Crystalline silica-induced pulmonary inflammation and autoimmunity in mature adult NZBW/f1 mice: age-related sensitivity and impact of omega-3 fatty acid intervention.

OBJECTIVE: Occupational exposure to respirable crystalline silica (cSiO2) has been linked to lupus development. Previous studies in young lupus-prone mice revealed that intranasal cSiO2 exposure triggered autoimmunity, preventable with docosahexaenoic acid (DHA). This study explores cSiO2 and DHA effects in mature lupus-prone adult mice, more representative of cSiO2-exposed worker age. METHODS: Female NZBWF1 mice (14-week old) were fed control (CON) or DHA-supplemented diets. After two weeks, mice were intranasally instilled saline (VEH) or 1 mg cSiO2 weekly for four weeks. Cohorts were then analyzed 1- and 5-weeks postinstillation for lung inflammation, cell counts, chemokines, histopathology, B- and T-cell infiltration, autoantibodies, and gene signatures, with results correlated to autoimmune glomerulonephritis onset. RESULTS: VEH/CON mice showed no pathology. cSiO2/CON mice displayed significant ectopic lymphoid tissue formation in lungs at 1 week, increasing by 5 weeks. cSiO2/CON lungs exhibited elevated cellularity, chemokines, CD3+ T-cells, CD45R + B-cells, IgG + plasma cells, gene expression, IgG autoantibodies, and glomerular hypertrophy. DHA supplementation mitigated all these effects. DISCUSSION: The mature adult NZBWF1 mouse used here represents a life-stage coincident with immunological tolerance breach and one that more appropriately represents the age (20-30 yr) of cSiO2-exposed workers. cSiO2-induced robust pulmonary inflammation, autoantibody responses, and glomerulonephritis in mature adult mice, surpassing effects observed previously in young adults. DHA at a human-equivalent dosage effectively countered cSiO2-induced inflammation/autoimmunity in mature mice, mirroring protective effects in young mice. CONCLUSION: These results highlight life-stage significance in this preclinical lupus model and underscore omega-3 fatty acids' therapeutic potential against toxicant-triggered autoimmune responses.

Influx, Persistence, and Recall of Eosinophils and GATA-3+ Innate Lymphoid Cells in the Nasal Mucosa of Mice Exposed and Reexposed to the Gaseous Air Pollutant Ozone.

Mice exposed to the air pollutant ozone develop eosinophilic rhinitis that is mediated by group 2, GATA-3+, innate lymphoid cells (ILC2s). In the present study, we determined the influx, persistence, and recall of nasal ILC2s and eosinophils in ozone-exposed mice. C57BL/6 (T/B cell sufficient, ILC sufficient), Rag2-/- (T/B cell deficient, ILC sufficient), and Rag2-/-Il2rg-/- (T/B cell deficient, ILC deficient) mice were exposed to 0 or 0.8 ppm ozone for 1 or 9 weekdays and killed 1 or 17 days postexposure. GATA-3+ lymphocytes were sparse in nasal tissue of air-exposed ILC-sufficient mice and absent in ILC-deficient mice. Nine-day, but not 1-day, ozone exposures induced nasal influxes of eosinophils and GATA-3+ lymphocytes in C57BL/6 and Rag2-/- mice but not in Rag2-/-Il2rg-/- mice. Eosinophils waned 17 days postexposure in ILC-sufficient strains of mice. GATA-3+ lymphocytes in C57BL/6 mice also attenuated after exposure but not in ILC-sufficient Rag2-/- mice. Eosinophils, but not GATA-3+ cells, increased rapidly with reexposure in ILC-sufficient mice. Type 2 immune-related messenger RNA expression correlated with cellular responses to ozone. These new findings in mice further elucidate the role of ILC2s in ozone-induced eosinophilic rhinitis and support epidemiologic associations between ozone exposure and eosinophilic inflammation in children.

Pathogenesis and Persistence of Increased Epithelial Mucosubstances in the Nasal Airways of Rats and Mice Episodically Exposed to Ethylene.

Rats repeatedly exposed to high airborne concentrations of ethylene develop eosinophilic rhinitis and mucous cell hyperplasia/hypertrophy (MCH) in nasal respiratory epithelium. Mechanisms underlying these lesions are not well understood to inform occupational exposure guidelines. In this study, we determined (1) the nasal histopathology in rats episodically exposed to ethylene, (2) the ethylene-induced nasal histopathology in similarly exposed mice, and (3) how innate lymphoid cells (ILCs) play a role in ethylene-induced MCH. Animals were exposed to 0 or 10,000 ppm ethylene, 6 h/d, 5 d/wk, for 2 weeks and sacrificed 1 day or 2 weeks postexposure. Others received three 2-week exposure blocks separated by 2-week intervals of no exposure. Episodic exposure was chosen to aid in distinguishing irritant from immune responses. Mucous cell hyperplasia/hypertrophy was induced by ethylene in both species. Rats developed a mild, but transient, eosinophilic rhinitis. Mucous cell hyperplasia/hypertrophy was transient in mice, but persistent in rats. Increases in epithelial mucosubstances after 2 weeks of exposure were only present in ILC-sufficient mice, but not in ILC-deficient mice suggesting that ILCs play a role in MCH and overexpression of genes associated with mucus production/secretion. These findings in animals suggest that inhaled ethylene does not act as a sensitizing agent and will not induce allergen-like nasal airway disease.

Serum-borne factors alter cerebrovascular endothelial microRNA expression following particulate matter exposure near an abandoned uranium mine on the Navajo Nation.

BACKGROUND: Commercial uranium mining on the Navajo Nation has subjected communities on tribal lands in the Southwestern United States to exposures from residual environmental contamination. Vascular health effects from these ongoing exposures are an active area of study. There is an association between residential mine-site proximity and circulating biomarkers in residents, however, the contribution of mine-site derived wind-blown dusts on vascular and other health outcomes is unknown. To assess neurovascular effects of mine-site derived dusts, we exposed mice using a novel exposure paradigm, the AirCARE1 mobile inhalation laboratory, located 2 km from an abandoned uranium mine, Claim 28 in Blue Gap Tachee, AZ. Mice were exposed to filtered air (FA) (n = 6) or concentrated ambient particulate matter (CAPs) (n = 5) for 2 wks for 4 h per day. RESULTS: To assess miRNA differential expression in cultured mouse cerebrovascular cells following particulate matter (PM) exposure (average: 96.6 ± 60.4 µg/m3 for all 4 h exposures), the serum cumulative inflammatory potential (SCIP) assay was employed. MiRNA sequencing was then performed in cultured mouse cerebrovascular endothelial cells (mCECs) to evaluate transcriptional changes. Results indicated 27 highly differentially expressed (p < 0.01) murine miRNAs, as measured in the SCIP assay. Gene ontology (GO) pathway analysis revealed notable alterations in GO enrichment related to the cytoplasm, protein binding and the cytosol, while significant KEGG pathways involved pathways in cancer, axon guidance and Wnt signaling. Expression of these 27 identified, differentially expressed murine miRNAs were then evaluated in the serum. Nine of these miRNAs (~ 30%) were significantly altered in the serum and 8 of those miRNAs demonstrated the same directional change (either upregulation or downregulation) as cellular miRNAs, as measured in the SCIP assay. Significantly upregulated miRNAs in the CAPs exposure group included miRNAs in the let-7a family. Overexpression of mmu-let-7a via transfection experiments, suggested that this miRNA may mediate mCEC barrier integrity following dust exposure. CONCLUSIONS: Our data suggest that mCEC miRNAs as measured in the SCIP assay show similarity to serum-borne miRNAs, as approximately 30% of highly differentially expressed cellular miRNAs in the SCIP assay were also found in the serum. While translocation of miRNAs via exosomes or an alternative mechanism is certainly possible, other yet-to-be-identified factors in the serum may be responsible for significant miRNA differential expression in endothelium following inhaled exposures. Additionally, the most highly upregulated murine miRNAs in the CAPs exposure group were in the let-7a family. These miRNAs play a prominent role in cell growth and differentiation and based on our transfection experiments, mmu-let-7a may contribute to cerebrovascular mCEC alterations following inhaled dust exposure.

Strain Differences in a Murine Model of Air Pollutant-induced Nonatopic Asthma and Rhinitis.

Ozone is an irritating gas found in photochemical smog. Epidemiological associations have been made between the onset of asthma and childhood exposures to increasing levels of ambient ozone (i.e., air pollutant-induced nonatopic asthma). Individuals, however, vary in their susceptibility to this outdoor air pollutant, which may be due, in part, to their genetic makeup. The present study was designed to test the hypothesis that there are murine strain-dependent differences in pulmonary and nasal pathologic responses to repeated ozone exposures. C57BL/6NTac and BALB/cNTac mice were exposed to 0 or 0.8 ppm ozone, 4 hr/day, for 9 consecutive weekdays. In both strains of mice, ozone induced eosinophilic inflammation and mucous cell metaplasia in the nasal and pulmonary airways. Lungs of ozone-exposed C57BL/6NTac mice, however, had greater eosinophilic inflammation, mucous cell metaplasia, and expression of genes related to type 2 immunity and airway mucus hypersecretion, as compared to similarly exposed BALB/cNTac mice. Ozone-exposed C57BL/6NTac mice also had greater eosinophilic rhinitis but a similar degree of mucous cell metaplasia in nasal epithelium, as ozone-exposed BALB/cNTac mice. These findings suggest that nonatopic individuals may differ in their inflammatory and epithelial responses to repeated ozone exposures that are due, in part, to genetic factors.

Innate Lymphoid Cells Mediate Pulmonary Eosinophilic Inflammation, Airway Mucous Cell Metaplasia, and Type 2 Immunity in Mice Exposed to Ozone.

Exposure to elevated levels of ambient ozone in photochemical smog is associated with eosinophilic airway inflammation and nonatopic asthma in children. In the present study, we determined the role of innate lymphoid cells (ILCs) in the pathogenesis of ozone-induced nonatopic asthma by using lymphoid cell-sufficient C57BL/6 mice, ILC-sufficient Rag2-/- mice (devoid of T and B cells), and ILC-deficient Rag2-/-Il2rg-/- mice (depleted of all lymphoid cells including ILCs). Mice were exposed to 0 or 0.8 parts per million ozone for 1 day or 9 consecutive weekdays (4 hr/day). A single exposure to ozone caused neutrophilic inflammation, airway epithelial injury, and reparative DNA synthesis in all strains of mice, irrespective of the presence or absence of ILCs. In contrast, 9-day exposures induced eosinophilic inflammation and mucous cell metaplasia only in the lungs of ILC-sufficient mice. Repeated ozone exposures also elicited increased messenger RNA expression of transcripts associated with type 2 immunity and airway mucus production in ILC-sufficient mice. ILC-deficient mice repeatedly exposed to ozone had no pulmonary pathology or increased gene expression related to type 2 immunity. These results suggest a new paradigm for the biologic mechanisms underlying the development of a phenotype of childhood nonatopic asthma that has been linked to ambient ozone exposures.

Ozone-Induced Type 2 Immunity in Nasal Airways. Development and Lymphoid Cell Dependence in Mice.

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.

Ozone-Induced Nasal Type 2 Immunity in Mice Is Dependent on Innate Lymphoid Cells.

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.

Subacute inhalation exposure to ozone induces systemic inflammation but not insulin resistance in a diabetic mouse model.

Epidemiological studies suggest that diabetics may be more susceptible to the adverse health effects from exposure to high ambient concentrations of ozone, the primary oxidant gas in photochemical smog. While increased morbidity and mortality from ozone inhalation has been linked to disruption of normal cardiovascular and airway functions, potential effects on glucose and insulin homeostasis are not understood. We tested the hypothesis that ozone exposure would worsen metabolic homeostasis in KKAy mice, a genetic diabetic animal model. Male KKAy mice were exposed to 0.5 ppm ozone for 13 consecutive weekdays, and then assessed for airway, adipose and systemic inflammation, glucose homeostasis, and insulin signaling. Ozone exposure increased plasma TNFα, as well as expression of VCAM-1, iNOS and IL-6 in both pulmonary and adipose tissues. Pro-inflammatory CD11b(+)Gr-1(lo)7/4(hi) macrophages were increased by 200% in adipose tissue, but unchanged in blood. Interestingly, glucose levels were not significantly different in the insulin tolerance test between air- and ozone-exposed mice, whereas fasting insulin levels and HOMA-IR in ozone-exposed animals were significantly reduced. These changes were accompanied by increased insulin signaling in skeletal muscle and liver, but not adipose tissues. Ozone also caused decrease in body weight and plasma leptin. Our results show that in addition to marked local and systemic inflammation, ozone increases insulin sensitivity that may be related to weight loss/leptin sensitization-dependent mechanisms in KKAy mice, warranting further study on the role of hyperglycemia in mediating cardiometabolic effects of ozone inhalation.

Repeated ozone exposure exacerbates insulin resistance and activates innate immune response in genetically susceptible mice.

BACKGROUND: Inhaled ozone (O3) has been demonstrated as a harmful pollutant and associated with chronic inflammatory diseases such as diabetes and vascular disorders. However, the underlying mechanisms by which O3 mediates harmful effects are poorly understood. OBJECTIVES: To investigate the effect of O3 exposure on glucose intolerance, immune activation and underlying mechanisms in a genetically susceptible mouse model. METHODS: Diabetes-prone KK mice were exposed to filtered air (FA), or O3 (0.5 ppm) for 13 consecutive weekdays (4 h/day). Insulin tolerance test (ITT) was performed following the last exposure. Plasma insulin, adiponectin, and leptin were measured by ELISA. Pathologic changes were examined by H&E and Oil-Red-O staining. Inflammatory responses were detected using flow cytometry and real-time PCR. RESULTS: KK mice exposed to O3 displayed an impaired insulin response. Plasma insulin and leptin levels were reduced in O3-exposed mice. Three-week exposure to O3 induced lung inflammation and increased monocytes/macrophages in both blood and visceral adipose tissue. Inflammatory monocytes/macrophages increased both systemically and locally. CD4 + T cell activation was also enhanced by the exposure of O3 although the relative percentage of CD4 + T cell decreased in blood and adipose tissue. Multiple inflammatory genes including CXCL-11, IFN-γ, TNFα, IL-12, and iNOS were up-regulated in visceral adipose tissue. Furthermore, the expression of oxidative stress-related genes such as Cox4, Cox5a, Scd1, Nrf1, and Nrf2, increased in visceral adipose tissue of O3-exposed mice. CONCLUSIONS: Repeated O3 inhalation induces oxidative stress, adipose inflammation and insulin resistance.

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

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-1ß, 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.

Subchronic intranasal lipopolysaccharide exposure induces pulmonary autoimmunity and glomerulonephritis in NZBWF1 mice.

Lupus, a systemic autoimmune disease shaped by gene-environment interplay, often progresses to endstage renal failure. While subchronic systemic exposure to bacterial lipopolysaccharide (LPS) triggers autoimmunity and glomerulonephritis in lupus-prone mice, it is unknown if inhaling LPS, which is common in certain occupations, can similarly trigger lupus. Here we determined how subchronic intranasal (IN) LPS instillation influences autoimmunity and glomerulonephritis development in lupusprone NZBWF1 female mice. Briefly, mice were IN-instilled with vehicle or E. coli LPS (0.8 µg/g) twice weekly for 5 wk, followed by necropsy. For systemic comparison, additional cohorts of mice were injected with LPS intraperitoneally (IP) using identical doses/timing. Lungs were assessed for inflammatory and autoimmune responses and then related to systemic autoimmunity and glomerulonephritis. IN/LPS exposure induced in the lung: i) leukocyte infiltration, ii)mRNA signatures for cytokines, chemokines, IFN-regulated, and cell death-related genes, iii) ectopic lymphoid tissue formation, and iv)diverse IgM and IgG autoantibodies (AAbs). Pulmonary effects coincided with enlarged spleens, elevated plasma IgG AAbs, and inflamed IgG-containing kidney glomeruli. In contrast, IP/LPS treatment induced systemic autoimmunity and glomerulonephritis without pulmonary manifestations. Taken together, these preclinical findings suggest the lung could serve as a critical nexus for triggering autoimmunity by respirable LPS in genetically predisposed individuals.

Engineered silica nanoparticles act as adjuvants to enhance allergic airway disease in mice.

BACKGROUND: With 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. METHODS: We 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. RESULTS: Co-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. CONCLUSIONS: These 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.

Ambient fine particulate matter and ozone exposures induce inflammation in epicardial and perirenal adipose tissues in rats fed a high fructose diet.

BACKGROUND: Inflammation and oxidative stress play critical roles in the pathogenesis of inhaled air pollutant-mediated metabolic disease. Inflammation in the adipose tissues niches are widely believed to exert important effects on organ dysfunction. Recent data from both human and animal models suggest a role for inflammation and oxidative stress in epicardial adipose tissue (EAT) as a risk factor for the development of cardiovascular disease. We hypothesized that inhalational exposure to concentrated ambient fine particulates (CAPs) and ozone (O3) exaggerates inflammation and oxidative stress in EAT and perirenal adipose tissue (PAT). METHODS: Eight- week-old Male Sprague-Dawley rats were fed a normal diet (ND) or high fructose diet (HFr) for 8 weeks, and then exposed to ambient AIR, CAPs at a mean of 356 µg/m3, O3 at 0.485 ppm, or CAPs (441 µg/m3) + O3 (0.497 ppm) in Dearborn, MI, 8 hours/day, 5 days/week, for 9 days over 2 weeks. RESULTS: EAT and PAT showed whitish color in gross, and less mitochondria, higher mRNA expression of white adipose specific and lower brown adipose specific genes than in brown adipose tissues. Exposure to CAPs and O3 resulted in the increase of macrophage infiltration in both EAT and PAT of HFr groups. Proinflammatory genes of Tnf-α, Mcp-1 and leptin were significantly upregulated while IL-10 and adiponectin, known as antiinflammatory genes, were reduced after the exposures. CAPs and O3 exposures also induced an increase in inducible nitric oxide synthase (iNOS) protein expression, and decrease in mitochondrial area in EAT and PAT. We also found significant increases in macrophages of HFr-O3 rats. The synergetic interaction of HFr and dirty air exposure on the inflammation was found in most of the experiments. Surprisingly, exposure to CAPs or O3 induced more significant inflammation and oxidative stress than co-exposure of CAPs and O3 in EAT and PAT. CONCLUSION: EAT and PAT are both white adipose tissues. Short-term exposure to CAPs and O3, especially with high fructose diet, induced inflammation and oxidative stress in EAT and PAT in rats. These findings may provide a link between air-pollution exposure and accelerated susceptibility to cardiovascular disease and metabolic complications.

Lipidome modulation by dietary omega-3 polyunsaturated fatty acid supplementation or selective soluble epoxide hydrolase inhibition suppresses rough LPS-accelerated glomerulonephritis in lupus-prone mice.

Introduction: Lipopolysaccharide (LPS)-accelerated autoimmune glomerulonephritis (GN) in NZBWF1 mice is a preclinical model potentially applicable for investigating lipidome-modulating interventions against lupus. LPS can be expressed as one of two chemotypes: smooth LPS (S-LPS) or rough LPS (R-LPS) which is devoid of O-antigen polysaccharide sidechain. Since these chemotypes differentially affect toll-like receptor 4 (TLR4)-mediated immune cell responses, these differences may influence GN induction. Methods: We initially compared the effects of subchronic intraperitoneal (i.p.) injection for 5 wk with 1) Salmonella S-LPS, 2) Salmonella R-LPS, or 3) saline vehicle (VEH) (Study 1) in female NZBWF1 mice. Based on the efficacy of R-LPS in inducing GN, we next used it to compare the impact of two lipidome-modulating interventions, ω-3 polyunsaturated fatty acid (PUFA) supplementation and soluble epoxide hydrolase (sEH) inhibition, on GN (Study 2). Specifically, effects of consuming ω-3 docosahexaenoic acid (DHA) (10 g/kg diet) and/or the sEH inhibitor 1-(4-trifluoro-methoxy-phenyl)-3-(1-propionylpiperidin-4-yl) urea (TPPU) (22.5 mg/kg diet ≈ 3 mg/kg/day) on R-LPS triggering were compared. Results: In Study 1, R-LPS induced robust elevations in blood urea nitrogen, proteinuria, and hematuria that were not evident in VEH- or S-LPS-treated mice. R-LPS-treated mice further exhibited kidney histopathology including robust hypertrophy, hyperplasia, thickened membranes, lymphocytic accumulation containing B and T cells, and glomerular IgG deposition consistent with GN that was not evident in VEH- or SLPS-treated groups. R-LPS but not S-LPS induced spleen enlargement with lymphoid hyperplasia and inflammatory cell recruitment in the liver. In Study 2, resultant blood fatty acid profiles and epoxy fatty acid concentrations reflected the anticipated DHA- and TPPU-mediated lipidome changes, respectively. The relative rank order of R-LPS-induced GN severity among groups fed experimental diets based on proteinuria, hematuria, histopathologic scoring, and glomerular IgG deposition was: VEH/CON< R-LPS/DHA ≈ R-LPS/TPPU<<< R-LPS/TPPU+DHA ≈ R-LPS/CON. In contrast, these interventions had modest-to- negligible effects on R-LPS-induced splenomegaly, plasma antibody responses, liver inflammation, and inflammation-associated kidney gene expression. Discussion: We show for the first time that absence of O-antigenic polysaccharide in R-LPS is critical to accelerated GN in lupus-prone mice. Furthermore, intervention by lipidome modulation through DHA feeding or sEH inhibition suppressed R-LPS-induced GN; however, these ameliorative effects were greatly diminished upon combining the treatments.

Combined adjuvant effects of ambient vapor-phase organic components and particulate matter potently promote allergic sensitization and Th2-skewing cytokine and chemokine milieux in mice: The importance of mechanistic multi-pollutant research.

Although exposure to ambient particulate matter (PM) is linked to asthma, the health effects of co-existing vapor-phase organic pollutants (vapor) and their combined effects with PM on this disease are poorly understood. We used a murine asthma model to test the hypothesis that exposure to vapor would enhance allergic sensitization and this effect would be further strengthened by co-existing PM. We found that vapor and PM each individually exerted adjuvant effects on OVA sensitization. Co-exposure to vapor and PM during sensitization further enhanced allergic lung inflammation and OVA-specific antibody production which was accompanied by pulmonary cytokine/chemokine milieu that favored T-helper 2 immunity (i.e. increased IL-4, downregulation of Il12a and Ifng, and upregulation of Ccl11 and Ccl8). TNFα, IL-6, Ccl12, Cxcl1 and detoxification/antioxidant enzyme responses in the lung were pollutant-dependent. Inhibition of lipopolysaccharide-induced IL-12 secretion from primary antigen-presenting dendritic cells correlated positively with vapor's oxidant potential. In conclusion, concurrent exposure to vapor and PM led to significantly exaggerated adjuvant effects on allergic lung inflammation which were more potent than that of each pollutant type alone. These findings suggest that the effects of multi-component air pollution on asthma may be significantly underestimated if research only focuses on a single air pollutant (e.g., PM).

Comparative effects of human-equivalent low, moderate, and high dose oral prednisone intake on autoimmunity and glucocorticoid-related toxicity in a murine model of environmental-triggered lupus.

Autoimmune diseases can be triggered by environmental toxicants such as crystalline silica dust (cSiO2). Here, we characterized the dose-dependent immunomodulation and toxicity of the glucocorticoid (GC) prednisone in a preclinical model that emulates onset and progression of cSiO2-triggered lupus. Two cohorts of 6-wk-old female NZBWF1 mice were fed either control AIN-93G diet or one of three AIN-93G diets containing prednisone at 5, 15, or 50 mg/kg diet which span human equivalent oral doses (HED) currently considered to be low (PL; 5 mg/d HED), moderate (PM; 14 mg/d HED), or high (PH; 46 mg/d HED), respectively. At 8 wk of age, mice were intranasally instilled with either saline vehicle or 1 mg cSiO2 once weekly for 4 wk. The experimental plan was to 1) terminate one cohort of mice (n=8/group) 14 wk after the last cSiO2 instillation for pathology and autoimmunity assessment and 2) to maintain a second cohort (n=9/group) to monitor glomerulonephritis development and survival. Mean blood concentrations of prednisone's principal active metabolite, prednisolone, in mice fed PL, PM, and PH diets were 27, 105, 151 ng/ml, respectively, which are consistent with levels observed in human blood ≤ 12 h after single bolus treatments with equivalent prednisone doses. Results from the first cohort revealed that consumption of PM, but not PL diet, significantly reduced cSiO2-induced pulmonary ectopic lymphoid structure formation, nuclear-specific AAb production, inflammation/autoimmune gene expression in the lung and kidney, splenomegaly, and glomerulonephritis in the kidney. Relative to GC-associated toxicity, PM diet, but not PL diet, elicited muscle wasting, but these diets did not affect bone density or cause glucosuria. Importantly, neither PM nor PL diet improved latency of cSiO2-accelerated death. PH-fed mice in both cohorts displayed robust GC-associated toxicity including body weight loss, reduced muscle mass, and extensive glucosuria 7 wk after the final cSiO2 instillation requiring their early removal from the study. Taken together, our results demonstrate that while moderate doses of prednisone can reduce important pathological endpoints of cSiO2-induced autoimmunity in lupus-prone mice, such as upstream ectopic lymphoid structure formation, these ameliorative effects come with unwanted GC toxicity, and, crucially, none of these three doses extended survival time.

Ambient particulate air pollution induces oxidative stress and alterations of mitochondria and gene expression in brown and white adipose tissues.

BACKGROUND: Prior studies have demonstrated a link between air pollution and metabolic diseases such as type II diabetes. Changes in adipose tissue and its mitochondrial content/function are closely associated with the development of insulin resistance and attendant metabolic complications. We investigated changes in adipose tissue structure and function in brown and white adipose depots in response to chronic ambient air pollutant exposure in a rodent model. METHODS: Male ApoE knockout (ApoE-/-) mice inhaled concentrated fine ambient PM (PM < 2.5 µm in aerodynamic diameter; PM2.5) or filtered air (FA) for 6 hours/day, 5 days/week, for 2 months. We examined superoxide production by dihydroethidium staining; inflammatory responses by immunohistochemistry; and changes in white and brown adipocyte-specific gene profiles by real-time PCR and mitochondria by transmission electron microscopy in response to PM2.5 exposure in different adipose depots of ApoE-/- mice to understand responses to chronic inhalational stimuli. RESULTS: Exposure to PM2.5 induced an increase in the production of reactive oxygen species (ROS) in brown adipose depots. Additionally, exposure to PM2.5 decreased expression of uncoupling protein 1 in brown adipose tissue as measured by immunohistochemistry and Western blot. Mitochondrial number was significantly reduced in white (WAT) and brown adipose tissues (BAT), while mitochondrial size was also reduced in BAT. In BAT, PM2.5 exposure down-regulated brown adipocyte-specific genes, while white adipocyte-specific genes were differentially up-regulated. CONCLUSIONS: PM2.5 exposure triggers oxidative stress in BAT, and results in key alterations in mitochondrial gene expression and mitochondrial alterations that are pronounced in BAT. We postulate that exposure to PM2.5 may induce imbalance between white and brown adipose tissue functionality and thereby predispose to metabolic dysfunction.

Rapid Induction of Pulmonary Inflammation, Autoimmune Gene Expression, and Ectopic Lymphoid Neogenesis Following Acute Silica Exposure in Lupus-Prone Mice.

Occupational exposure to crystalline silica (cSiO2) is etiologically associated with systemic lupus erythematosus (lupus) and other autoimmune diseases. cSiO2's autoimmune effects in humans can be mimicked chronically in female lupus-prone NZBWF1 mice following repeated exposure to the particle. However, the immediate and short-term effects of cSiO2 in this widely used model of autoimmune disease are not well-understood. In the present study, we tested the hypothesis that a single acute cSiO2 dose triggers early presentation of cellular, histopathological, transcriptomic, and protein biomarkers of inflammation and autoimmunity in lupus-prone mice. Eight-week old female NZBWF1 mice were intranasally instilled once with 2.5 mg cSiO2 or saline vehicle and necropsied at 1, 7, 14, 21, and 28 d post-instillation (PI). Analyses of bronchoalveolar lavage fluid (BALF) and lung tissue revealed that by 7 d PI, acute cSiO2 exposure persistently provoked: (i) robust recruitment of macrophages, neutrophils, and lymphocytes into the alveoli, (ii) cell death as reflected by increased protein, double-stranded DNA, and lactate dehydrogenase activity, (iii) elevated secretion of the cytokines IL-1α, IL-1ß, IL-18, TNF-α, IL-6, MCP-1, and B cell activation factor (BAFF), and (iv) upregulation of genes associated with chemokines, proinflammatory cytokines, lymphocyte activation, and type I interferon signaling. The appearance of these endpoints was subsequently followed by the emergence in the lung of organized CD3+ T cells (14 d PI) and CD45R+ B cells (21 d PI) that were indicative of ectopic lymphoid structure (ELS) development. Taken together, acute cSiO2 exposure triggered a rapid onset of autoimmune disease pathogenesis that was heralded in the lung by unresolved inflammation and cell death, proinflammatory cytokine production, chemokine-driven recruitment of leukocytes, an interferon response signature, B and T cell activation, and ELS neogenesis. This short-term murine model provides valuable new insight into potential early mechanisms of cSiO2-induced lupus flaring and, furthermore, offers a rapid venue for evaluating interventions against respirable particle-triggered inflammation and autoimmunity.

Omega-3 Polyunsaturated Fatty Acid Intervention Against Established Autoimmunity in a Murine Model of Toxicant-Triggered Lupus.

Workplace exposure to respirable crystalline silica dust (cSiO2) has been etiologically linked to the development of lupus and other human autoimmune diseases. Lupus triggering can be recapitulated in female NZBWF1 mice by four weekly intranasal instillations with 1 mg cSiO2. This elicits inflammatory/autoimmune gene expression and ectopic lymphoid structure (ELS) development in the lung within 1 week, ultimately driving early onset of systemic autoimmunity and glomerulonephritis. Intriguingly, dietary supplementation with docosahexaenoic acid (DHA), an ω-3 polyunsaturated fatty acid (PUFA) found in fish oil, beginning 2 week prior to cSiO2 challenge, prevented inflammation and autoimmune flaring in this novel model. However, it is not yet known how ω-3 PUFA intervention influences established autoimmunity in this murine model of toxicant-triggered lupus. Here we tested the hypothesis that DHA intervention after cSiO2-initiated intrapulmonary autoimmunity will suppress lupus progression in the NZBWF1 mouse. Six-week old NZWBF1 female mice were fed purified isocaloric diet for 2 weeks and then intranasally instilled with 1 mg cSiO2 or saline vehicle weekly for 4 consecutive weeks. One week after the final instillation, which marks onset of ELS formation, mice were fed diets supplemented with 0, 4, or 10 g/kg DHA. One cohort of mice (n = 8/group) was terminated 13 weeks after the last cSiO2 instillation and assessed for autoimmune hallmarks. A second cohort of mice (n = 8/group) remained on experimental diets and was monitored for proteinuria and moribund criteria to ascertain progression of glomerulonephritis and survival, respectively. DHA consumption dose-dependently increased ω-3 PUFA content in the plasma, lung, and kidney at the expense of the ω-6 PUFA arachidonic acid. Dietary intervention with high but not low DHA after cSiO2 treatment suppressed or delayed: (i) recruitment of T cells and B cells to the lung, (ii) development of pulmonary ELS, (iii) elevation of a wide spectrum of plasma autoantibodies associated with lupus and other autoimmune diseases, (iv) initiation and progression of glomerulonephritis, and (v) onset of the moribund state. Taken together, these preclinical findings suggest that DHA supplementation at a human caloric equivalent of 5 g/d was an effective therapeutic regimen for slowing progression of established autoimmunity triggered by the environmental toxicant cSiO2.