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.

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.

Divergent pathogenetic outcomes in BALB/c mice following Omicron subvariant infection.

Following the emergence of B.1.1.529 Omicron, the SARS-CoV-2 virus evolved into a significant number of sublineage variants that possessed numerous mutations throughout the genome, but particularly within the spike glycoprotein (S) gene. For example, the BQ.1.1 and the XBB.1 and XBB.1.5 subvariants contained 34 and 41 mutations in S, respectively. However, these variants elicited largely replication only or mild disease phenotypes in mice. To better model pathogenic outcomes and measure countermeasure performance, we developed mouse adapted versions (BQ.1.1 MA; XBB.1 MA; XBB.1.5 MA) that reflect more pathogenic acute phase pulmonary disease symptoms of SARS-CoV-2, as well as derivative strains expressing nano-luciferase (nLuc) in place of ORF7 (BQ.1.1 nLuc; XBB.1 nLuc; XBB.1.5 nLuc). Amongst the mouse adapted (MA) viruses, a wide range of disease outcomes were observed including mortality, weight loss, lung dysfunction, and tissue viral loads in the lung and nasal turbinates. Intriguingly, XBB.1 MA and XBB.1.5 MA strains, which contained identical mutations throughout except at position F486S/P in S, exhibited divergent disease outcomes in mice (Ao et al., 2023). XBB.1.5 MA infection was associated with significant weight loss and ∼45 % mortality across two independent studies, while XBB.1 MA infected animals suffered from mild weight loss and only 10 % mortality across the same two independent studies. Additionally, the development and use of nanoluciferase expressing strains provided moderate throughput for live virus neutralization assays. The availability of small animal models for the assessment of Omicron VOC disease potential will enable refined capacity to evaluate the efficacy of on market and pre-clinical therapeutics and interventions.

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.

Aerosol physicochemical determinants of carbon black and ozone inhalation co-exposure induced pulmonary toxicity.

Air pollution accounts for more than 7 million premature deaths worldwide. Using ultrafine carbon black (CB) and ozone (O3) as a model for an environmental co-exposure scenario, the dose response relationships in acute pulmonary injury and inflammation were determined by generating, characterizing, and comparing stable concentrations of CB aerosols (2.5, 5.0, 10.0 mg/m3), O3 (0.5, 1.0, 2.0 ppm) with mixture CB + O3 (2.5 + 0.5, 5.0 + 1.0, 10.0 + 2.0). C57BL6 male mice were exposed for 3 h by whole body inhalation and acute toxicity determined after 24 h. CB itself did not cause any alteration, however, a dose response in pulmonary injury/inflammation was observed with O3 and CB + O3. This increase in response with mixtures was not dependent on the uptake but was due to enhanced reactivity of the particles. Benchmark dose modeling showed several-fold increase in potency with CB + O3 compared with CB or O3 alone. Principal component analysis provided insight into response relationships between various doses and treatments. There was a significant correlation in lung responses with charge-based size distribution, total/alveolar deposition, oxidant generation, and antioxidant depletion potential. Lung tissue gene/protein response demonstrated distinct patterns that are better predicted by either particle dose/aerosol responses (interleukin-1ß, keratinocyte chemoattractant, transforming growth factor beta) or particle reactivity (thymic stromal lymphopoietin, interleukin-13, interleukin-6). Hierarchical clustering showed a distinct signature with high dose and a similarity in mRNA expression pattern of low and medium doses of CB + O3. In conclusion, we demonstrate that the biological outcomes from CB + O3 co-exposure are significantly greater than individual exposures over a range of aerosol concentrations and aerosol characteristics can predict biological outcome.

Prevention of bleomycin-induced lung fibrosis via inhibition of the MRTF/SRF transcription pathway.

Bleomycin-induced lung fibrosis is a debilitating disease, linked to high morbidity and mortality in chemotherapy patients. The MRTF/SRF transcription pathway has been proposed as a potential therapeutic target, as it is critical for myofibroblast differentiation, a hallmark of fibrosis. In human lung fibroblasts, the MRTF/SRF pathway inhibitor, CCG-257081, effectively decreased mRNA levels of downstream genes: smooth muscle actin and connective tissue growth factor, with IC50 s of 4 and 15 µM, respectively. The ability of CCG-257081 to prevent inflammation and fibrosis, measured via pulmonary collagen content and histopathology, was tested in a murine model of bleomycin-induced lung fibrosis. Animals were given intraperitoneal bleomycin for 4 weeks and concurrently dosed with CCG-257081 (0, 10, 30, and 100 mg/kg PO), a clinical anti-fibrotic (nintedanib) or the clinical standard of care (prednisolone). Mice treated with 100 mg/kg CCG-257081 gained weight vs. vehicle-treated control mice, while those receiving nintedanib and prednisolone lost significant weight. Hydroxyproline content and histological findings in tissue of animals on 100 mg/kg CCG-257081 were not significantly different from naive tissue, indicating successful prevention. Measures of tissue fibrosis were comparable between CCG-257081 and nintedanib, but only the MRTF/SRF inhibitor decreased plasminogen activator inhibitor-1 (PAI-1), a marker linked to fibrosis, in bronchoalveolar lavage fluid. In contrast, prednisolone led to marked increases in lung fibrosis by all metrics. This study demonstrates the potential use of MRTF/SRF inhibitors to prevent bleomycin-induced lung fibrosis in a clinically relevant model of the disease.

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.

Single cell analysis of docosahexaenoic acid suppression of sequential LPS-induced proinflammatory and interferon-regulated gene expression in the macrophage.

Preclinical and clinical studies suggest that consumption of long chain omega-3 polyunsaturated fatty acids (PUFAs) reduces severity of chronic inflammatory and autoimmune diseases. While these ameliorative effects are conventionally associated with downregulated expression of proinflammatory cytokine and chemokine genes, our laboratory has recently identified Type 1 interferon (IFN1)-regulated gene expression to be another key target of omega-3 PUFAs. Here we used single cell RNA sequencing (scRNAseq) to gain new mechanistic perspectives on how the omega-3 PUFA docosahexaenoic acid (DHA) influences TLR4-driven proinflammatory and IFN1-regulated gene expression in a novel self-renewing murine fetal liver-derived macrophage (FLM) model. FLMs were cultured with 25 µM DHA or vehicle for 24 h, treated with modest concentration of LPS (20 ng/ml) for 1 and 4 h, and then subjected to scRNAseq using the 10X Chromium System. At 0 h (i.e., in the absence of LPS), DHA increased expression of genes associated with the NRF2 antioxidant response (e.g. Sqstm1, Hmox1, Chchd10) and metal homeostasis (e.g.Mt1, Mt2, Ftl1, Fth1), both of which are consistent with DHA-induced polarization of FLMs to a more anti-inflammatory phenotype. At 1 h post-LPS treatment, DHA inhibited LPS-induced cholesterol synthesis genes (e.g. Scd1, Scd2, Pmvk, Cyp51, Hmgcs1, and Fdps) which potentially could contribute to interference with TLR4-mediated inflammatory signaling. At 4 h post-LPS treatment, LPS-treated FLMs reflected a more robust inflammatory response including upregulation of proinflammatory cytokine (e.g. Il1a, Il1b, Tnf) and chemokine (e.g.Ccl2, Ccl3, Ccl4, Ccl7) genes as well as IFN1-regulated genes (e.g. Irf7, Mx1, Oasl1, Ifit1), many of which were suppressed by DHA. Using single-cell regulatory network inference and clustering (SCENIC) to identify gene expression networks, we found DHA modestly downregulated LPS-induced expression of NF-κB-target genes. Importantly, LPS induced a subset of FLMs simultaneously expressing NF-κB- and IRF7/STAT1/STAT2-target genes that were conspicuously absent in DHA-pretreated FLMs. Thus, DHA potently targeted both the NF-κB and the IFN1 responses. Altogether, scRNAseq generated a valuable dataset that provides new insights into multiple overlapping mechanisms by which DHA may transcriptionally or post-transcriptionally regulate LPS-induced proinflammatory and IFN1-driven responses in macrophages.

A Locus on Chromosome 15 Contributes to Acute Ozone-induced Lung Injury in Collaborative Cross Mice.

Ozone (O3)-induced respiratory toxicity varies considerably within the human population and across inbred mouse strains, indicative of gene-environment interactions (GxE). Though previous studies have identified several quantitative trait loci (QTL) and candidate genes underlying responses to O3 exposure, precise mechanisms of susceptibility remain incompletely described. We sought to update our understanding of the genetic architecture of O3 responsiveness using the Collaborative Cross (CC) recombinant inbred mouse panel. We evaluated hallmark O3-induced inflammation and injury phenotypes in 56 CC strains after exposure to filtered air or 2 ppm O3, and performed focused genetic analysis of variation in lung injury, as reflected by protein in lung lavage fluid. Strain-dependent responses to O3 were clear, and QTL mapping revealed two novel loci on Chr (Chromosomes) 10 (peak, 26.2 Mb; 80% confidence interval [CI], 24.6-43.6 Mb) and 15 (peak, 47.1 Mb; 80% CI, 40.2-54.9 Mb), the latter surpassing the 95% significance threshold. At the Chr 15 locus, C57BL/6J and CAST/EiJ founder haplotypes were associated with higher lung injury responses compared with all other CC founder haplotypes. With further statistical analysis and a weight of evidence approach, we delimited the Chr 15 QTL to an ∼2 Mb region containing 21 genes (10 protein coding) and nominated three candidate genes, namely Oxr1, Rspo2, and Angpt1. Gene and protein expression data further supported Oxr1 and Angpt1 as priority candidate genes. In summary, we have shown that O3-induced lung injury is modulated by genetic variation, identified two high priority candidate genes, and demonstrated the value of the CC for detecting GxE.

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).

Neuroinflammatory and Neurometabolomic Consequences From Inhaled Wildfire Smoke-Derived Particulate Matter in the Western United States.

Utilizing a mobile laboratory located >300 km away from wildfire smoke (WFS) sources, this study examined the systemic immune response profile, with a focus on neuroinflammatory and neurometabolomic consequences, resulting from inhalation exposure to naturally occurring wildfires in California, Arizona, and Washington in 2020. After a 20-day (4 h/day) exposure period in a mobile laboratory stationed in New Mexico, WFS-derived particulate matter (WFPM) inhalation resulted in significant neuroinflammation while immune activity in the peripheral (lung, bone marrow) appeared to be resolved in C57BL/6 mice. Importantly, WFPM exposure increased cerebrovascular endothelial cell activation and expression of adhesion molecules (VCAM-1 and ICAM-1) in addition to increased glial activation and peripheral immune cell infiltration into the brain. Flow cytometry analysis revealed proinflammatory phenotypes of microglia and peripheral immune subsets in the brain of WFPM-exposed mice. Interestingly, endothelial cell neuroimmune activity was differentially associated with levels of PECAM-1 expression, suggesting that subsets of cerebrovascular endothelial cells were transitioning to resolution of inflammation following the 20-day exposure. Neurometabolites related to protection against aging, such as NAD+ and taurine, were decreased by WFPM exposure. Additionally, increased pathological amyloid-beta protein accumulation, a hallmark of neurodegeneration, was observed. Neuroinflammation, together with decreased levels of key neurometabolites, reflect a cluster of outcomes with important implications in priming inflammaging and aging-related neurodegenerative phenotypes.

Hyperspectral microscopy of subcutaneously released silver nanoparticles reveals sex differences in drug distribution.

Biomaterials have a great potential to improve human health, however in vitro and in vivo studies are necessary to provide information on their efficacy and safety. This study reports on a comprehensive evaluation of core-shell electrospun fibers loaded with silver nanoparticles (Ag NP) where the delivery rate was controlled by different sizes of Ag NP and thermoresponsive poly(n-isopropylacrylamide) (PNIPAM) hydrogel particles. Fiber meshes also contain zinc oxide nanoparticles (ZnO NP), to improve pore structure for controlled release of Ag NP. In vitro cytotoxicity studies using cultured human A549 epithelial cells demonstrated that the ZnO NP component, which is known to cause cytotoxicity, of the fiber meshes did cause measurable cell death. In vitro antibacterial efficacy of the fiber meshes was shown with rapid and efficient growth inhibition in E. coli bacterial culture. Fiber meshes were implanted subcutaneously for up to 27 days in male and female C57BL/6 mice to evaluate the in vivo drug release and biocompatibility. Hyperspectral microscopy was used as an advanced tool to determine precise location of released Ag NP into the skin compared to the conventional tissue staining methods. Results suggested that Ag NP were continuously released over 27 days of implantation in mice. Hyperspectral imaging revealed that released Ag NP dispersed in the dermis of male mice, however, Ag NP accumulated in the hair follicles of female mice (Figure). Mice implanted with fiber meshes containing ZnO NP had better hair regrowth and wound healing, which was in contrast to in vitro cytotoxicity results. These findings suggest that these newly developed fiber meshes can have unique long-term release of drugs loaded in the fiber core and appear to be biocompatible. The differences in the sex-bias outcome suggest the opportunity for development of sex-specific drug delivery systems.

Transcriptomics of single dose and repeated carbon black and ozone inhalation co-exposure highlight progressive pulmonary mitochondrial dysfunction.

BACKGROUND: Air pollution is a complex mixture of particles and gases, yet current regulations are based on single toxicant levels failing to consider potential interactive outcomes of co-exposures. We examined transcriptomic changes after inhalation co-exposure to a particulate and a gaseous component of air pollution and hypothesized that co-exposure would induce significantly greater impairments to mitochondrial bioenergetics. A whole-body inhalation exposure to ultrafine carbon black (CB), and ozone (O3) was performed, and the impact of single and multiple exposures was studied at relevant deposition levels. C57BL/6 mice were exposed to CB (10 mg/m3) and/or O3 (2 ppm) for 3 h (either a single exposure or four independent exposures). RNA was isolated from lungs and mRNA sequencing performed using the Illumina HiSeq. Lung pathology was evaluated by histology and immunohistochemistry. Electron transport chain (ETC) activities, electron flow, hydrogen peroxide production, and ATP content were assessed. RESULTS: Compared to individual exposure groups, co-exposure induced significantly greater neutrophils and protein levels in broncho-alveolar lavage fluid as well as a significant increase in mRNA expression of oxidative stress and inflammation related genes. Similarly, a significant increase in hydrogen peroxide production was observed after co-exposure. After single and four exposures, co-exposure revealed a greater number of differentially expressed genes (2251 and 4072, respectively). Of these genes, 1188 (single exposure) and 2061 (four exposures) were uniquely differentially expressed, with 35 mitochondrial ETC mRNA transcripts significantly impacted after four exposures. Both O3 and co-exposure treatment significantly reduced ETC maximal activity for complexes I (- 39.3% and - 36.2%, respectively) and IV (- 55.1% and - 57.1%, respectively). Only co-exposure reduced ATP Synthase activity (- 35.7%) and total ATP content (30%). Further, the ability for ATP Synthase to function is limited by reduced electron flow (- 25%) and translation of subunits, such as ATP5F1, following co-exposure. CONCLUSIONS: CB and O3 co-exposure cause unique transcriptomic changes in the lungs that are characterized by functional deficits to mitochondrial bioenergetics. Alterations to ATP Synthase function and mitochondrial electron flow underly a pathological adaptation to lung injury induced by co-exposure.

Oxidant-induced epithelial alarmin pathway mediates lung inflammation and functional decline following ultrafine carbon and ozone inhalation co-exposure.

Environmental inhalation exposures are inherently mixed (gases and particles), yet regulations are still based on single toxicant exposures. While the impacts of individual components of environmental pollution have received substantial attention, the impact of inhalation co-exposures is poorly understood. Here, we mechanistically investigated pulmonary inflammation and lung function decline after inhalation co-exposure and individual exposures to ozone (O3) and ultrafine carbon black (CB). Environmentally/occupationally relevant lung deposition levels in mice were achieved after inhalation of stable aerosols with similar aerodynamic and mass median distributions. X-ray photoemission spectroscopy detected increased surface oxygen contents on particles in co-exposure aerosols. Compared with individual exposures, co-exposure aerosols produced greater acellular and cellular oxidants detected by electron paramagnetic resonance (EPR) spectroscopy, and in vivo immune-spin trapping (IST), as well as synergistically increased lavage neutrophils, lavage proteins and inflammation related gene/protein expression. Co-exposure induced a significantly greater respiratory function decline compared to individual exposure. A synthetic catalase-superoxide dismutase mimetic (EUK-134) significantly blunted lung inflammation and respiratory function decline confirming the role of oxidant imbalance. We identified a significant induction of epithelial alarmin (thymic stromal lymphopoietin-TSLP)-dependent interleukin-13 pathway after co-exposure, associated with increased mucin and interferon gene expression. We provided evidence of interactive outcomes after air pollution constituent co-exposure and identified a key mechanistic pathway that can potentially explain epidemiological observation of lung function decline after an acute peak of air pollution. Developing and studying the co-exposure scenario in a standardized and controlled fashion will enable a better mechanistic understanding of how environmental exposures result in adverse outcomes.

Fenofibrate, a peroxisome proliferator-activated receptor-alpha agonist, blocks steatosis and alters the inflammatory response in a mouse model of inflammation-dioxin interaction.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (dioxin; TCDD) is an environmental contaminant that elicits a variety of toxic effects, many of which are mediated through activation of the aryl hydrocarbon receptor (AhR). Interaction between AhR and the peroxisome proliferator-activated receptor-alpha (PPAR-α), which regulates fatty acid metabolism, has been suggested. Furthermore, with recognition of the prevalence of inflammatory conditions, there is current interest in the potential for inflammatory stress to modulate the response to environmental agents. The aim of this work was to assess the interaction of TCDD with hepatic inflammation modulated by fenofibrate, a PPAR-α agonist. Female, C57BL/6 mice were treated orally with vehicle or fenofibrate (250 mg/kg) for 13 days, and then were given vehicle or 30 µg/kg TCDD. Four days later, the animals received an i.p. injection of lipopolysaccharide-galactosamine (LPS-GalN) (0.05x107 EU/kg and 500 mg/kg, respectively) to incite inflammation, or saline as vehicle control. After 4 h, the mice were euthanized, and blood and liver samples were collected for analysis. Livers of animals treated with TCDD with or without LPS-GalN had increased lipid deposition, and this effect was blocked by fenofibrate. In TCDD/LPS-GalN-treated mice, fenofibrate caused an increase in plasma activity of alanine aminotransferase, a marker of hepatocellular injury. TCDD reduced LPS-GalN-induced apoptosis, an effect that was prevented by fenofibrate pretreatment. LPS-GalN induced an increase in the concentration of interleukin-6 in plasma and accumulation of neutrophils in liver. TCDD exposure enhanced the former response and inhibited the latter one. These results suggest that fenofibrate counteracts the changes in lipid metabolism induced by TCDD but increases inflammation and liver injury in this model of inflammation-TCDD interaction.

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.

Respiratory and systemic impacts following MWCNT inhalation in B6C3F1/N mice.

BACKGROUND: A very pure multi-walled carbon nanotube (MWCNT) that was shown to have very low toxicity in vitro, was evaluated for lung and systemic effects and distribution following inhalation exposure. METHODS: B6C3F1/N mice were exposed to varying doses (0, 0.06, 0.2, and 0.6 mg/m3) of the (99.1% carbon) MWCNT by inhalation for 30 days (excluding weekends). Ten days following the last exposure, the lungs and spleen were harvested and processed for histology and immune cell population assessment. In addition, lung lavage cells and fluid were analyzed. Stimulated Raman scattering (SRS) was used to identify particles in the lungs, spleen, kidneys, liver, mediastinal and brachial lymph nodes, and olfactory bulb. Splenic tissue sections were stained with hematoxylin and eosin (H&E) for light microscopic histopathology assessment. Blood plasma was analyzed for cytokines and cathepsins. A section of the spleen was processed for RNA isolation and relative gene expression for 84 inflammation-related cytokines/chemokines. RESULTS: Following MWCNT exposure, particles were clearly evident in the lungs, spleens, lymph nodes and olfactory bulbs, (but not livers or kidneys) of exposed mice in a dose-dependent manner. Examination of the lavaged lung cells was unremarkable with no significant inflammation indicated at all particle doses. In contrast, histological examination of the spleen indicated the presence of apoptotic bodies within T cells regions of the white pulp area. Isolated splenic leukocytes had significant changes in various cells including an increased number of proinflammatory CD11b+Ly6C+ splenic cells. The gene expression studies confirmed this observation as several inflammation-related genes were upregulated particularly in the high dose exposure (0.6 mg/m3). Blood plasma evaluations showed a systemic down-regulation of inflammatory cytokines and a dose-dependent up-regulation of lysosomal cathepsins. CONCLUSIONS: The findings in the lungs were consistent with our hypothesis that this MWCNT exposure would result in minimal lung inflammation and injury. However, the low toxicity of the MWCNT to lung macrophages may have contributed to enhanced migration of the MWCNT to the spleen through the lymph nodes, resulting in splenic toxicity and systemic changes in inflammatory mediators.

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.