Dynamic regulation of airway surface liquid pH by TMEM16A and SLC26A4 in cystic fibrosis nasal epithelia with rare mutations.

In cystic fibrosis (CF), defects in the CF transmembrane conductance regulator (CFTR) channel lead to an acidic airway surface liquid (ASL), which compromises innate defence mechanisms, predisposing to pulmonary failure. Restoring ASL pH is a potential therapy for people with CF, particularly for those who cannot benefit from current highly effective modulator therapy. However, we lack a comprehensive understanding of the complex mechanisms underlying ASL pH regulation. The calcium-activated chloride channel, TMEM16A, and the anion exchanger, SLC26A4, have been proposed as targets for restoring ASL pH, but current results are contradictory and often utilise nonphysiological conditions. To provide better evidence for a role of these two proteins in ASL pH homeostasis, we developed an efficient CRISPR-Cas9-based approach to knock-out (KO) relevant transporters in primary airway basal cells lacking CFTR and then measured dynamic changes in ASL pH under thin-film conditions in fully differentiated airway cultures, which better simulate the in vivo situation. Unexpectantly, we found that both proteins regulated steady-state as well as agonist-stimulated ASL pH, but only under inflammatory conditions. Furthermore, we identified two Food and Drug Administration (FDA)-approved drugs which raised ASL pH by activating SLC26A4. While we identified a role for SLC26A4 in fluid absorption, KO had no effect on cyclic adenosine monophosphate (cAMP)-stimulated fluid secretion in airway organoids. Overall, we have identified a role of TMEM16A in ASL pH homeostasis and shown that both TMEM16A and SLC26A4 could be important alternative targets for ASL pH therapy in CF, particularly for those people who do not produce any functional CFTR.

Long-term expanding human airway organoids for disease modeling.

Organoids are self-organizing 3D structures grown from stem cells that recapitulate essential aspects of organ structure and function. Here, we describe a method to establish long-term-expanding human airway organoids from broncho-alveolar resections or lavage material. The pseudostratified airway organoids consist of basal cells, functional multi-ciliated cells, mucus-producing secretory cells, and CC10-secreting club cells. Airway organoids derived from cystic fibrosis (CF) patients allow assessment of CFTR function in an organoid swelling assay. Organoids established from lung cancer resections and metastasis biopsies retain tumor histopathology as well as cancer gene mutations and are amenable to drug screening. Respiratory syncytial virus (RSV) infection recapitulates central disease features, dramatically increases organoid cell motility via the non-structural viral NS2 protein, and preferentially recruits neutrophils upon co-culturing. We conclude that human airway organoids represent versatile models for the in vitro study of hereditary, malignant, and infectious pulmonary disease.

Recurrent Respiratory Syncytial Virus Infection in a CD14-Deficient Patient.

BACKGROUND: Recurrent respiratory syncytial virus (RSV) infection requiring hospitalization is rare and the underlying mechanism is unknown. We aimed to determine the role of CD14-mediated immunity in the pathogenesis of recurrent RSV infection. METHODS: We performed genotyping and longitudinal immunophenotyping of the first patient with a genetic CD14 deficiency who developed recurrent RSV infection. We analyzed gene expression profiles and interleukin (IL)-6 production by patient peripheral blood mononuclear cells in response to RSV pre- and post-fusion (F) protein. We generated CD14-deficient human nasal epithelial cells cultured at air-liquid interface (HNEC-ALI) of patient-derived cells and after CRISPR-based gene editing of control cells. We analyzed viral replication upon RSV infection. RESULTS: Sanger sequencing revealed a homozygous single-nucleotide deletion in CD14, resulting in absence of the CD14 protein in the index patient. In vitro, viral replication was similar in wild-type and CD14-/- HNEC-ALI. Loss of immune cell CD14 led to impaired cytokine and chemokine responses to RSV pre- and post-F protein, characterized by absence of IL-6 production. CONCLUSIONS: We report an association of recurrent RSV bronchiolitis with a loss of CD14 function in immune cells. Lack of CD14 function led to defective immune responses to RSV pre- and post-F protein without a change in viral replication.

Drug Repurposing for Cystic Fibrosis: Identification of Drugs That Induce CFTR-Independent Fluid Secretion in Nasal Organoids.

Individuals with cystic fibrosis (CF) suffer from severe respiratory disease due to a genetic defect in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which impairs airway epithelial ion and fluid secretion. New CFTR modulators that restore mutant CFTR function have been recently approved for a large group of people with CF (pwCF), but ~19% of pwCF cannot benefit from CFTR modulators Restoration of epithelial fluid secretion through non-CFTR pathways might be an effective treatment for all pwCF. Here, we developed a medium-throughput 384-well screening assay using nasal CF airway epithelial organoids, with the aim to repurpose FDA-approved drugs as modulators of non-CFTR-dependent epithelial fluid secretion. From a ~1400 FDA-approved drug library, we identified and validated 12 FDA-approved drugs that induced CFTR-independent fluid secretion. Among the hits were several cAMP-mediating drugs, including ß2-adrenergic agonists. The hits displayed no effects on chloride conductance measured in the Ussing chamber, and fluid secretion was not affected by TMEM16A, as demonstrated by knockout (KO) experiments in primary nasal epithelial cells. Altogether, our results demonstrate the use of primary nasal airway cells for medium-scale drug screening, target validation with a highly efficient protocol for generating CRISPR-Cas9 KO cells and identification of compounds which induce fluid secretion in a CFTR- and TMEM16A-indepent manner.

Aberrant epithelial differentiation by cigarette smoke dysregulates respiratory host defence.

It is currently unknown how cigarette smoke-induced airway remodelling affects highly expressed respiratory epithelial defence proteins and thereby mucosal host defence.Localisation of a selected set of highly expressed respiratory epithelial host defence proteins was assessed in well-differentiated primary bronchial epithelial cell (PBEC) cultures. Next, PBEC were cultured at the air-liquid interface, and during differentiation for 2-3 weeks exposed daily to whole cigarette smoke. Gene expression, protein levels and epithelial cell markers were subsequently assessed. In addition, functional activities and persistence of the cigarette smoke-induced effects upon cessation were determined.Expression of the polymeric immunoglobulin receptor, secretory leukocyte protease inhibitor and long and short PLUNC (palate, lung and nasal epithelium clone protein) was restricted to luminal cells and exposure of differentiating PBECs to cigarette smoke resulted in a selective reduction of the expression of these luminal cell-restricted respiratory host defence proteins compared to controls. This reduced expression was a consequence of cigarette smoke-impaired end-stage differentiation of epithelial cells, and accompanied by a significant decreased transepithelial transport of IgA and bacterial killing.These findings shed new light on the importance of airway epithelial cell differentiation in respiratory host defence and could provide an additional explanation for the increased susceptibility of smokers and patients with chronic obstructive pulmonary disease to respiratory infections.

Proinflammatory Cytokines Impair Vitamin D-Induced Host Defense in Cultured Airway Epithelial Cells.

Vitamin D is a regulator of host defense against infections and induces expression of the antimicrobial peptide hCAP18/LL-37. Vitamin D deficiency is associated with chronic inflammatory lung diseases and respiratory infections. However, it is incompletely understood if and how (chronic) airway inflammation affects vitamin D metabolism and action. We hypothesized that long-term exposure of primary bronchial epithelial cells to proinflammatory cytokines alters their vitamin D metabolism, antibacterial activity, and expression of hCAP18/LL-37. To investigate this, primary bronchial epithelial cells were differentiated at the air-liquid interface for 14 days in the presence of the proinflammatory cytokines, TNF-α and IL-1ß (TNF-α/IL-1ß), and subsequently exposed to vitamin D (inactive 25(OH)D3 and active 1,25(OH)2D3). Expression of hCAP18/LL-37, vitamin D receptor, and enzymes involved in vitamin D metabolism (CYP24A1 and CYP27B1) was determined using quantitative PCR, Western blot, and immunofluorescence staining. Furthermore, vitamin D-mediated antibacterial activity was assessed using nontypeable Haemophilus influenzae. We found that TNF-α/IL-1ß treatment reduced vitamin D-induced expression of hCAP18/LL-37 and killing of nontypeable H. influenzae. In addition, CYP24A1 (a vitamin D-degrading enzyme) was increased by TNF-α/IL-1ß, whereas CYP27B1 (that converts 25(OH)D3 to its active form) and vitamin D receptor expression remained unaffected. Furthermore, we have demonstrated that the TNF-α/IL-1ß-mediated induction of CYP24A1 was, at least in part, mediated by the transcription factor specific protein 1, and the epidermal growth factor receptor-mitogen-activated protein kinase pathway. These findings indicate that TNF-α/IL-1ß decreases vitamin D-mediated antibacterial activity and hCAP18/LL-37 expression via induction of CYP24A1 and suggest that chronic inflammation impairs protective responses induced by vitamin D.

Basal cells contribute to innate immunity of the airway epithelium through production of the antimicrobial protein RNase 7.

Basal cells play a critical role in the response of the airway epithelium to injury and are recently recognized to also contribute to epithelial immunity. Antimicrobial proteins and peptides are essential effector molecules in this airway epithelial innate immunity. However, little is known about the specific role of basal cells in antimicrobial protein and peptide production and about the regulation of the ubiquitous antimicrobial protein RNase 7. In this study, we report that basal cells are the principal cell type producing RNase 7 in cultured primary bronchial epithelial cells (PBEC). Exposure of submerged cultured PBEC (primarily consisting of basal cells) to the respiratory pathogen nontypeable Haemophilus influenzae resulted in a marked increase in expression of RNase 7, although this was not observed in differentiated air-liquid interface cultured PBEC. However, transient epithelial injury in air-liquid interface-cultured PBEC induced by cigarette smoke exposure led to epidermal growth factor receptor-mediated expression of RNase 7 in remaining basal cells. The selective induction of RNase 7 in basal cells by cigarette smoke was demonstrated using confocal microscopy and by examining isolated luminal and basal cell fractions. Taken together, these findings demonstrate a phenotype-specific innate immune activity of airway epithelial basal cells, which serves as a second line of airway epithelial defense that is induced by airway epithelial injury.

TNF-α and IL-1ß-activated human mesenchymal stromal cells increase airway epithelial wound healing in vitro via activation of the epidermal growth factor receptor.

BACKGROUND: Mesenchymal stromal cells (MSCs) are investigated for their potential to reduce inflammation and to repair damaged tissue. Inflammation and tissue damage are hallmarks of chronic obstructive pulmonary disease (COPD) and MSC infusion is a promising new treatment for COPD. Inflammatory mediators attract MSCs to sites of inflammation and affect their immune-modulatory properties, but little is known about their effect on regenerative properties of MSCs. This study investigates the effect of the pro-inflammatory cytokines TNF-α and IL-1ß on the regenerative potential of MSCs, using an in vitro wound healing model of airway epithelial cells. METHODS: Standardized circular wounds were created by scraping cultures of the airway epithelial cell line NCI-H292 and primary bronchial epithelial cells cultured at the air-liquid interface (ALI-PBEC), and subsequently incubated with MSC conditioned medium (MSC-CM) that was generated in presence or absence of TNF-α/IL-1ß. Remaining wound size was measured up to 72 h. Phosphorylation of ERK1/2 by MSC-CM was assessed using Western blot. Inhibitors for EGFR and c-Met signaling were used to investigate the contribution of these receptors to wound closure and to ERK1/2 phosphorylation. Transactivation of EGFR by MSC-CM was investigated using a TACE inhibitor, and RT-PCR was used to quantify mRNA expression of several growth factors in MSCs and NCI-H292. RESULTS: Stimulation of MSCs with the pro-inflammatory cytokines TNF-α and IL-1ß increased the mRNA expression of various growth factors by MCSs and enhanced the regenerative potential of MSCs in an in vitro model of airway epithelial injury using NCI-H292 airway epithelial cells. Conditioned medium from cytokine stimulated MSCs induced ERK1/2 phosphorylation in NCI-H292, predominantly via EGFR; it induced ADAM-mediated transactivation of EGFR, and it induced airway epithelial expression of several EGFR ligands. The contribution of activation of c-Met via HGF to increased repair could not be confirmed by inhibitor experiments. CONCLUSION: Our data imply that at sites of tissue damage, when inflammatory mediators are present, for example in lungs of COPD patients, MSCs become more potent inducers of repair, in addition to their well-known immune-modulatory properties.

Polymeric immunoglobulin receptor down-regulation in chronic obstructive pulmonary disease. Persistence in the cultured epithelium and role of transforming growth factor-ß.

RATIONALE: The generation of protective secretory IgA relies on the epithelial polymeric immunoglobulin receptor (pIgR). pIgR expression is reduced in chronic obstructive pulmonary disease (COPD), but correlation to disease severity and underlying mechanisms remains unknown. OBJECTIVES: To address the hypothesis that pIgR down-regulation in COPD concerns severe disease in relation to aberrant programming of the bronchial epithelium. METHODS: Surgical lung tissue and primary bronchial epithelium (cultured in air-liquid interface, ALI) obtained from a large series of patients (n = 116) were studied for pIgR expression and regulation. MEASUREMENTS AND MAIN RESULTS: pIgR immunostaining in the bronchial epithelium is decreased in severe COPD. In contrast, pIgR transcription was up-regulated in smokers with or without COPD. In ALI (vs. submerged) cultures, pIgR expression was strongly induced, whereas pIgR expression and IgA-transcytosis capacity were decreased in cultures from subjects with severe COPD as compared with control subjects. In addition, COPD cultures released more transforming growth factor-ß1 (TGF-ß1), reflecting increased epithelial TGF-ß1 immunostaining in COPD lung tissue. Finally, besides inducing epithelial dedifferentiation, exogenous TGF-ß1 dose-dependently inhibited pIgR production, whereas pIgR increased on blockade of TGF-ß1 activity during ALI differentiation. CONCLUSIONS: pIgR down-regulation in COPD correlates with disease severity, and the bronchial epithelium reconstituted in vitro from these patients retains its aberrant imprinting for pIgR expression. This study also links pIgR down-regulation to TGF-ß-driven reprogramming of the bronchial epithelium, which results in impaired lung IgA immunity in patients with COPD.

Role of activin-A in cigarette smoke-induced inflammation and COPD.

Activin-A is a pleiotropic cytokine belonging to the transforming growth factor-ß superfamily and has been implicated in asthma and pulmonary fibrosis. However, the role of activin-A and its endogenous inhibitor, follistatin, in the pathogenesis of chronic obstructive pulmonary disease (COPD) is unknown. We first quantified activin-A and follistatin in the lungs of air- or cigarette smoke-exposed mice and in the lungs of patients with COPD by immunohistochemistry, ELISA and quantitative real-time PCR. We subsequently studied the effect of cigarette smoke on primary human bronchial epithelial cells in vitro. Next, activin-A signalling was antagonised in vivo by administration of follistatin in mice exposed to air or cigarette smoke for 4 weeks. Protein levels of activin-A were increased in the airway epithelium of patients with COPD compared with never-smokers and smokers. Cigarette smoke-exposed human bronchial epithelial cells expressed higher levels of activin-A and lower levels of follistatin. Both mRNA and protein levels of activin-A were increased in the lungs of cigarette smoke-exposed mice, whereas follistatin levels were reduced upon cigarette smoke exposure. Importantly, administration of follistatin attenuated the cigarette smoke-induced increase of inflammatory cells and mediators in the bronchoalveolar lavage fluid in mice. These results suggest that an imbalance between activin-A and follistatin contributes to the pathogenesis of cigarette smoke-induced inflammation and COPD.

Protocol for generating airway organoids from 2D air liquid interface-differentiated nasal epithelia for use in a functional CFTR assay.

We present a protocol to generate organoids from air-liquid-interface (ALI)-differentiated nasal epithelia. We detail their application as cystic fibrosis (CF) disease model in the cystic fibrosis transmembrane conductance regulator (CFTR)-dependent forskolin-induced swelling (FIS) assay. We describe steps for isolation, expansion and cryostorage of nasal brushing-derived basal progenitor cells, and their differentiation in ALI cultures. Furthermore, we detail the conversion of differentiated epithelial fragments into organoids of healthy controls and CF subjects for validating CFTR function and modulator responses. For complete details on the use and execution of this protocol, please refer to Amatngalim et al.1.

Exploring intrinsic variability between cultured nasal and bronchial epithelia in cystic fibrosis.

The nasal and bronchial epithelium are unified parts of the respiratory tract that are affected in the monogenic disorder cystic fibrosis (CF). Recent studies have uncovered that nasal and bronchial tissues exhibit intrinsic variability, including differences in mucociliary cell composition and expression of unique transcriptional regulatory proteins which relate to germ layer origin. In the present study, we explored whether intrinsic differences between nasal and bronchial epithelial cells persist in cell cultures and affect epithelial cell functioning in CF. Comparison of air-liquid interface (ALI) differentiated epithelial cells from subjects with CF revealed distinct mucociliary differentiation states of nasal and bronchial cultures. Moreover, using RNA sequencing we identified cell type-specific signature transcription factors in differentiated nasal and bronchial epithelial cells, some of which were already poised for expression in basal progenitor cells as evidenced by ATAC sequencing. Analysis of differentiated nasal and bronchial epithelial 3D organoids revealed distinct capacities for fluid secretion, which was linked to differences in ciliated cell differentiation. In conclusion, we show that unique phenotypical and functional features of nasal and bronchial epithelial cells persist in cell culture models, which can be further used to investigate the effects of tissue-specific features on upper and lower respiratory disease development in CF.

Airway Epithelial Cultures of Children with Esophageal Atresia as a Model to Study Respiratory Tract Disorders.

Esophageal atresia (EA) is a rare birth defect in which respiratory tract disorders are a major cause of morbidity. It remains unclear whether respiratory tract disorders are in part caused by alterations in airway epithelial cell functions such as the activity of motile cilia. This can be studied using airway epithelial cell culture models of patients with EA. Therefore, the aim of this study was to evaluate the feasibility to culture and functionally characterize motile cilia function in the differentiated air-liquid interface cultured airway epithelial cells and 3D organoids derived from nasal brushings and bronchoalveolar lavage (BAL) fluid from children with EA. We demonstrate the feasibility of culturing differentiated airway epithelia and organoids of nasal brushings and BAL fluid of children with EA, which display normal motile cilia function. EA patient-derived airway epithelial cultures can be further used to examine whether alterations in epithelial functions contribute to respiratory disorders in EA.

Measuring cystic fibrosis drug responses in organoids derived from 2D differentiated nasal epithelia.

Cystic fibrosis is caused by genetic defects that impair the CFTR channel in airway epithelial cells. These defects may be overcome by specific CFTR modulating drugs, for which the efficacy can be predicted in a personalized manner using 3D nasal-brushing-derived airway organoids in a forskolin-induced swelling assay. Despite of this, previously described CFTR function assays in 3D airway organoids were not fully optimal, because of inefficient organoid differentiation and limited scalability. In this report, we therefore describe an alternative method of culturing nasal-brushing-derived airway organoids, which are created from an equally differentiated airway epithelial monolayer of a 2D air-liquid interface culture. In addition, we have defined organoid culture conditions, with the growth factor/cytokine combination neuregulin-1<i>ß</i> and interleukin-1<i>ß</i>, which enabled consistent detection of CFTR modulator responses in nasal-airway organoid cultures from subjects with cystic fibrosis.

CRISPR-Based Adenine Editors Correct Nonsense Mutations in a Cystic Fibrosis Organoid Biobank.

Adenine base editing (ABE) enables enzymatic conversion from A-T into G-C base pairs. ABE holds promise for clinical application, as it does not depend on the introduction of double-strand breaks, contrary to conventional CRISPR/Cas9-mediated genome engineering. Here, we describe a cystic fibrosis (CF) intestinal organoid biobank, representing 664 patients, of which ~20% can theoretically be repaired by ABE. We apply SpCas9-ABE (PAM recognition sequence: NGG) and xCas9-ABE (PAM recognition sequence: NGN) on four selected CF organoid samples. Genetic and functional repair was obtained in all four cases, while whole-genome sequencing (WGS) of corrected lines of two patients did not detect off-target mutations. These observations exemplify the value of large, patient-derived organoid biobanks representing hereditary disease and indicate that ABE may be safely applied in human cells.

High-resolution 3D imaging of fixed and cleared organoids.

In vitro 3D organoid systems have revolutionized the modeling of organ development and diseases in a dish. Fluorescence microscopy has contributed to the characterization of the cellular composition of organoids and demonstrated organoids' phenotypic resemblance to their original tissues. Here, we provide a detailed protocol for performing high-resolution 3D imaging of entire organoids harboring fluorescence reporters and upon immunolabeling. This method is applicable to a wide range of organoids of differing origins and of various sizes and shapes. We have successfully used it on human airway, colon, kidney, liver and breast tumor organoids, as well as on mouse mammary gland organoids. It includes a simple clearing method utilizing a homemade fructose-glycerol clearing agent that captures 3D organoids in full and enables marker quantification on a cell-by-cell basis. Sample preparation has been optimized for 3D imaging by confocal, super-resolution confocal, multiphoton and light-sheet microscopy. From organoid harvest to image analysis, the protocol takes 3 d.

Contribution of Host Defence Proteins and Peptides to Host-Microbiota Interactions in Chronic Inflammatory Lung Diseases.

The respiratory tract harbours a variety of microorganisms, collectively called the respiratory microbiota. Over the past few years, alterations in respiratory and gut microbiota composition have been associated with chronic inflammatory diseases of the lungs. How these changes influence disease development and progression is an active field of investigation. Identifying and understanding host-microbiota interactions and factors contributing to these interactions could promote the development of novel therapeutic strategies aimed at restoring host-microbiota homeostasis. In this review, we discuss recent literature on host-microbiota interactions in the respiratory tract, with a specific focus on the influence of endogenous host defence peptides and proteins (HDPs) on the composition of microbiota populations in vivo and explore possible HDPs-related therapeutic approaches targeting microbiota dysbiosis in chronic inflammatory lung diseases.

Antibacterial Defense of Human Airway Epithelial Cells from Chronic Obstructive Pulmonary Disease Patients Induced by Acute Exposure to Nontypeable Haemophilus influenzae: Modulation by Cigarette Smoke.

Antimicrobial proteins and peptides (AMPs) are a central component of the antibacterial activity of airway epithelial cells. It has been proposed that a decrease in antibacterial lung defense contributes to an increased susceptibility to microbial infection in smokers and patients with chronic obstructive pulmonary disease (COPD). However, whether reduced AMP expression in the epithelium contributes to this lower defense is largely unknown. We investigated the bacterial killing activity and expression of AMPs by air-liquid interface-cultured primary bronchial epithelial cells from COPD patients and non-COPD (ex-)smokers that were stimulated with nontypeable Haemophilus influenzae (NTHi). In addition, the effect of cigarette smoke on AMP expression and the activation of signaling pathways was determined. COPD cell cultures displayed reduced antibacterial activity, whereas smoke exposure suppressed the NTHi-induced expression of AMPs and further increased IL-8 expression in COPD and non-COPD cultures. Moreover, smoke exposure impaired NTHi-induced activation of NF-κB, but not MAP-kinase signaling. Our findings demonstrate that the antibacterial activity of cultured airway epithelial cells induced by acute bacterial exposure was reduced in COPD and suppressed by cigarette smoke, whereas inflammatory responses persisted. These findings help to explain the imbalance between protective antibacterial and destructive inflammatory innate immune responses in COPD.