Dietary docosahexaenoic acid supplementation inhibits acute pulmonary transcriptional and autoantibody responses to a single crystalline silica exposure in lupus-prone mice

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.

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.