TLR7 is expressed by support cells, but not sensory neurons, in ganglia.

BACKGROUND: Toll-like receptor 7 (TLR7) is an innate immune receptor that detects viral single-stranded RNA and triggers the production of proinflammatory cytokines and type 1 interferons in immune cells. TLR7 agonists also modulate sensory nerve function by increasing neuronal excitability, although studies are conflicting whether sensory neurons specifically express TLR7. This uncertainty has confounded the development of a mechanistic understanding of TLR7 function in nervous tissues. METHODS: TLR7 expression was tested using in situ hybridization with species-specific RNA probes in vagal and dorsal root sensory ganglia in wild-type and TLR7 knockout (KO) mice and in guinea pigs. Since TLR7 KO mice were generated by inserting an Escherichia coli lacZ gene in exon 3 of the mouse TLR7 gene, wild-type and TLR7 (KO) mouse vagal ganglia were also labeled for lacZ. In situ labeling was compared to immunohistochemistry using TLR7 antibody probes. The effects of influenza A infection on TLR7 expression in sensory ganglia and in the spleen were also assessed. RESULTS: In situ probes detected TLR7 in the spleen and in small support cells adjacent to sensory neurons in the dorsal root and vagal ganglia in wild-type mice and guinea pigs, but not in TLR7 KO mice. TLR7 was co-expressed with the macrophage marker Iba1 and the satellite glial cell marker GFAP, but not with the neuronal marker PGP9.5, indicating that TLR7 is not expressed by sensory nerves in either vagal or dorsal root ganglia in mice or guinea pigs. In contrast, TLR7 antibodies labeled small- and medium-sized neurons in wild-type and TLR7 KO mice in a TLR7-independent manner. Influenza A infection caused significant weight loss and upregulation of TLR7 in the spleens, but not in vagal ganglia, in mice. CONCLUSION: TLR7 is expressed by macrophages and satellite glial cells, but not neurons in sensory ganglia suggesting TLR7's neuromodulatory effects are mediated indirectly via activation of neuronally-associated support cells, not through activation of neurons directly. Our data also suggest TLR7's primary role in neuronal tissues is not related to antiviral immunity.

Airway Sensory Nerve Density Is Increased in Chronic Cough.

Rationale: Chronic cough is characterized by frequent urges to cough and a heightened sensitivity to inhaled irritants. Airway sensory nerves trigger cough. We hypothesized that sensory nerve density is increased in chronic cough, which may contribute to excessive and persistent coughing.Objectives: To measure airway nerve density (axonal length) and complexity (nerve branching, neuropeptide expression) in humans with and without chronic cough.Methods: Bronchoscopic human airway biopsies were immunolabeled for nerves and the sensory neuropeptide substance P. Eosinophil peroxidase was also quantified given previous reports showing associations between eosinophils and nerve density. Three-dimensional image z-stacks of epithelium and subepithelium were generated using confocal microscopy, and from these z-stacks, total nerve length, the number of nerve branch points, substance P expression, and eosinophil peroxidase were quantified within each airway compartment.Measurements and Main Results: Nerve length and the number of branch points were significantly increased in epithelium, but not subepithelium, in chronic cough compared with healthy airways. Substance P expression was scarce and was similar in chronic cough and healthy airways. Nerve length and branching were not associated with eosinophil peroxidase nor with demographics such as age and sex in either group.Conclusions: Airway epithelial sensory nerve density is increased in chronic cough, suggesting sensory neuroplasticity contributes to cough hypersensitivity.

Lung eosinophils increase vagus nerve-mediated airway reflex bronchoconstriction in mice.

Eosinophils mediate airway hyperresponsiveness by increasing vagally mediated reflex bronchoconstriction. Here, we tested whether circulating or airway eosinophils change nerve function. Airway resistance in response to aerosolized 5-hydroxytryptamine (5-HT, 10-300 mM) was measured in wild-type mice or transgenic mice that overexpress IL5 in T cells (+IL5T), overexpress IL5 in airway epithelium (+IL5AE), or overexpress IL5 but are devoid of eosinophils (+IL5AE/-Eos). Inflammatory cells in bronchoalveolar lavage (BAL), blood, and bone marrow were quantified. Blood eosinophils were increased in +IL5T and +IL5AE mice compared with wild-type mice. +IL5T mice had increased eosinophils in bone marrow while +IL5AE mice had increased eosinophils in BAL. Eosinophils surrounding large airways were significantly increased only in +IL5AE mice. With intact vagal innervation, aerosolized 5-HT significantly increased airway resistance in +IL5AE mice. 5-HT-induced bronchoconstriction was blocked by vagotomy or atropine, demonstrating that it was mediated via a vagal reflex. Airway resistance was not increased in +IL5AE/-Eos mice, demonstrating that it required lung eosinophils, but was not affected by increased bone marrow or blood eosinophils or by increased IL5 in the absence of eosinophils. Eosinophils did not change M3 function on airway smooth muscle, since airway responses to methacholine in vagotomized mice were not different among strains. Eosinophils surrounding large airways were sufficient, even in the absence of increased IL5 or external insult, to increase vagally mediated reflex bronchoconstriction. Specifically blocking or reducing eosinophils surrounding large airways may effectively inhibit reflex hyperresponsiveness mediated by vagus nerves in eosinophilic asthma.

Organophosphorus Pesticides Induce Cytokine Release from Differentiated Human THP1 Cells.

Epidemiologic studies link organophosphorus pesticides (OPs) to increased incidence of asthma. In guinea pigs, OP-induced airway hyperreactivity requires macrophages and TNF-α. Here, we determined whether OPs interact directly with macrophages to alter cytokine expression or release. Human THP1 cells were differentiated into macrophages and then exposed to parathion, chlorpyrifos, or diazinon, or their oxon, phosphate, or phosphorothioate metabolites for 24 hours in the absence or presence of reagents that block cholinergic receptors. TNF-α, IL-1ß, platelet-derived growth factor, and transforming growth factor-ß mRNA and protein were quantified by qPCR and ELISA, respectively. The effects of OPs on NF-κB, acetylcholinesterase, and intracellular calcium were also measured. Parent OPs and their oxon metabolites upregulated cytokine mRNA and stimulated cytokine release. TNF-α release, which was the most robust response, was triggered by parent, but not oxon, compounds. Cytokine expression was also increased by diethyl dithiophosphate but not diethyl thiophosphate or diethyl phosphate metabolites. Parent OPs, but not oxon metabolites, activated NF-κB. Parent and oxon metabolites decreased acetylcholinesterase activity, but comparable acetylcholinesterase inhibition by eserine did not mimic OP effects on cytokines. Consistent with the noncholinergic mechanisms of OP effects on macrophages, pharmacologic antagonism of muscarinic or nicotinic receptors did not prevent OP-induced cytokine expression or release. These data indicate that phosphorothioate OP compounds directly stimulate macrophages to release TNF-α, potentially via activation of NF-κB, and suggest that therapies that target NF-κB may prevent OP-induced airway hyperreactivity.

Ozone-induced eosinophil recruitment to airways is altered by antigen sensitization and tumor necrosis factor-α blockade.

Ozone is an atmospheric pollutant that causes lung inflammation and airway hyperresponsiveness. Ozone's effects occur in two distinct phases that are mediated by different populations of eosinophils. In the acute phase 1 day after exposure, mature airway-resident eosinophils alter parasympathetic nerve function that results in airway hyperresponsiveness. At this time point, the severity of hyperresponsiveness correlates with the number of eosinophils in close proximity to airway nerves, but not with eosinophils in bronchoalveolar lavage. Three days later, newly divided eosinophils are recruited to airways by a tumor necrosis factor-α-dependent mechanism. These new eosinophils paradoxically attenuate ozone-induced airway hyperresponsiveness. Ozone's effects on airway tissue eosinophils and nerve-associated eosinophils 3 days after exposure are unknown. Thus, we tested ozone's effects on eosinophils in airway subepithelium and around airway nerves 1 and 3 days after ozone in nonsensitized and ovalbumin-sensitized guinea pigs with or without the tumor necrosis factor-α antagonist, etanercept, and compared changes in eosinophils with ozone-induced airway hyperresponsiveness. More eosinophils were present in small, noncartilaginous airways and along small airway nerves compared to large cartilaginous airways in all treatment groups. The number of airway and nerve-associated eosinophils were unaffected 1 day after ozone exposure, whereas significantly fewer airway eosinophils were present 3 days later. Airway and nerve-associated eosinophils were also decreased in small airways 3 days after ozone in sensitized animals. These changes were blocked by etanercept. Airway eosinophils, but not nerve-associated or bronchoalveolar lavage eosinophils correlated with airway hyperresponsiveness 3 days after ozone. Our findings indicate ozone causes persistent alterations in airway eosinophils and reinforce the importance of characterizing eosinophils' effects within distinct airway compartments.

Newly divided eosinophils limit ozone-induced airway hyperreactivity in nonsensitized guinea pigs.

Ozone causes vagally mediated airway hyperreactivity and recruits inflammatory cells, including eosinophils, to lungs, where they mediate ozone-induced hyperreactivity 1 day after exposure but are paradoxically protective 3 days later. We aimed to test the role of newly divided eosinophils in ozone-induced airway hyperreactivity in sensitized and nonsensitized guinea pigs. Nonsensitized and sensitized guinea pigs were treated with 5-bromo-2-deoxyuridine (BrdU) to label newly divided cells and were exposed to air or ozone for 4 h. Later (1 or 3 days later), vagally induced bronchoconstriction was measured, and inflammatory cells were harvested from bone marrow, blood, and bronchoalveolar lavage. Ozone induced eosinophil hematopoiesis. One day after ozone, mature eosinophils dominate the inflammatory response and potentiate vagally induced bronchoconstriction. However, by 3 days, newly divided eosinophils have reached the lungs, where they inhibit ozone-induced airway hyperreactivity because depleting them with antibody to IL-5 or a TNF-α antagonist worsened vagally induced bronchoconstriction. In sensitized guinea pigs, both ozone-induced eosinophil hematopoiesis and subsequent recruitment of newly divided eosinophils to lungs 3 days later failed to occur. Thus mature eosinophils dominated the ozone-induced inflammatory response in sensitized guinea pigs. Depleting these mature eosinophils prevented ozone-induced airway hyperreactivity in sensitized animals. Ozone induces eosinophil hematopoiesis and recruitment to lungs, where 3 days later, newly divided eosinophils attenuate vagally mediated hyperreactivity. Ozone-induced hematopoiesis of beneficial eosinophils is blocked by a TNF-α antagonist or by prior sensitization. In these animals, mature eosinophils are associated with hyperreactivity. Thus interventions targeting eosinophils, although beneficial in atopic individuals, may delay resolution of airway hyperreactivity in nonatopic individuals.

Protective Role of Eosinophils and TNFa after Ozone Inhalation.

Introduction: Exposure to ozone induces deleterious responses in the airways that include shortness of breath, inflammation, and bronchoconstriction. People with asthma have increased airway sensitivity to ozone and other irritants. Dr. Allison Fryer and colleagues addressed how exposure to ozone affects the immune and physiological responses in guinea pigs. Guinea pigs are considered a useful animal model for studies of respiratory and physiological responses in humans; their response to airborne allergens is similar to that in humans and shares some features of allergic asthma. Fryer and colleagues had previously observed that within 24 hours of exposure, ozone not only induced bronchoconstriction but also stimulated the production of new cells in the bone marrow, where all white blood cells develop. As a result of ozone exposure, increased numbers of newly synthesized white blood cells, particularly eosinophils, moved into the blood and lungs. The central hypothesis of the current study was that newly synthesized eosinophils recruited to the lungs 3 days after ozone exposure were beneficial to the animals because they reduced ozoneinduced bronchoconstriction. The investigators also hypothesized that the beneficial effect seen in normal (nonsensitized) animals was lost in animals that had been injected with an allergen, ovalbumin (sensitized). They also planned to explore the effects of inhibitors of certain cytokines (cellsignaling molecules). Immune responses in sensitized animals are dominated by a Th2 pattern, which is characterized by the synthesis of cytokines (interleukin [IL]-4, IL-5, and IL-13) and the Th2 subset of CD4+ T lymphocytes and the cells they activate (predominantly eosinophils, and B lymphocytes that switch to making immunoglobulin E [IgE]). Thus, sensitized animals were used as a model of allergic humans, whose immune responses tend to be dominated by IgE. Approach: Fryer and colleagues exposed normal and sensitized (allergic) guinea pigs to 2 ppm ozone or filtered air for 4 hours and measured changes in cell numbers and airway responses 1 or 3 days later. They counted the numbers of eosinophils and other white blood cells (macrophages, neutrophils, and lymphocytes) in bone marrow, blood, and bronchoalveolar lung lavage fluid. The investigators also measured important physiological responses, including bronchoconstriction. Some animals were pretreated with etanercept and monoclonal anti-IL-5, which block tumor necrosis factor-a (TNFa) and IL-5, respectively. TNFa and IL-5 blockers have been used to treat patients with asthma. A key feature of the study was a technique to distinguish which white blood cells were synthesized after exposure from those that already existed, by injecting animals with bromodeoxyuridine (BrdU). BrdU is a thymidine analogue that is incorporated into the DNA of dividing cells, serving as a marker of newly produced cells. Therefore, a snapshot can be obtained of the proportion of newly synthesized (BrdU-positive) versus pre-existing (BrdU-negative) cell types. Key results: 1. Allergic and normal animals differed in the time course of bronchoconstriction and changes in cell types after ozone exposure. In normal animals, bronchoconstriction increased substantially at day 1 but decreased by day 3 after ozone exposure. In contrast, in allergic animals bronchoconstriction remained high at day 3. Ozone also increased the percentage of newly formed, BrdU2 positive eosinophils in the bone marrow and lungs of normal but not allergic animals. 2. Pretreatment with the TNFa blocker etanercept had complex effects, which differed between normal and allergic animals. In normal animals, etanercept decreased ozone-induced new synthesis of eosinophils in the bone marrow and blocked eosinophil migration to the lung; it also increased bronchoconstriction at day 3 (relative to day 1 without etanercept). In allergic animals, etanercept had no effect on any cell type in the bone marrow or lung after exposure to ozone and did not change bronchoconstriction compared with allergic animals not treated with etanercept. Etanercept tended to increase the numbers of blood monocytes and lymphocytes in air- and ozone-exposed normal and allergic animals at day 3, but had no effect on eosinophils in blood at this time point. This was one of the few statistically significant findings in the blood of exposed animals in the study. 3. Anti-IL-5 reduced bronchoconstriction at day 3 after exposure of allergic animals to ozone. In contrast, bronchoconstriction was greatly increased in normal animals treated with anti-IL-5. Conclusions: Fryer and colleagues explored the airway and cellular responses in guinea pigs exposed to ozone. The HEI Review Committee, which conducted an independent review of the study, agreed that the findings supported the authors' hypothesis (1) that exposure to ozone stimulates production of eosinophils in bone marrow, (2) that these newly formed eosinophils migrate to the lungs, and (3) that those eosinophils play a delayed but potentially beneficial role in reducing ozone-induced inflammation in the airways of healthy normal animals, but not in allergen-sensitized animals. The Committee also agreed that guinea pigs were a good model for studying responses to an allergen, because a major subtype of asthma (the high Th2 or allergic type) is associated with high levels of eosinophils in the blood. A novel finding was that the TNFa blocker etanercept decreased ozone-induced formation of eosinophils in the bone marrow and blocked eosinophil migration to the lung in normal animals. However, because injecting etanercept had little effect on eosinophils and did not decrease bronchoconstriction in allergic guinea pigs, the potential for treating patients with allergic asthma with TNFa blockers is uncertain. This is consistent with the poor performance of TNFa blockers in clinical studies of asthma treatment. Blocking the cytokine IL-5 with an anti-IL-5 antibody substantially decreased bronchoconstriction in sensitized animals. This suggests that therapies targeting IL-5 and eosinophils would be promising in at least some types of asthma. The Committee expressed caution toward experiments with cytokine blockers, both in animal models and humans, because such blockers are often not specific to a particular cell type and may differ at different sites in the body. Without further detailed confirmation of the effects of the blockers, interpreting these experiments can be challenging. The Committee concluded that the study by Fryer and colleagues raises several intriguing directions for future research, including exploring ways in which newly formed eosinophils differ from pre-existing ones, and how such findings apply to humans with allergy or asthma.

Interleukin-1ß mediates virus-induced m2 muscarinic receptor dysfunction and airway hyperreactivity.

Respiratory viral infections are associated with the majority of asthma attacks. Inhibitory M2 receptors on parasympathetic nerves, which normally limit acetylcholine (ACh) release, are dysfunctional after respiratory viral infection. Because IL-1ß is up-regulated during respiratory viral infections, we investigated whether IL-1ß mediates M2 receptor dysfunction during parainfluenza virus infection. Virus-infected guinea pigs were pretreated with the IL-1ß antagonist anakinra. In the absence of anakinra, viral infection increased bronchoconstriction in response to vagal stimulation but not to intravenous ACh, and neuronal M2 muscarinic receptors were dysfunctional. Pretreatment with anakinra prevented virus-induced increased bronchoconstriction and M2 receptor dysfunction. Anakinra did not change smooth muscle M3 muscarinic receptor response to ACh, lung viral loads, or blood and bronchoalveolar lavage leukocyte populations. Respiratory virus infection decreased M2 receptor mRNA expression in parasympathetic ganglia extracted from infected animals, and this was prevented by blocking IL-1ß or TNF-α. Treatment of SK-N-SH neuroblastoma cells or primary cultures of guinea pig parasympathetic neurons with IL-1ß directly decreased M2 receptor mRNA, and this was not synergistic with TNF-α treatment. Treating guinea pig trachea segment with TNF-α or IL-1ß in vitro increased tracheal contractions in response to activation of airway nerves by electrical field stimulation. Blocking IL-1ß during TNF-α treatment prevented this hyperresponsiveness. These data show that virus-induced hyperreactivity and M2 dysfunction involves IL-1ß and TNF-α, likely in sequence with TNF-α causing production of IL-1ß.

Macrophage TNF-α mediates parathion-induced airway hyperreactivity in guinea pigs.

Organophosphorus pesticides (OPs) are implicated in human asthma. We previously demonstrated that, at concentrations that do not inhibit acetylcholinesterase activity, the OP parathion causes airway hyperreactivity in guinea pigs as a result of functional loss of inhibitory M2 muscarinic receptors on parasympathetic nerves. Because macrophages are associated with asthma, we investigated whether macrophages mediate parathion-induced M2 receptor dysfunction and airway hyperreactivity. Airway physiology was measured in guinea pigs 24 h after a subcutaneous injection of parathion. Pretreatment with liposome-encapsulated clodronate induced alveolar macrophage apoptosis and prevented parathion-induced airway hyperreactivity in response to electrical stimulation of the vagus nerves. As determined by qPCR, TNF-α and IL-1ß mRNA levels were increased in alveolar macrophages isolated from parathion-treated guinea pigs. Parathion treatment of alveolar macrophages ex vivo did not significantly increase IL-1ß and TNF-α mRNA but did significantly increase TNF-α protein release. Consistent with these data, pretreatment with the TNF-α inhibitor etanercept but not the IL-1ß receptor inhibitor anakinra prevented parathion-induced airway hyperreactivity and protected M2 receptor function. These data suggest a novel mechanism of OP-induced airway hyperreactivity in which low-level parathion activates macrophages to release TNF-α-causing M2 receptor dysfunction and airway hyperreactivity. These observations have important implications regarding therapeutic approaches for treating respiratory disease associated with OP exposures.

ß2-Agonists inhibit TNF-α-induced ICAM-1 expression in human airway parasympathetic neurons.

BACKGROUND: Major basic protein released from eosinophils to airway parasympathetic nerves blocks inhibitory M(2) muscarinic receptors on the parasympathetic nerves, increasing acetylcholine release and potentiating reflex bronchoconstriction. Recruitment of eosinophils to airway parasympathetic neurons requires neural expression of both intercellular adhesion molecular-1 (ICAM-1) and eotaxin. We have shown that inflammatory cytokines induce eotaxin and ICAM-1 expression in parasympathetic neurons. OBJECTIVE: To test whether the ß(2) agonist albuterol, which is used to treat asthma, changes TNF-alpha-induced eotaxin and ICAM-1 expression in human parasympathetic neurons. METHODS: Parasympathetic neurons were isolated from human tracheas and grown in serum-free medium for one week. Cells were incubated with either (R)-albuterol (the active isomer), (S)-albuterol (the inactive isomer) or (R,S)-albuterol for 90 minutes before adding 2 ng/ml TNF-alpha for another 4 hours (for mRNA) or 24 hours (for protein). RESULTS AND CONCLUSIONS: Baseline expression of eotaxin and ICAM-1 were not changed by any isomer of albuterol as measured by real time RT-PCR. TNF-alpha induced ICAM-1 expression was significantly inhibited by (R)-albuterol in a dose dependent manner, but not by (S) or (R,S)-albuterol. Eotaxin expression was not changed by TNF-alpha or by any isomer of albuterol. The ß-receptor antagonist propranolol blocked the inhibitory effect of (R)-albuterol on TNF-alpha-induced ICAM-1 expression. CLINICAL IMPLICATION: The suppressive effect of (R)-albuterol on neural ICAM-1 expression may be an additional mechanism for decreasing bronchoconstriction, since it would decrease eosinophil recruitment to the airway nerves.

Eosinophils increase neuron branching in human and murine skin and in vitro.

Cutaneous nerves are increased in atopic dermatitis, and itch is a prominent symptom. We studied the functional interactions between eosinophils and nerves in human and mouse skin and in culture. We demonstrated that human atopic dermatitis skin has eosinophil granule proteins present in the same region as increased nerves. Transgenic mice in which interleukin-5 (IL-5) expression is driven by a keratin-14 (K14) promoter had many eosinophils in the epidermis, and the number of nerves was also significantly increased in the epidermis. In co-cultures, eosinophils dramatically increased branching of sensory neurons isolated from the dorsal root ganglia (DRG) of mice. This effect did not occur in DRG neurons co-cultured with mast cells or with dead eosinophils. Physical contact of the eosinophils with the neurons was not required, and the effect was not blocked by an antibody to nerve growth factor. DRG neurons express eotaxin-1, ICAM-1 and VCAM-1, which may be important in the recruitment, binding, and activation of eosinophils in the region of cutaneous nerves. These data indicate a pathophysiological role for eosinophils in cutaneous nerve growth in atopic dermatitis, and suggest they may present a therapeutic target in atopic dermatitis and other eosinophilic skin conditions with neuronal symptoms such as itch.

Role of TNF-α in virus-induced airway hyperresponsiveness and neuronal M2 muscarinic receptor dysfunction.

BACKGROUND AND PURPOSE: Infections with respiratory viruses induce exacerbations of asthma, increase acetylcholine release and potentiate vagally mediated bronchoconstriction by blocking inhibitory M2 muscarinic receptors on parasympathetic neurons. Here we test whether virus-induced M2 receptor dysfunction and airway hyperresponsiveness are tumour necrosis factor-alpha (TNF-α) dependent. EXPERIMENTAL APPROACH: Guinea pigs were pretreated with etanercept or phosphate-buffered saline 24 h before intranasal infection with parainfluenza. Four days later, pulmonary inflation pressure, heart rate and blood pressure were measured. M2 receptor function was assessed by the potentiation by gallamine (an M2 receptor antagonist) of bronchoconstriction caused by electrical stimulation of the vagus nerves and measured as increased pulmonary inflation pressure. Human airway epithelial cells were infected with influenza and TNF-α concentration in supernatant was measured before supernatant was applied to human neuroblastoma cells. M2 receptor expression in these neuroblastoma cells was measured by qRT-PCR. KEY RESULTS: Influenza-infected animals were hyperresponsive to vagal stimulation but not to intravenous ACh. Gallamine did not potentiate vagally induced bronchoconstriction in virus-infected animals, indicating M2 receptor dysfunction. Etanercept prevented virus-induced airway hyperresponsiveness and M2 receptor dysfunction, without changing lung viral titres. Etanercept caused a non-significant decrease in total cells, macrophages and neutrophils in bronchoalveolar lavage. Influenza infection significantly increased TNF-α release from isolated epithelial cells, sufficient to decrease M2 receptors in neuroblastoma cells. This ability of supernatants from infected epithelial cells to inhibit M2 receptor expression was blocked by etanercept. CONCLUSIONS AND IMPLICATIONS: TNF-α is a key mediator of virus-induced M2 muscarinic receptor dysfunction and airway hyperresponsiveness.

Toll-like receptor 7 agonists are potent and rapid bronchodilators in guinea pigs.

BACKGROUND: Respiratory tract viral infections result in asthma exacerbations. Toll-like receptor (TLR) 7 is a receptor for viral single-stranded RNA and is expressed at high levels in the lungs. OBJECTIVE: Because TLR7 polymorphisms are associated with asthma, we examined the effects of TLR7 agonists in guinea pig airways. METHODS: We induced bronchoconstriction in guinea pigs in vivo by means of electrical stimulation of the vagus nerve or intravenous administration of acetylcholine and measured the effect of a TLR7 agonist administered intravenously. We induced contraction of airway smooth muscle in segments of isolated guinea pig tracheas in vitro and measured the effect of TLR7 agonists, antagonists, and pharmacologic inhibitors of associated signaling pathways administered directly to the bath. RESULTS: TLR7 agonists acutely inhibited bronchoconstriction in vivo and relaxed contraction of airway smooth muscle in vitro within minutes of administration. Airway relaxation induced by the TLR7 agonist R837 (imiquimod) was partially blocked with a TLR7 antagonist and was also blocked by inhibitors of large-conductance, calcium-activated potassium channels; prostaglandin synthesis; and nitric oxide generation. Another TLR7 agonist, 21-mer single-stranded phosphorothioated polyuridylic acid (PolyUs), mediated relaxation that was completely blocked by a TLR7 antagonist. CONCLUSIONS: These data demonstrate a novel protective mechanism to limit bronchoconstriction and maintain airflow during respiratory tract viral infections. The fast time frame is inconsistent with canonical TLR7 signaling. R837 mediates bronchodilation by means of TLR7-dependent and TLR7-independent mechanisms, whereas PolyUs does so through only the TLR7-dependent mechanism. TLR7-independent mechanisms involve prostaglandins and large-conductance, calcium-activated potassium channels, whereas TLR7-dependent mechanisms involve nitric oxide. TLR7 is an attractive therapeutic target for its ability to reverse bronchoconstriction within minutes.

Organophosphorus pesticides decrease M2 muscarinic receptor function in guinea pig airway nerves via indirect mechanisms.

BACKGROUND: Epidemiological studies link organophosphorus pesticide (OP) exposures to asthma, and we have shown that the OPs chlorpyrifos, diazinon and parathion cause airway hyperreactivity in guinea pigs 24 hr after a single subcutaneous injection. OP-induced airway hyperreactivity involves M2 muscarinic receptor dysfunction on airway nerves independent of acetylcholinesterase (AChE) inhibition, but how OPs inhibit neuronal M2 receptors in airways is not known. In the central nervous system, OPs interact directly with neurons to alter muscarinic receptor function or expression; therefore, in this study we tested whether the OP parathion or its oxon metabolite, paraoxon, might decrease M2 receptor function on peripheral neurons via similar direct mechanisms. METHODOLOGY/PRINCIPAL FINDINGS: Intravenous administration of paraoxon, but not parathion, caused acute frequency-dependent potentiation of vagally-induced bronchoconstriction and increased electrical field stimulation (EFS)-induced contractions in isolated trachea independent of AChE inhibition. However, paraoxon had no effect on vagally-induced bradycardia in intact guinea pigs or EFS-induced contractions in isolated ileum, suggesting mechanisms other than pharmacologic antagonism of M2 receptors. Paraoxon did not alter M2 receptor expression in cultured cells at the mRNA or protein level as determined by quantitative RT-PCR and radio-ligand binding assays, respectively. Additionally, a biotin-labeled fluorophosphonate, which was used as a probe to identify molecular targets phosphorylated by OPs, did not phosphorylate proteins in guinea pig cardiac membranes that were recognized by M2 receptor antibodies. CONCLUSIONS/SIGNIFICANCE: These data indicate that neither direct pharmacologic antagonism nor downregulated expression of M2 receptors contributes to OP inhibition of M2 function in airway nerves, adding to the growing evidence of non-cholinergic mechanisms of OP neurotoxicity.

Retinoic acid prevents virus-induced airway hyperreactivity and M2 receptor dysfunction via anti-inflammatory and antiviral effects.

Inhibitory M(2) muscarinic receptors on airway parasympathetic nerves normally limit acetylcholine release. Viral infections decrease M(2) receptor function, increasing vagally mediated bronchoconstriction. Since retinoic acid deficiency causes M(2) receptor dysfunction, we tested whether retinoic acid would prevent virus-induced airway hyperreactivity and prevent M(2) receptor dysfunction. Guinea pigs infected with parainfluenza virus were hyperreactive to electrical stimulation of the vagus nerves, but not to intravenous acetylcholine, indicating that hyperreactivity was due to increased release of acetylcholine from parasympathetic nerves. The muscarinic agonist pilocarpine, which inhibits vagally mediated bronchoconstriction in control animals, no longer inhibited vagally induced bronchoconstriction, demonstrating M(2) receptor dysfunction. Treatment with all-trans retinoic acid (1 mg/kg) prevented virus-induced hyperreactivity and M(2) receptor dysfunction. However, retinoic acid also significantly reduced viral titers in the lungs and attenuated virus-induced lung inflammation. In vitro, retinoic acid decreased M(2) receptor mRNA expression in both human neuroblastoma cells and primary cultures of airway parasympathetic neurons. Thus, the protective effects of retinoic acid on airway function during viral infection appear to be due to anti-inflammatory and antiviral mechanisms, rather than to direct effects on M(2) receptor gene expression.

Etanercept prevents airway hyperresponsiveness by protecting neuronal M2 muscarinic receptors in antigen-challenged guinea pigs.

BACKGROUND AND PURPOSE: Increased tumour necrosis factor-alpha (TNF-alpha) is associated with airway hyperreactivity in antigen-challenged animals. In human asthmatics, TNF-alpha is increased and blocking it prevents airway hyperreactivity in some asthmatic patients. However, the mechanisms by which TNF-alpha mediates hyperreactivity are unknown. Airway hyperreactivity can be caused by dysfunction of neuronal M(2) muscarinic receptors that normally limit acetylcholine release from parasympathetic nerves. Here we test whether blocking TNF-alpha receptors with etanercept prevents M(2) receptor dysfunction and airway hyperreactivity in antigen-challenged guinea pigs. EXPERIMENTAL APPROACH: Ovalbumin-sensitized guinea pigs were challenged by inhalation of antigen. Some animals received etanercept (3 mg kg(-1) i.p.) 3 h before challenge. 24 h after challenge, airway hyperreactivity and M(2) receptor function were tested. Inflammatory cells in bronchoalveolar lavage, blood and lung were counted. TNF-alpha and its receptors were detected by real-time RT-PCR and immunocytochemistry in parasympathetic nerves from humans and guinea pigs and in human neuroblastoma cells. KEY RESULTS: Antigen-challenged animals were hyperreactive to vagal stimulation and neuronal M(2) receptors were dysfunctional. Both M(2) receptor dysfunction and airway hyperreactivity were prevented by etanercept. Etanercept reduced eosinophils around airway nerves, and in blood, bronchoalveolar lavage and airway smooth muscle. Also, TNF-alpha decreased M(2) receptor mRNA in human and guinea pig parasympathetic neurons. CONCLUSIONS AND IMPLICATIONS: Tumour necrosis factor-alpha may contribute to M(2) receptor dysfunction and airway hyperreactivity directly by decreasing receptor expression and indirectly by promoting recruitment of eosinophils, containing major basic protein, an M(2) antagonist. This suggests that etanercept may be beneficial in treatment of allergic asthma.

IL-1 receptors mediate persistent, but not acute, airway hyperreactivity to ozone in guinea pigs.

Ozone exposure in the lab and environment causes airway hyperreactivity lasting at least 3 days in humans and animals. In guinea pigs 1 day after ozone exposure, airway hyperreactivity is mediated by eosinophils that block neuronal M(2) muscarinic receptor function, thus increasing acetylcholine release from airway parasympathetic nerves. However, mechanisms of ozone-induced airway hyperreactivity change over time, so that depleting eosinophils 3 days after ozone makes airway hyperreactivity worse rather than better. Ozone exposure increases IL-1beta in bone marrow, which may contribute to acute and chronic airway hyperreactivity. To test whether IL-1beta mediates ozone-induced airway hyperreactivity 1 and 3 days after ozone exposure, guinea pigs were pretreated with an IL-1 receptor antagonist (anakinra, 30 mg/kg, intraperitoneally) 30 minutes before exposure to filtered air or to ozone (2 ppm, 4 h). One or three days after exposure, airway reactivity was measured in anesthetized guinea pigs. The IL-1 receptor antagonist prevented ozone-induced airway hyperreactivity 3 days, but not 1 day, after ozone exposure. Ozone-induced airway hyperreactivity was vagally mediated, since bronchoconstriction induced by intravenous acetylcholine was not changed by ozone. The IL-1 receptor antagonist selectively prevented ozone-induced reduction of eosinophils around nerves and prevented ozone-induced deposition of extracellular eosinophil major basic protein in airways. These data demonstrate that IL-1 mediates ozone-induced airway hyperreactivity at 3 days, but not 1 day, after ozone exposure. Furthermore, preventing hyperreactivity was accompanied by decreased eosinophil major basic protein deposition within the lung, suggesting that IL-1 affects eosinophil activation 3 days after ozone exposure.

Expression and regulation of intercellular adhesion molecule-1 on airway parasympathetic nerves.

BACKGROUND: Eosinophils cluster along airway nerves in patients with asthma and release eosinophil major basic protein, an antagonist of inhibitory M2 muscarinic receptors on nerves. Blocking M2 function increases bronchoconstriction, leading to airway hyperreactivity. Intercellular adhesion molecule-1 (ICAM-1) mediates eosinophil adhesion to nerves. OBJECTIVE: We investigated mechanisms of ICAM-1 expression by parasympathetic nerves. METHODS: ICAM-1 expression was examined by immunocytochemistry of lung sections from ovalbumin-sensitized and challenged guinea pigs. ICAM-1 was measured in parasympathetic nerves isolated from subjects and guinea pigs and in human neuroblastoma cells by real-time RT-PCR, immunocytochemistry, and Western blot. RESULTS: ICAM-1 was not detected in control airway parasympatheric nerves in vivo or in cultured cells. ICAM-1 was expressed throughout antigen-challenged guinea pig lung tissue and was selectively decreased by dexamethasone only in nerves. ICAM-1 was induced in human and guinea pig parasympathetic nerves by TNF-alpha and IFN-gamma and was inhibited by dexamethasone and by an inhibitor of nuclear factor-kappaB (NF-kappaB). In neuroblastoma cell lines TNF-alpha and IFN-gamma-induced ICAM-1 was blocked by an inhibitor of NF-kappaB but not by inhibitors of mitogen-activated protein kinases. Dexamethasone did not inhibit ICAM-1 expression in neuroblastoma cells. CONCLUSIONS: ICAM-1 induced in nerves by antigen challenge and proinflammatory cytokines is sensitive to dexamethasone. ICAM-1 expression is also sensitive to inhibitors of NF-kappaB. Neuroblastoma cells mimic many, but not all, characteristics of ICAM-1 expression in parasympathetic nerves. CLINICAL IMPLICATIONS: Dexamethasone and NF-kappaB inhibitors could prevent eosinophils from adhering to nerves by blocking ICAM-1 expression on parasympathetic nerves, thus protecting inhibitory M2 muscarinic receptors and making this pathway a potential target for asthma treatment.

Neuronal eotaxin and the effects of CCR3 antagonist on airway hyperreactivity and M2 receptor dysfunction.

Eosinophils cluster around airway nerves in patients with fatal asthma and in antigen-challenged animals. Activated eosinophils release major basic protein, which blocks inhibitory M2 muscarinic receptors (M2Rs) on nerves, increasing acetylcholine release and potentiating vagally mediated bronchoconstriction. We tested whether GW701897B, an antagonist of CCR3 (the receptor for eotaxin as well as a group of eosinophil active chemokines), affected vagal reactivity and M2R function in ovalbumin-challenged guinea pigs. Sensitized animals were treated with the CCR3 antagonist before inhaling ovalbumin. Antigen-challenged animals were hyperresponsive to vagal stimulation, but those that received the CCR3 antagonist were not. M2R function was lost in antigen-challenged animals, but not in those that received the CCR3 antagonist. Although the CCR3 antagonist did not decrease the number of eosinophils in lung tissues as assessed histologically, CCR3 antagonist prevented antigen-induced clustering of eosinophils along the nerves. Immunostaining revealed eotaxin in airway nerves and in cultured airway parasympathetic neurons from both guinea pigs and humans. Both IL-4 and IL-13 increased expression of eotaxin in cultured airway parasympathetic neurons as well as in human neuroblastoma cells. Thus, signaling via CCR3 mediates eosinophil recruitment to airway nerves and may be a prerequisite to blockade of inhibitory M2Rs by eosinophil major basic protein.

Disruption of Nrf2 enhances susceptibility to severe airway inflammation and asthma in mice.

Oxidative stress has been postulated to play an important role in the pathogenesis of asthma; although a defect in antioxidant responses has been speculated to exacerbate asthma severity, this has been difficult to demonstrate with certainty. Nuclear erythroid 2 p45-related factor 2 (Nrf2) is a redox-sensitive basic leucine zipper transcription factor that is involved in the transcriptional regulation of many antioxidant genes. We show that disruption of the Nrf2 gene leads to severe allergen-driven airway inflammation and hyperresponsiveness in mice. Enhanced asthmatic response as a result of ovalbumin sensitization and challenge in Nrf2-disrupted mice was associated with more pronounced mucus cell hyperplasia and infiltration of eosinophils into the lungs than seen in wild-type littermates. Nrf2 disruption resulted in an increased expression of the T helper type 2 cytokines interleukin (IL)-4 and IL-13 in bronchoalveolar lavage fluid and in splenocytes after allergen challenge. The enhanced severity of the asthmatic response from disruption of the Nrf2 pathway was a result of a lowered antioxidant status of the lungs caused by lower basal expression, as well as marked attenuation, of the transcriptional induction of multiple antioxidant genes. Our studies suggest that the responsiveness of Nrf2-directed antioxidant pathways may act as a major determinant of susceptibility to allergen-mediated asthma.