Data from: Dietary docosahexaenoic acid supplementation inhibits acute pulmonary transcriptional and autoantibody responses to a single crystalline silica exposure in lupus-prone mice
Data files
May 02, 2023 version files 814.80 KB
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Dryad_File_1_-_Normalized_log2_mRNA_Counts.xlsx
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Dryad_File_2_-_Autoantibody_Array_Data_(IgG).xlsx
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Dryad_File_3_-_Lung_Lipid_Results.xlsx
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README.md
Nov 05, 2023 version files 2.19 MB
Jan 27, 2024 version files 2.19 MB
Abstract
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.
README: 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: chauhanp@chop.edu
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: pestka@msu.edu
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: abby.benninghoff@usu.edu
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: favoroli@msu.edu
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: wagnerja@msu.edu
Author/Alternate Contact Information
Name: Ryan Lewandowski
ORCID: None
Institution: Michigan State University
Address: 1129 Farm Ln, Office 218, East Lansing, MI 48824
Email: lewando2@msu.edu
Author/Alternate Contact Information
Name: Dr. Lichchavi Rajasinghe
ORCID: 0000-0002-5136-7422
Institution: AstraZeneca
Address: 1800 Concord Pike, Wilmington, DE 19803
Email: lichchavi.rajasinghe@astrazeneca.com
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: harkemaj@msu.edu
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 >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 >0.58 or <-0.58) with BH q <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) (
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 (cSiO2). 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) cSiO2/Con, and 3) cSiO2/DHA. One cohort of mice was sacrificed at time = D7 (7 days post-cSiO2 instillation) and time = D28 (28 days post-cSiO2 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
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: 25
Missing data codes: None
Specialized formats of other abbreviations used:
Veh_Con - Veh/Con experimental group
cSiO2_Con - cSiO2/Con experimental group
cSiO2_DHA - cSiO2/DHA experimental group
D7 - 7 days post-cSiO2 instillation
D28 - 28 days post-cSiO2 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 - cSiO2/Con experimental group
cSiO2_DHA - cSiO2/DHA experimental group
D7 - 7 days post-cSiO2 instillation
D28 - 28 days post-cSiO2 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 - cSiO2/Con experimental group
cSiO2_DHA - cSiO2/DHA experimental group
D7 - 7 days post-cSiO2 instillation
D28 - 28 days post-cSiO2 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-cSiO2 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-cSiO2 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-cSiO2 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 - cSiO2/Con experimental group
cSiO2_DHA - cSiO2/DHA experimental group
D7 - 7 days post-cSiO2 instillation
D28 - 28 days post-cSiO2 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 - cSiO2/Con experimental group
cSiO2_DHA - cSiO2/DHA experimental group
D7 - 7 days post-cSiO2 instillation
D28 - 28 days post-cSiO2 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 - cSiO2/Con experimental group
cSiO2_DHA - cSiO2/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 - cSiO2/Con experimental group
cSiO2_DHA - cSiO2/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 - cSiO2/Con experimental group
cSiO2_DHA - cSiO2/DHA experimental group
D7 - 7 days post-cSiO2 instillation
D28 - 28 days post-cSiO2 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 - cSiO2/Con experimental group
cSiO2_DHA - cSiO2/DHA experimental group
D7 - 7 days post-cSiO2 instillation
D28 - 28 days post-cSiO2 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 - cSiO2/Con experimental group
cSiO2_DHA - cSiO2/DHA experimental group
D7 - 7 days post-cSiO2 instillation
D28 - 28 days post-cSiO2 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
Methods
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 >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: 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 >0.58 or <-0.58) with BH q <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) (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).
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 -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.