Ana I. Caceres

A sensory neuronal ion channel essential for airway inflammation and hyperreactivity in asthma

Asthma is an inflammatory disorder caused by airway exposures to allergens and chemical irritants. Studies focusing on immune, smooth muscle, and airway epithelial function revealed many aspects of the disease mechanism of asthma. However, the limited efficacies of immune-directed therapies suggest the involvement of additional mechanisms in asthmatic airway inflammation. TRPA1 is an irritant-sensing ion channel expressed in airway chemosensory nerves. TRPA1-activating stimuli such as cigarette smoke, chlorine, aldehydes, and scents are among the most prevalent triggers of asthma. Endogenous TRPA1 agonists, including reactive oxygen species and lipid peroxidation products, are potent drivers of allergen-induced airway inflammation in asthma. Here, we examined the role of TRPA1 in allergic asthma in the murine ovalbumin model. Strikingly, genetic ablation of TRPA1 inhibited allergen-induced leukocyte infiltration in the airways, reduced cytokine and mucus production, and almost completely abolished airway hyperreactivity to contractile stimuli. This phenotype is recapitulated by treatment of wild-type mice with HC-030031, a TRPA1 antagonist. HC-030031, when administered during airway allergen challenge, inhibited eosinophil infiltration and prevented the development of airway hyperreactivity. Trpa1−/− mice displayed deficiencies in chemically and allergen-induced neuropeptide release in the airways, providing a potential explanation for the impaired inflammatory response. Our data suggest that TRPA1 is a key integrator of interactions between the immune and nervous systems in the airways, driving asthmatic airway inflammation following inhaled allergen challenge. TRPA1 may represent a promising pharmacological target for the treatment of asthma and other allergic inflammatory conditions.

TRPA1 controls inflammation and pruritogen responses in allergic contact dermatitis

Allergic contact dermatitis is a common skin disease associated with inflammation and persistent pruritus. Transient receptor potential (TRP) ion channels in skin‐innervating sensory neurons mediate acute inflammatory and pruritic responses following exogenous stimulation and may contribute to allergic responses. Genetic ablation or pharmacological inhibition of TRPA1, but not TRPV1, inhibited skin edema, keratinocyte hyperplasia, nerve growth, leukocyte infiltration, and antihistamine‐resistant scratching behavior in mice exposed to the haptens, oxazolone and urushiol, the contact allergen of poison ivy. Hapten‐challenged skin of TRPA1‐deficient mice contained diminished levels of inflammatory cytokines, nerve growth factor, and endogenous pruritogens, such as substance P (SP) and serotonin. TRPA1‐deficient sensory neurons were defective in SP signaling, and SP‐induced scratching behavior was abolished in Trpa1–/– mice. SP receptor antagonists, such as aprepitant inhibited both hapten‐induced cutaneous inflammation and scratching behavior. These findings support a central role for TRPA1 and SP in the integration of immune and neuronal mechanisms leading to chronic inflammatory responses and pruritus associated with contact dermatitis.—Liu, B., Escalera, J., Balakrishna, S., Fan, L., Caceres, A. I., Robinson, E., Sui, A., McKay, M. C., McAlexander, M. A., Herrick, C. A., Jordt, S. E., TRPA1 controls inflammation and pruritogen responses in allergic contact dermatitis. FASEB J. 27, 3549–3563 (2013). www.fasebj.org

Transient receptor potential ankyrin 1 antagonists block the noxious effects of toxic industrial isocyanates and tear gases

The release of methyl isocyanate in Bhopal, India, caused the worst industrial accident in history. Exposures to industrial isocyanates induce lacrimation, pain, airway irritation, and edema. Similar responses are elicited by chemicals used as tear gases. Despite frequent exposures, the biological targets of isocyanates and tear gases in vivo have not been identified, precluding the development of effective counter measures. We use Ca2+ imaging and electrophysiology to show that the noxious effects of isocyanates and those of all major tear gas agents are caused by activation of Ca2+ influx and membrane currents in mustard oil‐sensitive sensory neurons. These responses are mediated by transient receptor potential ankyrin 1 (TRPA1), an ion channel serving as a detector for reactive chemicals. In mice, genetic ablation or pharmacological inhibition of TRPA1 dramatically reduces isocyanate‐ and tear gas‐induced nocifensive behavior after both ocular and cutaneous exposures. We conclude that isocyanates and tear gas agents target the same neuronal receptor, TRPA1. Treatment with TRPA1 antagonists may prevent and alleviate chemical irritation of the eyes, skin, and airways and reduce the adverse health effects of exposures to a wide range of toxic noxious chemicals.—Bessac, B. F., Sivula, M., vonHehn, C. A., Caceres, A. I., Escalera, J., Jordt, S.‐E. Transient receptor potential ankyrin 1 antagonists block the noxious effects of toxic industrial isocyanates and tear gases. FASEB J. 23, 1102–1114 (2009)

Vaccinia Virus E3 Protein Prevents the Antiviral Action of ISG15

The ubiquitin-like modifier ISG15 is one of the most predominant proteins induced by type I interferons (IFN). In this study, murine embryo fibroblast (MEFs) and mice lacking the gene were used to demonstrate a novel role of ISG15 as a host defense molecule against vaccinia virus (VACV) infection. In MEFs, the growth of replication competent Western Reserve (WR) VACV strain was affected by the absence of ISG15, but in addition, virus lacking E3 protein (VVΔE3L) that is unable to grow in ISG15+/+ cells replicated in ISG15-deficient cells. Inhibiting ISG15 with siRNA or promoting its expression in ISG15−/− cells with a lentivirus vector showed that VACV replication was controlled by ISG15. Immunoprecipitation analysis revealed that E3 binds ISG15 through its C-terminal domain. The VACV antiviral action of ISG15 and its interaction with E3 are events independent of PKR (double-stranded RNA-dependent protein kinase). In mice lacking ISG15, infection with VVΔE3L caused significant disease and mortality, an effect not observed in VVΔE3L-infected ISG15+/+ mice. Pathogenesis in ISG15-deficient mice infected with VVΔE3L or with an E3L deletion mutant virus lacking the C-terminal domain triggered an enhanced inflammatory response in the lungs compared with ISG15+/+-infected mice. These findings showed an anti-VACV function of ISG15, with the virus E3 protein suppressing the action of the ISG15 antiviral factor.

Ventilatory responses and carotid body function in adult rats perinatally exposed to hyperoxia

Hypoxia increases the release of neurotransmitters from chemoreceptor cells of the carotid body (CB) and the activity in the carotid sinus nerve (CSN) sensory fibers, elevating ventilatory drive. According to previous reports, perinatal hyperoxia causes CSN hypotrophy and varied diminishment of CB function and the hypoxic ventilatory response. The present study aimed to characterize the presumptive hyperoxic damage. Hyperoxic rats were born and reared for 28 days in 55%–60% O2; subsequent growth (to 3.5–4.5 months) was in a normal atmosphere. Hyperoxic and control rats (born and reared in a normal atmosphere) responded with a similar increase in ventilatory frequency to hypoxia and hypercapnia. In comparison with the controls, hyperoxic CBs showed (1) half the size, but comparable percentage area positive to tyrosine hydroxylase (chemoreceptor cells) in histological sections; (2) a twofold increase in dopamine (DA) concentration, but a 50% reduction in DA synthesis rate; (3) a 75% reduction in hypoxia‐evoked DA release, but normal high [K+]0‐evoked release; (4) a 75% reduction in the number of hypoxia‐sensitive CSN fibers (although responding units displayed a nearly normal hypoxic response); and (5) a smaller percentage of chemoreceptor cells that increased [Ca2+]1 in hypoxia, although responses were within the normal range. We conclude that perinatal hyperoxia causes atrophy of the CB–CSN complex, resulting in a smaller number of chemoreceptor cells and fibers. Additionally, hyperoxia damages O2‐sensing, but not exocytotic, machinery in most surviving chemoreceptor cells. Although hyperoxic CBs contain substantially smaller numbers of chemoreceptor cells/sensory fibers responsive to hypoxia they appear sufficient to evoke normal increases in ventilatory frequency.

Role of voltage‐dependent calcium channels in stimulus–secretion coupling in rabbit carotid body chemoreceptor cells

We have defined Ca2+ channel subtypes expressed in rabbit carotid body (CB) chemoreceptor cells and their participation in the stimulus‐evoked catecholamine (CA) release. Ca2+ currents (ICa) activated at –30 mV, peaked at +10 mV and were fully blocked by 200 μm Cd2+. L‐type channels (sensitive to 2 μm nisoldipine) activated at –30 mV and carried 21 ± 2% of total ICa. Non‐L‐type channels activated at potentials positive to –10 mV and carried: N channels (sensitive to 1 μmω‐conotoxin‐GVIA) 16 ± 1% of total ICa, P/Q channels (sensitive to 3 μmω‐conotoxin‐MVIIC after nisoldipine plus GVIA) 23 ± 3% of total ICa and R channels (resistant to all blockers combined) 40 ± 3% of total ICa. CA release induced by hypoxia, hypercapnic acidosis, dinitrophenol (DNP) and high K+o in the intact CB was inhibited by 79–98% by 200 μm Cd2+. Hypoxia, hypercapnic acidosis and DNP, depolarized chemoreceptor cells and eventually generated repetitive action potential discharge. Nisoldipine plus MVIIC nearly abolished the release of CAs induced by hypoxia and hypercapnic acidosis and reduced by 74% that induced by DNP. All these secretory responses were insensitive to GVIA. 30 and 100 mm K+o brought resting membrane potential (Em) of chemoreceptor cells (–48.1 ± 1.2 mV) to –22.5 and +7.2 mV, respectively. Thirty millimolar K+o‐evoked release was abolished by nisoldipine but that induced by 100 mm K+o was mediated by activation of L, N, and P/Q channels. Data show that tested stimuli depolarize rabbit CB chemoreceptor cells and elicit CA release through Ca2+ entry via voltage‐activated channels. Only L and P/Q channels are tightly coupled to the secretion of CA.

Molecular identification and functional role of voltage-gated sodium channels in rat carotid body chemoreceptor cells. Regulation of expression by chronic hypoxia in vivo

We have assessed the expression, molecular identification and functional role of Na+ channels (Nav) in carotid bodies (CB) obtained from normoxic and chronically hypoxic adult rats. Veratridine evoked release of catecholamines (CA) from an in vitro preparation of intact CBs obtained from normoxic animals, the response being Ca2+ and Na+‐dependent and sensitive to tetrodotoxin (TTX). TTX inhibited by 25–50% the CA release response evoked by graded hypoxia. Immunoblot assays demonstrated the presence of Navα‐subunit (c. 220 kDa) in crude homogenates from rat CBs, being evident an up‐regulation (60%) of this protein in the CBs obtained from chronically hypoxic rats (10% O2; 7 days). This up‐regulation was accompanied by an enhanced TTX‐sensitive release response to veratridine, and by an enhanced ventilatory response to acute hypoxic stimuli. RT‐PCR studies demonstrated the expression of mRNA for Nav1.1, Nav1.2, Nav1.3, Nav1.6 and Nav1.7 isoforms. At least three isoforms, Nav1.1, Nav1.3 and Nav1.6 co‐localized with tyrosine hydroxylase in all chemoreceptor cells. RT‐PCR and immunocytochemistry indicated that Nav1.1 isoform was up‐regulated by chronic hypoxia in chemoreceptor cells. We conclude that Nav up‐regulation represents an adaptive mechanism to increase chemoreceptor sensitivity during acclimatization to sustained hypoxia as evidenced by enhanced ventilatory responses to acute hypoxic tests.

IL-33/ST2 signaling excites sensory neurons and mediates itch response in a mouse model of poison ivy contact allergy

Significance In the United States, the most common cause of allergic contact dermatitis (ACD) is contact with poison ivy. Severe itch and skin inflammation are the major manifestations of poison ivy-induced ACD. In this study, we have established a critical role of IL-33/ST2 (interleukin 33/growth stimulation expressed gene 2) signaling in both itch and skin inflammation of poison ivy-induced ACD and revealed a previously unidentified interaction of IL-33/ST2 signaling with primary sensory neurons that may underlie the pruritic mechanisms of poison ivy-induced ACD. Blocking IL-33/ST2 signaling may represent a therapeutic approach to ameliorate itch and skin inflammation related to poison ivy dermatitis and, possibly, other chronic itch conditions in which IL-33/ST2 signaling may participate. Poison ivy-induced allergic contact dermatitis (ACD) is the most common environmental allergic condition in the United States. Case numbers of poison ivy ACD are increasing due to growing biomass and geographical expansion of poison ivy and increasing content of the allergen, urushiol, likely attributable to rising atmospheric CO2. Severe and treatment-resistant itch is the major complaint of affected patients. However, because of limited clinical data and poorly characterized models, the pruritic mechanisms in poison ivy ACD remain unknown. Here, we aim to identify the mechanisms of itch in a mouse model of poison ivy ACD by transcriptomics, neuronal imaging, and behavioral analysis. Using transcriptome microarray analysis, we identified IL-33 as a key cytokine up-regulated in the inflamed skin of urushiol-challenged mice. We further found that the IL-33 receptor, ST2, is expressed in small to medium-sized dorsal root ganglion (DRG) neurons, including neurons that innervate the skin. IL-33 induces Ca2+ influx into a subset of DRG neurons through neuronal ST2. Neutralizing antibodies against IL-33 or ST2 reduced scratching behavior and skin inflammation in urushiol-challenged mice. Injection of IL-33 into urushiol-challenged skin rapidly exacerbated itch-related scratching via ST2, in a histamine-independent manner. Targeted silencing of neuronal ST2 expression by intrathecal ST2 siRNA delivery significantly attenuated pruritic responses caused by urushiol-induced ACD. These results indicate that IL-33/ST2 signaling is functionally present in primary sensory neurons and contributes to pruritus in poison ivy ACD. Blocking IL-33/ST2 signaling may represent a therapeutic approach to ameliorate itch and skin inflammation related to poison ivy ACD.

EPAC signalling pathways are involved in low PO2 chemoreception in carotid body chemoreceptor cells

Chemoreceptor cells of the carotid bodies (CB) are activated by hypoxia and acidosis, responding with an increase in their rate of neurotransmitter release, which in turn increases the electrical activity in the carotid sinus nerve and evokes a homeostatic hyperventilation. Studies in isolated chemoreceptor cells have shown that moderate hypoxias (≈ 46 mmHg) produces smaller depolarisations and comparable Ca2+ transients but a much higher catecholamine (CA) release response in intact CBs than intense acidic/hypercapnic stimuli (20% CO2, pH 6.6). Similarly, intense hypoxia (≈ 20 mmHg) produces smaller depolarizations and Ca2+ transients in isolated chemoreceptor cells but a higher CA release response in intact CBs than a pure depolarizing stimulus (30–35 mm external K+). Studying the mechanisms responsible for these differences we have found the following. (1) Acidic hypercapnia inhibited ICa (∼60%; whole cell) and CA release (∼45%; intact CB) elicited by ionomycin and high K+. (2) Adenylate cyclase inhibition (SQ‐22536; 80 μm) inhibited the hypoxic release response (>50%) and did not affect acidic/hypercapnic release, evidencing that the high gain of hypoxia to elicit neurotransmitter release is cAMP dependent. (3) The last effect was independent of PKA activation, as three kinase inhibitors (H‐89, KT 5720 and Rp‐cAMP; ≥ 10 × IC50) did not alter the hypoxic release response. (4) The Epac (exchange protein activated by cAMP) activator (8‐pCPT‐2′‐O‐Me‐cAMP, 100 μm) reversed the effects of the cyclase inhibitor. (5) The Epac inhibitor brefeldin A (100 μm) inhibited (54%) hypoxic induced release. Our findings show for the first time that an Epac‐mediated pathway mediates O2 sensing/transduction in chemoreceptor cells.

Menthol Attenuates Respiratory Irritation and Elevates Blood Cotinine in Cigarette Smoke Exposed Mice

Addition of menthol to cigarettes may be associated with increased initiation of smoking. The potential mechanisms underlying this association are not known. Menthol, likely due to its effects on cold-sensing peripheral sensory neurons, is known to inhibit the sensation of irritation elicited by respiratory irritants. However, it remains unclear whether menthol modulates cigarette smoke irritancy and nicotine absorption during initial exposures to cigarettes, thereby facilitating smoking initiation. Using plethysmography in a C57Bl/6J mouse model, we examined the effects of L-menthol, the menthol isomer added to cigarettes, on the respiratory sensory irritation response to primary smoke irritants (acrolein and cyclohexanone) and smoke of Kentucky reference 2R4 cigarettes. We also studied L-menthol’s effect on blood levels of the nicotine metabolite, cotinine, immediately after exposure to cigarette smoke. L-menthol suppressed the irritation response to acrolein with an apparent IC₅₀ of 4 ppm. Suppression was observed even at acrolein levels well above those necessary to produce a maximal response. Cigarette smoke, at exposure levels of 10 mg/m³ or higher, caused an immediate and marked sensory irritation response in mice. This response was significantly suppressed by L-menthol even at smoke concentrations as high as 300 mg/m³. Counterirritation by L-menthol was abolished by treatment with a selective inhibitor of Transient Receptor Potential Melastatin 8 (TRPM8), the neuronal cold/menthol receptor. Inclusion of menthol in the cigarette smoke resulted in roughly a 1.5-fold increase in plasma cotinine levels over those observed in mice exposed to smoke without added menthol. These findings document that, L-menthol, through TRPM8, is a strong suppressor of respiratory irritation responses, even during highly noxious exposures to cigarette smoke or smoke irritants, and increases blood cotinine. Therefore, L-menthol, as a cigarette additive, may promote smoking initiation and nicotine addiction.