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Immortalization of human bronchial epithelial (hBE) cells often entails loss of differentiation. Bmi-1 is a protooncogene that maintains stem cells, and its expression creates cell lines that recapitulate normal cell structure and function. We introduced Bmi-1 and the catalytic subunit of telomerase (hTERT) into three non-cystic fibrosis (CF) and three [DELTA]F508 homozygous CF primary bronchial cell preparations. This treatment extended cell life span, although not as profoundly as viral oncogenes, and at passages 14 and 15, the new cell lines had a diploid karyotype. Ussing chamber analysis revealed variable transepithelial resistances, ranging from 200 to 1,200 [OMEGA] x [cm.sup.2]. In the non-CF cell lines, short-circuit currents were stimulated by forskolin and inhibited by CFTR(inh)-172 at levels mostly comparable to early passage primary cells. CF cell lines exhibited no forskolin-stimulated current and minimal CFrR(inh)-172 response. Amiloride-inhibitable and UTP-stimulated currents were present, but at lower and higher amplitudes than in primary cells, respectively. The cells exhibited a pseudostratified morphology, with prominent apical membrane polarization, few apoptotic bodies, numerous mucous secretory cells, and occasional ciliated cells. CF and non-CF cell lines produced similar levels of IL-8 at baseline and equally increased IL-8 secretion in response to IL-1[beta], TNF-[alpha], and the Toll-like receptor 2 agonist Pam3Cys. Although they have lower growth potential and more fastidious growth requirements than viral oncogene transformed cells, Bmi-1/hTERT airway epithelial cell lines will be useful for several avenues of investigation and will help fill gaps currently hindering CF research and therapeutic development. Bmi-1; cystic fibrosis transmembrane conductance regulator; inflammation; karyotype; Using chamber

Protein folding for export through the exocytic pathway of the eukaryotic cell is sensitive to the local composition of membrane trafficking components, and requires the assistance of multiple chaperones that define the folding buffer of the cell--the chaperome. Different cell types exploit the variable composition of the chaperome to maintain protein folding homeostasis or proteostasis--an adaptive, celltypespecific biological environment that works through the kinetic and thermodynamic properties of the protein fold to achieve functionality. Proteostasis is now recognized to be linked to the epigenome through histone deacetylase (HDAC)-mediated pathways. Perturbation of these relationships occurs in misfolding diseases, such as cystic fibrosis (CF), where a Phe508 deletion in the NBD1 domain of the multimembrane spanning chloride channel CF transmembrane conductance regulator prevents its delivery to the cell surface. It is now apparent that protein misfolding in disease and in aging can be altered by the use of pharmacological chaperones to stabilize the fold, and (or) by the manipulation of pathways that favor metabolic stability through the use of system specific proteostasis regulators. Newly identified mechanisms that efficiently correct CF suggest a multitarget solution to restore transport and channel function at the cell surface through epigenetic mechanism(s). These results suggest the existence of a tunable range of epigenetic and proteostatic environments that may be used to manage correction of misfolding disease phenotypes., doi: 10.1139/O09-905 W. Balch, D. Hutt, D. Herman, A. Koulov, P. Lapointe, J. Coppinger, S. Pankow, B. Lu, A. Rodriquez, S. Noel, J. Kelly, M. Gentzsch, J. Riordan, E. Sorscher, [...]

aaaaaaaaaaFulcher ML, Gabriel SE, Olsen JC, Tatreau JR, Gentzsch M, Livanos E, Saavedra MT, Salmon P, Randell SH. Novel human bronchial epithelial cell lines for cystic fibrosis research. Am J Physiol Lung Cell Mol Physiol 296: L82-L91, 2009. First published October 31, 2008; doi: 10.1 152/ajplung.903 14.2008.-Immortalization of hu- man bronchial epithelial (hBE) cells often entails loss of differentia- tion. Bmi-1 is a protooncogene that maintains stem cells, and its expression creates cell lines that recapitulate normal cell structure and function. We introduced Bmi- 1 and the catalytic subunit of telomerase (hTERT) into three non-cystic fibrosis (CF) and three SF508 homozy- gous CF primary bronchial cell preparations. This treatment extended cell life span, although not as profoundly as viral oncogenes, and at passages 14 and 15, the new cell lines had a diploid karyotype. Ussing chamber analysis revealed variable transepithelial resistances, ranging from 200 to 1,200 flcm2. In the non-CF cell lines, short-circuit currents were stimulated by forskolin and inhibited by CVFR(inh)- 172 at levels mostly comparable to early passage primary cells. CF cell lines exhibited no forskolin-stimulated current and minimal CFTR(inh)- 172 response. Amiloride-inhibitable and UTP-stimulated currents were present, but at lower and higher amplitudes than in primary cells, respectively. The cells exhibited a pseudostratified morphology, with prominent apical membrane polarization, few apoptotic bodies, numerous mucous secretory cells, and occasional ciliated cells. CF and non-CF cell lines produced similar levels of IL-8 at baseline and equally increased IL-8 secretion in response to IL-i ~, TNF-ci, and the Toll-like receptor 2 agonist Pam3Cys. Although they have lower growth potential and more fastidious growth requirements than viral oncogene transformed cells, Bmi-1IhTERT airway epithe- hal cell lines will be useful for several avenues of investigation and will help fill gaps currently hindering CF research and therapeutic development. [ABSTRACT FROM AUTHOR]

Rationale: Hyper IgE syndrome (STAT3-HIES), also known as Job's syndrome, is a rare immunodeficiency disease typically caused by dominant-negative STAT3 mutations. STAT3-HIES syndrome is characterized by chronic pulmonary infection and inflammation, suggesting impairment of pulmonary innate host defense., Objectives: To identify airway epithelial host defense defects consequent to STAT3 mutations that, in addition to reported mutant STAT3 immunologic abnormalities, produce pulmonary infection., Methods: STAT3-HIES sputum was evaluated for biochemical/biophysical properties. STAT3-HIES excised lungs were harvested for histology; bronchial brush samples were collected for RNA sequencing and in vitro culture. A STAT3-HIES-specific mutation (R382W), expressed by lentiviruses, and a STAT3 knockout, generated by CRISPR/Cas9, were maintained in normal human bronchial epithelia under basal or inflammatory (IL1β) conditions. Effects of STAT3 deficiency on transcriptomics, and epithelial ion channel, secretory, antimicrobial, and ciliary functions were assessed., Measurements and Main Results: Mucus concentrations and viscoelasticity were increased in STAT3-HIES sputum. STAT3-HIES excised lungs exhibited mucus obstruction and elevated IL1β expression. STAT3 deficiency impaired CFTR-dependent fluid and mucin secretion, inhibited expression of antimicrobial peptides, cytokines, and chemokines, and acidified airway surface liquid at baseline and post-IL1β exposure in vitro. Notably, mutant STAT3 suppressed IL1R1 expression. STAT3 mutations also inhibited ciliogenesis in vivo and impaired mucociliary transport in vitro, a process mediated via HES6 suppression. Administration of a γ-secretase inhibitor increased HES6 expression and improved ciliogenesis in STAT3 R382W mutant cells., Conclusions: STAT3 dysfunction leads to multi-component defects in airway epithelial innate defense, which, in conjunction with STAT3-HIES immune deficiency, contributes to chronic pulmonary infection.

Rescue of N1303K CFTR by highly effective modulator therapy (HEMT) is enabled by CF airway inflammation. These findings suggest that evaluation of HEMT for rare CFTR mutations must be performed under inflammatory conditions relevant to CF airways. https://bit.ly/3tTcoJE., Competing Interests: Conflict of interest: M. Gentzsch directs the CFTR Functional Analysis Core of the CF Foundation (CFF) Research Development Program BOUCHE19R0 and the CF Molecular/Functional Measurement Core of the NIH CFRTCC Program P30DK065988. M. Gentzsch and C.M.P. Ribeiro are recipients of a Boost Award from the School of Medicine at the University of North Carolina and have obtained support for registration (NACFC) or travel (ECFS Conference) to conferences. C.W. Kam reports grants from the CFF, Friends Fighting Cystic Fibrosis and the American Society of Health-System Pharmacists Foundation. C.J. McKinzie received funding from the CFF and consulting fees from Vertex Pharmaceuticals, Inc. K.A. Despotes received funding from the CFF. C.R. Esther Jr directs a National Resource Center Core that is funded by the CFF and obtained consulting fees from the CFF. B. Baker, D.M. Cholon, S.E. Boyles, and N.L. Quinney report no conflict of interest., (Copyright ©The authors 2024.)

People with cystic fibrosis (pwCF) suffer from chronic and recurring bacterial lung infections that begin very early in life and contribute to progressive lung failure. CF is caused by mutations in the CF transmembrane conductance regulator ( CFTR ) gene, which encodes an ion channel important for maintaining the proper hydration of pulmonary surfaces. When CFTR function is ablated or impaired, airways develop thickened, adherent mucus that contributes to a vicious cycle of infection and inflammation. Therapeutics for pwCF, called CFTR modulators, target the CFTR defect directly, restoring airway surface hydration and mucociliary clearance. However, even with CFTR modulator therapy, bacterial infections persist. To develop a relevant model of diseased airway epithelium, we established a primary human airway epithelium culture system with persistent Pseudomonas aeruginosa infection. We used this model to examine the effects of CFTR modulators on CFTR maturation, CFTR function, and bacterial persistence. We found that the presence of P. aeruginosa increased CFTR mRNA, protein, and function. We also found that CFTR modulators caused a decrease in P. aeruginosa burden. These results demonstrate the importance of including live bacteria to accurately model the CF lung, and that understanding the effects of infection on CFTR rescue by CFTR modulators is critical to evaluating and optimizing drug therapies for all pwCF.

Unlike solid organs, human airway epithelia derive their oxygen from inspired air rather than the vasculature. Many pulmonary diseases are associated with intraluminal airway obstruction caused by aspirated foreign bodies, virus infection, tumors, or mucus plugs intrinsic to airway disease, including cystic fibrosis (CF). Consistent with requirements for luminal O 2 , airway epithelia surrounding mucus plugs in chronic obstructive pulmonary disease (COPD) lungs are hypoxic. Despite these observations, the effects of chronic hypoxia (CH) on airway epithelial host defense functions relevant to pulmonary disease have not been investigated. Molecular characterization of resected human lungs from individuals with a spectrum of muco-obstructive lung diseases (MOLDs) or COVID-19 identified molecular features of chronic hypoxia, including increased EGLN3 expression, in epithelia lining mucus-obstructed airways. In vitro experiments using cultured chronically hypoxic airway epithelia revealed conversion to a glycolytic metabolic state with maintenance of cellular architecture. Chronically hypoxic airway epithelia unexpectedly exhibited increased MUC5B mucin production and increased transepithelial Na + and fluid absorption mediated by HIF1α/HIF2α-dependent up-regulation of β and γENaC (epithelial Na + channel) subunit expression. The combination of increased Na + absorption and MUC5B production generated hyperconcentrated mucus predicted to perpetuate obstruction. Single-cell and bulk RNA sequencing analyses of chronically hypoxic cultured airway epithelia revealed transcriptional changes involved in airway wall remodeling, destruction, and angiogenesis. These results were confirmed by RNA-in situ hybridization studies of lungs from individuals with MOLD. Our data suggest that chronic airway epithelial hypoxia may be central to the pathogenesis of persistent mucus accumulation in MOLDs and associated airway wall damage.

Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) channel that perturb anion transport across the epithelia of the airways and other organs. To treat cystic fibrosis, strategies that target mutant CFTR have been developed such as correctors that rescue folding and enhance transfer of CFTR to the apical membrane, and potentiators that increase CFTR channel activity. While there has been tremendous progress in development and approval of CFTR therapeutics for the most common (F508del) and several other CFTR mutations, around 10-20% of people with cystic fibrosis have rare mutations that are still without an effective treatment. In the current decade, there was an impressive evolution of patient-derived cell models for precision medicine. In cystic fibrosis, these models have played a crucial role in characterizing the molecular defects in CFTR mutants and identifying compounds that target these defects. Cells from nasal, bronchial, and rectal epithelia are most suitable to evaluate treatments that target CFTR. In vitro assays using cultures grown at an air-liquid interface or as organoids and spheroids allow the diagnosis of the CFTR defect and assessment of potential treatment strategies. An overview of currently established cell culture models and assays for personalized medicine approaches in cystic fibrosis will be provided in this review. These models allow theratyping of rare CFTR mutations with available modulator compounds to predict clinical efficacy. Besides evaluation of individual personalized responses to CFTR therapeutics, patient-derived culture models are valuable for testing responses to developmental treatments such as novel RNA- and DNA-based therapies., Competing Interests: Declaration of Competing Interest ASR and FA declare no conflict of interest. MG is an inventor of US patent No. 11357758 (Epithelial cell spheroids and methods of making and using the same)., (Copyright © 2022. Published by Elsevier B.V.)