Losartan ameliorates TGF-β1–induced CFTR dysfunction and improves correction by cystic fibrosis modulator therapies

Highly effective modulator therapies dramatically improve the prognosis for those with cystic fibrosis (CF). The triple combination of elexacaftor, tezacaftor, and ivacaftor (ETI) benefits many, but not all, of those with the most common F508del mutation in the CF transmembrane conductance regulator (CFTR). Here, we showed that poor sweat chloride concentration responses and lung function improvements upon initiation of ETI were associated with elevated levels of active TGF-β1 in the upper airway. Furthermore, TGF-β1 impaired the function of ETI-corrected F508del-CFTR, thereby increasing airway surface liquid (ASL) absorption rates and inducing mucus hyperconcentration in primary CF bronchial epithelial cells in vitro. TGF-β1 not only decreased CFTR mRNA, but was also associated with increases in the mRNA expression of TNFA and COX2 and TNF-α protein. Losartan improved TGF-β1–mediated inhibition of ETI-corrected F508del-CFTR function and reduced TNFA and COX2 mRNA and TNF-α protein expression. This likely occurred by improving correction of mutant CFTR rather than increasing its mRNA (without an effect on potentiation), thereby reversing the negative effects of TGF-β1 and improving ASL hydration in the CF airway epithelium in vitro. Importantly, these effects were independent of type 1 angiotensin II receptor inhibition.


ASL volume measurement
Meniscus scanning of high-resolution images of ALI cultures was used to estimate ASL volumes as previously described (9). The apical surface of ALI cultures was washed with DPBS and ASL volumes were measured after 24 hours. The basolateral media was replenished with media containing ETI + TGF-b1 ± losartan and ASL volumes were measured again after an additional 24 hours. DASL represents the difference between these two measurements with a more negative value indicating greater ASL absorption.

Mucus concentration (% solids) measurement
Mucus concentration (percent solids) was determined by published methods for measuring the wet and dry weights of mucus using a microbalance with accuracy to 100 ng (10,11). Mucus was lifted off the cultures with a laser-cut mesh that was left on the surface of ALI cultures for 10 minutes at 37°C in an incubator.

Quantitative PCR
CFBE cells and nasal epithelial cells (see below) were lysed, and total RNA isolated using the E.Z.N.A. ® Total RNA Kit (Omega Bio-tek, Norcross, GA, USA). Quantitative PCR (qPCR) was performed as described using TaqMan Gene Expression Assays (ThermoFisher Scientific, TGFBR2 (Hs00234253_m1), and TNFA (Hs00174128_m1) and normalized to reference gene GAPDH. microRNA was isolated from CFBE cells using the miRNeasy Micro Kit (Qiagen, Germantown, MD, USA). miR-145 expression was evaluated using TaqMan Advanced miRNA Assays (Assay ID 002278) and normalized to RNU6B (or U6 snRNA).

ELISA
Basolateral media was collected from CFBE cells 24 hours after treatment. Media samples were analyzed using the Ella Automated Immunoassay System and Simplex Plex cartridge-based immunoassay for TNFa (Bio-Techne Corp., Minneapolis, MN, USA). Activated and total TGF-b1 protein levels were measured from nasal epithelial lining fluid (see below) with a Simplex Plex cartridge-based immunoassay for TGF-b1 (Bio-Techne Corp.). Total TGF-b1 was measured by activating latent TGF-b1 with acidification and neutralization steps before running the assay.
Levels of active TGF-b1 are expressed as a ratio of active/total TGF-b1.

Study approval and enrollment criteria
The study protocol was approved by the University of Kansas Medical Center Institutional Review Board and informed consent was obtained from each participant.

Participants and Study Procedures
Those with CF age 18 years and older at the University of Kansas Health System on a stable dose of ETI for at least three months were enrolled. Participants were excluded if they had undergone treatment for a CF pulmonary exacerbation in the past month, were currently taking systemic corticosteroids or other anti-inflammatory medications, or had recent nose bleeding.
Demographics and clinical parameters were obtained from the electronic medical record. Lung function response to ETI was determined by the change, both relative and absolute, in the percent predicted forced expiratory volume in one second (ppFEV1) between the average of the three highest ppFEV1 measurements in the year preceding and year following initiation of ETI. Sweat was obtained through pilocarpine disc iontophoresis (Macroduct®, ELITechGroup, Logan, UT, USA). Sweat chloride levels were measured in a CLIA certified laboratory.

Nasal fluid collection (Leukosorb)
Collection of nasal epithelial lining fluid (ELF) was performed with pre-cut strips of synthetic absorptive filter (Leukosorb; Pall Corporation, Port Washington, NY, USA). Leukosorb strips were inserted under direct visual guidance into the nasal cavity of CF participants with the entire length of the strip applied laterally against the anterior inferior turbinate. Nose clips were used to ensure good contact with the mucosal surface for two minutes. After removal from the nose, Leukosorb strips were placed in an Eppendorf tube and stored at -80ºC before elution as previously described (12).

Nasal cells collection
Nasal cells were collected using sterile cytology brushes (Medical Packaging Corporation, Camarillo, CA, USA). The brushes were introduced under direct visual guidance into the nasal cavity of CF participants and were placed between the nasal septum and the inferior turbinate.
No anesthesia was used. The cells were harvested by a few careful backward-forward and rotary movements before twirling the brush into 5 mL of sterile PBS in a 15 mL tube to release the cells.
The same procedure was repeated 3 times in each nostril. Immediately after the harvest, the tube was centrifuged at 360 x g for 5 min at 4°C. The supernatant was discarded, and the remaining pellet was frozen at -80°C until qPCR experiments were performed.

Statistical analyses
Statistics: Demographics and baseline clinical characteristics were summarized with descriptive statistics. Pearson's or Spearman's correlation coefficient with one-tailed or two-tailed p values (see figure legends), depending on the distribution of the data was used to assess correlation between continuous variables. Two-tailed Student's t-test and one way ANOVA were used otherwise. All results were considered statistically significant if p < 0.05. All analysis was conducted using Prism (GraphPad Software, San Diego, CA, USA).

Study approval. The study protocol was approved by the University of Kansas Medical Center
Institutional Review Board and informed consent was obtained from each participant.