Dietary Docosahexaenoic Acid Prevents Silica-Induced Development of Pulmonary Ectopic Germinal Centers and Glomerulonephritis in the Lupus-Prone NZBWF1 Mouse

Abstract

Ectopic lymphoid structures (ELS) consist of B-cell and T-cell aggregates that are initiated de novo in inflamed tissues outside of secondary lymphoid organs. When organized within follicular dendritic cell (FDC) networks, ELS contain functional germinal centers that can yield autoantibody-secreting plasma cells and promote autoimmune disease. Intranasal instillation of lupus-prone mice with crystalline silica (cSiO2), a respirable particle linked to human lupus, triggers ELS formation in the lung, systemic autoantibodies, and early onset of glomerulonephritis. Here we tested the hypothesis that consumption of docosahexaenoic acid (DHA), an ω-3 polyunsaturated fatty acid with anti-inflammatory properties, influences the temporal profile of cSiO2-induced pulmonary ectopic germinal center formation and development of glomerulonephritis. Female NZBWF1 mice (6-wk old) were fed purified isocaloric diets supplemented with 0, 4, or 10 g/kg DHA - calorically equivalent to 0, 2, or 5 g DHA per day consumption by humans, respectively. Beginning at age 8 wk, mice were intranasally instilled with 1 mg cSiO2, or saline vehicle alone, once per wk, for 4 wk. Cohorts were sacrificed 1, 5, 9, or 13 wk post-instillation (PI) of the last cSiO2 dose, and lung and kidney lesions were investigated by histopathology. Tissue fatty acid analyses confirmed uniform dose-dependent DHA incorporation across all cohorts. As early as 1 wk PI, inflammation comprising of B (CD45R+) and T (CD3+) cell accumulation was observed in lungs of cSiO2-treated mice compared to vehicle controls; these responses intensified over time. Marked follicular dendritic cell (FDC; CD21+/CD35+) networking appeared at 9 and 13 wk PI. IgG+ plasma cells suggestive of mature germinal centers were evident at 13 wk. DHA supplementation dramatically suppressed cSiO2-triggered B-cell, T-cell, FDC, and IgG+ plasma cell appearance in the lungs as well as anti-dsDNA IgG in bronchial lavage fluid and plasma over the course of the experiment. cSiO2 induced glomerulonephritis with concomitant B-cell accumulation in the renal cortex at 13 wk PI but this response was abrogated by DHA feeding. Taken together, realistic dietary DHA supplementation prevented initiation and/or progression of ectopic lymphoid neogenesis, germinal center development, systemic autoantibody elevation, and resultant glomerulonephritis in this unique preclinical model of environment-triggered lupus.

Docosahexaenoic Acid Consumption Impedes Early Interferon- and Chemokine-Related Gene Expression While Suppressing Silica-Triggered Flaring of Murine Lupus

“…Lungs were analyzed at 1 (Lung.W1), 5 (Lung.W5), 9 (Lung.W9), and 13 (Lung.W13) weeks post cSiO 2 exposure; spleens (Spleen.W13) and kidneys (Kidney.W13) were analyzed at 13 weeks. These times correspond with pathological changes previously reported in NZBWF1 mice after cSiO 2 exposure preceding and through glomerulonephritis onset (15,16,31). Fatty acid concentrations in erythrocytes were analyzed by gas liquid chromatogorphy at OmegaQuant (Sioux Falls, SD).…”

“…This investigation used materials and methods that have been more fully described in two previous published studies by our laboratory (17,31). Experiments were approved by the Institutional Animal Care and Use Committee at Michigan State University (AUF #01/15-021-00).…”

“…Caudal lung lobes were removed, held in RNAlater (Thermo Fisher Scientific, Wilmington, DE) for 16 h at 4 • C, and then stored at −80 • C until RNA isolation. For the time course study (31), groups of mice were treated with VEH or cSiO 2 weekly for 4 weeks beginning at age 8 weeks, (Figure 1C). Afterward, cohorts were terminated at 1, 5, 9, and 13 weeks post final cSiO 2 exposure and organs collected and stored in RNAlater as described above.…”

“…Spearman rank correlations were done to assess overall patterns in the gene expression profiles compared to percent CD45R+ (B cells) and CD3+ (T cells) in lung tissues as markers for ectopic lymphoid tissue development (31) and with the percent of ω-3 highly unsaturated fatty acids (HUFA; fatty FIGURE 5 | Effect of DHA supplementation on cSiO 2 -induced transcriptional changes in lung tissues of mice 1, 5, 9, or 13 weeks post instillation. (A) Venn diagrams depicting overlap of genes differentially regulated by exposure to cSiO 2 compared to those differentially regulated by supplementation with DHA low or DHA high diets (FDR q < 0.05, 1.5-fold change).…”

“…Understanding how DHA influences cSiO 2 -induced transcription signatures in vivo could provide insights into the underlying mechanisms by which ω-3s interfere with lupus flaring. In this investigation, we employed tissues from two recent published studies (17,31) to test the hypothesis that DHA consumption interferes with upregulation of critical genes associated with cSiO 2triggered murine lupus. The results indicate that dietary DHA supplementation at clinically realistic levels impaired cSiO 2 -triggered expression of IFN-and chemokine-related genes FIGURE 2 | Acute transcriptional response of immune-associated genes in DHA-supplemented mice that received either a single or four weekly instillations of cSiO 2 .…”