NanoString autoimmune profiling panel normalized linear counts and summary of statistical analyses

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.

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.