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1.  Environmentally persistent free radicals amplify ultrafine particle mediated cellular oxidative stress and cytotoxicity 
Background
Combustion generated particulate matter is deposited in the respiratory tract and pose a hazard to the lungs through their potential to cause oxidative stress and inflammation. We have previously shown that combustion of fuels and chlorinated hydrocarbons produce semiquinone-type radicals that are stabilized on particle surfaces (i.e. environmentally persistent free radicals; EPFRs). Because the composition and properties of actual combustion-generated particles are complex, heterogeneous in origin, and vary from day-to-day, we have chosen to use surrogate particle systems. In particular, we have chosen to use the radical of 2-monochlorophenol (MCP230) as the EPFR because we have previously shown that it forms a EPFR on Cu(II)O surfaces and catalyzes formation of PCDD/F. To understand the physicochemical properties responsible for the adverse pulmonary effects of combustion by-products, we have exposed human bronchial epithelial cells (BEAS-2B) to MCP230 or the CuO/silica substrate. Our general hypothesis was that the EPFR-containing particle would have greater toxicity than the substrate species.
Results
Exposure of BEAS-2B cells to our combustion generated particle systems significantly increased reactive oxygen species (ROS) generation and decreased cellular antioxidants resulting in cell death. Resveratrol treatment reversed the decline in cellular glutathione (GSH), glutathione peroxidase (GPx), and superoxide dismutase (SOD) levels for both types of combustion-generated particle systems.
Conclusion
The enhanced cytotoxicity upon exposure to MCP230 correlated with its ability to generate more cellular oxidative stress and concurrently reduce the antioxidant defenses of the epithelial cells (i.e. reduced GSH, SOD activity, and GPx). The EPFRs in MCP230 also seem to be of greater biological concern due to their ability to induce lipid peroxidation. These results are consistent with the oxidizing nature of the CuO/silica ultrafine particles and the reducing nature and prolonged environmental and biological lifetimes of the EPFRs in MCP230.
doi:10.1186/1743-8977-6-11
PMCID: PMC2676242  PMID: 19374750
2.  Radical-Containing Ultrafine Particulate Matter Initiates Epithelial-to-Mesenchymal Transitions in Airway Epithelial Cells 
Environmentally persistent free radicals (EPFRs) in combustion-generated particulate matter (PM) are capable of inducing pulmonary pathologies and contributing to the development of environmental asthma. In vivo exposure of infant rats to EPFRs demonstrates their ability to induce airway hyperresponsiveness to methacholine, a hallmark of asthma. However, the mechanisms by which combustion-derived EPFRs elicit in vivo responses remain elusive. In this study, we used a chemically defined EPFR consisting of approximately 0.2 μm amorphrous silica containing 3% cupric oxide with the organic pollutant 1,2-dichlorobenzene (DCB-230). DCB-230 possesses similar radical content to urban-collected EPFRs but offers several advantages, including lack of contaminants and chemical uniformity. DCB-230 was readily taken up by BEAS-2B and at high doses (200 μg/cm2) caused substantial necrosis. At low doses (20 μg/cm2), DCB-230 particles caused lysosomal membrane permeabilization, oxidative stress, and lipid peroxidation within 24 hours of exposure. During this period, BEAS-2B underwent epithelial-to-mesenchymal transition (EMT), including loss of epithelial cell morphology, decreased E-cadherin expression, and increased α–smooth muscle actin (α-SMA) and collagen I production. Similar results were observed in neonatal air–liquid interface culture (i.e., disruption of epithelial integrity and EMT). Acute exposure of infant mice to DCB-230 resulted in EMT, as confirmed by lineage tracing studies and evidenced by coexpression of epithelial E-cadherin and mesenchymal α-SMA proteins in airway cells and increased SNAI1 expression in the lungs. EMT in neonatal mouse lungs after EPFR exposure may provide an explanation for epidemiological evidence supporting PM exposure and increased risk of asthma.
doi:10.1165/rcmb.2012-0052OC
PMCID: PMC3604062  PMID: 23087054
particulate matter; epithelial–mesenchymal transition; environmental asthma; pediatric
3.  Environmentally persistent free radicals induce airway hyperresponsiveness in neonatal rat lungs 
Background
Increased asthma risk/exacerbation in children and infants is associated with exposure to elevated levels of ultrafine particulate matter (PM). The presence of a newly realized class of pollutants, environmentally persistent free radicals (EPFRs), in PM from combustion sources suggests a potentially unrecognized risk factor for the development and/or exacerbation of asthma.
Methods
Neonatal rats (7-days of age) were exposed to EPFR-containing combustion generated ultrafine particles (CGUFP), non-EPFR containing CGUFP, or air for 20 minutes per day for one week. Pulmonary function was assessed in exposed rats and age matched controls. Lavage fluid was isolated and assayed for cellularity and cytokines and in vivo indicators of oxidative stress. Pulmonary histopathology and characterization of differential protein expression in lung homogenates was also performed.
Results
Neonates exposed to EPFR-containing CGUFP developed significant pulmonary inflammation, and airway hyperreactivity. This correlated with increased levels of oxidative stress in the lungs. Using differential two-dimensional electrophoresis, we identified 16 differentially expressed proteins between control and CGUFP exposed groups. In the rats exposed to EPFR-containing CGUFP; peroxiredoxin-6, cofilin1, and annexin A8 were upregulated.
Conclusions
Exposure of neonates to EPFR-containing CGUFP induced pulmonary oxidative stress and lung dysfunction. This correlated with alterations in the expression of various proteins associated with the response to oxidative stress and the regulation of glucocorticoid receptor translocation in T lymphocytes.
doi:10.1186/1743-8977-8-11
PMCID: PMC3061909  PMID: 21388553
4.  Exposure to combustion generated environmentally persistent free radicals enhances severity of influenza virus infection 
Background
Exposures to elevated levels of particulate matter (PM) enhance severity of influenza virus infection in infants. The biological mechanism responsible for this phenomenon is unknown. The recent identification of environmentally persistent free radicals (EPFRs) associated with PM from a variety of combustion sources suggests its role in the enhancement of influenza disease severity.
Methods
Neonatal mice (< seven days of age) were exposed to DCB230 (combustion derived PM with a chemisorbed EPFR), DCB50 (non-EPFR PM sample), or air for 30 minutes/day for seven consecutive days. Four days post-exposure, neonates were infected with influenza intranasally at 1.25 TCID50/neonate. Neonates were assessed for morbidity (% weight gain, peak pulmonary viral load, and viral clearance) and percent survival. Lungs were isolated and assessed for oxidative stress (8-isoprostanes and glutathione levels), adaptive immune response to influenza, and regulatory T cells (Tregs). The role of the EPFR was also assessed by use of transgenic mice expressing human superoxide dismutase 2.
Results
Neonates exposed to EPFRs had significantly enhanced morbidity and decreased survival following influenza infection. Increased oxidative stress was also observed in EPFR exposed neonates. This correlated with increased pulmonary Tregs and dampened protective T cell responses to influenza infection. Reduction of EPFR-induced oxidative stress attenuated these effects.
Conclusions
Neonatal exposure to EPFR containing PM resulted in pulmonary oxidative stress and enhanced influenza disease severity. EPFR-induced oxidative stress resulted in increased presence of Tregs in the lungs and subsequent suppression of adaptive immune response to influenza.
Electronic supplementary material
The online version of this article (doi:10.1186/s12989-014-0057-1) contains supplementary material, which is available to authorized users.
doi:10.1186/s12989-014-0057-1
PMCID: PMC4222384  PMID: 25358535
Particulate matter; EPFR; Influenza; Infant; Neonate; Air pollution; DCB230; Oxidative stress
5.  Maternal exposure to combustion generated PM inhibits pulmonary Th1 maturation and concomitantly enhances postnatal asthma development in offspring 
Background
Epidemiological studies suggest that maternal exposure to environmental hazards, such as particulate matter, is associated with increased incidence of asthma in childhood. We hypothesized that maternal exposure to combustion derived ultrafine particles containing persistent free radicals (MCP230) disrupts the development of the infant immune system and results in aberrant immune responses to allergens and enhances asthma severity.
Methods
Pregnant C57/BL6 mice received MCP230 or saline by oropharyngeal aspiration on gestational days 10 and 17. Three days after the second administration, blood was collected from MCP230 or saline treated dams and 8-isoprostanes in the serum were measured to assess maternal oxidative stress. Pulmonary T cell populations were assayed in the infant mice at six days, three and six weeks of postnatal age. When the infant mice matured to adults (i.e. six weeks of age), an asthma model was established with ovalbumin (OVA). Airway inflammation, mucus production and airway hyperresponsiveness were then examined.
Results
Maternal exposure to MCP230 induced systemic oxidative stress. The development of pulmonary T helper (Th1/Th2/Th17) and T regulatory (Treg) cells were inhibited in the infant offspring from MCP230-exposed dams. As the offspring matured, the development of Th2 and Treg cells recovered and eventually became equivalent to that of offspring from non-exposed dams. However, Th1 and Th17 cells remained attenuated through 6 weeks of age. Following OVA sensitization and challenge, mice from MCP230-exposed dams exhibited greater airway hyperresponsiveness, eosinophilia and pulmonary Th2 responses compared to offspring from non-exposed dams.
Conclusions
Our data suggest that maternal exposure to MCP230 enhances postnatal asthma development in mice, which might be related to the inhibition of pulmonary Th1 maturation and systemic oxidative stress in the dams.
doi:10.1186/1743-8977-10-29
PMCID: PMC3717277  PMID: 23856009
Maternal exposure; Particulate matter; Offspring; Asthma
6.  Environmentally persistent free radicals decrease cardiac function before and after ischemia/reperfusion injury in vivo 
Exposure to airborne particles is associated with increased cardiovascular morbidity and mortality. During the combustion of chlorine-containing hazardous materials and fuels, chlorinated hydrocarbons chemisorb to the surface of transition metal-oxide-containing particles, reduce the metal, and form an organic free radical. These radical-particle systems can survive in the environment for days and are called environmentally persistent free radicals (EPFRs). This study determined whether EPFRs could decrease left ventricular function before and after ischemia and reperfusion (I/R) in vivo. Male Brown Norway rats were dosed (8 mg/kg, i.t.) 24 hr prior to testing with particles containing the EPFR of 1, 2-dichlorobenzene (DCB230). DCB230 treatment decreased systolic and diastolic function. DCB230 also produced pulmonary and cardiac inflammation. After ischemia, systolic, but not diastolic function was significantly decreased in DCB230-treated rats. Ventricular function was not affected by I/R in control rats. There was greater oxidative stress in the heart and increased 8-isoprostane (biomarker of oxidative stress) in the plasma of treated vs control rats after I/R. These data demonstrate for the first time that DCB230 can produce inflammation and significantly decrease cardiac function at baseline and after I/R in vivo. Furthermore, these data suggest that EPFRs may be a risk factor for cardiac toxicity in healthy individuals and individuals with ischemic heart disease. Potential mechanisms involving cytokines/chemokines and/or oxidative stress are discussed.
doi:10.3109/10799893.2011.555767
PMCID: PMC3152960  PMID: 21385100
inflammation; cytokines; chemokines; pressure-volume; particles; oxidative stress
7.  Model combustion-generated particulate matter containing persistent free radicals redox cycle to produce reactive oxygen species 
Chemical research in toxicology  2013;26(12):1862-1871.
Particular matter (PM) is emitted during thermal decomposition of waste. During this process, aromatic compounds chemisorb to the surface of metal-oxide-containing PM, forming a surface-stabilized environmentally persistent free radical (EPFR). We hypothesized that EPFR-containing PM redox cycle to produce ROS and that this redox cycle is maintained in biological environments. To test our hypothesis, we incubated model EPFRs with the fluorescent probe dihydrorhodamine (DHR). Marked increases in DHR fluorescence were observed. Using a more specific assay, hydroxyl radicals (•OH) were also detected, and their level was further increased by co-treatment with thiols or ascorbic acid (AA), known components of epithelial lining fluid. Next, we incubated our model EPFR in bronchoalveolar lavage fluid (BALF) or serum. Detection of EPFRs and •OH verified that PM generate ROS in biological fluids. Moreover, incubation of pulmonary epithelial cells with EPFR-containing PM increased •OH levels compared to PM lacking EPFRs. Finally, measurements of oxidant injury in neonatal rats exposed to EPFRs by inhalation suggested that EPFRs induce an oxidant injury within lung lining fluid and that the lung responds by increasing antioxidant levels. In summary, our EPFR-containing PM redox cycle to produce ROS, and these ROS are maintained in biological fluids and environments. Moreover, these ROS may modulate toxic responses of PM in biological tissues such as the lung.
doi:10.1021/tx400227s
PMCID: PMC4112599  PMID: 24224526
Particulate matter; reactive oxygen species; combustion; free radicals; epithelial cells
8.  Effect of Copper Oxide Concentration on the Formation and Persistency of Environmentally Persistent Free Radicals (EPFRs) in Particulates 
Environmental Science & Technology  2014;48(4):2212-2217.
Environmentally persistent free radicals (EPFRs) are formed by the chemisorption of substituted aromatics on metal oxide surfaces in both combustion sources and superfund sites. The current study reports the dependency of EPFR yields and their persistency on metal loading in particles (0.25, 0.5, 0.75, 1, 2, and 5% CuO/silica). The EPFRs were generated through exposure of particles to three adsorbate vapors at 230 °C: phenol, 2-monochlorophenol (2-MCP), and dichlorobenzene (DCBz). Adsorption resulted in the formation of surface-bound phenoxyl- and semiquinoine-type radicals with characteristic EPR spectra displaying a g value ranging from ∼2.0037 to 2.006. The highest EPFR yield was observed for CuO concentrations between 1 and 3% in relation to MCP and phenol adsorption. However, radical density, which is expressed as the number of radicals per copper atom, was highest at 0.75–1% CuO loading. For 1,2-dichlorobenzene adsorption, radical concentration increased linearly with decreasing copper content. At the same time, a qualitative change in the radicals formed was observed—from semiquinone to chlorophenoxyl radicals. The two longest lifetimes, 25 and 23 h, were observed for phenoxyl-type radicals on 0.5% CuO and chlorophenoxyl-type radicals on 0.75% CuO, respectively.
doi:10.1021/es404013g
PMCID: PMC3983329  PMID: 24437381
9.  Environmentally Persistent Free Radicals (EPFRs) - 2. Are Free Hydroxyl Radicals Generated in Aqueous Solutions? 
Environmental science & technology  2011;45(21):9232-9239.
A chemical spin trap, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), in conjunction with electron paramagnetic resonance (EPR) spectroscopy was employed to measure the production of hydroxyl radical (.OH) in aqueous suspensions of 5% Cu(II)O/silica (3.9% Cu) particles containing environmentally persistent free radicals (EPFRs) of 2-monochlorophenol (2-MCP). The results indicate: 1) a significant differences in accumulated DMPO-OH adducts between EPFR containing particles and non-EPFR control samples, 2) a strong correlation between the concentration of DMPO-OH adducts and EPFRs per gram of particles, and 3) a slow, constant growth of DMPO-OH concentration over a period of days in solution containing 50 μg/ml EPFRs particles + DMPO (150 mM) + reagent balanced by 200 μl phosphate buffered (pH = 7.4) saline. However, failure to form secondary radicals using standard scavengers, such as ethanol, dimethylsulfoxide, sodium formate, and sodium azide, suggests free hydroxyl radicals may not have been generated in solution. This suggests surface-bound, rather than free, hydroxyl radicals were generated by a surface catalyzed-redox cycle involving both the EPFRs and Cu(II)O. Toxicological studies clearly indicate these bound free radicals promote various types of cardiovascular and pulmonary disease normally attributed to unbound free radicals; however, the exact chemical mechanism deserves further study in light of the implication of formation of bound, rather than free, hydroxyl radicals.
doi:10.1021/es201702q
PMCID: PMC3236504  PMID: 21942783
Spin trapping; fine particles; combustion; thermal treatment; biological activity; redox cycling; reactive oxygen species (ROS)
10.  Lifetime of combustion-generated environmentally persistent free radicals on Zn(II)O and other transition metal oxides 
Previous studies indicated that Environmentally Persistent Free Radicals (EPFRs) are formed in the post-flame, cool zone of combustion. They result from the chemisorption of gas-phase products of incomplete combustion (particularly hydroxyl- and chlorine-substituted aromatics) on Cu(II)O, Fe(III)2O3, and Ni(II)O domains of particulate matter (fly ash or soot particles). This study reports our detailed laboratory investigation on the lifetime of EPFRs on Zn(II)O/silica surface. Similarly, as in the case of other transition metals, chemisorption of the adsorbate on the Zn(II)O surface and subsequent transfer of electron from the adsorbate to the metal forms a surface-bound EPFR and a reduced metal ion center. The EPFRs are stabilized by their interaction with the metal oxide domain surface. The half-lives of EPFRs formed on Zn(II)O domains were the longest observed among the transition metal oxides studied and ranged from 3 to 73 days. These half-lives were an order of magnitude longer than those formed on nickel and iron oxides, and were 2 orders of magnitude longer compared to the EPFRs on copper oxide which have half-lives only on the order of hours. The longest-lived radicals on Zn(II)O correspond to the persistency in ambient air particles of almost a year. The half-life of EPFRs was found to correlate with the standard reduction potential of the associated metal.
doi:10.1039/c2em30545c
PMCID: PMC4303464  PMID: 22990982
11.  In Vitro and In Vivo Assessment of Pulmonary Risk Associated with Exposure to Combustion Generated Fine Particles 
Strong correlations exist between exposure to PM2.5 and adverse pulmonary effects. PM2.5 consists of fine (≤2.5 μm) and ultrafine (≤0.1 μm) particles with ultrafine particles accounting for >70% of the total particles. Environmentally persistent free radicals (EPFRs) have recently been identified in airborne PM2.5. To determine the adverse pulmonary effects of EPFRs associated with exposure to elevated levels of PM2.5, we engineered 2.5 μm surrogate EPFR-particle systems. We demonstrated that EPFRs generated greater oxidative stress in vitro, which was partly responsible for the enhanced cytotoxicity following exposure. In vivo studies using rats exposed to EPFRs containing particles demonstrated minimal adverse pulmonary effects. Additional studies revealed that fine particles failed to reach the alveolar region. Overall, our study implies qualitative differences between the health effects of PM size fractions.
doi:10.1016/j.etap.2009.12.007
PMCID: PMC2848491  PMID: 20369027
Fine particles; HEp-2 cells; Persistent free radical; Oxidative stress; Resveratrol
12.  Breast regression protein-39 (BRP-39) promotes dendritic cell maturation in vitro and enhances Th2 inflammation in murine model of asthma 
Acta Pharmacologica Sinica  2012;33(12):1525-1532.
Aim:
To determine the roles of breast regression protein-39 (BRP-39) in regulating dendritic cell maturation and in pathology of acute asthma.
Methods:
Mouse bone marrow-derived dendritic cells (BMDCs) were prepared, and infected with adenovirus over-expressing BRP-39. Ovalbumin (OVA)-induced murine model of acute asthma was made in female BALB/c mice by sensitizing and challenging with chicken OVA and Imject Alum. The transfected BMDCs were adoptively transferred into OVA-treated mice via intravenous injection. Airway hyperresponsiveness (AHR), inflammation and pulmonary histopathology were characterized.
Results:
The expression of BRP-39 mRNA and protein was significantly increased in lung tissues of OVA-treated mice. The BMDCs infected with adenovirus BRP-39 exhibited greater maturation and higher activity in vitro. Adoptive transfer of the cells into OVA-treated mice significantly augmented OVA-induced AHR and eosinophilic inflammation. Meanwhile, BRP-39 further enhanced the production of OVA-induced Th2 cytokines IL-4, IL-5 and IL-13, but significantly attenuated OVA-induced IFN-γ production in bronchoalveolar lavage fluid.
Conclusion:
In OVA-induced murine model of acute asthma, BRP-39 is over-expressed in lung tissue and augments Th2 inflammatory response and AHR. BRP-39 promotes dendritic cell maturation in vitro. Therefore, BRP-39 may be a potential therapeutic target of asthma.
doi:10.1038/aps.2012.154
PMCID: PMC4001840  PMID: 23178461
asthma; ovalbumin; bone marrow-derived dendritic cells (BMDCs); breast regression protein-39 (BRP-39); YKL-40; Th2 inflammation; airway hyperresponsiveness; bronchoalveolar lavage fluid
13.  Detection of Environmentally Persistent Free Radicals at a Superfund Wood Treating Site 
Environmental science & technology  2011;45(15):6356-6365.
Environmentally persistent free radicals (EPFRs) have previously been observed in association with combustion-generated particles and airborne PM2.5 (particulate matter, d < 2.5um). The purpose of this study was to determine if similar radicals were present in soils and sediments at Superfund sites. The site was a former wood treating facility containing pentachlorophenol (PCP) as a major contaminant. Both contaminated and non-contaminated (just outside the contaminated area) soil samples were collected. The samples were subjected to the conventional humic substances (HS) extraction procedure. Electron paramagnetic resonance (EPR) spectroscopy was used to measure the EPFR concentrations and determine their structure for each sample fraction. Analyses revealed a ~30× higher EPFR concentration in the PCP contaminated soils (20.2 × 1017 spins/g) than in the non-contaminated soil (0.7 × 1017 spins/g). Almost 90% of the EPFR signal originated from the Minerals/Clays/Humins fraction. GC-MS analyses revealed ~6500 ppm of PCP in the contaminated soil samples and none detected in the background samples. Inductively coupled plasma-atomic emission spectrophotometry (ICP-AES) analyses revealed ~7× higher concentrations of redox-active transition metals, in the contaminated soils than the non-contaminated soil. Vapor phase and liquid phase dosing of the clays/minerals/humins fraction of the soil with PCP resulted in an EPR signal identical to that observed in the contaminated soil, strongly suggesting the observed EPFR is pentachlorophenoxyl radical. Chemisorption and electron transfer from PCP to transition metals and other electron sinks in the soil are proposed to be responsible for EPFR formation.
doi:10.1021/es2012947
PMCID: PMC3167238  PMID: 21732664
14.  Candida soluble cell wall β-glucan facilitates ovalbumin-induced allergic airway inflammation in mice: Possible role of antigen-presenting cells 
Respiratory Research  2009;10(1):68.
Background
Although fungi have been implicated as initiating/deteriorating factors for allergic asthma, their contributing components have not been fully elucidated. We previously isolated soluble β-glucan from Candida albicans (CSBG) (Ohno et al., 2007). In the present study, the effects of CSBG exposure on airway immunopathology in the presence or absence of other immunogenic allergen was investigated in vivo, and their cellular mechanisms were analyzed both in vivo and in vitro.
Methods
In vivo, ICR mice were divided into 4 experimental groups: vehicle, CSBG (25 μg/animal), ovalbumin (OVA: 2 μg/animal), and CSBG + OVA were repeatedly administered intratracheally. The bronchoalveolar lavage cellular profile, lung histology, levels of cytokines and chemokines in the lung homogenates, the expression pattern of antigen-presenting cell (APC)-related molecules in the lung digests, and serum immunoglobulin values were studied. In vitro, the impacts of CSBG (0–12.5 μg/ml) on the phenotype and function of immune cells such as splenocytes and bone marrow-derived dendritic cells (BMDCs) were evaluated in terms of cell proliferation, the surface expression of APC-related molecules, and OVA-mediated T-cell proliferating activity.
Results
In vivo, repeated pulmonary exposure to CSBG induced neutrophilic airway inflammation in the absence of OVA, and markedly exacerbated OVA-related eosinophilic airway inflammation with mucus metaplasia in mice, which was concomitant with the amplified lung expression of Th2 cytokines and IL-17A and chemokines related to allergic response. Exposure to CSBG plus OVA increased the number of cells bearing MHC class II with or without CD80 in the lung compared to that of others. In vitro, CSBG significantly augmented splenocyte proliferation in the presence or absence of OVA. Further, CSBG increased the expression of APC-related molecules such as CD80, CD86, and DEC205 on BMDCs and amplified OVA-mediated T-cell proliferation through BMDCs.
Conclusion
CSBG potentiates allergic airway inflammation with maladaptive Th immunity, and this potentiation was associated with the enhanced activation of APCs including DC.
doi:10.1186/1465-9921-10-68
PMCID: PMC2731726  PMID: 19619338
15.  Inhibition of Th2 Adaptive Immune Responses and Pulmonary Inflammation by Leukocyte Ig-like Receptor B4 on Dendritic Cells1,2 
We previously established that the inhibitory receptor LILRB4 mitigates LPS-induced, neutrophil-dependent pathologic effector mechanisms in inflammation. We now report that LILRB4 on dendritic cells (DCs) counterregulates development of an adaptive Th2 immune response and ensuing inflammation in a model of allergic pulmonary inflammation initiated by inhalation sensitization with OVA and LPS followed by airway challenge with OVA. We found that Lilrb4−/− mice had significantly exacerbated eosinophilic pulmonary inflammation as assessed in bronchoalveolar lavage and lung tissue, as well as elevated levels of OVA-specific IgE and Th2 cytokines produced by OVA-restimulated lymph node cells. LILRB4 was preferentially expressed on MHC class IIhighCD86high OVA-bearing DCs in lung-draining lymph nodes after sensitization or challenge. Moreover, the lymph nodes of Lilrb4−/− mice had significantly more of these mature DCs after challenge with OVA, which was accompanied by significantly more IL-4-producing lymphocytes, compared with Lilrb4+/+ mice. Sensitization of naïve Lilrb4+/+ mice by transfer of OVA-LPS-pulsed Lilrb4−/− bone marrow-derived DCs (BMDCs) was sufficient to confer exacerbated allergic lung pathology upon challenge with OVA, compared with mice that received Lilrb4+/+ BMDCs. Our findings establish that maturation and migration of pulmonary DCs to lymph nodes in response to Ag and an innate immune stimulus is associated with upregulated expression of LILRB4. In addition, this receptor attenuates the number of these mature DCs and attendant IL-4-producing lymphocytes in the lymph nodes, and accordingly, the ability of DCs to elicit pathologic Th2 pulmonary inflammation.
doi:10.4049/jimmunol.0900877
PMCID: PMC2836496  PMID: 19966208
Dendritic Cells; Inflammation; Allergy; Lung
16.  Antigen-Specific IgG ameliorates allergic airway inflammation via Fcγ receptor IIB on dendritic cells 
Respiratory Research  2011;12(1):42.
Background
There have been few reports on the role of Fc receptors (FcRs) and immunoglobulin G (IgG) in asthma. The purpose of this study is to clarify the role of inhibitory FcRs and antigen presenting cells (APCs) in pathogenesis of asthma and to evaluate antigen-transporting and presenting capacity by APCs in the tracheobronchial mucosa.
Methods
In FcγRIIB deficient (KO) and C57BL/6 (WT) mice, the effects of intratracheal instillation of antigen-specific IgG were analysed using the model with sensitization and airborne challenge with ovalbumin (OVA). Thoracic lymph nodes instilled with fluorescein-conjugated OVA were analysed by fluorescence microscopy. Moreover, we analysed the CD11c+ MHC class II+ cells which intaken fluorescein-conjugated OVA in thoracic lymph nodes by flow cytometry. Also, lung-derived CD11c+ APCs were analysed by flow cytometry. Effects of anti-OVA IgG1 on bone marrow dendritic cells (BMDCs) in vitro were also analysed. Moreover, in FcγRIIB KO mice intravenously transplanted dendritic cells (DCs) differentiated from BMDCs of WT mice, the effects of intratracheal instillation of anti-OVA IgG were evaluated by bronchoalveolar lavage (BAL).
Results
In WT mice, total cells and eosinophils in BAL fluid reduced after instillation with anti-OVA IgG1. Anti-OVA IgG1 suppressed airway inflammation in hyperresponsiveness and histology. In addition, the number of the fluorescein-conjugated OVA in CD11c+ MHC class II+ cells of thoracic lymph nodes with anti-OVA IgG1 instillation decreased compared with PBS. Also, MHC class II expression on lung-derived CD11c+ APCs with anti-OVA IgG1 instillation reduced. Moreover, in vitro, we showed that BMDCs with anti-OVA IgG1 significantly decreased the T cell proliferation. Finally, we demonstrated that the lacking effects of anti-OVA IgG1 on airway inflammation on FcγRIIB KO mice were restored with WT-derived BMDCs transplanted intravenously.
Conclusion
Antigen-specific IgG ameliorates allergic airway inflammation via FcγRIIB on DCs.
doi:10.1186/1465-9921-12-42
PMCID: PMC3079623  PMID: 21477339
17.  Topographical and Biological Evidence Revealed FTY720-Mediated Anergy-Polarization of Mouse Bone Marrow-Derived Dendritic Cells In Vitro 
PLoS ONE  2012;7(5):e34830.
Abnormal inflammations are central therapeutic targets in numerous infectious and autoimmune diseases. Dendritic cells (DCs) are involved in these inflammations, serving as both antigen presenters and proinflammatory cytokine providers. As an immuno-suppressor applied to the therapies of multiple sclerosis and allograft transplantation, fingolimod (FTY720) was shown to affect DC migration and its crosstalk with T cells. We posit FTY720 can induce an anergy-polarized phenotype switch on DCs in vitro, especially upon endotoxic activation. A lipopolysaccharide (LPS)-induced mouse bone marrow-derived dendritic cell (BMDC) activation model was employed to test FTY720-induced phenotypic changes on immature and mature DCs. Specifically, methods for morphology, nanostructure, cytokine production, phagocytosis, endocytosis and specific antigen presentation studies were used. FTY720 induced significant alterations of surface markers, as well as decline of shape indices, cell volume, surface roughness in LPS-activated mature BMDCs. These phenotypic, morphological and topographical changes were accompanied by FTY720-mediated down-regulation of proinflammatory cytokines, including IL-6, TNF-α, IL-12 and MCP-1. Together with suppressed nitric oxide (NO) production and CCR7 transcription in FTY720-treated BMDCs with or without LPS activation, an inhibitory mechanism of NO and cytokine reciprocal activation was suggested. This implication was supported by the impaired phagocytotic, endocytotic and specific antigen presentation abilities observed in the FTY720-treated BMDCs. In conclusion, we demonstrated FTY720 can induce anergy-polarization in both immature and LPS-activated mature BMDCs. A possible mechanism is FTY720-mediated reciprocal suppression on the intrinsic activation pathway and cytokine production with endpoint exhibitions on phagocytosis, endocytosis, antigen presentation as well as cellular morphology and topography.
doi:10.1371/journal.pone.0034830
PMCID: PMC3365054  PMID: 22693544
18.  Inhaled dsRNA and rhinovirus evoke neutrophilic exacerbation and lung expression of thymic stromal lymphopoietin in allergic mice with established experimental asthma 
Allergy  2013;69(3):348-358.
Background
Rhinovirus infection or dsRNA stimulation increased thymic stromal lymphopoietin (TSLP), an upstream pro-allergic cytokine, in asthmatic bronchial epithelial cells. We hypothesized that dsRNA challenges superimposed on established experimental allergic asthma constitute a useful exacerbation model. We further hypothesized that TSLP is induced at dsRNA- and rhinoviral infection-induced exacerbations.
Methods
Allergic mice were challenged with OVA followed by three daily intranasal challenges with dsRNA or saline. Bronchoalveolar lavage fluid (BALF) was analysed for total protein, lactate dehydrogenase (LDH), CXCL1/KC, CCL2/MCP-1 and differential cell counts. Lung tissue histology, neutrophils and TSLP, TNF-α, IFN-β and IFN-λ mRNA were examined. Alternatively, allergen-challenged mice received intranasal rhinovirus-(RV)-1B followed by lung TSLP immunostaining.
Results
In mice with allergic airway inflammation, dsRNA challenges caused a significant exacerbation increasing lung tissue inflammation score and tissue neutrophilia. Bronchoalveolar lavage fluid neutrophils, total protein, LDH, CXCL1/KC and CCL2/MCP-1 were also increased (P < 0.01), and so were lung tissue expressions of TNF-α, IFN-λ and TSLP (P < 0.01), but IFN-β was not increased. TSLP, IFN-λ and LDH were not increased by allergen or dsRNA challenges alone, but increased exclusively at exacerbations. RV1B infection-induced exacerbation also increased lung tissue TSLP (P < 0.05).
Conclusions
dsRNA-induced exacerbation in mice with experimental asthma involved general inflammation, cytokines and interferons, in agreement with previous observations in exacerbating human asthma. Additionally, both dsRNA and RV1B infection increased lung TSLP exclusively at exacerbations. Our data suggest that dsRNA challenges superimposed on allergic inflammation are suited for pharmacological studies of asthma exacerbations including the regulation of lung tissue TSLP, TNF-α, IFN-β and IFN-λ.
doi:10.1111/all.12329
PMCID: PMC4223976  PMID: 24283976
allergy; asthma; exacerbation; neutrophils; TLR3
19.  Pulmonary and Systemic Expression of Monocyte Chemotactic Proteins in Preterm Sheep Fetuses Exposed to LPS Induced Chorioamnionitis 
Pediatric research  2010;68(3):210-215.
Monocyte chemoattractant proteins (MCP-1 and MCP-2) mediate monocyte and T-lymphocyte chemotaxis, and IL-1 contributes to the pathogenesis of chorioamnionitis-induced lung inflammation and fetal inflammatory responses. We tested the hypothesis that IL-1 mediates the systemic and pulmonary induction of MCP-1 and MCP-2 in response to lipopolysaccharide (LPS) induced chorioamnionitis. MCP-1 mRNA, MCP-2 mRNA and MCP-1 protein expression were measured in two models: 1) intra-amniotic LPS and 2) intra-amniotic recombinant sheep IL-1α given at varying intervals prior to preterm delivery at 124d gestational age. Intra-amniotic LPS or IL-1α induced MCP-1 mRNA and protein and MCP-2 mRNA in fetal lung many fold at 1-2d. LPS induced intense MCP-1 expression in sub-epithelial mesenchymal cells and interstitial inflammatory cells in the lung. Inhibition of IL-1 signaling with recombinant human IL-1 receptor antagonist (rhIL-1ra) did not attenuate LPS induced increase in MCP-1 or MCP-2 expression. MCP-1 and MCP-2 were not induced in liver or chorioamnion, but MCP-1 increased in cord plasma. LPS or IL-1 can induce robust expression of MCP-1 or MCP-2 in the fetal lung. LPS induction of MCP-1 is not IL-1 dependent in fetal sheep. MCP-1 and MCP-2 may be significant contributors to fetal inflammation.
doi:10.1203/PDR.0b013e3181e9c556
PMCID: PMC3123719  PMID: 20703142
20.  Environmentally Persistent Free Radicals (EPFRs). 1. Generation of Reactive Oxygen Species in Aqueous Solutions 
Environmental science & technology  2011;45(19):8559-8566.
Reactive oxygen species (ROS) generated by environmentally persistent free radicals (EPFRs) of 2-monochlorophenol, associated with CuO/silica particles, were detected using the chemical spin trap, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), in conjunction with Electron Paramagnetic Resonance (EPR) spectroscopy. Yields of hydroxyl radical (.OH), superoxide anion radical (O2.−), and hydrogen peroxide (H2O2) generated by EPFR-particle systems are reported. Failure to trap superoxide radicals in aqueous solvent, formed from the reaction of EPFRs with molecular oxygen, results from the fast transformation of the superoxide to hydrogen peroxide. However, formation of superoxide as an intermediate product in hydroxyl radical formation in aprotic solutions of dimethyl sulfoxide (DMSO) and acetonitrile (AcN) was observed. Experiments with superoxide dismutase (SOD) and catalase (CAT) confirmed the formation of superoxide and hydrogen peroxide, respectively, in the presence of EPFRs. The large number of hydroxyl radicals formed per EPFR and monotonic increase of the DMPO-OH spin adduct concentration with the incubation time suggest a catalytic cycle of ROS formation.
doi:10.1021/es201309c
PMCID: PMC3230252  PMID: 21823585
Fly-ash; PM2.5; fine particles; combustion; thermal treatment; hazardous waste; hazardous materials; Superfund sites
21.  Exacerbation of allergic inflammation in mice exposed to diesel exhaust particles prior to viral infection 
Background
Viral infections and exposure to oxidant air pollutants are two of the most important inducers of asthma exacerbation. Our previous studies have demonstrated that exposure to diesel exhaust increases the susceptibility to influenza virus infections both in epithelial cells in vitro and in mice in vivo. Therefore, we examined whether in the setting of allergic asthma, exposure to oxidant air pollutants enhances the susceptibility to respiratory virus infections, which in turn leads to increased virus-induced exacerbation of asthma. Ovalbumin-sensitized (OVA) male C57BL/6 mice were instilled with diesel exhaust particles (DEP) or saline and 24 hours later infected with influenza A/PR/8. Animals were sacrificed 24 hours post-infection and analyzed for markers of lung injury, allergic inflammation, and pro-inflammatory cytokine production.
Results
Exposure to DEP or infection with influenza alone had no significant effects on markers of injury or allergic inflammation. However, OVA-sensitized mice that were exposed to DEP and subsequently infected with influenza showed increased levels of eosinophils in lung lavage and tissue. In addition Th2-type cytokines, such as IL-4 and IL-13, and markers of eosinophil chemotaxis, such as CCL11 and CCR3, were increased in OVA-sensitized mice exposed to DEP prior to infection with influenza. These mice also showed increased levels of IL-1α, but not IL-10, RANTES, and MCP-1 in lung homogenates.
Conclusion
These data suggest that in the setting of allergic asthma, exposure to diesel exhaust could enhance virus-induced exacerbation of allergic inflammation.
doi:10.1186/1743-8977-6-22
PMCID: PMC2739151  PMID: 19682371
22.  Protease-activated receptor 2 activation of myeloid dendritic cells regulates allergic airway inflammation 
Respiratory Research  2011;12(1):122.
Background
A common characteristic of allergens is that they contain proteases that can activate protease-activated receptor (PAR-2); however the mechanism by which PAR-2 regulates allergic airway inflammation is unclear.
Methods
Mice (wild type and PAR-2-deficient) were sensitized using German cockroach (GC) feces (frass), the isolated protease from GC frass, or through adoptive transfer of GC frass-treated bone marrow-derived dendritic cells (BMDC) and measurements of airway inflammation (cellular infiltration, cytokine expression, and mucin production), serum IgE levels and airway hyperresponsiveness (AHR) were assessed. BMDC were cultured, treated with GC frass and assessed for cytokine production. PAR-2 expression on pulmonary mDCs was determined by flow cytometry.
Results
Exposure to GC frass induced AHR and airway inflammation in wild type mice; however PAR-2-deficient mice had significantly attenuated responses. To directly investigate the role of the protease, we isolated the protease from GC frass and administered the endotoxin-free protease into the airways of mice in the presence of OVA. GC frass proteases were sufficient to promote the development of AHR, serum IgE, and Th2 cytokine production. PAR-2 expression on mDC was upregulated following GC frass exposure, but the presence of a functional PAR-2 did not alter antigen uptake. To determine if PAR-2 activation led to differential cytokine production, we cultured BMDC in the presence of GM-CSF and treated these cells ex vivo with GC frass. PAR-2-deficient BMDC released significantly less IL-6, IL-23 and TNFα compared to BMDC from wild type mice, suggesting PAR-2 activation was important in Th2/Th17 skewing cytokine production. To determine the role for PAR-2 on mDCs on the initiation of allergic airway inflammation, BMDCs from wild type and PAR-2-deficient mice were treated in the presence or absence of GC frass and then adoptively transferred into the airway of wild type mice. Importantly, GC frass-stimulated wild type BMDCs were sufficient to induce AHR and allergic airway inflammation, while GC frass-stimulated PAR-2-deficient BMDC had attenuated responses.
Conclusions
Together these data suggest an important role for allergen activation of PAR-2 on mDCs in mediating Th2/Th17 cytokine production and allergic airway responses.
doi:10.1186/1465-9921-12-122
PMCID: PMC3184630  PMID: 21936897
23.  Mast cells that reside at different locations in the jejunum of mice infected with Trichinella spiralis exhibit sequential changes in their granule ultrastructure and chymase phenotype 
The Journal of Cell Biology  1996;135(1):279-290.
Whether or not a nontransformed, mature mouse mast cell (MC) or its committed progenitor can change its granule protease phenotype during inflammatory responses, has not been determined. To address this issue, the granule morphology and protease content of the MC in the jejunum of BALB/c mice exposed to Trichinella spiralis were assessed during the course of the infection. Within 1 wk after helminth infection of the mice, increased numbers of MC appeared in the crypts at the base of the villi, and by wk 2 the number of MC throughout the villi increased by approximately 25-fold. Shortly after the peak of the mastocytosis, the intraepithelial population of MC disappeared, followed by a progressive loss of lamina propria MC. The presence of stellate-shaped granules containing crystalline structures in intraepithelial MC at the height of infection and the retention of such granules with fragmented crystals in lamina propria MC during resolution of the mastocytosis suggest that MC migrate during the various phases of the inflammation. As assessed by immunohistochemical analyses of serial sections, predominant chymase phenotypes were observed at the height of the infection in the muscle that expressed mouse MC protease (mMCP) 5 without mMCP-1 or mMCP-2 and in the epithelium that expressed mMCP-1 and mMCP-2 without mMCP-5. Accompanying these two MC populations were transitional forms in the submucosa that expressed mMCP-2 and mMCP-5 without mMCP-1 and in the lamina propria that expressed mMCP-2 alone. These data suggest that jejunal MC sequentially express mMCP-2, cease expressing mMCP-5, and finally express mMCP-1 as the cells progressively appear in the submucosa, lamina propria, and epithelium, respectively. In the recovery phase of the disease, MC sequentially cease expressing mMCP-1, express mMCP-5, and finally cease expressing mMCP-2 as they present at the tips of the villi, the base of the villi, and the submucosa, respectively. That MC can reversibly alter their protease phenotypes suggests that a static nomenclature with fixed functional implications is inadequate to describe MC populations during an inflammatory process within a particular tissue.
PMCID: PMC2121032  PMID: 8858180
24.  Serum amyloid A inhibits dendritic cell apoptosis to induce glucocorticoid resistance in CD4+ T cells 
Cell Death & Disease  2013;4(9):e786-.
Mediators produced by the airway epithelium control the activation, recruitment, and survival of pulmonary dendritic cells (DC) that present antigen to CD4+ T cells during the genesis and exacerbation of allergic asthma. The epithelial-derived acute phase protein, serum amyloid A (SAA), induces DC maturation and TH17 polarization. TH17 responses are associated with severe forms of allergic asthma that are poorly controlled by corticosteroids. We sought to determine whether SAA would enhance the survival of DC during serum starvation and could then contribute to the development of a glucocorticoid-resistant phenotype in CD4+ T cells. Bone marrow-derived dendritic cells (BMDC) that were serum starved in the presence of SAA were protected from activation of caspase-3 and released less lactate dehydrogenase. In comparison with untreated serum-starved BMDC, treatment with SAA downregulated mRNA expression of the pro-apoptotic molecule Bim, increased production of the pro-survival heat shock protein 70 (HSP70), and induced secretion of pro-inflammatory cytokines. SAA-treated BMDC that were serum starved for 48 h remained capable of presenting antigen and induced OTII CD4+ T cells to secrete IL-17A, IL-17F, IL-21, IL-22, and IFNγ in the presence of ovalbumin. IL-17A, IL-17F, IL-21, and IFNγ production occurred even when the CD4+ T cells were treated with dexamethasone (Dex), whereas glucocorticoid treatment abolished cytokine secretion by T cells cocultured with untreated serum-starved BMDC. Measurement of Dex-responsive gene expression demonstrated CD4+ T cells as the target of glucocorticoid hyperresponsiveness manifest as a consequence of BMDC stimulation by SAA. Finally, allergic airway disease induced by SAA and antigen inhalation was unresponsive to Dex treatment. Our results indicate that apo-SAA affects DC to both prolong their viability and increase their inflammatory potential under apoptosis-inducing conditions. These findings reveal mechanisms through which SAA enhances the CD4+ T-cell-stimulating capacity of antigen-presenting cells that may actively participate in the pathogenicity of glucocorticoid-resistant lung disease.
doi:10.1038/cddis.2013.327
PMCID: PMC3789185  PMID: 24008730
dendritic cell; HSP70; apoptosis; glucocorticoid resistance
25.  Small interfering RNA against CD86 during allergen challenge blocks experimental allergic asthma 
Respiratory Research  2014;15(1):132.
Background
CD86-CD28 interaction has been suggested as the principal costimulatory pathway for the activation and differentiation of naïve T cells in allergic inflammation. However, it remains uncertain whether this pathway also has an essential role in the effector phase. We sought to determine the contribution of CD86 on dendritic cells in the reactivation of allergen-specific Th2 cells.
Methods
We investigated the effects of the downregulation of CD86 by short interfering RNAs (siRNAs) on Th2 cytokine production in the effector phase in vitro and on asthma phenotypes in ovalbumin (OVA)-sensitized and -challenged mice.
Results
Treatment of bone marrow-derived dendritic cells (BMDCs) with CD86 siRNA attenuated LPS-induced upregulation of CD86. CD86 siRNA treatment impaired BMDCs’ ability to activate OVA-specific Th2 cells. Intratracheal administration of CD86 siRNA during OVA challenge downregulated CD86 expression in the airway mucosa. CD86 siRNA treatment ameliorated OVA-induced airway eosinophilia, airway hyperresponsiveness, and the elevations of OVA-specific IgE in the sera and IL-5, IL-13, and CCL17 in the bronchoalveolar lavage fluid, but not the goblet cell hyperplasia.
Conclusion
These results suggest that local administration of CD86 siRNA during the effector phase ameliorates lines of asthma phenotypes. Targeting airway dendritic cells with siRNA suppresses airway inflammation and hyperresponsiveness in an experimental model of allergic asthma.
doi:10.1186/s12931-014-0132-z
PMCID: PMC4216659  PMID: 25344652

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