"shocking" the system to achieve efficient gene targeting in primary human airway epithelia

The ability to knock out specific genes to gain an understanding of their function is a mainstay of modern research, and the use of technologies to achieve this goal to study pulmonary biology is no exception. Homologous recombination in embryonic stem cells, antisense oligonucleotides, sh/siRNA, and CRISPR-mediated deletions are widely known and used. Most of these methods will perform well in various model systems and cell lines, with the classic method, homologous recombination in embryonic stem cells, being highly useful in animals. However, for translational questions, interspecies and/or cell line differences often require confirmation of functional findings in primary human cells. Primary human airway cultures are a highly valued model to study pulmonary biology, and the application of robust gene knockdown technologies to these cells is a valuable method to foster our knowledge in this field. Unfortunately, as with other primary cell models, primary airway cells generally remain persistently defiant to easy genetic manipulation

X-box-binding protein 1 and innate immune responses of human cystic fibrosis alveolar macrophages

Rationale: Alveolar macrophages (AMs) play a key role in host defense to inhaled bacterial pathogens, in part by secreting inflammatory mediators. Cystic fibrosis (CF) airways exhibit a persistent, robust inflammatory response that may contribute to the pathophysiology of CF. Recent findings have linked endoplasmic reticulum stress responses mediated by inositol-requiring enzyme 1a-dependentmessengerRNAsplicing (activation) ofX-box-binding protein-1 (XBP-1s) to inflammation in peripheral macrophages. However, the role of XBP-1s in CF AMfunction is not known. Objectives: To evaluate inflammatory responses of AMs from chronically infected/inflamed human CF lungs and test whether XBP-1s is required for AM-mediated inflammation. Methods: Basal and LPS-induced inflammatory responses were evaluated in primary cultures of non-CF versus CF AMs. XBP-1s was measured and its function was evaluated in AMs using 8-formyl-7-hydroxy-4-methylcoumarin (4μ8C), an inhibitor of inositolrequiring enzyme 1a-dependent XBP-1s, and in THP-1 cells stably expressing XBP-1 shRNA, XBP-1s, or a dominant-negative XBP-1. Measurements and Main Results: CF AMs exhibited exaggerated basal and LPS-induced production of tumor necrosis factor-a and IL-6, and these responses were coupled to increased levels of XBP-1s. In non-CF and CF AMs, LPS-induced cytokine production was blunted by 4μ8C. A role for XBP-1s in AM inflammatory responses was further established by data from dTHP-1 cells indicating that expression of XBP-1 shRNA reduced XBP-1s levels and LPS-induced inflammatory responses; and LPS-induced inflammation was upregulated by expression of XBP-1s and inhibited by dominantnegative XBP-1. Conclusions: These findings suggest that AMs contribute to the robust inflammation of CF airways via an up-regulation of XBP-1smediated cytokine production

Revisiting Host-Pathogen Interactions in Cystic Fibrosis Lungs in the Era of CFTR Modulators

Cystic fibrosis transmembrane conductance regulator (CFTR) modulators, a new series of therapeutics that correct and potentiate some classes of mutations of the CFTR, have provided a great therapeutic advantage to people with cystic fibrosis (pwCF). The main hindrances of the present CFTR modulators are related to their limitations in reducing chronic lung bacterial infection and inflammation, the main causes of pulmonary tissue damage and progressive respiratory insufficiency, particularly in adults with CF. Here, the most debated issues of the pulmonary bacterial infection and inflammatory processes in pwCF are revisited. Special attention is given to the mechanisms favoring the bacterial infection of pwCF, the progressive adaptation of Pseudomonas aeruginosa and its interplay with Staphylococcus aureus, the cross-talk among bacteria, the bronchial epithelial cells and the phagocytes of the host immune defenses. The most recent findings of the effect of CFTR modulators on bacterial infection and the inflammatory process are also presented to provide critical hints towards the identification of relevant therapeutic targets to overcome the respiratory pathology of pwCF

Chronic airway infection/inflammation induces a Ca2+ i-dependent hyperinflammatory response in human cystic fibrosis airway epithelia

Hyperinflammatory responses to infection have been postulated as a component of cystic fibrosis (CF) lung disease. Studies have united intracellular calcium (Ca2+i) mobilization with inflammatory responses in several systems. We have reported that the pro-inflammatory mediator bradykinin (BK) promotes larger Ca2+ i signals in CF compared with normal bronchial epithelia, a response that reflects endeplasmic reticulum (ER)/Ca2+ store expansion induced by chronic luminal airway infection/inflammation. The present study investigated whether CF airway epithelia were hyperinflammatory and, if so, whether the hyperinflammatory CF phenotype was united to larger Ca2+ stores in the ER. We found that ΔF508 CF bronchial epithelia were hyperinflammatory as defined by an increased basal and mucosal BK-induced interleukin (IL)-8 secretion. However, the CF hyperinflammation expressed in short-term. (6-11-day-old) primary cultures of ΔF508 bronchial epithelia was lost in long-term (30-40-day-old) primary cultures of ΔF508 bronchial epithelia, indicating this response was independent of mutant cystic fibrosis transmembrane conductance regulator. Exposure of 30-40-day-old cultures of normal airway epithelia to supernatant from mucopurulent material (SMM) from CF airways reproduced the increased basal and mucosal BK-stimulated IL-8 secretion of short-term CF cultures. The BK-triggered increased IL-8 secretion in SMM-treated cultures was mediated by an increased Ca2+i mobilization consequent to an ER expansion associated with increases in protein synthesis (total, cytokines, and antimicrobial factors). The increased ER-dependent, Ca2+i-mediated hyperinflammatory epithelial response may represent a general beneficial airway epithelial adaptation to transient luminal infection. However, in CF airways, the Ca2+ i-mediated hyperinflammation may be ineffective in promoting the eradication of infection in thickened mucus and, consequently, may have adverse effects in the lung

Azithromycin treatment alters gene expression in inflammatory, lipid metabolism, and cell cycle pathways in well-differentiated human airway epithelia

Prolonged macrolide antibiotic therapy at low doses improves clinical outcome in patients affected with diffuse panbronchiolitis and cystic fibrosis. Consensus is building that the therapeutic effects are due to anti-inflammatory, rather than anti-microbial activities, but the mode of action is likely complex. To gain insights into how the macrolide azithromycin (AZT) modulates inflammatory responses in airways, well-differentiated primary cultures of human airway epithelia were exposed to AZT alone, an inflammatory stimulus consisting of soluble factors from cystic fibrosis airways, or AZT followed by the inflammatory stimulus. RNA microarrays were conducted to identify global and specific gene expression changes. Analysis of gene expression changes revealed that the AZT treatment alone altered the gene profile of the cells, primarily by significantly increasing the expression of lipid/cholesterol genes and decreasing the expression of cell cycle/ mitosis genes. The increase in cholesterol biosynthetic genes was confirmed by increased filipin staining, an index of free cholesterol, after AZT treatment. AZT also affected genes with inflammatory annotations, but the effect was variable (both up- and down-regulation) and gene specific. AZT pretreatment prevented the up-regulation of some genes, such as MUC5AC and MMP9, triggered by the inflammatory stimulus, but the up-regulation of other inflammatory genes, e.g., cytokines and chemokines, such as interleukin-8, was not affected. On the other hand, HLA genes were increased by AZT. Notably, secreted IL-8 protein levels did not reflect mRNA levels, and were, in fact, higher after AZT pretreatment in cultures exposed to the inflammatory stimulus, suggesting that AZT can affect inflammatory pathways other than by altering gene expression. These findings suggest that the specific effects of AZT on inflamed and non-inflamed airway epithelia are likely relevant to its clinical activity, and their apparent complexity may help explain the diverse immunomodulatory roles of macrolides

SPLUNC1 degradation by the cystic fibrosis mucosal environment drives airway surface liquid dehydration

The multi-organ disease cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane regulator gene (CFTR) that lead to diminished transepithelial anion transport. CF lungs are characterised by airway surface liquid (ASL) dehydration, chronic infection/inflammation and neutrophilia. Dysfunctional CFTR may upregulate the epithelial Na+ channel (ENaC), further exacerbating dehydration. We previously demonstrated that short palate lung and nasal epithelial clone 1 (SPLUNC1) negatively regulates ENaC in normal airway epithelia. Here, we used pulmonary tissue samples, sputum and human bronchial epithelial cells (HBECs) to determine whether SPLUNC1 could regulate ENaC in a CF-like environment. We found reduced endogenous SPLUNC1 in CF secretions, and rapid degradation of recombinant SPLUNC1 (rSPLUNC1) by CF secretions. Normal sputum, containing SPLUNC1 and SPLUNC1-derived peptides, inhibited ENaC in both normal and CF HBECs. Conversely, CF sputum activated ENaC, and rSPLUNC1 could not reverse this phenomenon. Additionally, we observed upregulation of ENaC protein levels in human CF bronchi. Unlike SPLUNC1, the novel SPLUNC1-derived peptide SPX-101 resisted protease degradation, bound apically to HBECs, inhibited ENaC and prevented ASL dehydration following extended pre-incubation with CF sputum. Our data indicate that CF mucosal secretions drive ASL hyperabsorption and that protease-resistant peptides, e.g. SPX-101, can reverse this effect to rehydrate CF ASL

Mucin production and hydration responses to mucopurulent materials in normal versus cystic fibrosis airway epithelia

Rationale: Cystic fibrosis (CF) airways disease produces a mucoobstructive lung phenotype characterized by airways mucus plugging, epithelial mucous cell metaplasia/hyperplasia, chronic infection, and inflammation. Simultaneous biochemical and functional in vivo studies of mucin synthesis and secretion from CF airways are not available. In vitro translational models may quantitate differential CF versus normal mucin and fluid secretory responses to infectious/inflammatory stimuli. Objectives: We tested the hypothesis that CF airways exhibit defective epithelial fluid, but not mucin, secretory responses to bacterial/inflammatory host products. Methods: Well-differentiated primary human bronchial epithelial cultures were exposed to supernatant from mucopurulent material (SMM) from human CF airways as a test of bacterial/inflammatory host product stimulus. Human bronchial epithelia (HBE) with normal CF transmembrane conductance regulator function were compared with DF508/DF508 CF HBE. Measurements and Main Results: Acute (up to 60 min) SMM exposure promoted mucin secretion, but mucins were degraded by the proteolytic enzymes present in SMM. Chronic SMM exposure induced upregulation of mucin synthesis and storage and generated absolute increases in basal and stimulated mucin release in normal and CF cultures. These responses were similar in normal and CF cultures. In contrast, SMM produced a coordinated CF transmembrane conductance regulator-mediated Cl secretory response in normal HBE, but not in CF HBE. The absence of the fluid secretory response in CF produced quantitatively more dehydrated mucus. Conclusions: Our study reveals the interplay between regulation of mucin and fluid secretion rates in inflamed versus noninflamed conditions and why a hyperconcentrated mucus is produced in CF airways

XBP1s regulates MUC5B in a promoter variant-dependent pathway in idiopathic pulmonary fibrosis airway epithelia

Rationale: The goal was to connect elements of idiopathic pulmonary fibrosis (IPF) pathogenesis, including chronic endoplasmic reticulum stress in respiratory epithelia associated with injury/inflammation and remodeling, distal airway mucus obstruction and honeycomb cyst formation with accumulation of MUC5B (mucin 5B), and associations between IPF risk and polymorphisms in the MUC5B promoter. Objectives: To test whether the endoplasmic reticulum (ER) stress sensor protein ERN2 (ER-to-nucleus signaling 2) and its downstream effector, the spliced form of XBP1S (X-box-binding protein 1), regulate MUC5B expression and differentially activate the MUC5B promoter variant in respiratory epithelia. Methods: Primary human airway epithelial (HAE) cells, transgenic mouse models, human IPF lung tissues, and cell lines expressing XBP1S and MUC5B promoters were used to explore relationships between the ERN2/XBP1S pathway and MUC5B. An inhibitor of the pathway, KIRA6, and XBP1 CRISPR-Cas9 were used in HAE cells to explore therapeutic potential. Measurements and Main Results: ERN2 regulated MUC5B and MUC5AC mRNAs. Downstream XBP1S selectively promoted MUC5B expression in vitro and in distal murine airway epithelia in vivo. XBP1S bound to the proximal region of the MUC5B promoter and differentially upregulated MUC5B expression in the context of the MUC5B promoter rs35705950 variant. High levels of ERN2 and XBP1S were associated with excessive MUC5B mRNAs in distal airways of human IPF lungs. Cytokine-induced MUC5B expression in HAE cells was inhibited by KIRA6 and XBP1 CRISPR-Cas9. Conclusions: A positive feedback bistable ERN2-XBP1S pathway regulates MUC5B-dominated mucus obstruction in IPF, providing an unfolded protein response-dependent mechanism linking the MUC5B promoter rs35705950 polymorphism with IPF pathogenesis. Inhibiting ERN2-dependent pathways/elements may provide a therapeutic option for IPF

IL-1β dominates the promucin secretory cytokine profile in cystic fibrosis

Cystic fibrosis (CF) lung disease is characterized by early and persistent mucus accumulation and neutrophilic inflammation in the distal airways. Identification of the factors in CF mucopurulent secretions that perpetuate CF mucoinflammation may provide strategies for novel CF pharmacotherapies. We show that IL-1β, with IL-1α, dominated the mucin prosecretory activities of supernatants of airway mucopurulent secretions (SAMS). Like SAMS, IL-1β alone induced MUC5B and MUC5AC protein secretion and mucus hyperconcentration in CF human bronchial epithelial (HBE) cells. Mechanistically, IL-1β induced the sterile α motif-pointed domain containing ETS transcription factor (SPDEF) and downstream endoplasmic reticulum to nucleus signaling 2 (ERN2) to upregulate mucin gene expression. Increased mRNA levels of IL1B, SPDEF, and ERN2 were associated with increased MUC5B and MUC5AC expression in the distal airways of excised CF lungs. Administration of an IL-1 receptor antagonist (IL-1Ra) blocked SAMS-induced expression of mucins and proinflammatory mediators in CF HBE cells. In conclusion, IL-1α and IL-1β are upstream components of a signaling pathway, including IL-1R1 and downstream SPDEF and ERN2, that generate a positive feedback cycle capable of producing persistent mucus hyperconcentration and IL-1α and/or IL-1β-mediated neutrophilic inflammation in the absence of infection in CF airways. Targeting this pathway therapeutically may ameliorate mucus obstruction and inflammation-induced structural damage in young CF children