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.

Centrality of Myeloid-Lineage Phagocytes in Particle-Triggered Inflammation and Autoimmunity.

Exposure to exogenous particles found as airborne contaminants or endogenous particles that form by crystallization of certain nutrients can activate inflammatory pathways and potentially accelerate autoimmunity onset and progression in genetically predisposed individuals. The first line of innate immunological defense against particles are myeloid-lineage phagocytes, namely macrophages and neutrophils, which recognize/internalize the particles, release inflammatory mediators, undergo programmed/unprogrammed death, and recruit/activate other leukocytes to clear the particles and resolve inflammation. However, immunogenic cell death and release of damage-associated molecules, collectively referred to as "danger signals," coupled with failure to efficiently clear dead/dying cells, can elicit unresolved inflammation, accumulation of self-antigens, and adaptive leukocyte recruitment/activation. Collectively, these events can promote loss of immunological self-tolerance and onset/progression of autoimmunity. This review discusses critical molecular mechanisms by which exogenous particles (i.e., silica, asbestos, carbon nanotubes, titanium dioxide, aluminum-containing salts) and endogenous particles (i.e., monosodium urate, cholesterol crystals, calcium-containing salts) may promote unresolved inflammation and autoimmunity by inducing toxic responses in myeloid-lineage phagocytes with emphases on inflammasome activation and necrotic and programmed cell death pathways. A prototypical example is occupational exposure to respirable crystalline silica, which is etiologically linked to systemic lupus erythematosus (SLE) and other human autoimmune diseases. Importantly, airway instillation of SLE-prone mice with crystalline silica elicits severe pulmonary pathology involving accumulation of particle-laden alveolar macrophages, dying and dead cells, nuclear and cytoplasmic debris, and neutrophilic inflammation that drive cytokine, chemokine, and interferon-regulated gene expression. Silica-induced immunogenic cell death and danger signal release triggers accumulation of T and B cells, along with IgG-secreting plasma cells, indicative of ectopic lymphoid tissue neogenesis, and broad-spectrum autoantibody production in the lung. These events drive early autoimmunity onset and accelerate end-stage autoimmune glomerulonephritis. Intriguingly, dietary supplementation with ω-3 fatty acids have been demonstrated to be an intervention against silica-triggered murine autoimmunity. Taken together, further insight into how particles drive immunogenic cell death and danger signaling in myeloid-lineage phagocytes and how these responses are influenced by the genome will be essential for identification of novel interventions for preventing and treating inflammatory and autoimmune diseases associated with these agents.

Silica Induction of Diverse Inflammatory Proteome in Lungs of Lupus-Prone Mice Quelled by Dietary Docosahexaenoic Acid Supplementation.

Repeated short-term intranasal instillation of lupus-prone mice with crystalline silica (cSiO2) induces inflammatory gene expression and ectopic lymphoid neogenesis in the lung, leading to early onset of systemic autoimmunity and rapid progression to glomerulonephritis. These responses are suppressed by dietary supplementation with the ω-3 polyunsaturated fatty acid docosahexaenoic acid (DHA). Here, we tested the hypothesis that dietary DHA supplementation suppresses cSiO2-induced inflammatory proteins in bronchoalveolar alveolar lavage fluid (BALF) and plasma of lupus-prone mice. Archived tissue fluid samples were used from a prior investigation in which 6 wk-old lupus-prone female NZBWF1 mice were fed isocaloric diets containing 0 or 10 g/kg DHA for 2 wks and then intranasally instilled with 1 mg cSiO2 or vehicle once weekly for 4 wks. Cohorts were terminated at 1, 5, 9 or 13 wk post-instillation (PI). BALF and plasma from each cohort were analyzed by high density multiplex array profiling of 200 inflammatory proteins. cSiO2 time-dependently induced increases in the BALF protein signatures that were highly reflective of unresolved lung inflammation, although responses in the plasma were much less robust. Induced proteins in BALF included chemokines (e.g., MIP-2, MCP-5), enzymes (e.g., MMP-10, granzyme B), adhesion molecules (e.g., sE-selectin, sVCAM-1), co-stimulatory molecules (e.g., sCD40L, sCD48), TNF superfamily proteins (e.g., sTNFRI, sBAFF-R), growth factors (e.g., IGF-1, IGFBP-3), and signal transduction proteins (e.g., MFG-E8, FcgRIIB), many of which were blocked or delayed by DHA supplementation. The BALF inflammatory proteome correlated positively with prior measurements of gene expression, pulmonary ectopic lymphoid tissue neogenesis, and induction of autoantibodies in the lungs of the control and treatment groups. Ingenuity Pathway Analysis (IPA) revealed that IL-1ß, TNF-α, and IL-6 were among the top upstream regulators of the cSiO2-induced protein response. Furthermore, DHA's effects were associated with downregulation of cSiO2-induced pathways involving i) inhibition of ARE-mediated mRNA decay, ii) bacterial and viral pattern recognition receptor activation, or iii) TREM1, STAT3, NF-κB, and VEGF signaling and with upregulation of PPAR, LXR/RXR and PPARα/RXRα signaling. Altogether, these preclinical findings further support the contention that dietary DHA supplementation could be applicable as an intervention against inflammation-driven autoimmune triggering by cSiO2 or potentially other environmental agents.

Omega-3 Docosahexaenoic Acid (DHA) Impedes Silica-Induced Macrophage Corpse Accumulation by Attenuating Cell Death and Potentiating Efferocytosis.

Airway exposure of lupus-prone NZBWF1 mice to crystalline silica (cSiO2), a known trigger of human autoimmune disease, elicits sterile inflammation and alveolar macrophage death in the lung that, in turn, induces early autoimmune onset and accelerates lupus progression to fatal glomerulonephritis. Dietary supplementation with docosahexaenoic acid (DHA), a marine ω-3 polyunsaturated fatty acid (PUFA), markedly ameliorates cSiO2-triggered pulmonary, systemic, and renal manifestations of lupus. Here, we tested the hypothesis that DHA influences both cSiO2-induced death and efferocytotic clearance of resultant cell corpses using three murine macrophage models: (i) primary alveolar macrophages (AM) isolated from NZBWF1 mice; (ii) self-renewing AM-like Max Planck Institute (MPI) cells isolated from fetuses of C57BL/6 mice, and (iii) RAW 264.7 murine macrophages, a virus-transformed cell line derived from BALB/c mice stably transfected with the inflammasome adaptor protein ASC (RAW-ASC). Incubation with cSiO2 at 25 and 50 µg/ml for 6 h was found to dose-dependently induce cell death (p < 0.05) in all three models as determined by both acridine orange/propidium iodide staining and release of lactate dehydrogenase into cell culture supernatant. Pre-incubation with DHA at a physiologically relevant concentration (25 µM) significantly reduced cSiO2-induced death (p < 0.05) in all three models. Cell death induction by cSiO2 alone and its suppression by DHA were primarily associated with caspase-3/7 activation, suggestive of apoptosis, in AM, MPI, and RAW-ASC cells. Fluorescence microscopy revealed that all three macrophage models were similarly capable of efferocytosing RAW-ASC target cell corpses. Furthermore, MPI effector cells could likewise engulf RAW-ASC target cell corpses elicited by treatment with staurosporine (apoptosis), LPS, and nigericin (pyroptosis), or cSiO2. Pre-incubation of RAW-ASC target cells with 25 µM DHA prior to death induced by these agents significantly enhanced their efferocytosis (p < 0.05) by MPI effector cells. In contrast, pre-incubating MPI effector cells with DHA did not affect engulfment of RAW-ASC target cells pre-incubated with vehicle. Taken together, these findings indicate that DHA at a physiologically relevant concentration was capable of attenuating macrophage death and could potentiate efferocytosis, with the net effect of reducing accumulation of cell corpses capable of eliciting autoimmunity.

Omega-3 fatty acid intake suppresses induction of diverse autoantibody repertoire by crystalline silica in lupus-prone mice.

Inhalation of crystalline silica (cSiO2) in the workplace is etiologically linked to lupus and other autoimmune diseases. Exposing lupus-prone NZBWF1 mice to respirable cSiO2 unleashes a vicious cycle of inflammation and cell death in the lung that triggers interferon-regulated gene expression, ectopic lymphoid structure (ELS) development, elevation of local and systemic autoantibodies (AAbs), and glomerulonephritis. However, cSiO2-induced inflammation and onset of autoimmunity can be prevented by inclusion of the ω-3 polyunsaturated fatty acid docosahexaenoic acid (DHA) into the diet of these mice. Since cSiO2 both causes cell death and interferes with efferocytosis, secondary necrosis of residual cell corpses might provide a rich and varied autoantigen (AAg) source in the lung. While it is known that the particle induces anti-nuclear and anti-dsDNA AAbs in NZBWF1 mice, the full extent of the cSiO2-induced AAb response relative to specificity and isotype is not yet understood. The purpose of this study was to test the hypotheses that cSiO2 exposure induces a wide spectrum of AAbs in the pulmonary and systemic compartments, and that dietary DHA intervention prevents these changes. Archived tissue fluid samples were obtained from a prior study in which NZBWF1 mice were fed purified isocaloric diets containing no DHA (control) or DHA corresponding calorically to human doses of 2 and 5 g/day. Mice were intranasally instilled with 1 mg cSiO2 or saline vehicle weekly for 4 weeks, then groups euthanized 1, 5, 9, or 13 weeks post-instillation (PI) of the last cSiO2 dose. Bronchoalveolar lavage fluid (BALF) and plasma from each time point were subjected to AAb profiling using a microarray containing 122 AAgs. cSiO2 triggered robust IgG and IgM AAb responses against lupus-associated AAgs, including DNA, histones, ribonucleoprotein, Smith antigen, Ro/SSA, La/SSB, and complement as early as 1 week PI in BALF and 5 weeks PI in plasma, peaking at 9 and 13 weeks PI, respectively. Importantly, cSiO2 also induced AAbs to AAgs associated with rheumatoid arthritis (collagen II, fibrinogen IV, fibrinogen S, fibronectin, and vimentin), Sjögren's syndrome (α-fodrin), systemic sclerosis (topoisomerase I), vasculitis (MPO and PR3), myositis (Mi-2, TIF1-γ, MDA5), autoimmune hepatitis (LC-1), and celiac disease (TTG). cSiO2 elicited comparable but more modest IgA AAb responses in BALF and plasma. cSiO2-induced AAb production was strongly associated with time dependent inflammatory/autoimmune gene expression, ELS development, and glomerulonephritis. AAb responses were dose-dependently suppressed by DHA supplementation and negatively correlated with the ω-3 index, an erythrocyte biomarker of ω-3 content in tissue phospholipids. Taken together, these findings suggest that cSiO2 exposure elicits a diverse multi-isotype repertoire of AAbs, many of which have been reported in individuals with lupus and other autoimmune diseases. Furthermore, induction of this broad AAb spectrum could be impeded by increasing ω-3 tissue content via dietary DHA supplementation.

Docosahexaenoic Acid Suppresses Silica-Induced Inflammasome Activation and IL-1 Cytokine Release by Interfering With Priming Signal.

Occupational exposure to respirable crystalline silica (cSiO2) has been etiologically linked to human autoimmunity. Intranasal instillation with cSiO2 triggers profuse inflammation in the lung and onset of autoimmunity in lupus-prone mice; however, dietary supplementation with the omega-3 polyunsaturated fatty acid docosahexaenoic acid (DHA) abrogates these responses. Inflammasome activation, IL-1 cytokine release, and death in alveolar macrophages following cSiO2 exposure are early and critical events that likely contribute to triggering premature autoimmune pathogenesis by this particle. Here we tested the hypothesis that DHA suppresses cSiO2-induced NLRP3 inflammasome activation, IL-1 cytokine release, and cell death in the macrophage. The model used was the murine macrophage RAW 264.7 cell line stably transfected with the inflammasome adapter protein ASC (RAW-ASC). Following priming with LPS, both the canonical activator nigericin and cSiO2 elicited robust inflammasome activation in RAW-ASC cells, as reflected by IL-1ß release and caspase-1 activation. These responses were greatly diminished or absent in wild-type RAW cells. In contrast to IL-1ß, cSiO2 induced IL-1α release in both RAW-ASC and to a lesser extent in RAW-WT cells after LPS priming. cSiO2-driven effects in RAW-ASC cells were confirmed in bone-marrow derived macrophages. Pre-incubating RAW-ASC cells with 10 and 25 µM DHA for 24 h enriched this fatty acid in the phospholipids by 15- and 25-fold, respectively, at the expense of oleic acid. DHA pre-incubation suppressed inflammasome activation and release of IL-1ß and IL-1α by nigericin, cSiO2, and two other crystals - monosodium urate and alum. DHA's suppressive effects were linked to inhibition of LPS-induced Nlrp3, Il1b, and Il1a transcription, potentially through the activation of PPARγ. Finally, nigericin-induced death was inflammasome-dependent, indicative of pyroptosis, and could be inhibited by DHA pretreatment. In contrast, cSiO2-induced death was inflammasome-independent and not inhibited by DHA. Taken together, these findings indicate that DHA suppresses cSiO2-induced inflammasome activation and IL-1 cytokine release in macrophages by acting at the level of priming, but was not protective against cSiO2-induced cell death.

Correction: Silica-Triggered Autoimmunity in Lupus-Prone Mice Blocked by Docosahexaenoic Acid Consumption.

[This corrects the article DOI: 10.1371/journal.pone.0160622.].

Mapping of Dynamic Transcriptome Changes Associated With Silica-Triggered Autoimmune Pathogenesis in the Lupus-Prone NZBWF1 Mouse.

Crystalline silica (cSiO2) is a widely recognized environmental trigger of autoimmune disease. In the lupus-prone female NZBWF1 mouse, airway exposure to cSiO2 triggers pulmonary ectopic lymphoid neogenesis, systemic autoantibody elevation, and glomerulonephritis. Here we tested the hypothesis that upregulation of adaptive immune function genes in the lung precedes cSiO2-triggering of autoimmune disease in this model. The study include three groups of mice, as follows: (1) necropsied 1 d after a single intranasal instillation of 1 mg cSiO2 or vehicle, (2) necropsied 1 d after four weekly single instillations of 1 mg cSiO2 or vehicle, or (3) necropsied 1, 5, 9, or 13 weeks after four weekly single instillations of 1 mg cSiO2 or vehicle. NanoString nCounter analysis revealed modest transcriptional changes associated with innate and adaptive immune response as early as 1 d after a single cSiO2 instillation. These responses were greatly expanded after four weekly cSiO2 instillations. Concurrent with ectopic lymphoid neogenesis, dramatic increases in mRNAs associated with chemokine release, cytokine production, sustained interferon activity, complement activation, and adhesion molecules were observed. As disease progressed, expression of these genes persisted and was further amplified. Consistent with autoimmune pathogenesis, the time between 5 and 9 weeks post-instillation reflected an important transition period where considerable immune gene upregulation in the lung was observed. Upon termination of the chronic study (13 weeks), cSiO2-induced changes in transcriptome signatures were similarly robust in kidney as compared to the lung, but more modest in spleen. Transcriptomic signatures in lung and kidney were indicative of infiltration and/or expansion of neutrophils, macrophages, dendritic cells, B cells, and T cells that corresponded with accelerated autoimmune pathogenesis. Taken together, airway exposure to cSiO2 elicited aberrant mRNA signatures for both innate and adaptive immunity that were consistent with establishment of the lung as the central autoimmune nexus for launching systemic autoimmunity and ultimately, kidney injury.

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

Exposure of lupus-prone female NZBWF1 mice to respirable crystalline silica (cSiO2), a known human autoimmune trigger, initiates loss of tolerance, rapid progression of autoimmunity, and early onset of glomerulonephritis. We have previously demonstrated that dietary supplementation with the ω-3 polyunsaturated fatty acid docosahexaenoic acid (DHA) suppresses autoimmune pathogenesis and nephritis in this unique model of lupus flaring. In this report, we utilized tissues from prior studies to test the hypothesis that DHA consumption interferes with upregulation of critical genes associated with cSiO2-triggered murine lupus. A NanoString nCounter platform targeting 770 immune-related genes was used to assess the effects cSiO2 on mRNA signatures over time in female NZBWF1 mice consuming control (CON) diets compared to mice fed diets containing DHA at an amount calorically equivalent to human consumption of 2 g per day (DHA low) or 5 g per day (DHA high). Experimental groups of mice were sacrificed: (1) 1 d after a single intranasal instillation of 1 mg cSiO2 or vehicle, (2) 1 d after four weekly single instillations of vehicle or 1 mg cSiO2, and (3) 1, 5, 9, and 13 weeks after four weekly single instillations of vehicle or 1 mg cSiO2. Genes associated with inflammation as well as innate and adaptive immunity were markedly upregulated in lungs of CON-fed mice 1 d after four weekly cSiO2 doses but were significantly suppressed in mice fed DHA high diets. Importantly, mRNA signatures in lungs of cSiO2-treated CON-fed mice over 13 weeks reflected progressive amplification of interferon (IFN)- and chemokine-related gene pathways. While these responses in the DHA low group were suppressed primarily at week 5, significant downregulation was observed at weeks 1, 5, 9, and 13 in mice fed the DHA high diet. At week 13, cSiO2 treatment of CON-fed mice affected 214 genes in kidney tissue associated with inflammation, innate/adaptive immunity, IFN, chemokines, and antigen processing, mostly by upregulation; however, feeding DHA dose-dependently suppressed these responses. Taken together, dietary DHA intake in lupus-prone mice impeded cSiO2-triggered mRNA signatures known to be involved in ectopic lymphoid tissue neogenesis, systemic autoimmunity, and glomerulonephritis.

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

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.

Correction: Silica-Triggered Autoimmunity in Lupus-Prone Mice Blocked by Docosahexaenoic Acid Consumption.

[This corrects the article DOI: 10.1371/journal.pone.0160622.].

Silica-Triggered Autoimmunity in Lupus-Prone Mice Blocked by Docosahexaenoic Acid Consumption.

Occupational exposure to respirable crystalline silica (cSiO2, quartz) is etiologically linked to systemic lupus erythematosus (lupus) and other human autoimmune diseases (ADs). In the female NZBWF1 mouse, a widely used animal model that is genetically prone to lupus, short-term repeated intranasal exposure to cSiO2 triggers premature initiation of autoimmune responses in the lungs and kidneys. In contrast to cSiO2's triggering action, consumption of the ω-3 polyunsaturated fatty acid docosahexaenoic acid (DHA) prevents spontaneous onset of autoimmunity in this mouse strain. The aim of this study was to test the hypothesis that consumption of DHA will prevent cSiO2-triggered autoimmunity in the female NZBWF1 mouse. Mice (6 wk old) were fed isocaloric AIN-93G diets containing 0.0, 0.4, 1.2 or 2.4% DHA. Two wk after initiating feeding, mice were intranasally instilled with 1 mg cSiO2 once per wk for 4 wk and maintained on experimental diets for an additional 12 wk. Mice were then sacrificed and the lung, blood and kidney assessed for markers of inflammation and autoimmunity. DHA was incorporated into lung, red blood cells and kidney from diet in a concentration-dependent fashion. Dietary DHA dose-dependently suppressed cSiO2-triggered perivascular leukocyte infiltration and ectopic lymphoid tissue neogenesis in the lung. DHA consumption concurrently inhibited cSiO2-driven elevation of proinflammatory cytokines, B-cell proliferation factors, IgG and anti-dsDNA Ig in both bronchoalveolar lavage fluid and plasma. DHA's prophylactic effects were further mirrored in reduced proteinuria and glomerulonephritis in cSiO2-treated mice. Taken together, these results reveal that DHA consumption suppresses cSiO2 triggering of autoimmunity in female NZBWF1 mice as manifested in the lung, blood and kidney. Our findings provide novel insight into how dietary modulation of the lipidome might be used to prevent or delay triggering of AD by cSiO2. Such knowledge opens the possibility of developing practical, low-cost preventative strategies to reduce the risk of initiating AD and subsequent flaring in cSiO2-exposed individuals. Additional research in this model is required to establish the mechanisms by which DHA suppresses cSiO2-induced autoimmunity and to ascertain unique lipidome signatures predictive of susceptibility to cSiO2-triggered AD.

Silica Triggers Inflammation and Ectopic Lymphoid Neogenesis in the Lungs in Parallel with Accelerated Onset of Systemic Autoimmunity and Glomerulonephritis in the Lupus-Prone NZBWF1 Mouse.

Genetic predisposition and environmental factors influence the development of human autoimmune disease. Occupational exposure to crystalline silica (cSiO2) has been etiologically linked to increased incidence of autoimmunity, including systemic lupus erythematosus (SLE), but the underlying mechanisms are poorly understood. The purpose of this study was to test the hypothesis that early repeated short-term cSiO2 exposure will modulate both latency and severity of autoimmunity in the lupus-prone female NZBWF1 mouse. Weekly intranasal exposure to cSiO2 (0.25 and 1.0 mg) for 4 wk beginning at 9 wk of age both reduced latency and increased intensity of glomerulonephritis. cSiO2 elicited robust inflammatory responses in the lungs as evidenced by extensive perivascular and peribronchial lymphoplasmacytic infiltration consisting of IgG-producing plasma cells, and CD45R+ and CD3+ lymphocytes that were highly suggestive of ectopic lymphoid tissue (ELT). In addition, there were elevated concentrations of immunoglobulins and the cytokines MCP-1, TNF-α and IL-6 in bronchoalveolar lavage fluid. cSiO2-associated kidney and lung effects paralleled dose-dependent elevations of autoantibodies and proinflammatory cytokines in plasma. Taken together, cSiO2-induced pulmonary inflammation and ectopic lymphoid neogenesis in the NZBWF1 mouse corresponded closely to systemic inflammatory and autoimmune responses as well as the early initiation of pathological outcomes in the kidney. These findings suggest that following airway exposure to crystalline silica, in mice genetically prone to SLE, the lung serves as a platform for triggering systemic autoimmunity and glomerulonephritis.

Comparative effects of n-3, n-6 and n-9 unsaturated fatty acid-rich diet consumption on lupus nephritis, autoantibody production and CD4+ T cell-related gene responses in the autoimmune NZBWF1 mouse.

Mortality from systemic lupus erythematosus (SLE), a prototypical autoimmune disease, correlates with the onset and severity of kidney glomerulonephritis. There are both preclinical and clinical evidence that SLE patients may benefit from consumption of n-3 polyunsaturated fatty acids (PUFA) found in fish oil, but the mechanisms remain unclear. Here we employed the NZBWF1 SLE mouse model to compare the effects of dietary lipids on the onset and severity of autoimmune glomerulonephritis after consuming: 1) n-3 PUFA-rich diet containing docosahexaenoic acid-enriched fish oil (DFO), 2) n-6 PUFA-rich Western-type diet containing corn oil (CRN) or 3) n-9 monounsaturated fatty acid (MUFA)-rich Mediterranean-type diet containing high oleic safflower oil (HOS). Elevated plasma autoantibodies, proteinuria and glomerulonephritis were evident in mice fed either the n-6 PUFA or n-9 MUFA diets, however, all three endpoints were markedly attenuated in mice that consumed the n-3 PUFA diet until 34 wk of age. A focused PCR array was used to relate these findings to the expression of 84 genes associated with CD4+ T cell function in the spleen and kidney both prior to and after the onset of the autoimmune nephritis. n-3 PUFA suppression of autoimmunity in NZBWF1 mice was found to co-occur with a generalized downregulation of CD4+ T cell-related genes in kidney and/or spleen at wk 34. These genes were associated with the inflammatory response, antigen presentation, T cell activation, B cell activation/differentiation and leukocyte recruitment. Quantitative RT-PCR of representative affected genes confirmed that n-3 PUFA consumption was associated with reduced expression of CD80, CTLA-4, IL-10, IL-18, CCL-5, CXCR3, IL-6, TNF-α and osteopontin mRNAs in kidney and/or spleens as compared to mice fed n-6 PUFA or n-9 MUFA diets. Remarkably, many of the genes identified in this study are currently under consideration as biomarkers and/or biotherapeutic targets for SLE and other autoimmune diseases.

Peptide YY3-36 and 5-hydroxytryptamine mediate emesis induction by trichothecene deoxynivalenol (vomitoxin).

Deoxynivalenol (DON, vomitoxin), a trichothecene mycotoxin produced by Fusarium sp. that frequently occurs in cereal grains, has been associated with human and animal food poisoning. Although a common hallmark of DON-induced toxicity is the rapid onset of emesis, the mechanisms for this adverse effect are not fully understood. Recently, our laboratory has demonstrated that the mink (Neovison vison) is a suitable small animal model for investigating trichothecene-induced emesis. The goal of this study was to use this model to determine the roles of two gut satiety hormones, peptide YY3-36 (PYY3-36) and cholecystokinin (CCK), and the neurotransmitter 5-hydroxytryptamine (5-HT) in DON-induced emesis. Following ip exposure to DON at 0.1 and 0.25mg/kg bw, emesis induction ensued within 15-30min and then persisted up to 120min. Plasma DON measurement revealed that this emesis period correlated with the rapid distribution and clearance of the toxin. Significant elevations in both plasma PYY3-36 (30-60min) and 5-HT (60min) but not CCK were observed during emesis. Pretreatment with the neuropeptide Y2 receptor antagonist JNJ-31020028 attenuated DON- and PYY-induced emesis, whereas the CCK1 receptor antagonist devezapide did not alter DON's emetic effects. The 5-HT3 receptor antagonist granisetron completely suppressed induction of vomiting by DON and the 5-HT inducer cisplatin. Granisetron pretreatment also partially blocked PYY3-36-induced emesis, suggesting a potential upstream role for this gut satiety hormone in 5-HT release. Taken together, the results suggest that both PYY3-36 and 5-HT play contributory roles in DON-induced emesis.

Comparison of emetic potencies of the 8-ketotrichothecenes deoxynivalenol, 15-acetyldeoxynivalenol, 3-acetyldeoxynivalenol, fusarenon X, and nivalenol.

Although the acute toxic effects of trichothecene mycotoxin deoxynivalenol (DON or vomitoxin), a known cause of human food poisoning, have been well characterized in several animal species, much less is known about closely related 8-ketotrichothecenes that similarly occur in cereal grains colonized by toxigenic fusaria. To address this, we compared potencies of DON, 15-acetyldeoxynivalenol (15-ADON), 3-acetyldeoxynivalenol (3-ADON), fusarenon X (FX), and nivalenol (NIV) in the mink emesis model following intraperitoneal (ip) and oral administration. All five congeners dose-dependently induced emesis by both administration methods. With increasing doses, there were marked decreases in latency to emesis with corresponding increases in emesis duration and number of emetic events. The effective doses resulting in emetic events in 50% of the animals for ip exposure to DON, 15-ADON, 3-ADON, FX, and NIV were 80, 170, 180, 70, and 60 µg/kg bw, respectively, and for oral exposure, they were 30, 40, 290, 30, and 250 µg/kg bw, respectively. The emetic potency of DON determined here was comparable to that reported in analogous studies conducted in pigs and dogs, suggesting that the mink is a suitable small animal model for investigating acute trichothecene toxicity. The use of a mouse pica model, based on the consumption of kaolin, was also evaluated as a possible surrogate for studying emesis but was found unsuitable. From a public health perspective, comparative emetic potency data derived from small animal models such as the mink should be useful for establishing toxic equivalency factors for DON and other trichothecenes.