(416) Smooth muscle-specific CFTR loss induces airway hyperreactivity in response to TGFβ

Background: Though airway smooth muscle (ASM) function is known to be abnormal in CF, it was unclear if this was an inherent defect or secondary to abnormal mucus, inflammation, and infection. People with CF have higher prevalence of airway hyperresponsiveness (AHR), which is associated with faster lung function decline. Work in the CF neonatal pig model suggests that CFTR loss induces ASM hypercontractility [1]. Previously, we demonstrated that exposure to TGFβ, a known genetic modifier of CF lung disease, induces greater AHR in CF mice compared to controls [2]. In this study, we hypothesized that smooth muscle-specific loss of CFTR would increase sensitivity to the AHR effects of pulmonary TGFβ in a transgenic murine model.

Methods: To create a model with smooth muscle-specific loss of Cftr function, conditional Cftr KO mice (Cftr^fl/fl, [3]) were bred to Sm22-Cre mice expressing Cre in smooth muscle (Jackson Labs #017491). Littermates without the Cre transgene (Cftr^fl/fl) were used as controls. Controls and smooth muscle-specific Cftr KO mice (Cftr^fl/fl;Sm-Cre) were intratracheally treated with an adenoviral vector containing the TGFβ1 transgene (Ad-TGFβ) or an Empty vector control and analyzed after 3 days. Pulmonary mechanics were obtained with the FlexiVent system. Lungs and bronchoalveolar lavage (BAL) fluid were collected for histology, cell counts, and TGFβ ELISA analysis. AHR was determined by obtaining pulmonary resistance measurements at increasing doses of nebulized methacholine; area under the curve was calculated. Analyses were performed by one-way ANOVA with Tukey’s multiple comparisons test.

Results: Low-dose, subacute TGFβ exposure caused similar weight loss, increased BAL cellularity, and increased BAL TGFβ levels in both control and Cftr^fl/fl;Sm-Cre mice. Baseline resistance was similar in control and Cftr^fl/fl;Sm-Cre mice and unchanged in Ad-TGFβ exposed mice of either genotype. TGFβ exposure did not induce increased ASM area in either group as determined by αSMA staining. Despite these similarities, Cftr^fl/fl;Sm-Cre mice demonstrated TGFβ-induced AHR in response to methacholine treatment (p=0.0138), while control mice did not develop TGFβ-induced AHR.

Conclusions: We have previously shown that CF mice are more sensitive to TGFβ-induced AHR than non-CF mice [2]. Here, we further demonstrate that loss of CFTR function specifically in smooth muscle is sufficient to replicate this TGFβ-driven enhanced AHR. Thus, this ASM abnormality is not secondary to effects of CFTR functional loss in the airway epithelium. Instead, CF ASM has an inherent functional defect that may drive AHR in people with CF that contributes to disease modification by TGFβ. Targeting this abnormality could improve pulmonary and quality of life outcomes for people with CF.

Acknowledgements: This work was supported by the NIH (K08HL151762), CFF (KRAMER21AO-KB; NAREN19R0). Cftr^fl/fl mice obtained from the Case Western Reserve CF Mouse Models Core.

References: 1. Cook DP, Rector MV, Bouzek DC, Michalski AS, Gansemer ND, Reznikov LR, et al. CFTR in Sarcoplasmic Reticulum of Airway Smooth Muscle. Am J Respir Crit Care Med. 2016;193(4):417-26.

2. Kramer EL, Madala SK, Hudock KM, Davidson C, Clancy JP. Subacute TGFβ Exposure Drives Airway Hyperresponsiveness in CF Mice through the PI3K Pathway. Am J Resp Cell Mol Bio. 2020;62(5):657-67.

3. Hodges CA, Cotton CU, Palmert MR, Drumm ML. Generation of a conditional null allele for Cftr in mice. Genesis. 2008;46(10):546-52.