrings
 software nSolver v3.0.22 with the Advanced Analysis Module v2.0.
 Background subtraction was performed using the eight negative controls
 included with the module. Genes with counts below a threshold of 2 of the
 mean background signal were excluded from subsequent analysis.
 Differential gene expression analyses were performed as previously
 described (6). Three pairwise comparisons within each time point (day 7
 and day 28) were determined as follows: cSiO2/Con vs Veh/Con, cSiO2/DHA vs
 Veh/Con, and cSiO2/Con vs cSiO2/DHA. A statistically significant
 difference in gene expression was defined as 1.5-fold change in expression
 (log2 &gt;0.58 or &lt;-0.58) with BH q &lt;0.05. BioVenn was used to
 create Venn diagrams of significant differentially expressed genes (7)
 using log2 transcript count data for DEGs.ClustVis (8) was used to perform
 unsupervised hierarchical cluster analyses (HCC) and principal component
 analyses (PCA) (). Values in heat
 maps were centered by row; imputation was used to estimate missing values.
 Euclidean distance and Ward linkage were used to cluster rows. The volcano
 plot was created using the R Shiny online tool
 (). HIGH THROUGHPUT AUTOANTIBODY (AAb)
 MICROARRAY PROFILING High-throughput profiling of IgG AAb against a broad
 range of autoantigens (AAgs) was performed at the Microarray and Immune
 Phenotyping Core Facility at The University of Texas Southwestern Medical
 Center using AAg coated protein arrays as described previously (5).
 Briefly, BALF samples (n=8 samples/gp) were first treated with DNAse I to
 remove free-DNA. Then samples were diluted 1:25 and hybridized to protein
 array plates coated with 122 antigens and 6 controls. The antibodies
 binding with the antigens on plate were detected with Cy3-conjugated
 anti-mouse IgG(1:2000, Jackson ImmunoResearch Laboratories, PA) and
 fluorescent images captured with a Genepix 4200A scanner (Molecular
 Devices, CA). Fluorescent images were transformed to signal intensity
 values using GenePix 7.0 software and background subtracted and normalized
 to internal controls for IgG. The processed signal intensity value for
 each AAb was reported as antibody score (Ab-score), which is expressed
 based on the normalized signal intensity and signal-to-noise ratio (SNR)
 using the formula: Ab-score = log2(NSI x SNR + 1). Normalized and unit
 variance-scaled Ab-score values were represented with heat maps. FATTY
 ACID ANALYSIS Lung tissues (n=2 samples/gp) were kept at -80C until the
 fatty acid analysis. As previously described, gas chromatography (GC) with
 flame ionization detection was used for fatty acid analysis at Omega
 Quant, LLC (Sioux Falls, SD). (4). Fatty acids were identified by
 comparison with a standard mixture of fatty acids (GLC 782, NuCheck Prep)
 and an internal standard (C23:0 FAME, NuCheck Prep). Di-C23:0 PL was used
 to calculate recovery efficiency of the assay and applied to all fatty
 acids. The following 24 fatty acids (by class) were identified: saturated
 (14:0, 16:0, 18:0, 20:0, 22:0 24:0); cis monounsaturated (16:1, 18:1,
 20:1, 24:1); trans (16:1, 18:1, 18:2 ), cis n-6 polyunsaturated (18:2,
 18:3, 20:2, 20:3, 20:4, 22:4, 22:5); cis n-3 polyunsaturated (18:3, 20:5,
 22:5, 22:6). The composition of fatty acids expressed as a percentage of
 total identified fatty acids. Instrument- or software-specific information
 needed to interpret the data: NanoString nSolver Advanced Analysis
 software v3.0.22 Environmental/experimental conditions: Eight-week old
 female lupus-prone NZBWF1 mice were intranasally instilled with saline
 vehicle (Veh) or 2.5 mg crystalline silica (cSiO<sub>2</sub>).
 Mice were maintained on control AIN-93G diet (Con) or diet amended with
 the omega-3 fatty acid DHA (DHA). The experiment groups (n = 8 mice/group)
 are as follows: 1) Veh/Con, 2) cSiO<sub>2</sub>/Con, and 3)
 cSiO<sub>2</sub>/DHA. One cohort of mice was sacrificed at
 time = D7 (7 days post-cSiO<sub>2</sub> instillation) and time
 = D28 (28 days post-cSiO<sub>2</sub> instillation). Describe
 any quality-assurance procedures performed on the data: People involved
 with sample collection, processing, analysis, and/or submission: Dr.
 Preeti Chauhan, Dr. Abby Benninghoff, Dr. Olivia Favor, Dr. James Wagner,
 Ryan Lewandowski, Dr. Lichchavi Rajasinghe, Dr. Jack Harkema, and Dr.
 James Pestka # DATA-SPECIFIC INFORMATION FOR: Normalized log2 mRNA Counts
 Number of variables: Two variables - 1) silica exposure and 2)
 experimental diet (CON, DHA) Variable list: Veh - saline vehicle
 cSiO<sub>2</sub> - crystalline silica, 2.5 mg Con - control
 AIN-93G diet DHA - DHA-amended diet (10 g/kg diet) ## SHEET 1 - Key
 Description: Contains Sample ID numbers and corresponding experimental
 treatment groups with timepoints. Number of cases/rows: 25 Missing data
 codes: None Specialized formats of other abbreviations used: Veh_Con -
 Veh/Con experimental group cSiO2_Con - cSiO<sub>2</sub>/Con
 experimental group cSiO2_DHA - cSiO<sub>2</sub>/DHA
 experimental group D7 - 7 days post-cSiO<sub>2</sub>
 instillation D28 - 28 days post-cSiO<sub>2</sub> instillation
 ## SHEET 2 - Normalized log2 mRNA counts Description: Contains normalized
 log2 mRNA counts for 750 genes in the lung. Number of cases/rows: 753
 Missing data codes: None Specialized formats of other abbreviations used:
 Veh_Con - Veh/Con experimental group cSiO2_Con -
 cSiO<sub>2</sub>/Con experimental group cSiO2_DHA -
 cSiO<sub>2</sub>/DHA experimental group D7 - 7 days
 post-cSiO<sub>2</sub> instillation D28 - 28 days
 post-cSiO<sub>2</sub> instillation ## SHEET 3 - Linear counts
 Description: Contains linear mRNA counts for 750 genes in the lung.
 Calculated from Sheet 2 by using the formula: Linear mRNA count = 2^(log2
 mRNA count). Number of cases/rows: 753 Missing data codes: None
 Specialized formats of other abbreviations used: Veh_Con - Veh/Con
 experimental group cSiO2_Con - cSiO<sub>2</sub>/Con
 experimental group cSiO2_DHA - cSiO<sub>2</sub>/DHA
 experimental group D7 - 7 days post-cSiO<sub>2</sub>
 instillation D28 - 28 days post-cSiO<sub>2</sub> instillation
 ## SHEET 4 - Reference average Description: Contains individual and
 average linear mRNA counts for all samples in the Veh/Con experimental
 group at 7 days post-cSiO<sub>2</sub> instillation for 750
 genes in the lung. Number of cases/rows: 753 Missing data codes: None
 Specialized formats of other abbreviations used: Veh_Con - Veh/Con
 experimental group D7 - 7 days post-cSiO<sub>2</sub>
 instillation ## SHEET 5 - Average for all cells Description: Contains
 copies of average linear mRNA counts from Sheet 4 in all columns
 corresponding to experimental groups. This sheet was added to make the
 subsequent Excel calculations easier. Number of cases/rows: 753 Missing
 data codes: None Specialized formats of other abbreviations used: AVG
 Veh_Con - average linear mRNA counts from the Veh/Con experimental group
 at 7 days post-cSiO2 instillation D7 - 7 days
 post-cSiO<sub>2</sub> instillation ## SHEET 6 - Ratio sample
 vs avg ref Description: Contains ratios between individual linear mRNA
 counts from Sheet 3 and average linear mRNA counts from Sheet 5 for 750
 genes in the lung. Number of cases/rows: 753 Missing data codes: None
 Specialized formats of other abbreviations used: Veh_Con - Veh/Con
 experimental group cSiO2_Con - cSiO<sub>2</sub>/Con
 experimental group cSiO2_DHA - cSiO<sub>2</sub>/DHA
 experimental group D7 - 7 days post-cSiO<sub>2</sub>
 instillation D28 - 28 days post-cSiO<sub>2</sub> instillation
 ## SHEET 7 - Log2 ratio sample vs avg ref Description: Contains log2
 ratios for 750 genes in the lung. Calculated from Sheet 6 by using the
 formula: log2 ratio = log2(ratio sample vs avg ref) Number of cases/rows:
 753 Missing data codes: None Specialized formats of other abbreviations
 used: Veh_Con - Veh/Con experimental group cSiO2_Con -
 cSiO<sub>2</sub>/Con experimental group cSiO2_DHA -
 cSiO<sub>2</sub>/DHA experimental group D7 - 7 days
 post-cSiO<sub>2</sub> instillation D28 - 28 days
 post-cSiO<sub>2</sub> instillation # DATA-SPECIFIC INFORMATION
 FOR: Autoantibody Array Data (IgG) Number of variables: Two variables - 1)
 silica exposure and 2) experimental diet (CON, DHA) Variable list: Veh -
 saline vehicle cSiO2 - crystalline silica, 2.5 mg Con - control AIN-93G
 diet DHA - DHA-amended diet (10 g/kg diet) ## SHEET 1 - Key Description:
 Contains Sample ID numbers and corresponding experimental treatment groups
 Number of cases/rows: 25 Missing data codes: None Specialized formats of
 other abbreviations used: Veh_Con - Veh/Con experimental group cSiO2_Con -
 cSiO<sub>2</sub>/Con experimental group cSiO2_DHA -
 cSiO<sub>2</sub>/DHA experimental group ## SHEET 2 -
 Autoantibody array data (IgG) Description: Contains antibody scores
 (Ab-scores) for 120 IgG autoantibody classes in the bronchoalveolar lavage
 fluid (BALF) Number of cases/rows: 130 Missing data codes: None
 Specialized formats of other abbreviations used: Veh_Con - Veh/Con
 experimental group cSiO2_Con - cSiO<sub>2</sub>/Con
 experimental group cSiO2_DHA - cSiO<sub>2</sub>/DHA
 experimental group # DATA-SPECIFIC INFORMATION FOR: Lung Lipid Results
 Number of variables: Two variables - 1) silica exposure and 2)
 experimental diet (CON, DHA) Variable list: Veh - saline vehicle cSiO2 -
 crystalline silica, 2.5 mg Con - control AIN-93G diet DHA - DHA-amended
 diet (10 g/kg diet) ## SHEET 1 - Key Description: Contains Sample ID
 numbers and corresponding experimental treatment groups with timepoints
 Number of cases/rows: 13 Missing data codes: None Specialized formats of
 other abbreviations used: Veh_Con - Veh/Con experimental group cSiO2_Con -
 cSiO<sub>2</sub>/Con experimental group cSiO2_DHA -
 cSiO<sub>2</sub>/DHA experimental group D7 - 7 days
 post-cSiO<sub>2</sub> instillation D28 - 28 days
 post-cSiO<sub>2</sub> instillation ## SHEET 2 - Conc. Sample
 Receipt, Results Description: Contains masses of individual and total
 fatty acids in the lung in units of ug fatty acid per mg of lung tissue
 Number of cases/rows: 13 Missing data codes: N/A - not applicable
 Specialized formats of other abbreviations used: Veh_Con - Veh/Con
 experimental group cSiO2_Con - cSiO<sub>2</sub>/Con
 experimental group cSiO2_DHA - cSiO<sub>2</sub>/DHA
 experimental group D7 - 7 days post-cSiO<sub>2</sub>
 instillation D28 - 28 days post-cSiO<sub>2</sub> instillation
 OQ - OmegaQuant ## SHEET 3 - % Conc. Sample Receipt, Results Description:
 Contains composition of fatty acids expressed as percentage of total
 identified fatty acids in the lung Number of cases/rows: 13 Missing data
 codes: N/A - not applicable Specialized formats of other abbreviations
 used: Veh_Con - Veh/Con experimental group cSiO2_Con -
 cSiO<sub>2</sub>/Con experimental group cSiO2_DHA -
 cSiO<sub>2</sub>/DHA experimental group D7 - 7 days
 post-cSiO<sub>2</sub> instillation D28 - 28 days
 post-cSiO<sub>2</sub> instillation OQ - OmegaQuant #
 Sharing/access Information Licenses/restrictions placed on the data: None
 Links to publications that cite or use the data: doi:
 10.3389/fimmu.2024.1275265 Links to other publicly accessible locations of
 the data: None Links/relationships to ancillary data sets: None Was data
 derived from another source? No Recommended citation for this dataset: Not
 at this time