NanoString autoimmune profiling panel normalized linear counts and summary of statistical analyses
Data files
Oct 05, 2023 version files 2.22 MB
Abstract
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 (>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.
README
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 >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 >0.58 or <-0.58) with BH q <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 >0.5 or <-0.5 and p<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 < -0.585
Green = Log2 fold change > 0.585
Yellow = BH.p-value < 0.05
Methods
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 >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 >0.58 or <-0.58) with BH q <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. 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 >0.5 or <-0.5 and p<0.05.