Acid-Sensing Ion Channel 1a Contributes to Airway Hyperreactivity in Mice

Abstract

Neurons innervating the airways contribute to airway hyperreactivity (AHR), a hallmark feature of asthma. Several observations suggested that acid-sensing ion channels (ASICs), neuronal cation channels activated by protons, might contribute to AHR. For example, ASICs are found in vagal sensory neurons that innervate airways, and asthmatic airways can become acidic. Moreover, airway acidification activates ASIC currents and depolarizes neurons innervating airways. We found ASIC1a protein in vagal ganglia neurons, but not airway epithelium or smooth muscle. We induced AHR by sensitizing mice to ovalbumin and found that ASIC1a-/- mice failed to exhibit AHR despite a robust inflammatory response. Loss of ASIC1a also decreased bronchoalveolar lavage fluid levels of substance P, a sensory neuropeptide secreted from vagal sensory neurons that contributes to AHR. These findings suggest that ASIC1a is an important mediator of AHR and raise the possibility that inhibiting ASIC channels might be beneficial in asthma.

Fig 1. ASIC1a is present in vagal ganglia and expression in the airway is non-specific or negligible.

A) Images of wild-type (WT) and ASIC1a^-/- mouse vagal ganglia. ASIC1a immunostaining is in green, and DIC indicates differential interference contrast images. Scale bar in left and middle panels is 60 μm; scale bar in the right panel is 40 μm. B) Western blot of ASIC1a in the vagal ganglia. Brain is a positive control. For vagal ganglia, trachea, and lung, 40 μg of protein from tissues of 3 WT mice were pooled. For brain, 10 μg of protein was loaded. C) Images of wild-type (WT) and ASIC1a^-/- mouse lung cross-sections. ASIC1a immunostaining is shown in green, and DIC indicates differential interference contrast images. Scale bar in left and middle panels is 50 μm; scale bar in the right panel is 30 μm. Asterisks indicate airways; arrowheads show epithelia; arrows identify smooth muscle. D) Images of wild-type (WT) and ASIC1a^-/- mouse cultured airway epithelia. DAPI staining is blue (nuclei), ASIC1a immunostaining is in green, ulex europaeus agglutinin (UEA) staining is red (mucin-producing cells), and DIC indicates differential interference contrast images. Scale bar is 30 μm. Abbreviations: WT, wild-type; ASIC, acid-sensing ion channel; DIC, differential interference contrast. UEA, ulex europaeus agglutinin; DAPI, 4',6-diamidino-2-phenylindole. Staining of airways and cultures occurred using same procedures and same conditions as the vagal ganglia.

Fig 2. Loss of ASIC1a prevents airway hyperreactivity.

A) Male mice (8–9 weeks-old) were sensitized by intraperitoneal injection of 10 μg of OVA (Sigma) mixed with 1 mg of alum in 0.9% saline on days 0 and 7. Control mice received saline with 1 mg of alum at day 0 and 7. On days 14–16, mice were nebulized with either 1% OVA or 0.9% saline for 40 min in a chamber. B) Airway resistance (R) was measured by flexiVent in OVA-sensitized wild-type and ASIC1a^-/- mice before and following administration of increasing doses of methacholine. Data are mean±SEM. WT + Sal, n = 7 mice; WT + OVA, n = 6 mice; ASIC1a^-/- + Sal, n = 8 mice; ASIC1a^-/- + OVA, n = 8 mice. * indicates p = 0.043. C) Ratio of airway resistance after administration of 50 mg/ml methacholine in OVA-sensitized mice compared to non-sensitized mice. A ratio of 1 indicates that airway resistance of OVA-sensitized and non-sensitized mice was the same. * indicates p = 0.012. Ratios for ASIC1a^-/- mice were not statistically different from one (p = 0.18). D) Baseline airway resistance (R) prior to administering methacholine. p = 0.89. E) Airway measurements obtained from micro-CT scans. Data are mean±SEM area for 35 different airways. Airways are shown according to size. References to abbreviations and methods are in the Methods section. WT, n = 4 mice; ASIC1a^-/-, n = 5 mice. p = 0.35. F) Number of cells in bronchoalveolar lavage fluid from non-sensitized and sensitized mice. For WT + Sal vs. WT + OVA, * indicates p = 0.004; for ASIC1a^-/- + Sal vs. ASIC1a^-/- + OVA, * indicates p = 0.006. G) The percentage of granulocytes in bronchoalveolar lavage fluid. For WT + Sal vs. WT + OVA, * indicates p<0.0001; for ASIC1a^-/- + Sal vs. ASIC1a^-/- + OVA, * indicates p<0.0001. H) Levels of IL4 in bronchoalveolar lavage fluid. For WT + Sal vs. WT + OVA, * indicates p = 0.03; for ASIC1a^-/- + Sal vs. ASIC1a^-/- + OVA, * indicates p = 0.018. I) Levels of IL5 in bronchoalveolar lavage fluid. For WT + Sal vs. WT + OVA, * indicates p = 0.049; for ASIC1a^-/- + Sal vs. ASIC1a^-/- + OVA, * indicates p = 0.005. A Pearson’s normality test showed that IL5 values in the ASIC1a^-/- OVA-sensitized mice do not differ from a normal distribution. (J) Levels of IL13 in bronchoalveolar lavage fluid. For WT + Sal vs. WT + OVA, p = 0.054; for ASIC1a^-/- + Sal vs. ASIC1a^-/- + OVA, * indicates p = 0.013; for WT + OVA vs. ASIC1a^-/- + OVA, # indicates p = 0.036. A Pearson’s normality test showed that IL13 values in the ASIC1a^-/- OVA-sensitized mice do not differ from a normal distribution. K) muc5AC mRNA in mouse airways. For WT + Sal vs. WT + OVA, * indicates p<0.0001; for ASIC1a^-/- + Sal vs. ASIC1a^-/- + OVA, * indicates p = 0.018. For all panels, individual points represent data collected from a single mouse. Bars and whiskers indicate mean±SEM. Abbreviations: OVA, ovalbumin; Sal, saline; WT, wild-type; ASIC, acid-sensing ion channel; MCh, methacholine.

Fig 3. OVA-sensitization induces similar bronchovascular inflammation in wild-type and ASIC1a^-/- mice.

A) Representative hematoxylin and eosin staining of mouse lung sections. Asterisks indicate airways; arrows indicate examples of bronchovascular inflammation. Scale bar for top panels indicates 700 μm; for lower panels bar indicates 140 μm. B) Bronchovascular inflammation score. Bronchovascular inflammation severity was scored as follows: 1, within normal limits; 2, focal solitary cells with uncommon aggregates; 3, multifocal nominal to moderate sized aggregates; 4, moderate to high cellularity, multifocal large cellular aggregates that may be expansive into adjacent tissues. The following scores were assigned for bronchovascular inflammation distribution: 1, within normal limits; 2, minor to localized aggregates, <33% of lung; 3, multifocal aggregates, 33–66% of lung; 4, aggregates coalescing to widespread, >66% of lung. For severity score: WT + Sal vs. WT + OVA, * indicates p = 0.0002; for ASIC1a^-/- + Sal vs. ASIC1a^-/- + OVA, * indicates p = 0.007. For distribution score: WT + Sal vs. WT + OVA, * indicates p = 0.005; for ASIC1a^-/- + Sal vs. ASIC1a^-/- + OVA, * indicates p = 0.001. C) Substance P measured by ELISA in the bronchoalveolar lavage fluid as a test of sensory nerve activity. For WT + Sal vs. WT + OVA, * indicates p = 0.05; for WT + OVA vs. ASIC1a^-/- + OVA, # indicates p = 0.03. For panels B and C, each symbol indicates data from an individual mouse. Bars and whiskers indicate mean±SEM. Abbreviations: OVA, ovalbumin; Sal, saline; WT, wild-type; ASIC, acid-sensing ion channel.