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

Abstract

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.

Fig. 1.

Decreased inflammatory response to inhaled OVA in TRPA1-deficient mice. (A) Reduced leukocyte infiltration in airways of OVA-challenged Trpa1^−/− mice. Cell differentials are shown for total cells, eosinophils, macrophages, lymphocytes, and neutrophils in BALF collected from vehicle (veh, PBS)- and OVA-challenged Trpa1^+/+ and Trpa1^−/− mice. Animal groups: Trpa1^+/+ OVA: n = 8, Trpa1^−/− OVA: n = 8, Trpa1^+/+ veh: n = 7, Trpa1^−/− veh: n = 6. ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001. (B) Normal inflammatory leukocyte infiltration in OVA-challenged Trpv1^−/− mice. BALF leukocyte cell differentials are shown for vehicle (veh, PBS)- and OVA-challenged Trpv1^+/+ and Trpv1^−/− mice. Animal groups: Trpv1^+/+ OVA: n = 6, Trpv1^−/− OVA: n = 4, Trpv1^+/+ veh: n = 6, Trpv1^−/− veh: n = 4. (C) Comparison of airway resistance (R) in OVA-challenged Trpa1^+/+ (blue) and Trpa1^−/− mice (red), as well as vehicle (veh)-treated Trpa1^+/+ (green) and Trpa1^−/− (purple) mice, measured by forced oscillation in response to increasing dosages of acetylcholine. Animal groups: Trpa1^+/+ OVA: n = 4, Trpa1^−/− OVA: n = 4, Trpa1^+/+ veh: n = 6, Trpa1^−/− veh: n = 6. (∗, α = 0.05; ∗∗, α = 0.01; ∗∗∗, α = 0.001). (D) Induction of OVA-reactive Ig E in OVA-challenged wild-type and TRPA1-deficient mice, as determined by ELISA. Animal groups as in Fig. 1A. (E) Density of inflammation in H&E-stained airway sections from OVA-challenged Trpa1^+/+ and Trpa1^−/− mice, scored by counting of inflammatory cells near bronchial bundles (n = 4 mice per group).

Fig. 2.

Impaired induction of mucin, cytokines, and chemokines in OVA-challenged airways of Trpa1-deficient mice. (A) Relative quantities (RQ) of mucin5ac gene transcript, determined by Taqman qPCR of whole mouse lung cDNA. Mucin5ac induction is diminished in Trpa1^−/− OVA mice. GAPDH transcript levels were used for normalization as endogenous control. Animal groups: Trpa1^+/+ veh: n = 4, Trpa1^−/− veh: n = 4, Trpa1^+/+ OVA: n = 6, Trpa1^−/− OVA: n = 7. ∗, P < 0.05. (B) Relative quantities (RQ) of interleukin 5 (IL-5) gene transcript, as determined by Taqman real-time quantitative PCR of whole mouse lung cDNA. OVA-challenged Trpa1^−/− mice show no significant changes in IL-5 transcription compared with vehicle-treated mice. GAPDH transcript levels were used for normalization as endogenous control. Animal groups: Trpa1^+/+ veh: n = 4, Trpa1^−/− veh: n = 4, Trpa1^+/+ OVA: n = 6, Trpa1^−/− OVA: n = 6. ∗∗∗, P < 0.001. (C) Comparison of cytokine and chemokine levels in BALF of OVA-challenged Trpa1^+/+ (white) and Trpa1^−/− (black) mice, as measured by Luminex peptide analysis. Groups: Trpa1^+/+ n = 8–10, Trpa1^−/− n = 8–10 for each analyte. ∗∗, P < 0.01; ∗∗∗, P < 0.001

Fig. 3.

Decreased inflammatory response in Balb/C mice treated with the TRPA1 antagonist HC-030031 during the OVA airway challenge phase. (A) Cell differentials for total leukocytes, eosinophils, macrophages, lymphocytes, and neutrophils in BALF collected from vehicle (veh, PBS)- or OVA-challenged mice injected i.p. with HC-030031 or with methyl cellulose (MC) during the airway challenge phase. Animal groups: MC veh: n = 8, HC-030031 veh: n = 9, MC OVA, n = 10, HC-030031 OVA: n = 8. ∗, P < 0.05. (B) Comparison of airway hyperresponsiveness to i.v. acetylcholine in vehicle (veh, PBS) - or OVA-challenged mice injected i.p. with HC-030031 or with just methyl cellulose (MC) during OVA airway challenge. Animal groups: MC veh: n = 7, HC-030031 veh: n = 7, MC OVA, n = 7, HC-030031 OVA: n = 6 (∗, α = 0.05; ∗∗, α = 0.01; ∗∗∗, α = 0.001). (C) Decreased lung mucin5ac transcription in OVA-challenged mice treated with TRPA1 antagonist HC-030031, determined by Taqman q PCR. RQ of mucin5ac transcript are shown for vehicle (veh.) or OVA-challenged mice injected i.p. with HC-030031 or with just methyl cellulose (MC) during OVA airway challenge. GAPDH transcript levels were used for normalization as endogenous control. Animal groups: MC veh: n = 4, HC-030031 veh: n = 4, MC OVA, n = 8, HC-030031 OVA: n = 8. ∗, P < 0.05. (D) Cytokine and eotaxin levels in bronchoalveolar lavage fluid (BALF) of OVA-challenged Balb/C mice treated with TRPA1 antagonist HC-030031 (black) or carrier methyl cellulose (white). (n = 4 mice/group) ∗, P < 0.05; ∗∗, P < 0.01. (E) Density of inflammation in H&E-stained airway sections from OVA-challenged HC-030031-treated and −untreated (MC) Balb/C mice, scored by counting of inflammatory cells near bronchial bundles (n = 4 mice per group).

Fig. 4.

Role of TRPA1 in chemically induced and inflammatory neuropeptide release in the airways, measured by EIA. (A) Diminished chemically induced release of CGRP in Trpa1^−/− mice following lung exposure to CN (2-chloroacetophenone) during BAL. Averaged CGRP levels in BAL fluid of TRPA1^+/+ mice, treated with PBS (n = 4) or 4 mM CN (n = 6), and TRPA1^−/− mice treated with 4 mM CN (n = 5) are shown. ∗, P < 0.05. (B) Diminished chemically induced release of Substance P (SP) in Trpa1^−/− mice following lung exposure to CN (2-chloroacetophenone) during BAL. Treatments and mouse groups as in Fig. 4A. (C) Diminished chemically induced release of neurokinin A (NK-A) in Trpa1^−/− mice following lung exposure to CN (2-chloroacetophenone) during BAL. Treatments and mouse groups as in Fig. 4A. (D) Diminished CN-induced release of SP in mice injected i.p. with TRPA1 antagonist HC-030031. Averaged SP concentrations are shown in BAL fluid of Balb/C mice treated with PBS (n = 4), 200 μM CN and methylcellulose vehicle (MC CN, n = 4), or with 200 μM CN and HC-030031 (HC CN, n = 4) are shown. ∗, P < 0.05. (E) Reduced level of neurokinin A in BAL fluid of OVA-challenged Trpa1^−/− mice. NK-A levels were compared by EIA in BAL fluid of vehicle-treated wild-type mice (Trpa1^+/+ veh, n = 11), vehicle-treated Trpa1^−/− mice (n = 7), OVA-challenged wild-type mice (Trpa1^+/+ OVA, n = 12), and OVA-challenged Trpa1^−/− mice (n = 12). ∗, α<0.05. (F) Reduction of NK-A in BAL fluid of OVA-challenged Balb/C mice due to injection of TRPA1-antagonist HC-030031. NK-A levels were compared by EIA in BAL fluid of methyl cellulose and vehicle-treated mice (MC veh, n = 4), HC-030031- and vehicle-treated (HC veh, n = 4), methyl cellulose treated and OVA-challenged (MC OVA, n = 7), and HC-030031-treated and OVA-challenged mice (HC OVA, n = 8). ∗, α<0.05