Environmentally persistent free radicals induce airway hyperresponsiveness in neonatal rat lungs

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Methods</p> <p>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 <it>in vivo </it>indicators of oxidative stress. Pulmonary histopathology and characterization of differential protein expression in lung homogenates was also performed.</p> <p>Results</p> <p>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.</p> <p>Conclusions</p> <p>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.</p

Environmentally persistent free radicals amplify ultrafine particle mediated cellular oxidative stress and cytotoxicity

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>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.</p> <p>Conclusion</p> <p>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.</p

Inchoate CD8\u3csup\u3e+\u3c/sup\u3e T cell responses in neonatal mice permit influenza-induced persistent pulmonary dysfunction

Influenza infection remains a significant cause of pulmonary morbidity and mortality worldwide, with the highest hospitalization and mortality rates occurring in infants and elder adults. The mechanisms inducing this considerable morbidity and mortality are largely unknown. To address this question, we established a neonatal mouse model of influenza infection to test the hypothesis that the immaturity of the neonatal immune system is responsible for the severe pulmonary disease observed in infants. Seven-day-old mice were infected with influenza A virus (H1N1) and allowed to mature. As adults, these mice showed enhanced airway hyperreactivity, chronic pulmonary inflammation, and diffuse emphysematous-type lesions in the lungs. The adaptive immune responses of the neonates were much weaker than those of adults. This insufficiency appeared to be in both magnitude and functionality and was most apparent in the CD8 + T cell population. To determine the role of neonatal CD8 + T cells in disease outcome, adult, naive CD8+ T cells were adoptively transferred into neonates before infection. Neonatal mice receiving the adult CD8+ T cells had significantly lower pulmonary viral titers and greatly improved pulmonary function as adults (airway resistance similar to SHAM). Additional adoptive transfer studies using adult CD8+ T cells from IFN-γ-deficient mice demonstrated the importance of IFN-γ from CD8+ T cells in controlling the infection and in determining disease outcome. Our data indicate that neonates are more vulnerable to severe infections due to immaturity of their immune system and emphasize the importance of vaccination in infants. Copyright © 2008 by The American Association of Immunologists, Inc

TRPA1 and CGRP antagonists counteract vesicant-induced skin injury and inflammation

The skin is highly sensitive to the chemical warfare agent in mustard gas, sulfur mustard (SM) that initiates a delayed injury response characterized by erythema, inflammation and severe vesication (blistering). Although SM poses a continuing threat, used as recently as in the Syrian conflict, no mechanism-based antidotes against SM are available. Recent studies demonstrated that Transient Receptor Potential Ankyrin 1 (TRPA1), a chemosensory cation channel in sensory nerves innervating the skin, is activated by SM and 2‐chloroethyl ethyl sulfide (CEES), an SM analog, in vitro, suggesting it may promote vesicant injury. Here, we investigated the effects of TRPA1 inhibitors, and an inhibitor of Calcitonin Gene Related Peptide (CGRP), a neurogenic inflammatory peptide released upon TRPA1 activation, in a CEES-induced mouse ear vesicant model (CEES-MEVM). TRPA1 inhibitors (HC-030031 and A-967079) and a CGRP inhibitor (MK-8825) reduced skin edema, pro-inflammatory cytokines (IL-1β, CXCL1/KC), MMP-9, a protease implicated in skin damage, and improved histopathological outcomes. These findings suggest that TRPA1 and neurogenic inflammation contribute to the deleterious effects of vesicants in vivo, activated either directly by alkylation, or indirectly, by reactive intermediates or pro-inflammatory mediators. TRPA1 and CGRP inhibitors represent new leads that could be considered for validation and further development in other vesicant injury models

Inchoate CD8 +

Influenza infection remains a significant cause of pulmonary morbidity and mortality worldwide, with the highest hospitalization and mortality rates occurring in infants and elder adults. The mechanisms inducing this considerable morbidity and mortality are largely unknown. To address this question, we established a neonatal mouse model of influenza infection to test the hypothesis that the immaturity of the neonatal immune system is responsible for the severe pulmonary disease observed in infants. Seven-day-old mice were infected with influenza A virus (H1N1) and allowed to mature. As adults, these mice showed enhanced airway hyperreactivity, chronic pulmonary inflammation, and diffuse emphysematous-type lesions in the lungs. The adaptive immune responses of the neonates were much weaker than those of adults. This insufficiency appeared to be in both magnitude and functionality and was most apparent in the CD8 + T cell population. To determine the role of neonatal CD8 + T cells in disease outcome, adult, naive CD8+ T cells were adoptively transferred into neonates before infection. Neonatal mice receiving the adult CD8+ T cells had significantly lower pulmonary viral titers and greatly improved pulmonary function as adults (airway resistance similar to SHAM). Additional adoptive transfer studies using adult CD8+ T cells from IFN-γ-deficient mice demonstrated the importance of IFN-γ from CD8+ T cells in controlling the infection and in determining disease outcome. Our data indicate that neonates are more vulnerable to severe infections due to immaturity of their immune system and emphasize the importance of vaccination in infants. Copyright © 2008 by The American Association of Immunologists, Inc

Oxidized Phospholipid OxPAPC Activates TRPA1 and Contributes to Chronic Inflammatory Pain in Mice.

Oxidation products of the naturally occurring phospholipid 1-palmitoyl-2-arachidonoyl-sn-glycerol-3-phosphatidylcholine (PAPC), which are known as OxPAPC, accumulate in atherosclerotic lesions and at other sites of inflammation in conditions such as septic inflammation and acute lung injury to exert pro- or anti-inflammatory effects. It is currently unknown whether OxPAPC also contributes to inflammatory pain and peripheral neuronal excitability in these conditions. Here, we observed that OxPAPC dose-dependently and selectively activated human TRPA1 nociceptive ion channels expressed in HEK293 cells in vitro, without any effect on other TRP channels, including TRPV1, TRPV4 and TRPM8. OxPAPC agonist activity was dependent on essential cysteine and lysine residues within the N-terminus of the TRPA1 channel protein. OxPAPC activated calcium influx into a subset of mouse sensory neurons which were also sensitive to the TRPA1 agonist mustard oil. Neuronal OxPAPC responses were largely abolished in neurons isolated from TRPA1-deficient mice. Intraplantar injection of OxPAPC into the mouse hind paw induced acute pain and persistent mechanical hyperalgesia and this effect was attenuated by the TRPA1 inhibitor, HC-030031. More importantly, we found levels of OxPAPC to be significantly increased in inflamed tissue in a mouse model of chronic inflammatory pain, identified by the binding of an OxPAPC-specific antibody. These findings suggest that TRPA1 is a molecular target for OxPAPC and OxPAPC may contribute to chronic inflammatory pain through TRPA1 activation. Targeting against OxPAPC and TRPA1 signaling pathway may be promising in inflammatory pain treatment

OxPAPC-induced TRPA1 activation is independent of EP2 and DP receptors in both HEK293 cells and native DRG neurons.

<p>(A) Summary of OxPAPC-induced Ca²⁺ responses in HEK293 cells expressing hTRPA1. Cells were first superfused with vehicle only (0.05% DMSO), AH6809 (10 μM), PF04418948 (20 nM) or ruthenium red (10 μM) for 5 min before recording started and then recorded in the continued presence of above treatments. Cells were challenged with OxPAPC (30 μM) and subsequently with mustard oil (MO, 70 μM) and ionomycin (1 μM). Responses of > 50 cells were averaged from each group. (B) Average peak amplitudes of OxPAPC-induced Ca²⁺ responses in HEK293 cells as shown in (A). (C) Percentages of mouse DRG neurons responding to OxPAPC in vehicle only, or in the presence of AH6809 (10 μM) or PF04418948 (20 nM). Neurons were pretreated with the antagonists for 5 min before recording and recorded in the continued presence of the treatments. Cells were challenged with OxPAPC (10 μM) for 100 s and subsequently with KCl (40 mM) for 40 s. n = 5–8 tests/group, averages from 100–200 neurons per group. (D) Maximal Fura-2 emission ratio amplitudes of OxPAPC-induced Ca²⁺ responses in mouse DRG neurons recorded in (C). **p < 0.01 <i>vs</i>. Veh, NS: no significance (p > 0.05).</p

Covalent modification sites in TRPA1 are essential for OxPAPC sensitivity.

<p>(A) Representative Ca²⁺ imaging traces showing responses of hTRPA1 and hTRPA1 mutant (3CK mutant)-expressing HEK293 cells following application of OxPAPC (10 μg/ml) and carvacrol (300 μM). (B) Summary of the Ca²⁺ responses induced by OxPAPC and mustard oil (MO, 70 μM). Increase in [Ca²⁺]_i is displayed as percentage of [Ca²⁺]_i activated by a saturating dose of carvacrol (300 μM). n = 5–6 tests/group. Each test contains up to 30 cells. **p < 0.01 <i>vs</i>. hTRPA1 group.</p