Arhgef12 drives IL17A-induced airway contractility and airway hyperresponsiveness in mice Valerie Fong, … , Y.S. Prakash, Mallar Bhattacharya JCI Insight. 2018;3(21):e123578. https://doi.org/10.1172/jci.insight.123578. Research Article Muscle biology Pulmonology Patients with severe, treatment-refractory asthma are at risk for death from acute exacerbations. The cytokine IL17A has been associated with airway inflammation in severe asthma, and novel therapeutic targets within this pathway are urgently needed. We recently showed that IL17A increases airway contractility by activating the procontractile GTPase RhoA. Here, we explore the therapeutic potential of targeting the RhoA pathway activated by IL17A by inhibiting RhoA guanine nucleotide exchange factors (RhoGEFs), intracellular activators of RhoA. We first used a ribosomal pulldown approach to profile mouse airway smooth muscle by qPCR and identified Arhgef12 as highly expressed among a panel of RhoGEFs. ARHGEF12 was also the most highly expressed RhoGEF in patients with asthma, as found by RNA sequencing. Tracheal rings from Arhgef12-KO mice and WT rings treated with a RhoGEF inhibitor had evidence of decreased contractility and RhoA activation in response to IL17A treatment. In a house dust mite model of allergic sensitization, Arhgef12-KO mice had decreased airway hyperresponsiveness without effects on airway inflammation. Taken together, our results show that Arhgef12 is necessary for IL17A-induced airway contractility and identify a therapeutic target for severe asthma. Find the latest version: https://jci.me/123578/pdf RESEARCH ARTICLE Arhgef12 drives IL17A-induced airway contractility and airway hyperresponsiveness in mice Valerie Fong,1 Austin Hsu,2 Esther Wu,1 Agnieszka P. Looney,1 Previn Ganesan,1 Xin Ren,1 Dean Sheppard,1 Sarah A. Wicher,3 Michael A. Thompson,4 Rodney D. Britt Jr.,5,6 Y.S. Prakash,3,4 and Mallar Bhattacharya1 1Division of Pulmonary, Critical Care, Allergy, and Sleep, Department of Medicine, University of California, San Francisco, San Francisco, California, USA. 2Gladstone Institute of Cardiovascular Disease, San Francisco, California, USA. 3Department of Physiology and Biomedical Engineering and 4Department of Anesthesiology and Perioperative Medicine, Mayo Clinic, Rochester, Minnesota, USA. 5Center for Perinatal Research, The Research Institute at Nationwide Children’s Hospital, Columbus, Ohio, USA. 6Department of Pediatrics, The Ohio State University, Columbus, Ohio, USA. Patients with severe, treatment-refractory asthma are at risk for death from acute exacerbations. The cytokine IL17A has been associated with airway inflammation in severe asthma, and novel therapeutic targets within this pathway are urgently needed. We recently showed that IL17A increases airway contractility by activating the procontractile GTPase RhoA. Here, we explore the therapeutic potential of targeting the RhoA pathway activated by IL17A by inhibiting RhoA guanine nucleotide exchange factors (RhoGEFs), intracellular activators of RhoA. We first used a ribosomal pulldown approach to profile mouse airway smooth muscle by qPCR and identified Arhgef12 as highly expressed among a panel of RhoGEFs. ARHGEF12 was also the most highly expressed RhoGEF in patients with asthma, as found by RNA sequencing. Tracheal rings from Arhgef12- KO mice and WT rings treated with a RhoGEF inhibitor had evidence of decreased contractility and RhoA activation in response to IL17A treatment. In a house dust mite model of allergic sensitization, Arhgef12-KO mice had decreased airway hyperresponsiveness without effects on airway inflammation. Taken together, our results show that Arhgef12 is necessary for IL17A-induced airway contractility and identify a therapeutic target for severe asthma. Introduction Ten percent of patients with asthma have severe disease (1, 2). These patients account for the majority of morbidity and mortality associated with asthma and are exacerbation-prone despite maximal therapy. Fur- thermore, despite recent advances in inflammatory profiling and targeted treatments for mild-to-moderate asthmatics, progress has been limited for patients with severe disease. Therefore, a major goal of current translational research is to identify therapeutic targets that are relevant to severe asthma (1, 3–5). Authorship note: VF and AH A core challenge of treating patients with severe asthma is their limited treatment response to corticoste- contributed equally to this work. roids. Th17 cells producing the cytokine IL17A have been implicated in steroid-refractory asthma by several Conflict of interest: The authors have preclinical and clinical studies (6–12). Furthermore, there is evidence that IL17A may drive disease activ- declared that no conflict of interest ity most directly in patients with baseline airway hyperresponsiveness (AHR): in a 12-week clinical trial, exists. anti-IL17A antibody treatment improved symptom scores only in the subgroup of patients who, in baseline License: Copyright 2018, American pulmonary function testing, exhibited reversibility of airway obstruction with bronchodilator treatment (13). Society for Clinical Investigation. Since bronchodilator reversibility suggests airway contractility as a root cause of poor control in this sub- Submitted: July 20, 2018 group, we reasoned that finding therapeutic targets downstream of IL17A, specifically in airway smooth Accepted: September 26, 2018 muscle, should yield therapeutic targets relevant to this subgroup of severe asthmatics. Published: November 2, 2018 One such downstream target is the small GTPase RhoA. We recently showed that IL17A secreted by Th17 cells activates and increases airway contractility in mouse models via activation of the small GTPase Reference information: JCI Insight. 2018;3(21):e123578. RhoA (12). RhoA is a positive regulator of airway smooth muscle contraction via multiple pathways, https://doi.org/10.1172/jci. including the effector Rho kinase, which augments myosin phosphorylation, and actin cytoskeletal effects insight.123578. that enable increased force transmission across the tissue (12, 14–17). Given these multiple procontractile insight.jci.org https://doi.org/10.1172/jci.insight.123578 1 RESEARCH ARTICLE Figure 1. Riboprofiling identifies Arhgef12 as the most highly expressed RhoA guanine exchange factor in airway smooth muscle. (A) Schematic of smooth muscle RNA riboprofiling of trachea from acta2-CreERT2/Rosa26loxpSTOPloxpRpl10a-mCherry mice. B( ) Immunofluorescence of trachea from acta2-CreERT2/Rosa26loxpSTOPloxpRpl10a-mCherry mice. Scale bar: 200 μm. (C) Validation of smooth muscle specificity of riboprofiling by qPCR of smooth muscle and non–smooth muscle genes (n = 4 mice). PD, pulldown. (D) Riboprofiling of mouse tracheal RhoGEFs in healthy mice (n = 4 mice). (E) RhoGEF expression in airway smooth muscle from asthma patients (n = 7) and healthy controls (n = 12). Where 2 samples were available (4 of 7 asthma patients), counts were averaged. Box plots show minimum, maximum, and median, with 25th to 75th percentile range. *P < 0.01 by 1-tailed Student’s t test. effects of RhoA activation, we reasoned that RhoA guanine nucleotide exchange factors (RhoGEFs) (18, 19), activators of RhoA that catalyze GTP loading, should represent an upstream chokepoint for broad- based targeting of the contractile response to IL17A. While vascular smooth muscle RhoGEF activity has been identified as a potential target for treat- ment of hypertension (20), the relative expression in airway smooth muscle of individual genes among the large family of RhoGEFs is unknown. Our approach was to employ a smooth muscle–specific ribosomal pulldown to profile airway smooth muscle RhoGEF expression. This screen led to the identification of Arhgef12, which we found was necessary for IL17A-mediated RhoA activation and airway contractility as well as allergic AHR in vivo. Taken together, our findings encourage the development of ARHGEF12 inhibition as a therapy we believe to be novel for severe asthma. insight.jci.org https://doi.org/10.1172/jci.insight.123578 2 RESEARCH ARTICLE Table 1. Patients with a clinical diagnosis of asthma Age (yr) Sex BMI Relevant medications Lung function 65 F 22 Ipratropium, prednisone FEV1% 70; FEV1/FVC 0.66; B/D >15% 54 M 38 Protonix FEV1% 58; FEV1/FVC 0.64; B/D >15% 73 F 31 Albuterol FEV1% 64; B/D >15% 75 M Albuterol, ipratropium FEV1% 48 56 F 24 Albuterol, Pulmicort FEV1% 70; FEV1/FVC 0.68; B/D >15% 68 F 39 Advair FEV1% 71; FEV1/FVC 0.61 59 M 24 Albuterol, montelukast FEV1% 61; FEV1/FVC 0.58 F, female; M, male; B/D, percentage change in FEV1 with administration of bronchodilator. Results A major challenge to analysis of gene expression of airway smooth muscle is its poor suitability for flow cytometry–based sorting, given a lack of established cell surface markers. To overcome this limitation, we performed smooth muscle riboprofiling by pulldown of smooth muscle polysomes from fresh tracheal lysates using α–smooth muscle actin-CreERT2 (21) mice crossed with a ribotag allele, Rosa26-loxp-STOP- loxp-Rpl10a-mcherry (22). The mRNA thus isolated represents the actively translating mRNA in airway smooth muscle (Figure 1, A and B). Comparison by qPCR of expression of smooth muscle and non– smooth muscle genes before and after pulldown demonstrated the specificity of the pulldown (Figure 1C). We then performed a qPCR screen of tracheal mRNA isolated by smooth muscle ribotag pulldown to determine highly expressed RhoGEFs in airway smooth muscle in healthy mice. To compile a list