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.

BackgroundWith the increase in production and use of engineered nanoparticles (NP; ≤ 100 nm), safety concerns have risen about the potential health effects of occupational or environmental NP exposure. Results of animal toxicology studies suggest that inhalation of NP may cause pulmonary injury with subsequent acute or chronic inflammation. People with chronic respiratory diseases like asthma or allergic rhinitis may be even more susceptible to toxic effects of inhaled NP. Few studies, however, have investigated adverse effects of inhaled NP that may enhance the development of allergic airway disease.MethodsWe investigated the potential of polyethylene glycol coated amorphous silica NP (SNP; 90 nm diameter) to promote allergic airway disease when co-exposed during sensitization with an allergen. BALB/c mice were sensitized by intranasal instillation with 0.02% ovalbumin (OVA; allergen) or saline (control), and co-exposed to 0, 10, 100, or 400 μg of SNP. OVA-sensitized mice were then challenged intranasally with 0.5% OVA 14 and 15 days after sensitization, and all animals were sacrificed a day after the last OVA challenge. Blood and bronchoalveolar lavage fluid (BALF) were collected, and pulmonary tissue was processed for histopathology and biochemical and molecular analyses.ResultsCo-exposure to SNP during OVA sensitization caused a dose-dependent enhancement of allergic airway disease upon challenge with OVA alone. This adjuvant-like effect was manifested by significantly greater OVA-specific serum IgE, airway eosinophil infiltration, mucous cell metaplasia, and Th2 and Th17 cytokine gene and protein expression, as compared to mice that were sensitized to OVA without SNP. In saline controls, SNP exposure did cause a moderate increase in airway neutrophils at the highest doses.ConclusionsThese results suggest that airway exposure to engineered SNP could enhance allergen sensitization and foster greater manifestation of allergic airway disease upon secondary allergen exposures. Whereas SNP caused innate immune responses at high doses in non-allergic mice, the adjuvant effects of SNP were found at lower doses in allergic mice and were Th2/Th17 related. In conclusion, these findings in mice suggest that individuals exposed to SNP might be more prone to manifest allergic airway disease, due to adjuvant-like properties of SNP.

Epidemiological studies suggest that elevated ambient concentrations of ozone are associated with activation of eosinophils in the nasal airways of atopic and nonatopic children. Mice repeatedly exposed to ozone develop eosinophilic rhinitis and type 2 immune responses. In this study, we determined the role of innate lymphoid cells (ILCs) in the pathogenesis of ozone-induced eosinophilic rhinitis by using lymphoid-sufficient C57BL/6 mice, Rag2(-/-) mice that are devoid of T cells and B cells, and Rag2(-/-)Il2rg(-/-) mice that are depleted of all lymphoid cells including ILCs. The animals were exposed to 0 or 0.8 ppm ozone for 9 consecutive weekdays (4 h/d). Mice were killed 24 hours after exposure, and nasal tissues were selected for histopathology and gene expression analysis. ILC-sufficient C57BL/6 and Rag2(-/-) mice exposed to ozone developed marked eosinophilic rhinitis and epithelial remodeling (e.g., epithelial hyperplasia and mucous cell metaplasia). Chitinase-like proteins and alarmins (IL-33, IL-25, and thymic stromal lymphopoietin) were also increased morphometrically in the nasal epithelium of ozone-exposed C57BL/6 and Rag2(-/-) mice. Ozone exposure elicited increased expression of Il4, Il5, Il13, St2, eotaxin, MCP-2, Gob5, Arg1, Fizz1, and Ym2 mRNA in C57BL/6 and Rag2(-/-) mice. In contrast, ozone-exposed ILC-deficient Rag2(-/-)Il2rg(-/-) mice had no nasal lesions or overexpression of Th2- or ILC2-related transcripts. These results indicate that ozone-induced eosinophilic rhinitis, nasal epithelial remodeling, and type 2 immune activation are dependent on ILCs. To the best of our knowledge, this is the first study to demonstrate that ILCs play an important role in the nasal pathology induced by repeated ozone exposure.

Inhalation exposures to ozone commonly encountered in photochemical smog cause airway injury and inflammation. Elevated ambient ozone concentrations have been epidemiologically associated with nasal airway activation of neutrophils and eosinophils. In the present study, we elucidated the temporal onset and lymphoid cell dependency of eosinophilic rhinitis and associated epithelial changes in mice repeatedly exposed to ozone. Lymphoid cell-sufficient C57BL/6 mice were exposed to 0 or 0.5 parts per million (ppm) ozone for 1, 2, 4, or 9 consecutive weekdays (4 h/d). Lymphoid cell-deficient, Rag2(-/-)Il2rg(-/-) mice were similarly exposed for 9 weekdays. Nasal tissues were taken at 2 or 24 hours after exposure for morphometric and gene expression analyses. C57BL/6 mice exposed to ozone for 1 day had acute neutrophilic rhinitis, with airway epithelial necrosis and overexpression of mucosal Ccl2 (MCP-1), Ccl11 (eotaxin), Cxcl1 (KC), Cxcl2 (MIP-2), Hmox1, Il1b, Il5, Il6, Il13, and Tnf mRNA. In contrast, 9-day ozone exposure elicited type 2 immune responses in C57BL/6 mice, with mucosal mRNA overexpression of Arg1, Ccl8 (MCP-2), Ccl11, Chil4 (Ym2), Clca1 (Gob5), Il5, Il10, and Il13; increased density of mucosal eosinophils; and nasal epithelial remodeling (e.g., hyperplasia/hypertrophy, mucous cell metaplasia, hyalinosis, and increased YM1/YM2 proteins). Rag2(-/-)Il2rg(-/-) mice exposed to ozone for 9 days, however, had no nasal pathology or overexpression of transcripts related to type 2 immunity. These results provide a plausible paradigm for the activation of eosinophilic inflammation and type 2 immunity found in the nasal airways of nonatopic individuals subjected to episodic exposures to high ambient ozone.

Our findings in mice suggest that the severity and character of allergic airway disease are age dependent, with a bias towards a Th17 immune response with aging. Elderly, asthmatics may be prone to develop severe allergic airway inflammation with a mixed Th2/Th17 immune response.