Targeting acetylcholine receptor M3 prevents the ...

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Targeting acetylcholine receptor M3 prevents the progression of airway hyperreactivity in a mouse model of childhood asthma Kruti R. Patel,*,1 Yan Bai,†,1 Kenneth G. Trieu,† Juliana Barrios,‡ and Xingbin Ai†,2

*Division of Neurology and †Division of Pulmonary and Critical Care Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts, USA; and ‡Pulmonary Division, Boston University School of Medicine, Boston, Massachusetts, USA

Asthma often progresses into adulthood from early-life episodes of adverse environmental exposures. However, how the injury to developing lungs contributes to the pathophysiology of persistent asthma remains poorly understood. In this study, we identified an age-related mechanism along the cholinergic nerve–airway smooth muscle (ASM) axis that underlies prolonged airway hyperreactivity (AHR) in mice. We showed that ASM continued to mature until ∼3 wk after birth. Coinciding with postnatal ASM maturation, there was a critical time window for the development of ASM hypercontractility after cholinergic stimulation. We found that allergen exposure in neonatal mice, but not in adult mice, elevated the level and activity of cholinergic nerves (termed neuroplasticity). We demonstrated that cholinergic neuroplasticity is necessary for the induction of persistent AHR after neonatal exposure during rescue assays in mice deficient in neuroplasticity. In addition, early intervention with cholinergic receptor muscarinic (ChRM)-3 blocker reversed the progression of AHR in the neonatal exposure model, whereas b2-adrenoceptor agonists had no such effect. Together, our findings demonstrate a functional relationship between cholinergic neuroplasticity and ASM contractile phenotypes that operates uniquely in early life to induce persistent AHR after allergen exposure. Targeting ChRM3 may have disease-modifying benefits in childhood asthma.—Patel, K. R., Bai, Y., Trieu, K. G., Barrios, J., Ai, X. Targeting acetylcholine receptor M3 prevents the progression of airway hyperreactivity in a mouse model of childhood asthma. FASEB J. 31, 4335–4346 (2017). www.fasebj.org

ABSTRACT:

KEY WORDS:

allergen

•

neuroplasticity

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b2AR agonist

Despite the unprecedented strides of modern medicine, the prevalence of asthma is still on the rise worldwide. Asthma often starts in childhood; ;9.5% of children are affected. In more than half of the affected children, it progresses into adulthood (1, 2). Asthma is characterized by excessive airway narrowing in response to a variety of stimuli, even in the absence of disease exacerbation and inflammation. Commonly prescribed drugs include longacting b-agonists and high doses of corticosteroids. Newly developed therapeutics, such as antibodies against IgE and IL-5, have also been introduced into the clinic. ABBREVIATIONS: aSMA, a-smooth muscle actin; b2AR, b2-adrenoceptor;

AHR, airway hyperreactivity; ASM, airway smooth muscle; BAL, bronchoalveolar lavage; ChRM3, acetylcholine receptor muscarinic 3; 4-DAMP, 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide; HDM, house dust mite; MCh, methacholine; NT4, neurotrophin 4; Ova, ovalbumin; PCLS, precisioncut lung slice; VAChT, vesicular acetylcholine transporter 1 2

These authors contributed equally to this work. Correspondence: Division of Pulmonary and Critical Care Medicine, Brigham and Women’s Hospital, Harvard Medical School, 75 Francis Ave., Boston, MA 02115, USA. E-mail: [email protected]

doi: 10.1096/fj.201700186R This article includes supplemental data. Please visit http://www.fasebj.org to obtain this information.

0892-6638/17/0031-4335 © FASEB

However, the treatment strategy so far remains limited to symptomatic relief, carries substantial risks of adverse side effects, especially in pediatric patients with asthma and is ineffective in about a third of patients (3–5). In addition, the treatment has no beneficial effect on the progression of asthma from childhood to adulthood (6). In patients with progressive asthma and even in adults who outgrow earlier symptoms, the decline of long-term lung function is strongly associated with age onset and severity of asthma in childhood (7). These clinical findings highlight the unmet medical need for defeating childhood asthma. Risk factors for asthma include exposure to environmental insults, such as allergens, cigarette smoke, respiratory viral infection, and air pollutants, during infancy and early childhood when the lung is still developing (1, 2). It is speculated that early-life environmental insults disrupt postnatal development of the lung and thus contribute to the pathophysiology of persistent asthma. However, because of a lack of noninvasive methods to fully assess the structure and function of lungs in young children, the exact molecular nature of the developmental abnormalities after early-life environmental

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exposures and how these changes link to asthma later on remain poorly understood. Exposure to environmental insults in adult mice causes transient AHR that is associated with inflammation (8). In contrast, neonatal animal models of adverse exposures replicate prolonged airway dysfunction that outlasts inflammation (8–10) and thus provide invaluable tools for the study of the involvement of developmental factors in the pathogenesis of persistent asthma. Characterization of neonatal models in rodents and nonhuman primates identifies an increase in airway innervation (termed neuroplasticity) (8, 11–13). The neuroplasticity in mice after early-life allergen exposure is caused by an increase in the level of neurotrophin (NT)-4 and, in turn, upregulation of parasympathetic (cholinergic) innervation of ASM (8, 14). NT42/2 mice that were deficient in the neuroplasticity were protected from AHR without any defects in allergic inflammation and ASM hyperplasia after early-life allergen exposure (8). Notably, mice that were exposed to an allergen as neonates maintained AHR for a minimum of 5 wk, whereas neuroplasticity and inflammation were resolved over a relatively short period (8). These findings suggest that the early action of neural signals has a profound impact on long-term contractile phenotypes of ASM. In accordance with our findings in the neonatal mouse model, increased levels of neurotrophin family members, including NT4, are positively associated with respiratory viral infection in infants and asthma severity in children (15, 16). Although the status of airway innervation in pediatric patients remains to be characterized, these clinical findings support the hypothesis that aberrant neural development in lungs may be involved in the pathogenesis of persistent asthma after early-life episodes of adverse environmental exposures. ASM is innervated mostly by sensory and parasympathetic nerves that function in a neural circuit to regulate breathing and airway contraction (17, 18). In the neonatal mouse model, we found that early-life allergen exposure increased the abundance of parasympathetic, cholinergic nerves marked by vesicular acetylcholine transporter (VAChT) (14), whereas it had no effect on the level of sensory innervation assayed by immunostaining for calcitonin gene-related peptide (8). Cholinergic nerves release the neurotransmitter acetylcholine, which is a potent and most physiologically relevant stimulus for ASM contraction in vivo (17–19). Activation of acetylcholine receptor muscarinic (ChRM)-3 by acetylcholine not only triggers Ca2+ influx into ASM cells to induce contraction, but also activates multiple intracellular pathways to regulate the proliferation of ASM and gene expression (19, 20). Mice deficient in ChRM3 have reduced ASM mass and are resistant to ASM hyperplasia after allergen exposure (21). These findings support a critical role of acetylcholineactivated ChRM3 signaling in ASM development and allergen-induced ASM dysfunction. Building on our previous findings that the nerve– ASM axis may uniquely operate in early life to regulate ASM contractility, we assessed the functional and temporal relationships between cholinergic neuroplasticity and ASM hypercontractility in the neonatal mouse model of allergen exposure. We discovered that 4336

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cholinergic neural stimulation plays a critical role in the contractile phenotype of ASM during postnatal maturation. With age, ASM becomes resistant to cholinergic stimulation. The postnatal maturation of ASM provides a time window for susceptibility to the induction of AHR in response to cholinergic stimulation and at the same time, an opportunity for therapeutic intervention with anti-ChRM3 treatment. MATERIALS AND METHODS Mice and study approval Wild-type and NT42/2 (002497) mice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). All the mouse lines were in a C57BL/6 background. All mouse experiments were approved by the Institutional Animal Care and Use Committee at Brigham & Women’s Hospital, Harvard Medical School. Neonatal allergen exposure and treatment models For ovalbumin (Ova) exposure, mice were sensitized at postnatal day (P)5 and -10 by intraperitoneal injection of 10 mg Ova (A5503; Sigma-Aldrich, St. Louis, MO, USA) in Imject alum (77161; Thermo Fisher Scientific, Waltham, MA, USA) (8). Mice were then challenged daily from P18 to -20 with aerosolized 3% Ova solution for 10 min (8). Control mice were challenged with saline. For exposure to house dust mite (HDM) allergen, mice were given HDM (5 mg intranasally, XPB70D3A2.5; Greer Labs, Lenoir, NC, USA) at P5, -10, and -15 followed by daily intranasal instillations (15 mg) between P17 and -20. Control mice received saline intranasally. For methacholine (MCh) exposure, mice were nebulized for 10 min daily between P15 and -20 with 30 mg/ml of MCh (A2251; Sigma-Aldrich). Control mice were nebulized with saline. For treatment models, mice were given the CHRM3 blocker daily, 4-DAMP (50 ml, 100 mM; Tocris Bioscience, Bristol, United Kingdom) or the b2AR agonist, formoterol (50 ml, 5 nM, F9552; Sigma-Aldrich) via intratracheal delivery between P15 and P20 during Ova exposure or between P21 and -25 after Ova exposure. Controls were littermates that were challenged with PBS. Mice were euthanized at P21 and at P56 for harvest of bronchoalveolar lavage (BAL) fluid and lung tissue. Ova and MCh exposure in adult mice Adult mice (3 mo of age) were sensitized at d 0 and 7 by intraperitoneal injection of 25 mg Ova in Imject alum followed by 5 consecutive challenges with 3% aerosolized Ova solution starting at d 14, 10 min/d. For MCh exposure, adult mice were nebulized with MCh (30 mg/ml), 10 min/d for 6 consecutive days. Mice were euthanized at d 7 and 25 for tissue harvest and precision-cut lung slice (PCLS) collection. Controls were challenged with PBS. Airway contraction and protein assays of lung slices Mouse lung slices were prepared from agarose-inflated lungs according to established protocols (8). Contraction assays were performed after lung slices were maintained overnight at 37°C and 5% CO2 in DMEM/F-12 (1:1; Thermo Fisher Scientific) supplemented with antibiotics. PCLSs were stimulated with

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increasing doses of MCh or endothelin (E-7764; SigmaAldrich). The airway luminal area before stimulation and 5 min after stimulation was imaged with an inverted microscope (DMI6000B; Leica Microsystems, Buffalo Grove, IL, USA). Midsized airways with a baseline luminal area between 14,000 and 20,000 mm2 were selected for imaging. For ex vivo MCh treatment, PCLSs from P18, -21, -25, and adult mice were incubated 48 h in the culture medium with 100 mM MCh. After washing, lung slices were maintained in culture medium overnight in the absence of MCh followed by contraction assays. The airway luminal area was measured with ImageJ (National Institutes of Health, Bethesda, MD, USA) and normalized to the baseline value. For protein assays, lung slices were prepared from P21 and adult mice that had been nebulized with saline or MCh. After overnight recovery, lung slices were stimulated with 100 mM MCh for 5 min before Western blot assays for myosin light chain (MLC)-2. Intracellular Ca2+ imaging in ASM of PCLSs PCLSs were loaded with Ca2+ indicator, Oregon green 488 (BAPTA-1-AM; Thermo Fisher Scientific), as previously described (22). Fluorescence images were recorded at 30 images/s with a custom-built 2-photon laser scanning microscope and analyzed by Video Savant software (IO Industries, London, ON, Canada) with custom-written scripts. Western blot assays Protein samples were prepared from mouse lungs and mouse lung slices by tissue homogenization in RIPA buffer containing protease inhibitors (Roche, Basel, Switzerland) and phosphatase inhibitors (524627; EMD Millipore, Billerica, MA, USA) before Western blot assays. For assays of lung slices, 10 slices from each adult mouse and 15 slices from each P21 mouse were pooled to compose 1 sample. Primary antibodies include rabbit antiVAChT (1:100, AB68986; Abcam, Cambridge, MA, USA), rabbit anti-a smooth muscle actin (SMA, 1:5000, ab124964; Abcam), and mouse anti-GAPDH (1:2000, AB8245; Abcam), rabbit antiMLC-2 1:1000, 3672; Cell Signaling Technology, Danvers, MA, USA), rabbit anti-phospho MLC-2 (Ser19) (p-MLC2, 1:500, 3674; Cell Signaling Technology). Horseradish peroxidase– conjugated secondary antibodies include: goat anti-rabbit (1:1000, 1:5000, sc-2004; Santa Cruz Biotechnology, Dallas, TX, USA) goat anti-mouse (1:3000, 554002; BD Biosciences, San Jose, CA, USA). The antigen-antibody complex was detected by chemoluminescent substrate (Thermo Fisher Scientific). Signal intensity was measured with NIH ImageJ. Data were normalized to the GAPDH levels or to the total MLC levels. Histology and immunohistochemistry Paraffin-embedded sections of right cranial lobes were collected for antibody staining with biotinylated mouse anti-aSMA (1:25, MS-113-B; Thermo Fisher Scientific). Sections were counterstained with Hematoxylin QS (H-3404; Vector Laboratories, Burlingame, CA, USA). The immunoreactive area was quantified by using NIH ImageJ and normalized to the circumference of the airway. Statistics All data are represented as means 6 SEM from a minimum of 3 independent experiments. Statistical analysis was performed with the 2-tailed Student’s t test for comparisons between CHOLINERGIC NEURAL INDUCTION OF PERSISTENT AHR

2 conditions and with 2-way ANOVA for comparisons between multiple variances (Prism 5; GraphPad Software, La Jolla, CA, USA). A value of P , 0.05 was deemed statistically significant.

RESULTS Cholinergic stimulation of developing airways, but not mature airways, was sufficient to induce persistent AHR in vivo We showed in previous studies that allergen exposure during the first 3 wk after birth causes cholinergic hyperinnervation (8, 14). In addition, mice that had been exposed to allergens as neonates exhibited persistent AHR that outlasted neuroplasticity and inflammation (8). Since acetylcholine is a potent inducer of ASM contraction, we speculated that deregulated cholinergic stimulation alone contributes to persistent AHR. To test this hypothesis, mice were subjected to daily nebulization with MCh between P15 and -20 before the assessment of airway reactivity at P21 and after 5 wk at P56 in lung slices (Fig. 1A). Lung slices permit direct assessment of ASM contractility without confounding factors, such as mucus, and thus provided an invaluable ex vivo system for our study. The slices were washed and allowed to recover overnight after preparation. The recovery diminished any residual MCh activities in P21 lung slices, because the biologic effect of MCh lasts for about 2.5 h (23). Compared to saline nebulization, MCh nebulization significantly increased airway contractility at P21 (Fig. 1B), and the increase in contractility was maintained at P56 (Fig. 1C). We found no airway inflammation after MCh nebulization by differential immune cell counts in BAL collected at P21 (Supplemental Fig. 1A). This finding was consistent with previous reports that MCh alone had no effect on inflammation (21). In addition, aSMA staining showed no change in ASM mass in MCh-nebulized mice at P21 compared with saline controls (Fig. 1D). To test whether cholinergic stimulation had an agedependent effect on airway contractility, adult mice were similarly nebulized with MCh between d 1 and 6 (Fig. 1E). Airway contractility was assessed in lung slices prepared at d 7 and after 18 d at d 25. In contrast to neonatal nebulization, MCh nebulization in adult mice had no effect on airway contractility at both time points (Fig. 1F, G). These findings revealed an age-related role of cholinergic stimulation in airway contractility that was mediated through mechanisms independent of inflammation and ASM hyperplasia. The susceptibility to AHR was critically dependent on a postnatal time window To identify the time window of the susceptibility to airway hypercontractility induced by cholinergic stimulation, lung slices were prepared from neonatal and adult mice. Lung slices contain live nerve endings; however, they are inactive without electrochemical stimulation. Therefore, MCh treatment induces airway contraction free of the endogenous neural source of acetylcholine. After MCh


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Figure 1. MCh nebulization in early life, but not in adult life, was sufficient to induce persistent airway hypercontractility. A) Experimental scheme of MCh nebulization from P15 to -20. Lungs were harvested at P21 and -56. B) Measurement of airway contraction in response to MCh using lung slices from P21 mice nebulized with PBS or MCh. C ) Measurement of airway contraction in response to MCh in lung slices from mice at P56 that had been exposed to PBS or MCh as neonates. D) Representative images aSMA staining of midsized airways in PBS- and MCh-exposed mice at P21. Each column represents data from 3 airways of each mouse and a total of 3 mice. E ) Scheme of MCh nebulization in adult mice between experimental d 1 and 6. Lungs were harvested at d 7 and 15. F ) Measurement of airway contraction in response to MCh using lung slices of adult mice nebulized with PBS or MCh at d 7. G) Measurement of airway contraction in response to MCh using lung slices from adult mice at d 25 that had been exposed to PBS or MCh. Each point in contraction assays represents data from 6 to 12 airways of 3 mice. N.s., not significant. **P , 0.01.

treatment in culture for 48 h, lung slices were recovered overnight before the measurement of airway contraction (Fig. 2A). To eliminate any concern of residual MCh in lung slices, we used a physiologically irrelevant agonist, endothelin, to induce ASM contraction. Compared to untreated, age-matched controls, we found that MCh treatment was sufficient to induce airway hypercontractility in lung slices collected at P18 and P21 (Fig. 2B, C). However, the inductive effect of MCh was no longer evident after P25 (Fig. 2D, E). In addition, MCh treatment had no effect on the smooth muscle mass in both P21 and adult lung slices measured by Western blot assays for aSMA (Fig. 2F–I). To provide insights into age-related effects of MCh stimulation on airway contractility, we analyzed phosphorylation of MLC2 (p-MLC2), a key regulator of smooth muscle contractility in neonatal and adult airways. For this assay, lung slices were collected from P21 and adult mice that had been nebulized with MCh for 6 consecutive d (Fig. 3A, E). After recovery for 24 h to remove any residual MCh signaling activities, the slices were acutely stimulated with MCh for 5 min, followed by Western blot assays for p-MLC2. Notably, the lung slices contain both vascular and airway smooth muscle. However, only ASM contracts in response to MCh (24). Therefore, changes in the level of p-MLC2 in response to acute MCh stimulation were contributed by ASM. Samples that were never treated with MCh (the PBS-PBS group) from both age groups had baseline p-MLC2 levels (Fig. 3B, C, F, G), which likely 4338

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reflected certain degrees of vascular smooth muscle contraction to maintain the vascular tone. Acute MCh stimulation of lung slices from mice with no prior MCh nebulization (the PBS-MCh group) elicited no increase in p-MLC2 levels above the baseline. This lack of reaction may be explained by the low number of ASM cells in lung slices, and their responses were beyond detection by Western blot assays. However, prior MCh nebulization in neonatal mice significantly increased the response of ASM to acute MCh stimulation (the MCh-MCh group), shown by a 5-fold increase in the level of p-MLC2 compared to controls without acute MCh stimulation (the MCh-PBS group; Fig. 3B, D). In contrast, prior MCh nebulization in adult mice had no effect on p-MLC2 levels in response to acute MCh stimulation (Fig. 3F, H). Together, these findings provide evidence that MCh stimulation has an age-dependent effect on airway contractility. Although MCh treatment of immature ASM robustly elevates the contractile responses of ASM to agonists, this effect diminishes with age. At ;P25, airway contractility became resistant to changes in response to cholinergic stimulation. The contractile phenotype of ASM continued to mature until ∼P21 We hypothesized that the susceptibility to airway hypercontractility in response to cholinergic stimulation is



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Figure 2. Ex vivo MCh treatment of lung slices age-dependently induced airway hypercontractility. A) Experimental scheme of MCh treatment of lung slices prepared from neonatal and adult mice. B–E) Control and MCh-treated lung slices at P18 (B), P21 (C), P25 (D), and adult (E) stages were analyzed for airway reactivity in response to endothelin (End). Each point represents data from 10 airways of 3 mice. F ) Western blot analysis of aSMA in control and MCh-treated lung slices from mice at P21. GAPDH was loading control. G) Data from F were quantified and normalized. H ) Western blot analysis of aSMA in control and MCh-treated lung slices from adult mice. GAPDH was loading control. I) Data from H were quantified and normalized. Each column represents mean 6 SEM of 15–30 slices from 3 mice. N.s., not significant. *P , 0.05.

associated with ASM maturation. However, there is little information regarding the extent of ASM maturation after birth. To address this fundamental question, we characterized Ca2+ signaling in postnatal ASM as a physiologic marker of contractility (25). Because of technical difficulties in preparation of lung slices from mice younger than 2 wk of age, Ca2+ signaling in ASM was assayed at P14 and older ages. MCh stimulation increased the frequency of Ca2+ oscillations in ASM in a dose-dependent manner at all tested ages, consistent with Ca2+ oscillation as a functional measurement of ASM contractility (Fig. 4A). We found that the frequency of Ca2+ oscillations in ASM increased with age until it reached the adult level at ;P21 (Fig. 4B). Therefore, postnatal maturation of ASM provides a time window for ASM to develop hypercontractile phenotypes after cholinergic stimulation. ASM becomes resistant to changes in contractile phenotypes after maturation at P25 (Fig. 2D). NT4-dependent cholinergic hyperinnervation was essential for AHR after early-life allergen exposure We went on to test whether cholinergic stimulation was necessary for airway hypercontractility after early-life allergen exposure. As a first model, we chose NT42/2 mice. NT42/2 mice were protected from early-life CHOLINERGIC NEURAL INDUCTION OF PERSISTENT AHR

allergen-induced increases in airway innervation and AHR without any defects in allergic inflammation (8). We tested whether a lack of AHR in NT42/2 mice after early-life allergen exposure was caused by defective cholinergic innervation. We compared the level of a specific cholinergic nerve marker, VAChT, between wild-type and NT42/2 mice at P21, with and without allergen exposure (Fig. 5A). Western blot assays showed that the baseline level of VAChT was similar between wild-type and NT42/2 mice (Fig. 5B, E). Ova exposure significantly increased the level of VAChT by more than 50% in wild-type mice at P21 (Fig. 5B). Similar increases in VAChT levels were found in a neonatal model of HDM allergen exposure (Fig. 5D, E). In contrast, Ova and HDM exposure had no effect on baseline VAChT levels in NT42/2 mice (Fig. 5B, E). In addition to cholinergic nerves that produce acetylcholine, the epithelium was reported as a non-neuronal source of acetylcholine (20). However, we found no change in low levels of VAChT mRNA expression and other genes involved in acetylcholine biosynthesis and degradation in the airway epithelium after Ova exposure (Supplemental Fig. 2). In addition, the Ntrk2 receptor for NT4 is selectively expressed by nerves (8). Therefore, the increase in VAChT levels after early-life allergen exposure was most likely caused by NT4-dependent increases in cholinergic nerves. Furthermore, we employed electrical field stimulation (EFS) of lung slices to evaluate changes in


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Figure 3. MCh treatment plays an age-dependent role in regulating the contractile responses of ASM. A) Experimental scheme of in vivo MCh nebulization of neonatal mice between P15 and -20. Control mice were nebulized with PBS. Lung slices were collected at P21, left to recover overnight, and then acutely stimulated with PBS (saline control) or MCh (100 mM) for 5 min. B) Western blot analysis of p-MLC2 in lung slices from 4 experimental conditions: baseline control (PBS-PBS), with only acute MCh stimulation (PBS-MCh), MCh nebulization without acute MCh stimulation (MCh-PBS), and MCh nebulization with acute MCh stimulation (MCh-MCh). C, D) Data from B were quantified and normalized to the total MLC2. E ) Experimental scheme of in vivo saline (PBS) or MCh nebulization of adult mice between d 1 and 6. Lung slices were prepared at D7. After recovery, lung slices were acutely stimulated with MCh for 5 min before protein analysis. F ) Western blot analysis of p-MLC2 in lung slices from control (PBS) and MCh-nebulized adult mice, with and without acute MCh stimulation. G, H) Data from F were quantified and normalized to the total MLC2. Each column represents means 6 SEM of 10–15 slices from each mouse, 5–7 mice per experimental group in 2 independent experiments. N.s., not significant. *P , 0.05.

cholinergic innervation in NT42/2 mice. Notably, EFS triggers the release of acetylcholine from cholinergic nerves and, in turn, airway contraction, as EFS-induced airway contraction was completely blocked by 4-DAMP, a highly selective inhibitor of ChRM3 in ASM (Supplemental Fig. 3) (26). We found that EFS elicited more robust airway contraction in lung slices from Ovaexposed wild-type mice (24.7 6 0.9%) than saline controls at P21 (13.3 6 1.3%; P , 0.05; Fig. 5C), whereas Ova exposure had no effect on baseline contraction of lung slices from NT42/2 mice [9.1 6 0.2% (saline) vs. 7.1 6 2.8 (Ova)]. Together, NT4 is essential for the increase in cholinergic neural stimulation of ASM after early-life allergen exposure. We then assessed whether MCh nebulization would restore airway hypercontractility in Ova-exposed NT42/2 mice. For this assay, NT42/2 mice were nebulized with MCh between P15 and P20 during Ova exposure (Fig. 6A). Airway reactivity was measured in lung slices prepared at P21 and P56. MCh nebulization had no effect on allergic inflammation assayed by differential BAL counts (Supplemental Fig. 1B). In addition, MCh nebulization induced no further increases in ASM mass beyond the effect of Ova exposure (Fig. 6B). Therefore, MCh nebulization alone had no effect on ASM mass, consistent with the observation in wild-type mice (Fig. 1D). However, MCh nebulization restored airway 4340

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hypercontractility in Ova-exposed NT42/2 mice at P21 and P56 (Fig. 6C, D). Together, our findings demonstrated an essential role of NT4 in cholinergic hyperinnervation of ASM that causes airway hypercontractility after early-life allergen exposure.

Blockade of ChRM3 in early life prevented AHR after allergen exposure As a second model, we tested whether pharmaceutical blockade of cholinergic signaling during early-life Ova exposure prevented airway hypercontractility in wildtype mice. For this, we treated Ova-exposed wild-type mice daily with the ChRM3 blocker, 4-DAMP between P15 and -20 at a dosage that was shown to be effective in vivo (Fig. 7A) (27). 4-DAMP treatment had no effect on allergic inflammation (Supplemental Fig. 1C). We assessed the effect of 4-DAMP on airway contraction in lung slices. We chose endothelin as the agonist for assays at P21 to eliminate possible remaining biologic activities of 4-DAMP. 4-DAMP completely prevented early-life Ova-induced airway hypercontractility at P21 (Fig. 7B) and -56 (Fig. 7C). Out of 4 muscarinic receptors, 2 major receptors expressed in the airway are ChRM2 and -3. Although 4-DAMP may have off-target effects on ChRM2, previous studies showed that ChRM2 plays a dispensable



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Figure 4. Assessment of Ca2+ signaling in ASM indicated postnatal maturation of the contractile phenotype around P21. A) Representative traces of MCh-induced oscillatory elevation of intracellular Ca2+ levels in ASM from P14 to adulthood. B) The dose curve of Ca2+ oscillation frequency in response to increasing concentrations of MCh at P14, -18, -21, and adulthood. Each curve represents the data from 4 to 6 airways from 2 to 3 mice.

role in ASM contractility (21). Therefore, 4-DAMP blockade of airway hypercontractility in Ova-exposed mice is likely mediated by its activity against ChRM3. These findings, together with the results in NT42/2 mice, provide evidence that deregulated cholinergic stimulation after early-life allergen exposure is necessary for airway hypercontractility.

Early-life introduction of a ChRM3 blocker after allergen exposure reversed the progression of AHR ASM was susceptible to changes in contractility by cholinergic stimulation until P25 (Figs. 2 and 3). The time window between P21 and P25 provided an opportunity to

Figure 5. NT4 was essential for the increase in cholinergic hyperinnervation after early-life allergen exposure. A) Experimental scheme of neonatal Ova sensitization and challenge. Control mice were challenged with saline. Mice were analyzed at P21. B) Western blot analysis for VAChT levels in lungs of wild-type and NT42/2 mice with and without Ova exposure. Data were quantified by densitometry and normalized to GAPDH as loading control. Data represent the average and SEM of 6–9 mice from 3 independent experiments. C ) Representative trace showing contractile responses to 5 s electric field stimulation of lung slices from wild-type mice (n = 7 airways, 3 mice) and NT42/2 mice (n = 6 airways, 3 mice), with and without Ova exposure. D) Scheme of early life exposure to house dust mite (HDM) allergen. E ) Western blot analysis for VAChT levels in lungs of wild-type and NT42/2 mice with and without HDM exposure. Data were quantified by densitometry and normalized to GAPDH as loading control. Data represent the average and SEM of 2–3 experiments with 2–3 mice/ group for each experiment. *P , 0.05, **P , 0.01, ***P , 0.001.

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Figure 6. MCh nebulization restored airway hypercontractility in NT42/2 mice after early-life Ova exposure. A) Experimental scheme of MCh nebulization in NT42/2 mice during Ova exposure. Mice were analyzed at P21 and -56. B) Representative images of aSMA staining of midsized airways using lung sections from Ova or PBS exposed NT42/2 mice, with or without MCh nebulization at P21. Smooth muscle mass was quantified by normalizing the aSMA-immunoreactive area to the circumference of the airway. C ) Measurement of airway contraction using lung slices of Ova-exposed NT42/2 mice at P21 with or without MCh nebulization. NT42/2 mice that were challenged with PBS were baseline controls. D) Measurement of airway contraction using lung slices of NT42/2 mice at P56 that had been exposed to Ova as neonates, with and without MCh nebulization. NT42/2 mice at P56 that were challenged with saline as neonates were baseline controls. Data represent the results of 12 airways from 3 mice. *P , 0.05, **P , 0.01, ***P , 0.001.

test the therapeutic efficacies of anticholinergic drugs in mice with established AHR. For this assay, 4-DAMP was administered to mice between P21 and -25 after the last Ova challenge (Fig. 7D). As a control, formoterol, the b2AR agonist and the long-lasting bronchodilator, was similarly administered. The dosage of formoterol was tested effective in mice at P21 (Supplemental Fig. 4). Airway contractility was measured at P56 using lung slices. 4-DAMP treatment completely reversed airway hypercontractility to baseline in mice that had been exposed to Ova (Fig. 7E). In contrast, formoterol treatment had no long-term beneficial effects (Fig. 7F). These findings provide evidence that anticholinergic treatment offers disease-modifying benefits in addition to temporal effects of airway relaxation.

DISCUSSION Deregulated parasympathetic neural activities have been documented in patients with asthma and animal models. In addition to its contribution to disease exacerbation, our study provides evidence of its role in the development of persistent airway dysfunction after early episodes of adverse environmental exposures. Our findings support a 4342

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model of the cholinergic nerve–ASM axis in the disease process (Fig. 8). In our model, cholinergic innervation of the airway is established during embryonic and postnatal development to regulate normal development of ASM (Fig. 8A) (21). After allergen exposure in early life, ASM becomes hyperinnervated by cholinergic nerves (Fig. 8B). Excessive acetylcholine release from cholinergic nerves deregulates ChRM3 signaling in immature ASM, which in turn aberrantly alters the expression of downstream genes involved in the contractile phenotype of ASM. As a result, ASM becomes hypercontractile and drives persistent AHR. To present progressive AHR after adverse exposures in early life, blockade of ChRM3 signaling during the critical time window of ASM maturation blocks aberrant changes in gene expression and ultimately reverses the hypercontractile phenotype of ASM (Fig. 8C). Notably, allergen exposure in early life serves as an initial trigger of persistent AHR by causing cholinergic hyperinnervation and aberrant ChRM3 signaling. After these initial processes, the hypercontractile phenotype of ASM outlasts cholinergic neuroplasticity and inflammation. Our findings that the cholinergic nerve–ASM axis plays an age-dependent role in airway hyperreactivity lay the foundation for future mechanistic investigation. To mediate the long-term effect of cholinergic stimulation on the



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Figure 7. Blockade of cholinergic signaling in the neonatal mouse model of allergen exposure had long-term beneficial effects on airway reactivity. A) Experimental scheme of intratracheal delivery of 4-DAMP between P15 to -20 during Ova exposure. Mice were analyzed at P21 and -56. B, C ) Measurement of airway contraction in response to endothelin (End) using lung slices from PBS or Ova-exposed mice at P21 (B) and P56 (C ) with or without 4-DAMP treatment. D) Experimental scheme of intratracheal delivery of 4-DAMP or formoterol to mice between P21 and -25 after the last Ova challenge. Mice were analyzed at P56. E ) Measurement of airway contraction in response to MCh using lung slices from P56 mice that had been exposed to Ova with or without a follow-up 4-DAMP treatment. Significant differences between mice exposed to Ova as neonates and mice exposed to Ova followed by 4-DAMP treatment are marked. F ) Measurement of airway contraction using lung slices prepared from P56 mice that had been exposed to Ova as neonates with a follow-up treatment of 4-DAMP or formoterol. The dashed line shows the same results of airway contraction using lung slices from P56 mice that had been exposed to Ova. Results of 4-DAMP treatment were plotted (E , F) to allow direct comparison between 4-DAMP and formoterol. Each point represents data from 12 airways from 3 mice for each group. *P , 0.05, **P , 0.01, ***P , 0.001.

contractile phenotypes of ASM, one possibility is that changes in gene expression are involved. These genes may include candidates that regulate Ca2+ signaling, sensitization of contractile apparatus for force generation, and actin filament assembly to transmit the force from the contractile unit to extracellular matrix. For example, we showed that pretreatment of neonatal lung slices with MCh caused augmented MLC phosphorylation in response to bronchoconstrictive stimuli. These findings are in line with changes in genes involved in Ca2+ signaling, RhoA-Rho associated protein kinase, or PKC pathways that are known to differentially regulate the MLC kinase and MLC phosphatase activities. Long-term changes in candidate gene expression may be caused by integrating developmental genes in a positive regulatory loop or epigenetic modification (or both) that uniquely occurs in immature ASM after ChRM3 signaling. Given ample examples identified in developmental and stem cell biology, CHOLINERGIC NEURAL INDUCTION OF PERSISTENT AHR

age-related differences in the susceptibility to epigenetic modification are evident. The other possibility is that cholinergic simulation in early life triggers feed-forward interactions between ASM and cells in proximity, such as fibroblast cells that are known to secret a variety of matrix proteins and growth factors in response to contraction. Notably, the gene expression and cell–cell interaction mechanisms are not mutually exclusive. Administration of the ChRM3 antagonist during the time window of ASM maturation not only prevented AHR but also reversed AHR in mice with established phenotypes. These findings suggest that in addition to its effect on airway relaxation, anticholinergic treatment may have potential therapeutic benefits by modifying the progression of asthma. In comparison, the mainstream drug for childhood asthma, formoterol, had no such long-term effects in the neonatal mouse model, which is consistent with clinical data (6). The differences in the therapeutic efficacy


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Figure 8. A model of the cholinergic nerve-ASM axis in persistent ASM hypercontractility after early-life insults. A) During embryogenesis and postnatal growth, ASM becomes innervated by parasympathetic nerves. Acetylcholine secreted from parasympathetic nerves signals through ChRM3 in ASM cells to regulate ASM development. B) Allergen exposure in early life causes an increase in parasympathetic innervation of ASM and elevates the level of acetylcholine release. Deregulated ChRM3 signaling deregulates the expression of genes involved in functional maturation of ASM, which in turn causes the hypercontractile phenotype of ASM. C ) Blockade of aberrant ChRM3 signaling before ASM maturation prevents detrimental changes in gene expression and thereby reverses ASM hypercontractility triggered by early-life allergen exposure.

between ChRM3 blockers and b2AR agonists may be 4-fold. First, these 2 signaling pathways only partially interact (19). Therefore, the action of b2AR agonists may counteract only some downstream mediators of the ChRM3 pathway while sparing others. For example, b2AR activation triggers bronchodilation through the cAMP and PKA pathway and in turn inhibits Ca2+ signaling and sensitization of the contractile apparatus (28, 29). However, stimulation of ChRM3 by its agonists not only activates these cAMP-regulated pathways but also triggers a noncanonical RhoA-Rho-associated protein kinase pathway via receptor phosphorylation and subsequently increases Ca2+ sensitivity and actin filament assembly (30, 31). In addition, ChRM3 activation may augment TGF-b1-induced expression of contractile proteins in ASM (32). Second, overstimulation of ChRM3 after early-life allergen exposure may promote the desensitization of b2AR in ASM, thereby reducing the efficacy of formoterol (33). Third, recent studies have identified a proinflammatory role of b2AR agonists through the b-arrestin pathway that in the long term has adverse effects on asthma outcomes (34). Fourth, ChRM3 is expressed by ASM and other cell types in airways and may contribute to multiple aspects of the pathophysiology of asthma (20), whereas the primary effect of b2AR agonists is restricted to ASM. In support of this argument, a recent clinical study suggested that methacholine directly induces matrix remodeling by fibroblast cells without the involvement of allergic inflammation (35). Notably, we found in both neonatal and adult models of allergen exposure that collagen deposition is transient and dependent on allergic inflammation, rather than cholinergic stimulation (Supplemental Fig. 5). This finding differs from the clinical observation in patients with asthma (35). The discrepancy is most likely related to the fact that the tested patients had existing disease conditions, whereas we studied naive mice. In the context of an increasing body of evidence that suggests long-acting cholinergic antagonist as an effective add-on therapy (36–39), our findings of the therapeutic advantage of anticholinergic medication have clinical 4344

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implications in the management of pediatric asthma. It is important to point out that clinical trials of anticholinergic add-on therapy in patients with pediatric asthma between 6 and 11 yr of age evaluated the improvement of airflow obstruction and clinical symptoms up to 6 mo after treatment (38). Our findings in mice suggest that the diseasemodifying benefits of anticholinergic treatment in asthma require a longer duration of treatment. In addition, whether ASM continues to mature after 6 yr of age in humans is unknown. Anticholinergic drugs have been proven safe. Whether their application to younger pediatric patients with asthma may produce better long-term benefits warrants future studies. We used a histologic marker, VAChT, in combination with EFS to assess changes in cholinergic neural regulation of ASM contraction. Both assays provided consistent evidence of aberrant cholinergic neural stimulation of ASM in our neonatal mouse model. In contrast, allergen exposure in adult mice had no effect on the expression of VAChT (Supplemental Fig. 6). These findings highlight the agerelated differences in neuroplasticity. Notably, the lack of changes in VAChT levels in the adult mouse model is by no means a direct measurement of the cholinergic neural activity, as cholinergic nerves are regulated by a complex neural circuit and regulatory mechanisms shown in previous studies by vagal resection and the ChRM2 pathway (40–42). However, independent of cholinergic dysfunction in adult animal models, our findings indicate that mature ASM loses the ability to develop persistent changes in the contractile phenotype in response to cholinergic stimulation. We demonstrated that age-dependent cholinergic neuroplasticity needs NT4. NT4 is expressed by both ASM and pulmonary mast cells (14). In our previous study, we showed that, although ASM-derived NT4 was responsible for ASM innervation during development, allergen exposure activated mast cells that degranulated to release NT4 and thus became the major source of NT4 for neuroplasticity (14). The number of mast cells in lungs reduces dramatically with age (14, 43), which may explain why allergen-induced neuroplasticity only occurs at young



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ages. Mast cells in human lungs also express NT4 (14). In addition, patients with asthma have increased infiltration of mast cells in the lungs (44, 45). Therefore, mast cellmediated neuroplasticity may occur in children with asthma similar to the neonatal mouse model of allergen exposure. In summary, this study provides evidence that allergeninduced plasticity along the cholinergic nerve-ASM axis serves as an age-specific mechanism underlying persistent AHR after early-life allergen exposure. Our findings suggest that anticholinergic treatment may be worth further evaluation as a disease modifying therapy in pediatric patients with asthma. ACKNOWLEDGMENTS This work is supported by an American Asthma Foundation Award 12-0086, and U.S. National Institutes of Health (NIH) National Heart, Lung and Blood Institute Grants 1R01HL116163 and 1R01HL132991 (to X.A.) and F31 Training Grant HL007035 (to J.B.). The authors declare no conflicts of interest.

AUTHOR CONTRIBUTIONS K. R. Patel, Y. Bai, and X. Ai conceived the project and designed the experiments; X. Ai supervised the project; K. R. Patel, Y. Bai, K. G. Trieu, and J. Barrios performed the experiments; K. R. Patel, Y. Bai, and X. Ai compiled the data; K. G. Trieu and Y. Bai prepared the figures; K. R. Patel, Y. Bai, and X. Ai wrote the manuscript; and all authors provided feedback on the manuscript. REFERENCES 1. Maddox, L., and Schwartz, D. A. (2002) The pathophysiology of asthma. Annu. Rev. Med. 53, 477–498 2. Stern, D. A., Morgan, W. J., Halonen, M., Wright, A. L., and Martinez, F. D. (2008) Wheezing and bronchial hyper-responsiveness in early childhood as predictors of newly diagnosed asthma in early adulthood: a longitudinal birth-cohort study. Lancet 372, 1058–1064 3. Chapman, K. R., Boulet, L. P., Rea, R. M., and Franssen, E. (2008) Suboptimal asthma control: prevalence, detection and consequences in general practice. Eur. Respir. J. 31, 320–325 4. Buehring, B., Viswanathan, R., Binkley, N., and Busse, W. (2013) Glucocorticoid-induced osteoporosis: an update on effects and management. J. Allergy Clin. Immunol. 132, 1019–1030 5. Townley, R. G., Agrawal, S., and Sapkota, K. (2010) Omalizumab for pediatric asthma. Expert Opin. Biol. Ther. 10, 1595–1608 6. Guilbert, T. W., Morgan, W. J., Zeiger, R. S., Mauger, D. T., Boehmer, S. J., Szefler, S. J., Bacharier, L. B., Lemanske, R. F., Jr., Strunk, R. C., Allen, D. B., Bloomberg, G. R., Heldt, G., Krawiec, M., Larsen, G., Liu, A. H., Chinchilli, V. M., Sorkness, C. A., Taussig, L. M., and Martinez, F. D. (2006) Long-term inhaled corticosteroids in preschool children at high risk for asthma. N. Engl. J. Med. 354, 1985–1997 7. McGeachie, M. J., Yates, K. P., Zhou, X., Guo, F., Sternberg, A. L., Van Natta, M. L., Wise, R. A., Szefler, S. J., Sharma, S., Kho, A. T., Cho, M. H., Croteau-Chonka, D. C., Castaldi, P. J., Jain, G., Sanyal, A., Zhan, Y., Lajoie, B. R., Dekker, J., Stamatoyannopoulos, J., Covar, R. A., Zeiger, R. S., Adkinson, N. F., Williams, P. V., Kelly, H. W., Grasemann, H., Vonk, J. M., Koppelman, G. H., Postma, D. S., Raby, B. A., Houston, I., Lu, Q., Fuhlbrigge, A. L., Tantisira, K. G., Silverman, E. K., Tonascia, J., Weiss, S. T., and Strunk, R. C.; CAMP Research Group. (2016) Patterns of growth and decline in lung function in persistent childhood asthma. N. Engl. J. Med. 374, 1842–1852 CHOLINERGIC NEURAL INDUCTION OF PERSISTENT AHR

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Received for publication March 3, 2017. Accepted for publication May 30, 2017.


PATEL ET AL.

Targeting acetylcholine receptor M3 prevents the progression of airway hyperreactivity in a mouse model of childhood asthma Kruti R. Patel, Yan Bai, Kenneth G. Trieu, et al. FASEB J 2017 31: 4335-4346 originally published online June 15, 2017 Access the most recent version at doi:10.1096/fj.201700186R

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SUPPLEMENTARY FIGURES

Supplementary Figure 1. Cholinergic signaling had no effect on allergic inflammation. Inflammation was assessed by differential immune cell counts in bronchoalveolar lavage. Percentages of macrophages (Mac), eosinophils (Eos), lymphocytes (Lymph), and neutrophils (Neut) are shown. (A) Wild type mice were nebulized with PBS or Mch between P15-P20. Mice were analyzed at P21. (B) PBS- and OVA-exposed NT4-/- mice were nebulized with saline or Mch between P15-P20. Mice were analyzed at P21. (C) PBS- and OVA-exposed wild type mice were treated with and without 4-DAMP between P15-P20. Mice were analyzed at P21. Bar graphs represent mean±SEM of data from at least 4 mice for each group. *P