Rationale: Changes in airway epithelial cell differentiation, driven in part by IL-13, are important in asthma. Micro-RNAs (miRNAs) regulate cell differentiation in many systems and could contribute to epithelial abnormalities in asthma. Objectives: To determine whether airway epithelial miRNA expression is altered in asthma and identify IL-13-regulated miRNAs. Methods:We used miRNA microarrays to analyze bronchial epithelial brushings from 16 steroid-naive subjects with asthma before and after inhaled corticosteroids, 19 steroid-using subjects with asthma, and 12 healthy control subjects, and the effects of IL-13 and corticosteroids on cultured bronchial epithelial cells. We used quantitative polymerase chain reaction to confirm selected microarray results. Measurements and Main Results: Most (12 of 16) steroid-naive subjects with asthma had a markedly abnormal pattern of bronchial epithelial miRNA expression by microarray analysis. Compared with control subjects, 217 miRNAs were differentially expressed in steroid-naive subjects with asthma and 200 in steroid-using subjects with asthma (false discovery rate , 0.05). Treatment with inhaled corticosteroids had modest effects on miRNA expression in steroid-naive asthma, inducing a statistically significant (false discovery rate , 0.05) change for only nine miRNAs. qPCR analysis confirmed differential expression of 22 miRNAs that were highly differentially expressed by microarrays. IL-13 stimulation recapitulated changes in many differentially expressed miRNAs, including four members of the miR-34/ 449 family, and these changes in miR-34/449 family members were resistant to corticosteroids. Conclusions: Dramatic alterations of airway epithelial cell miRNA levels are a common feature of asthma. These alterations are only modestly corrected by inhaled corticosteroids. IL-13 effects may account for some of these alterations, including repression of miR-34/449 family members that have established roles in airway epithelial cell differentiation. Clinical trial registered with www.clinicaltrials.gov (NCT 00595153).Keywords: miRNA; asthma; airway epithelium Asthma is an inflammatory condition of the lung characterized by episodic airway obstruction and remodeling of airway tissues (1). The airway epithelium is increasingly recognized as an important participant in asthma pathophysiology (2, 3). Airway epithelial cells produce mucus, which is a major contributor to airway obstruction in fatal asthma (4), and even patients with stable mild and moderate asthma have increased epithelial mucin stores (5). Previous human, animal model, and cell culture studies demonstrate that the Th2 cytokine IL-13 is a critical inducer of airway epithelial abnormalities in asthma (6-8). IL-13 stimulation of epithelial cells leads to an increase in the number of mucous cells (mucous metaplasia) and a decrease in the number of ciliated cells (9). IL-13 stimulation of airway epithelial cells activates signal transduction and transcription factor 6, leading to changes in expression of mRNAs that...
Statistics. Twogroup comparisons were performed using the 2tailed Student's t test (normally distributed data) or the MannWhitney U test. A P value of less than 0.05 was considered statistically significant.Study approval. The UCSF Committee on Human Research approved the use of human airway sections and HBE cells. Written consent was not required, as materials were leftover clinical samples obtained from deidentified individuals.
Edited by Thomas Sö llnerW1282X is the fifth most common cystic fibrosis transmembrane regulator (CFTR) mutation that causes cystic fibrosis. Here, we investigated the utility of a small molecule corrector/ potentiator strategy, as used for ⌬F508-CFTR, to produce functional rescue of the truncated translation product of the W1282X mutation, CFTR 1281 , without the need for readthrough. In transfected cell systems, certain potentiators and correctors, including VX-809 and VX-770, increased CFTR 1281 activity. To identify novel correctors and potentiators with potentially greater efficacy on CFTR 1281 , functional screens were done of ϳ30,000 synthetic small molecules and drugs/nutraceuticals in CFTR 1281 -transfected cells. Corrector scaffolds of 1-arylpyrazole-4-arylsulfonyl-piperazine and spiro-piperidine-quinazolinone classes were identified with up to ϳ5-fold greater efficacy than VX-809, some of which were selective for CFTR 1281 , whereas others also corrected ⌬F508-CFTR. Several novel potentiator scaffolds were identified with efficacy comparable with VX-770; remarkably, a phenylsulfonamide-pyrrolopyridine acted synergistically with VX-770 to increase CFTR 1281 function ϳ8-fold over that of VX-770 alone, normalizing CFTR 1281 channel activity to that of wild type CFTR. Corrector and potentiator combinations were tested in primary cultures and conditionally reprogrammed cells generated from nasal brushings from one W1282X homozygous subject. Although robust chloride conductance was seen with correctors and potentiators in homozygous ⌬F508 cells, increased chloride conductance was not found in W1282X cells despite the presence of adequate transcript levels. Notwithstanding the negative data in W1282X cells from one human subject, we speculate that corrector and potentiator combinations may have therapeutic efficacy in cystic fibrosis caused by the W1282X mutation, although additional studies are needed on human cells from W1282X subjects.
ISG activation is prominent in asthma, independent of viral transcripts, orthogonal to type 2 inflammation, and associated with distinct clinical features. ER stress is associated with both type 2 inflammation and ISG expression.
BACKGROUND. Chronic obstructive pulmonary disease (COPD) is a heterogeneous smoking-related disease characterized by airway obstruction and inflammation. This inflammation may persist even after smoking cessation and responds variably to corticosteroids. Personalizing treatment to biologically similar “molecular phenotypes” may improve therapeutic efficacy in COPD. IL-17A is involved in neutrophilic inflammation and corticosteroid resistance, and thus may be particularly important in a COPD molecular phenotype.METHODS. We generated a gene expression signature of IL-17A response in bronchial airway epithelial brushings from smokers with and without COPD (n = 238), and validated it using data from 2 randomized trials of IL-17 blockade in psoriasis. This IL-17 signature was related to clinical and pathologic characteristics in 2 additional human studies of COPD: (a) SPIROMICS (n = 47), which included former and current smokers with COPD, and (b) GLUCOLD (n = 79), in which COPD participants were randomized to placebo or corticosteroids.RESULTS. The IL-17 signature was associated with an inflammatory profile characteristic of an IL-17 response, including increased airway neutrophils and macrophages. In SPIROMICS the signature was associated with increased airway obstruction and functional small airways disease on quantitative chest CT. In GLUCOLD the signature was associated with decreased response to corticosteroids, irrespective of airway eosinophilic or type 2 inflammation.CONCLUSION. These data suggest that a gene signature of IL-17 airway epithelial response distinguishes a biologically, radiographically, and clinically distinct COPD subgroup that may benefit from personalized therapy.TRIAL REGISTRATION. ClinicalTrials.gov NCT01969344.FUNDING. Primary support from the NIH, grants K23HL123778, K12HL11999, U19AI077439, DK072517, U01HL137880, K24HL137013 and R01HL121774 and contracts HHSN268200900013C, HHSN268200900014C, HHSN268200900015C, HHSN268200900016C, HHSN268200900017C, HHSN268200900018C, HHSN268200900019C and HHSN268200900020C.
Pendrin (SLC26A4) is a Cl 2 /anion exchanger expressed in the epithelium of inflamed airways where it is thought to facilitate Cl 2 absorption and HCO 3 2 secretion. Studies using pendrin knockout mice and airway epithelial cells from hearing-impaired subjects with pendrin loss of function suggest involvement of pendrin in inflammatory lung diseases, including cystic fibrosis (CF), perhaps by regulation of airway surface liquid (ASL) volume. Here we identified small-molecule pendrin inhibitors and demonstrated their efficacy in increasing ASL volume. A cell-based, functional high-throughput screen of ∼36,000 synthetic small molecules produced 3 chemical classes of inhibitors of human pendrin. After structure-activity studies, tetrahydropyrazolopyridine and pyrazolothiophenesulfonamide compounds reversibly inhibited pendrin-facilitated Cl 2 exchange with SCN 2 , I 2 , NO 3 2 , and HCO 3 2 with drug concentration causing 50% inhibition down to ∼2.5 mM. In well-differentiated primary cultures of human airway epithelial cells from non-CF and CF subjects, treatment with IL-13, which causes inflammation with strong pendrin up-regulation, strongly increased Cl 2 /HCO 3 2 exchange and the increase was blocked by pendrin inhibition. Pendrin inhibition significantly increased ASL depth (by ∼8 mm) in IL-13-treated non-CF and CF cells but not in untreated cells. These studies implicate the involvement of pendrin-facilitated Cl 2 / HCO 3 2 in the regulation of ASL volume and suggest the utility of pendrin inhibitors in inflammatory lung diseases, including CF
Available CFTR modulators provide no therapeutic benefit for cystic fibrosis (CF) caused by many lossof-function mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, including N1303K. We previously introduced the concept of 'co-potentiators' (combinationpotentiators) to rescue CFTR function in some minimal function CFTR mutants. Herein, a screen of ~120,000 drug-like synthetic small molecules identified active co-potentiators of pyrazoloquinoline, piperidine-pyridoindole, tetrahydroquinoline and phenylazepine classes, with EC 50 down to ~300 nM following initial structure-activity studies. Increased CFTR chloride conductance by up to 8-fold was observed when a co-potentiator (termed 'Class II potentiator') was used with a classical potentiator ('Class I potentiator') such as VX-770 or GLPG1837. To investigate the range of CFTR mutations benefitted by co-potentiators, 14 CF-associated CFTR mutations were studied in transfected cell models. Co-potentiator efficacy was found for CFTR missense, deletion and nonsense mutations in nucleotide binding domain-2 (NBD2), including W1282X, N1303K, c.3700A > G and Q1313X (with corrector for some mutations). In contrast, CFTR mutations G85E, R334W, R347P, V520F, R560T, A561E, M1101K and R1162X showed no co-potentiator activity, even with corrector. Co-potentiator efficacy was confirmed in primary human bronchial epithelial cell cultures generated from a N1303K homozygous CF subject. The Class II potentiators identified here may have clinical benefit for CF caused by mutations in the NBD2 domain of CFTR. Cystic fibrosis (CF) is caused by loss of function mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) protein, a cAMP-activated chloride channel 1. More than 2000 CF-causing CFTR variants have been identified (http://genet.sickkids.on.ca/Home.html). CFTR modulators have been developed that rescue defective cellular processing and cell-surface targeting of mutant CFTRs (correctors) and defective channel gating (potentiators) to restore CFTR anion transport 1-3. The potentiator Kalydeco (ivacaftor/VX-770) has been approved for CF subjects with gating mutations, including G551D-CFTR and now 38 additional mutations 2. The corrector/potentiator combinations Orkambi (VX-770 plus lumacaftor/VX-809) and Symdeko (VX-770 plus tezacaftor/VX-661) have been approved for CF subjects that are homozygous for the most common CF-causing CFTR mutation, F508del, or who have one F508del allele and a residual function CFTR mutation 2. Trikafta, a triple drug combination consisting of two correctors and one potentiator, has recently been approved for CF subjects with one or two F508del alleles 2,4-6. Trikafta and future CFTR modulators may benefit up to 90% of CF subjects 2. Therapeutic approaches are needed for CFTR mutations that are unlikely to benefit from existing modulators-the so-called 'remaining 10%' 2,7,8. Non-responsive minimal function CFTR mutations are distributed throughout the CFTR protein and are associated with low CF...
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