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

Abstract

Introduction: Workplace exposure to respirable crystalline silica (cSiO₂) has been epidemiologically linked to lupus. Consistent with this, repeated subchronic intranasal cSiO₂ instillation in lupus-prone NZBWF1 mice induces inflammation-/autoimmune-related gene expression, ectopic lymphoid tissue (ELT), autoantibody (AAb) production in the lung within 5 to 13 wk followed systemic AAb increases and accelerated onset and progression of glomerulonephritis within 13 to 17 wk. Interestingly, dietary docosahexaenoic acid (DHA) supplementation suppresses these pathologic effects, but the underlying molecular mechanisms remain unclear.

Methods: This study aimed to test the hypothesis that dietary DHA supplementation impacts acute transcriptional and autoantibody responses in the lungs of female NZBWF1 mice 1 and 4 wk after a single high-dose cSiO₂ challenge. Groups of mice were initially fed a control (Con) diet or a DHA-containing diet (10 g/kg). Cohorts of Con- and DHA-fed were subjected to a single intranasal instillation of 2.5 mg cSiO₂ in a saline vehicle (Veh), while a Con-fed cohort was instilled with Veh only. At 1 and 4 wk post-instillation (PI), we compared cSiO₂'s effects on innate-/autoimmune-related gene expression and autoantibody (AAb) in lavage fluid/lungs of Con- and DHA-fed mice and related these findings to inflammatory cell profiles, histopathology, cell death, and cytokine/chemokine production.

Results: DHA partially alleviated cSiO₂-induced alterations in total immune cell and lymphocyte counts in lung lavage fluid. cSiO₂-triggered dead cell accumulation and levels of inflammation-associated cytokines and IFN-stimulated chemokines were more pronounced in Con-fed mice than DHA-fed mice. Targeted multiplex transcriptome analysis revealed substantial upregulation of genes associated with autoimmune pathways in Con-fed mice in response to cSiO₂ that were suppressed in DHA-fed mice. Pathway analysis indicated that DHA inhibited cSiO₂ induction of proinflammatory and IFN-regulated gene networks, affecting key upstream regulators (e.g., TNFα, IL-1β, IFNAR, and IFNγ). Finally, cSiO₂-triggered AAb responses were suppressed in DHA-fed mice.

Discussion: Taken together, DHA mitigated cSiO₂-induced upregulation of pathways associated with proinflammatory and IFN-regulated gene responses within 1 wk and reduced AAb responses by 4 wk. These findings suggest that the acute short-term model employed here holds substantial promise for efficient elucidation of the molecular mechanisms through which omega-3 PUFAs exert protective effects against cSiO₂-induced autoimmunity.

Keywords: autoantibody; autoimmune disease; crystalline silica; docosahexaenoic acid (DHA); inflammation; lung pathology; omega-3 fatty acid; systemic lupus erythematosus.

Figure 1

Experimental design (A) and confirmation of docosahexaenoic acid (DHA) incorporation into the lung phospholipids of DHA-fed mice (B). (A) Female NZBWF1 mice were fed Con or DHA-enriched diets beginning at 6 wk of age (n=16 per group). At 8 wk of age, mice in the Veh/Con group were instilled with 25 μl PBS (Veh), whereas mice in groups cSiO₂/Con and cSiO₂/DHA were instilled with 25 μl PBS containing 2.5 mg cSiO₂. Cohorts of mice were terminated at 7 d and 28 d post-instillation (PI) with cSiO₂, and BALF and lung tissues were collected. BALF was used for differential cell count and AAb analysis. Lung tissue was used for fatty acid analysis, cytokine/chemokine ELISA, mRNA analyses, and TUNEL immunohistochemistry. (B) Consumption of a DHA-amended diet increases DHA and eicosapentaenoic acid (EPA) in lung phospholipids at the expense of adrenic acid (ADR) and arachidonic acid (ARA) at 7 d and 28 d post-cSiO₂ instillation. Data reflect the percentage of total fatty acids in the lungs and are mean ± SEM (n=2).

Figure 2

DHA supplementation suppresses cSiO₂-induced total cell and lymphocyte accumulation in the bronchoalveolar lavage fluid. BALF was collected at 7 d and 28 d PI, and total cell, monocytes/macrophages, neutrophils, and lymphocyte counts were determined. Individual data are shown with the mean ± SEM (n=8). Within each time point, groups without the same letter are significantly different (p ≤ 0.05).

Figure 3

DHA supplementation inhibits cSiO₂-induced cytokine and chemokine elevation in the lung tissue. Selected cytokines and chemokines were assessed in lung homogenates by multiplexed ELISA. Individual data are shown with the mean ± SEM (n=4). Within each time point, groups without the same letter are significantly different (p ≤ 0.05).

Figure 4

DHA intake attenuates cSiO₂-induced cell death in the lung at 7 d PI. (A–C) TUNEL immunostaining of representative tissue sections from the left lung of (A) Veh/Con, (B) cSiO₂/Con, and (C) cSiO₂/DHA treated mice. a, alveolar parenchyma; b, bronchiolar airway. The scale bar indicates 200 µm. (D) Digital morphometry revealed less TUNEL positivity in the lungs of DHA-fed mice 7 d after cSiO₂ treatment. Individual data are shown with the mean ± SEM (n=6). Within each time point, groups without the same letter are significantly different (p ≤ 0.05).

Figure 5

DHA supplementation impedes cSiO₂-induced modulation of autoimmune-related genes in the lung. Lung RNA was subjected to NanoString analysis using the nCounter Autoimmune Gene Expression panel. (A) Proportional Venn diagrams depict numbers of genes differentially regulated in for all pairwise comparisons among treatment/diet groups (FDR p <0.05, 1.5-fold change) at 7 or 28 d PI. (B) Principal component analysis (PCA) of normalized transcript counts in lung tissue of Veh/Con, cSiO₂/Con, and cSiO₂/DHA treated mice at 7 or 28 d PI. Ellipses indicate the 95% confidence interval. (C) Volcano plots of differentially expressed genes (FDR p <0.05, 1.5-fold change) in lung tissues of mice for all pairwise comparisons among treatment/diet groups at 7 or 28 d PI. Red, genes up-regulated; blue, genes significantly down-regulated; gray, genes not significantly affected.

Figure 6

DHA consumption interferes with cSiO₂-induced modulation of inflammatory- and autoimmune-related pathways in the lung. (A) Hierarchical clustering heatmap (Euclidean distance method) depicting pathway Z scores for inflammatory- and autoimmune-related pathways determined using nSolver analysis. (B) Radar and (C) Box plots of Z score data demonstrating DHA suppression of selected cSiO₂-induced autoimmune pathways. Box plots represent the min to the max with the median (n=4). Within each time point, groups with different letters are significantly different (p ≤ 0.05).

Figure 7

DHA supplementation suppresses cSiO₂-induced IFN-regulated gene expression in the lung. Gene expression data were obtained using the autoimmune profile panel subjected to NanoString analysis. (A) Protein-protein interaction (PPI) network of commonly upregulated DEGs expressed was created using STRING. The interactions were visualized with a high confidence >0.7. Proteins were clustered using the MCL algorithm. IFN-regulated genes (red nodes) were among the top genes affected by DHA. (B) Hierarchical clustering heatmap (Euclidean distance method) showing cSiO₂-induced IFN-regulated genes that are significantly affected (FDR p<0.05) by DHA. Values are shown as the log₂ ratio with respect to 7 d PI, Veh/Con group. (C) DHA diet suppresses the expression of cSiO₂-induced genes in the lungs. Data are shown as log₂ ratio ± SEM (n=4) for cSiO₂-exposed mice fed Con (cSiO₂/Con) or DHA diet (cSiO₂/DHA) for either time point with respect to the day 7 Veh/Con group. Within each time point, * indicates significantly different compared to time-matched Veh/Con or # indicates significantly different compared to time-matched cSiO₂/Con (FDR p ≤ 0.05).

Figure 8

DHA supplementation suppresses cSiO₂-triggered expression of diverse autoimmune-pathway-related genes. Hierarchical clustering heatmap (Euclidean distance method) shows the relative expression of genes that were differentially regulated by DHA at either 7 or 28 d PI. Values are shown as the log₂ ratios of gene expression with respect to the 7 d PI, Veh/Con mice. Annotations to the left of the heatmap indicate membership of each gene in selected autoimmune pathways.

Figure 9

DHA intake quells responses to top cSiO₂-induced upstream regulators at 7 and 28 d PI. Top upstream regulators in cSiO₂/Con and cSiO₂/DHA groups in the lung predicted by Ingenuity Pathway Analysis (IPA). A positive Z-score indicates activation and a negative Z-score indicates inhibition. Activation and inhibition scores in the cSiO₂/Con group were markedly lower in the corresponding cSiO₂/DHA for the same week.

Figure 10

DHA intake suppresses cSiO₂-induced autoantibody (AAb) responses in the lung. (A) Hierarchical clustering (Euclidean distance method) heat map of Ab-score values (row centered, variance stabilized) for expression of selected AAbs of the IgG isotype in BALF at 28 d PI as determined by microarray. (B) ∑Ab-scores of selected AAbs of the IgG isotype in BALF at 28 d PI. Data are mean ± SEM (n=8). Groups without the same letter are significantly different (p ≤ 0.05).

Figure 11

DHA supplementation suppressed IgG AAbs specific to cSiO₂-killed cells (A), staurosporine-killed cells (B), and nucleosomes (C) in BALF and plasma. BALF and plasma of Veh/Con, cSiO₂/Con, and cSiO₂/DHA mice sacrificed at 28 d PI were pooled, and IgG AAbs were measured by ELISA.

Figure 12

Hypothetical mechanisms for acute cSiO₂-induced inflammation and autoimmunity targeted by DHA intervention. As first responders to inhaled particles, alveolar macrophages (AM) phagocytose cSiO₂ and respond by 1) releasing IL-1α and IL-1β that drive further transcription, translation, and secretion of cytokines/chemokines promoting myeloid/lymphoid cell recruitment, 2) dying by necroptosis, apoptosis, and pyroptosis and releasing cSiO₂ for further uptake (pink dashed arrows), and/or 3) sequestering cSiO₂ and slowly clearing it from the lung via the mucociliary escalator. If resultant cell corpses are not immediately cleared by efferocytosis other AMs, they undergo secondary necrosis, releasing 1) damage-associated molecular patterns (DAMPs) that activate receptor-driven production of cytokines, chemokines, and IFNs and 2) autoantigens (AAgs) that promote antigen presentation and B/T cell activation. Collectively, these actions drive the development of pulmonary ectopic lymphoid tissues (ELT) that serve as sites for autoantibody (AAb) production against diverse AAgs, including nuclear, ribosomal, mitochondrial, and complement proteins. Red asterisks indicate targets of DHA suppression of early acute cSiO₂-induced pulmonary inflammation, autoimmune-related gene expression, and AAb production in lupus-prone NZBWF1 mice that were identified in this investigation. Created with Biorender.com.