DOI: 10.5061/dryad.8pk0p2nt6
title: NanoString autoimmune profiling panel normalized linear counts and summary of statistical analyses

description: Occupational exposure to respirable crystalline silica (cSiO2) is linked
 to the development of lupus. Preclinical studies have revealed weekly
 repeated intranasal exposure to 1 mg cSiO2 in young (8-11 wk-old) female
 NZBWF1 lupus-prone mice, a life-stage equivalent to 12–20-yr-old humans,
 triggers autoimmunity in the lungs and kidneys that is prevented by
 dietary supplementation with the omega-3 fatty acid docosahexaenoic acid
 (DHA). Methods: Here, we characterized cSiO2's and DHA's effects
 in mature adult (16–19-wk-old) female NZBWF1 mice, an age period that
 coincides with the onset of immunological tolerance breach and that is
 more representative of the age (&gt;20-yr-old) of cSiO2-exposed
 workers. We fed mice either a control diet (CON) or diet amended with DHA
 calorically equivalent to a human daily dose of 5 g. After 2 wk, we
 intranasally instilled them with either saline vehicle (VEH) or 1 mg of
 cSiO2 weekly for 4 wk. Cohorts were terminated 1 and 5 wk after the final
 installation. Lungs were then analyzed for inflammatory cell counts,
 chemokines, histopathology, B-and T-cell infiltration, autoantibody
 profile, and inflammatory/autoimmune gene signatures and results further
 related to autoimmune glomerulonephritis onset. Results: VEH/CON mice
 displayed no lung or kidney pathology at either time point. In contrast,
 cSiO2/CON mice exhibited mild ectopic lymphoid tissue (ELT) formation in
 the lungs at 1 wk, which increased significantly by 5 wk. Lungs from
 cSiO2/CON mice also showed elevations in BALF cellularity, chemokine
 production, CD3 + T-cells, CD45R + B-cells, IgG + plasma cells,
 inflammatory/autoimmune gene expression, IgG autoantibodies. cSiO2/CON
 mice had visible glomerular hypertrophy and IgG deposition. Dietary DHA
 supplementation suppressed all these endpoints. Discussion: Consistent
 with young mice, intranasal cSiO2 exposure in mature adult NZBWF1
 lupusprone mice elicited early pulmonary inflammation that served as a
 nexus for autoimmunity, suggesting these life-stage differences are not
 critical for cSiO2-triggered autoimmune response in this preclinical
 model. DHA supplementation at a translationally relevant human dosage
 effectively mitigated cSiO2-induced inflammation/autoimmunity in mature
 adult mice, resembling the protective effects observed in young mice.
 Together these findings further highlight the therapeutic potential of
 omega-3 fatty acids in mitigating toxicant-triggered autoimmune responses.
description: RNA was extracted from lung tissue (1- and 5-wk PI) with RNeasy
 Mini Kits with DNase treatment (Qiagen, Valencia, CA). RNA was dissolved
 in nuclease-free water, quantified with Qubit (Thermo Fisher Scientific),
 and integrity verified with a TapeStation (Agilent Technologies). Samples
 (RNA integrity &gt;8) were analyzed with NanoString Autoimmune Gene
 Expression assay (XT-CSO-MAIP1-12, NanoString Technologies, Seattle, WA)
 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. Gene expression data were analyzed as
 previously described. Briefly, raw gene expression data were analyzed
 using NanoString’s software nSolver v3.0.22 with the Advanced Analysis
 Module v2.0. For differential gene expression, a statistically significant
 difference in gene expression was defined as a 1.5-fold change in
 expression (log2 &gt;0.58 or &lt;-0.58) with BH q &lt;0.05.
 Two pairwise comparisons within each timepoint (1- and 5-wk PI) were
 determined a priori, as follows: cSiO<sub>2</sub>/CON vs
 VEH/CON and cSiO<sub>2</sub>/CON vs cSiO2/DHA.
 To assess the impact of cSiO<sub>2</sub> exposure and
 experimental diets on annotated gene sets, global and directed
 significance scores were calculated for each pathway at each time point,
 as previously described. The global score estimates the cumulative
 evidence for the differential expression of genes in a pathway. As a
 complementary method for comparing pathways and discriminating between
 experimental groups, pathway Z scores were calculated as the Z-scaled
 first principal component of the pathway genes’ normalized expression.
 ClustVis was used to perform unsupervised hierarchical cluster analyses
 (HCC) using log2 transcript count data for DEGs. Spearman rank
 correlations were performed to examine overall patterns in the gene
 expression profiles using the pathway Z score compared to other biomarkers
 of disease in lung tissues at 1- and 5-wk PI. A significant correlation
 was inferred when ρ &gt;0.5 or &lt;-0.5 and
 p&lt;0.05.
description: This readme file was generated on 2023-05-03 by Lauren Heine GENERAL
 INFORMATION Title of Dataset: Normalized linear counts from NanoString
 autoimmune profiling panel and summary of statistical analyses
 Author/Principal Investigator Information Name: Lauren Heine ORCID:
 0000-0002-5373-2665 Institution: Michigan State University Address: 1129
 Farm Ln, Office 213, East Lansing, MI 48824 Email: heinelau@msu.edu
 Author/Associate or Co-investigator Information Name: Dr. James Pestka
 ORCID: 0000-0003-4689-2756 Institution: Michigan State University Address:
 4176 Biomedical Physical Sciences, East Lansing, MI 48824 Email:
 pestka@msu.edu Date of data collection: 2022-06-24 - 2022-12-31 Geographic
 location of data collection: Michigan State University, East Lansing, MI
 48823 Information about funding sources that supported the collection of
 the data: National Institute of Environmental Health Sciences, ES027353
 SHARING/ACCESS INFORMATION Licenses/restrictions placed on the data: None
 Links to publications that cite or use the data: Currently in submission
 process 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: Not at this time. DATA & FILE OVERVIEW File List: File 1
 - Normalized Linear Counts from NanoString Autoimmunity Profiling Panel:
 contains normalized linear counts for 770 genes in lung tissue separated
 by timepoint (1 or 5 wk PI). File 2 - Summary of Statistical Analyses:
 contains log2-fold change values, p-values, and statistical comparisons
 between exposure/treatment groups for 770 genes separated by timepoint (1
 or 5 wk PI). 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 RNA was extracted from lung tissue (1- and 5-wk
 PI) with RNeasy Mini Kits with DNase treatment (Qiagen, Valencia, CA). RNA
 was dissolved in nuclease-free water, quantified with Qubit (Thermo Fisher
 Scientific), and integrity verified with a TapeStation (Agilent
 Technologies). Samples (RNA integrity &gt;8) were analyzed with
 NanoString Autoimmune Gene Expression assay (XT-CSO-MAIP1-12, NanoString
 Technologies, Seattle, WA) 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. Methods for processing data: Gene expression data was
 analyzed as performed as previously described (14, 22, 31, 32). Briefly,
 raw gene expression data were analyzed using NanoString’s software nSolver
 v3.0.22 with the Advanced Analysis Module v2.0. For differential gene
 expression, 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. Two pairwise comparisons within
 each timepoint (1 and 5 wk PI) were determined a priori, as follows:
 cSiO2/CON vs VEH/CON and cSiO2/CON vs cSiO2/DHA. To assess the impact of
 cSiO2 exposure and experimental diets on annotated gene sets, global and
 directed significance scores were calculated for each pathway at each time
 point, as previously described (32). The global score estimates the
 cumulative evidence for the differential expression of genes in a pathway.
 As a complementary method for comparing pathways and discriminating
 between experimental groups, pathway Z scores were calculated as the
 Z-scaled first principal component of the pathway genes’ normalized
 expression. ClustVis (33) was used to perform unsupervised hierarchical
 cluster analyses (HCC) using log2 transcript count data for DEGs. Spearman
 rank correlations were performed to examine overall patterns in the gene
 expression profiles using the pathway Z score compared to other biomarkers
 of disease in lung tissues at 1- and 5-wk PI. A significant correlation
 was inferred when ρ &gt;0.5 or &lt;-0.5 and p&lt;0.05.
 Instrument- or software-specific information needed to interpret the data:
 NanoString nSolver Advanced Analysis software v3.0.22 Standards and
 calibration information, if appropriate: Data normalization was performed
 on background-subtracted samples using internal positive controls and
 selected housekeeping genes that were identified with the geNorm algorithm
 (https://genorm.cmgg.be/). Environmental/experimental conditions: Female
 lupus-prone NZBWF1 mice were intranasally instilled with saline vehicle
 (VEH) or 1 mg crystalline silica (cSiO2) once per week for 4 consecutive
 weeks. Mice were maintained on either control AIN-93G diet (CON) or a
 DHA-amended diet (5 g/d). Mice were scarificed at either 1 or 5 wk
 following the last cSiO2 instillation. The experiment groups at each
 timepoint are as follows: 1) VEH/CON, 2) cSiO2/CON, 3) cSiO2/DHA. Describe
 any quality-assurance procedures performed on the data: People involved
 with sample collection, processing, analysis and/or submission: Lauren
 Heine, Dr. Abby Benninghoff, and Dr. James Pestka DATA-SPECIFIC
 INFORMATION FOR: Normalized Linear Counts from NanoString Autoimmunity
 Profiling Panel Number of variables: Three variables - 1) cSiO2 exposure,
 2) DHA treatment, and 3) time post final cSiO2 instillation Number of
 cases/rows: 752 Variable List: VEH - saline vehicle cSiO2 - crystalline
 silica, 1 mg CON - control AIN-93G diet DHA - DHA-amended diet (5 g/d
 diet) 1 wk PI - 1 wk post final cSiO2 instillation 5 wk PI - 5 wk post
 final cSiO2 instillation Missing data codes: None Specialized formats or
 other abbreviations used: None DATA-SPECIFIC INFORMATION FOR: Summary of
 Statistical Analyses Number of variables: Three variables - 1) cSiO2
 exposure, 2) DHA treatment, and 3) time post final cSiO2 instillation
 Number of cases/rows: 752 Variable List: VEH - saline vehicle cSiO2 -
 crystalline silica, 1 mg CON - control AIN-93G diet DHA - DHA-amended diet
 (5 g/d diet) 1 wk PI - 1 wk post final cSiO2 instillation 5 wk PI - 5 wk
 post final cSiO2 instillation Missing data codes: None Specialized formats
 or other abbreviations used: Conditional formatting: Red = Log2 fold
 change &lt; -0.585 Green = Log2 fold change &gt; 0.585 Yellow =
 BH.p-value &lt; 0.05