Pulmonary Epithelium Cell Fate Determination: COPD, Lung Cancer, or Both.

The concurrence of chronic obstructive pulmonary disease (COPD) and lung cancer has been widely reported and extensively addressed by pulmonologists and oncologists. However, most studies have focused on shared risk factors, DNA damage pathways, immune microenvironments, inflammation, and imbalanced proteases/antiproteases. In the present review, we explored the association between COPD and lung cancer in terms of airway pluripotent cell fate determination and discussed the various cell types and signaling pathways involved in the maintenance of lung epithelium homeostasis, and their involvement in the pathogenesis of co-occurrence of COPD and lung cancer.

Influence of intersignaling crosstalk on the intracellular localization of YAP/TAZ in lung cells.

A cell is a dynamic system in which various processes occur simultaneously. In particular, intra- and intercellular signaling pathway crosstalk has a significant impact on a cell's life cycle, differentiation, proliferation, growth, regeneration, and, consequently, on the normal functioning of an entire organ. Hippo signaling and YAP/TAZ nucleocytoplasmic shuttling play a pivotal role in normal development, homeostasis, and tissue regeneration, particularly in lung cells. Intersignaling communication has a significant impact on the core components of the Hippo pathway and on YAP/TAZ localization. This review describes the crosstalk between Hippo signaling and key lung signaling pathways (WNT, SHH, TGFß, Notch, Rho, and mTOR) using lung cells as an example and highlights the remaining unanswered questions.

AP-3-dependent targeting of flippase ATP8A1 to lamellar bodies suppresses activation of YAP in alveolar epithelial type 2 cells.

Lamellar bodies (LBs) are lysosome-related organelles (LROs) of surfactant-producing alveolar type 2 (AT2) cells of the distal lung epithelium. Trafficking pathways to LBs have been understudied but are likely critical to AT2 cell homeostasis given associations between genetic defects of endosome to LRO trafficking and pulmonary fibrosis in Hermansky Pudlak syndrome (HPS). Our prior studies uncovered a role for AP-3, defective in HPS type 2, in trafficking Peroxiredoxin-6 to LBs. We now show that the P4-type ATPase ATP8A1 is sorted by AP-3 from early endosomes to LBs through recognition of a C-terminal dileucine-based signal. Disruption of the AP-3/ATP8A1 interaction causes ATP8A1 accumulation in early sorting and/or recycling endosomes, enhancing phosphatidylserine exposure on the cytosolic leaflet. This in turn promotes activation of Yes-activating protein, a transcriptional coactivator, augmenting cell migration and AT2 cell numbers. Together, these studies illuminate a mechanism whereby loss of AP-3-mediated trafficking contributes to a toxic gain-of-function that results in enhanced and sustained activation of a repair pathway associated with pulmonary fibrosis.

Redox-Dependent Activation of Lung Epithelial STAT3 Is Required for Inducible Protection against Bacterial Pneumonia.

The lung epithelium is dynamic, capable of considerable structural and functional plasticity in response to pathogen challenges. Our laboratory has demonstrated that an inhaled combination of a Toll-like receptor (TLR) 2/6 agonist and a TLR9 agonist (Pam2ODN) results in robust protection against otherwise lethal pneumonias. We have previously shown that intact epithelial TLR signaling and generation of multisource epithelial reactive oxygen species (ROS) are required for inducible protection. Further investigating the mechanisms underlying this phenomenon of inducible resistance, reverse-phase protein array analysis demonstrated robust STAT3 (signal transducer and activator of transcription 3) phosphorylation following treatment of lung epithelial cells. We show here that Pam2ODN-induced STAT3 phosphorylation is IL-6-independent. We further found that therapeutic epithelial STAT3 activation is required for inducible protection against Pseudomonas aeruginosa pneumonia. Additional studies showed that inhibiting epithelial dual oxidases or scavenging ROS significantly reduced the Pam2ODN induction of STAT3 phosphorylation, suggesting a proximal role for ROS in inducible STAT3 activation. Dissecting these mechanisms, we analyzed the contributions of redox-sensitive kinases and found that Pam2ODN activated epithelial growth factor receptor in an ROS-dependent manner that is required for therapeutically inducible STAT3 activation. Taken together, we demonstrate that epithelial STAT3 is imperative for Pam2ODN's function and describe a novel redox-based mechanism for its activation. These key mechanistic insights may facilitate strategies to leverage inducible epithelial resistance to protect susceptible patients during periods of peak vulnerability.

CALU-3 lung cells three-dimensionally assembled onto CellFate® matrix present angiotensin-converting enzyme-2 activity.

Currently, there is a great need for the development of three-dimensional (3D) in vitro lung models. Particularly, the production of a suitable 3D model of pulmonary epithelium for understanding the pathophysiology of diseases such as the COVID-19 must consider the tissue architecture and presence, for example, of the angiotensin-converting enzyme-2 (ACE-2) in the cells. Different polymeric membranes are being used to support cell culturing, especially of lung cells, however, there is still no information about the culture of these cells onto bacterial nanocellulose (BNC) matrices. We have used the BNC matrix CellFate® as a support for the assembly of a 3D in vitro model of lung epithelium, composed of human lung fibroblasts (HLF) and lung adenocarcinoma cells (CALU-3). CellFate® matrices were made from bacterial fermentation resulting in a natural and biocompatible biopolymer. Cells were cultured onto CellFate® and maintained in a 5% CO2 humidified atmosphere at 37°C. Cell viability was assessed by the resazurin method The samples were, then, exposed to the air-liquid interface (ALI), and histologically analyzed. ACE-2 activity was verified on the hydrolyze of the fluorogenic substrate Mca-APK(Dnp)-OH, and its presence was evaluated by flow cytometry. The expression of the anionic transporter SLCO3A1 was evaluated by qPCR. Cell viability analysis indicates that CellFate® was not toxic to these cells. By flow cytometry, the presence of the ACE-2 was identified in the CALU-3 cells surface corroborating the results obtained from enzymatic activity analysis. The SLCO3A1 transporter expression was identified in cells cultured onto CellFate®, but not in cells cultured onto the transwell (control). CALU-3 cells cultivated onto CellFate® resulted in a pseudostratified organization, a typical morphology of the human respiratory tract epithelium. The current model opens perspectives for studies involving physiological characterization, improving its relevance for the understanding of the pathophysiology of diseases as well as the response to drugs.

The common ABCA3E292V variant disrupts AT2 cell quality control and increases susceptibility to lung injury and aberrant remodeling.

ATP-binding cassette class A3 (ABCA3) is a lipid transporter that plays a critical role in pulmonary surfactant function. The substitution of valine for glutamic acid at codon 292 (E292V) produces a hypomorphic variant that accounts for a significant portion of ABCA3 mutations associated with lung disorders spanning from neonatal respiratory distress syndrome and childhood interstitial lung disease to diffuse parenchymal lung disease (DPLD) in adults including pulmonary fibrosis. The mechanisms by which this and similar ABCA3 mutations disrupt alveolar type 2 (AT2) cell homeostasis and cause DPLD are largely unclear. The present study, informed by a patient homozygous for the E292V variant, used an in vitro and a preclinical murine model to evaluate the mechanisms by which E292V expression promotes aberrant lung injury and parenchymal remodeling. Cell lines stably expressing enhanced green fluorescent protein (EGFP)-tagged ABCA3 isoforms show a functional deficiency of the ABCA3E292V variant as a lipid transporter. AT2 cells isolated from mice constitutively homozygous for ABCA3E292V demonstrate the presence of small electron-dense lamellar bodies, time-dependent alterations in macroautophagy, and induction of apoptosis. These changes in AT2 cell homeostasis are accompanied by a spontaneous lung phenotype consisting of both age-dependent inflammation and fibrillary collagen deposition in alveolar septa. Older ABCA3E292V mice exhibit increased vulnerability to exogenous lung injury by bleomycin. Collectively, these findings support the hypothesis that the ABCA3E292V variant is a susceptibility factor for lung injury through effects on surfactant deficiency and impaired AT2 cell autophagy.

First contact: the role of respiratory cilia in host-pathogen interactions in the airways.

Respiratory cilia are the driving force of the mucociliary escalator, working in conjunction with secreted airway mucus to clear inhaled debris and pathogens from the conducting airways. Respiratory cilia are also one of the first contact points between host and inhaled pathogens. Impaired ciliary function is a common pathological feature in patients with chronic airway diseases, increasing susceptibility to respiratory infections. Common respiratory pathogens, including viruses, bacteria, and fungi, have been shown to target cilia and/or ciliated airway epithelial cells, resulting in a disruption of mucociliary clearance that may facilitate host infection. Despite being an integral component of airway innate immunity, the role of respiratory cilia and their clinical significance during airway infections are still poorly understood. This review examines the expression, structure, and function of respiratory cilia during pathogenic infection of the airways. This review also discusses specific known points of interaction of bacteria, fungi, and viruses with respiratory cilia function. The emerging biological functions of motile cilia relating to intracellular signaling and their potential immunoregulatory roles during infection will also be discussed.

Contribution of mast cells in irritant-induced airway epithelial barrier impairment in vitro.

The airway epithelium is continuously exposed to environmental irritants, which can cause adverse effects such as irritant-induced asthma (IIA). Mast cells are located near airway epithelia and are able to respond to a variety of stimuli. We aimed to investigate whether mast cells influence the response of the epithelium upon irritant exposure. Two cell lines and three different seeding conditions, that is, bronchial epithelial cells (16HBE) only, 16HBE with mast cells (HMC-1's) basolaterally, and 16HBE with HMC-1's apically, were established. Upon exposure to the environmental irritants, graphene (G), graphene oxide (GO), diesel exhaust particles (DEPs) or hypochlorite (ClO-), transepithelial electrical resistance (TEER) and paracellular flux of fluorescent-labeled dextrans were determined, along with the release of mediators. Identical experiments were conducted with the Ca2+ ionophore ionomycin. Exposure to G and GO induced a significant and permanent decrease of approximately 70% in TEER after 3 h of exposure, whereas DEP and ClO- exposure resulted in a transient decrease of approximately 20% in TEER. This response pattern was similar in all the different seeding conditions. After 24 h of exposure, fluorescein isothiocyanate-dextran transport was 10-fold greater for G and 5-fold greater for GO in each of the tested seeding conditions, while DEP and ClO- induced no change compared to the control. Upon exposure to the irritants, 16HBE did not release thymic stromal lymphopoietin, interleukin 33 (IL-33), or IL-1α, and HMC-1 cells did not release histamine, IL-6, or IL-8. Epithelial barrier integrity upon treatment with ionomycin was not affected by the presence of HMC-1 cells. A limited amount of IL-6 and IL-8 was released by ionomycin-exposed HMC-1 cells. To conclude, we found that the studied environmental irritants do not directly or indirectly activate HMC-1 cells. These mast cells did not influence the epithelial barrier function upon environmental exposure, and thus currently do not provide additional information for the underlying mechanism of IIA.

Epithelial IL-6 trans-signaling defines a new asthma phenotype with increased airway inflammation.

BACKGROUND: Although several studies link high levels of IL-6 and soluble IL-6 receptor (sIL-6R) to asthma severity and decreased lung function, the role of IL-6 trans-signaling (IL-6TS) in asthmatic patients is unclear. OBJECTIVE: We sought to explore the association between epithelial IL-6TS pathway activation and molecular and clinical phenotypes in asthmatic patients. METHODS: An IL-6TS gene signature obtained from air-liquid interface cultures of human bronchial epithelial cells stimulated with IL-6 and sIL-6R was used to stratify lung epithelial transcriptomic data (Unbiased Biomarkers in Prediction of Respiratory Disease Outcomes [U-BIOPRED] cohorts) by means of hierarchical clustering. IL-6TS-specific protein markers were used to stratify sputum biomarker data (Wessex cohort). Molecular phenotyping was based on transcriptional profiling of epithelial brushings, pathway analysis, and immunohistochemical analysis of bronchial biopsy specimens. RESULTS: Activation of IL-6TS in air-liquid interface cultures reduced epithelial integrity and induced a specific gene signature enriched in genes associated with airway remodeling. The IL-6TS signature identified a subset of patients with IL-6TS-high asthma with increased epithelial expression of IL-6TS-inducible genes in the absence of systemic inflammation. The IL-6TS-high subset had an overrepresentation of frequent exacerbators, blood eosinophilia, and submucosal infiltration of T cells and macrophages. In bronchial brushings Toll-like receptor pathway genes were upregulated, whereas expression of cell junction genes was reduced. Sputum sIL-6R and IL-6 levels correlated with sputum markers of remodeling and innate immune activation, in particular YKL-40, matrix metalloproteinase 3, macrophage inflammatory protein 1ß, IL-8, and IL-1ß. CONCLUSIONS: Local lung epithelial IL-6TS activation in the absence of type 2 airway inflammation defines a novel subset of asthmatic patients and might drive airway inflammation and epithelial dysfunction in these patients.

Organic Cation Transporters in the Lung-Current and Emerging (Patho)Physiological and Pharmacological Concepts.

Organic cation transporters (OCT) 1, 2 and 3 and novel organic cation transporters (OCTN) 1 and 2 of the solute carrier 22 (SLC22) family are involved in the cellular transport of endogenous compounds such as neurotransmitters, l-carnitine and ergothioneine. OCT/Ns have also been implicated in the transport of xenobiotics across various biological barriers, for example biguanides and histamine receptor antagonists. In addition, several drugs used in the treatment of respiratory disorders are cations at physiological pH and potential substrates of OCT/Ns. OCT/Ns may also be associated with the development of chronic lung diseases such as allergic asthma and chronic obstructive pulmonary disease (COPD) and, thus, are possible new drug targets. As part of the Special Issue "Physiology, Biochemistry and Pharmacology of Transporters for Organic Cations", this review provides an overview of recent findings on the (patho)physiological and pharmacological functions of organic cation transporters in the lung.

Palmitic Acid-Rich High-Fat Diet Exacerbates Experimental Pulmonary Fibrosis by Modulating Endoplasmic Reticulum Stress.

The impact of lipotoxicity on the development of lung fibrosis is unclear. Saturated fatty acids, such as palmitic acid (PA), activate endoplasmic reticulum (ER) stress, a cellular stress response associated with the development of idiopathic pulmonary fibrosis (IPF). We tested the hypothesis that PA increases susceptibility to lung epithelial cell death and experimental fibrosis by modulating ER stress. Total liquid chromatography and mass spectrometry were used to measure fatty acid content in IPF lungs. Wild-type mice were fed a high-fat diet (HFD) rich in PA or a standard diet and subjected to bleomycin-induced lung injury. Lung fibrosis was determined by hydroxyproline content. Mouse lung epithelial cells were treated with PA. ER stress and cell death were assessed by Western blotting, TUNEL staining, and cell viability assays. IPF lungs had a higher level of PA compared with controls. Bleomycin-exposed mice fed an HFD had significantly increased pulmonary fibrosis associated with increased cell death and ER stress compared with those fed a standard diet. PA increased apoptosis and activation of the unfolded protein response in lung epithelial cells. This was attenuated by genetic deletion and chemical inhibition of CD36, a fatty acid transporter. In conclusion, consumption of an HFD rich in saturated fat increases susceptibility to lung fibrosis and ER stress, and PA mediates lung epithelial cell death and ER stress via CD36. These findings demonstrate that lipotoxicity may have a significant impact on the development of lung injury and fibrosis by enhancing pro-death ER stress pathways.

Coordinate regulation of ELF5 and EHF at the chr11p13 CF modifier region.

E74-like factor 5 (ELF5) and ETS-homologous factor (EHF) are epithelial selective ETS family transcription factors (TFs) encoded by genes at chr11p13, a region associated with cystic fibrosis (CF) lung disease severity. EHF controls many key processes in lung epithelial function so its regulatory mechanisms are important. Using CRISPR/Cas9 technology, we removed three key cis-regulatory elements (CREs) from the chr11p13 region and also activated multiple open chromatin sites with CRISPRa in airway epithelial cells. Deletion of the CREs caused subtle changes in chromatin architecture and site-specific increases in EHF and ELF5. CRISPRa had most effect on ELF5 transcription. ELF5 levels are low in airway cells but higher in LNCaP (prostate) and T47D (breast) cancer cells. ATAC-seq in these lines revealed novel peaks of open chromatin at the 5' end of chr11p13 associated with an expressed ELF5 gene. Furthermore, 4C-seq assays identified direct interactions between the active ELF5 promoter and sites within the EHF locus, suggesting coordinate regulation between these TFs. ChIP-seq for ELF5 in T47D cells revealed ELF5 occupancy within EHF introns 1 and 6, and siRNA-mediated depletion of ELF5 enhanced EHF expression. These results define a new role for ELF5 in lung epithelial biology.

Activation of the mTORC1/PGC-1 axis promotes mitochondrial biogenesis and induces cellular senescence in the lung epithelium.

Cellular senescence is a biological process by which cells lose their capacity to proliferate yet remain metabolically active. Although originally considered a protective mechanism to limit the formation of cancer, it is now appreciated that cellular senescence also contributes to the development of disease, including common respiratory ailments such as chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis. While many factors have been linked to the development of cellular senescence, mitochondrial dysfunction has emerged as an important causative factor. In this study, we uncovered that the mitochondrial biogenesis pathway driven by the mammalian target of rapamycin/peroxisome proliferator-activated receptor-γ complex 1α/ß (mTOR/PGC-1α/ß) axis is markedly upregulated in senescent lung epithelial cells. Using two different models, we show that activation of this pathway is associated with other features characteristic of enhanced mitochondrial biogenesis, including elevated number of mitochondrion per cell, increased oxidative phosphorylation, and augmented mitochondrial reactive oxygen species (ROS) production. Furthermore, we found that pharmacological inhibition of the mTORC1 complex with rapamycin not only restored mitochondrial homeostasis but also reduced cellular senescence to bleomycin in lung epithelial cells. Likewise, mitochondrial-specific antioxidant therapy also effectively inhibited mTORC1 activation in these cells while concomitantly reducing mitochondrial biogenesis and cellular senescence. In summary, this study provides a mechanistic link between mitochondrial biogenesis and cellular senescence in lung epithelium and suggests that strategies aimed at blocking the mTORC1/PGC-1α/ß axis or reducing ROS-induced molecular damage could be effective in the treatment of senescence-associated lung diseases.

Quantitative biokinetics over a 28 day period of freshly generated, pristine, 20 nm titanium dioxide nanoparticle aerosols in healthy adult rats after a single two-hour inhalation exposure.

BACKGROUND: Industrially produced quantities of TiO2 nanoparticles are steadily rising, leading to an increasing risk of inhalation exposure for both professionals and consumers. Particle inhalation can result in inflammatory and allergic responses, and there are concerns about other negative health effects from either acute or chronic low-dose exposure. RESULTS: To study the fate of inhaled TiO2-NP, adult rats were exposed to 2-h intra-tracheal inhalations of 48V-radiolabeled, 20 nm TiO2-NP aerosols (deposited NP-mass 1.4 ± 0.5 µg). At five time points (1 h, 4 h, 24 h, 7d, 28d) post-exposure, a complete balance of the [48V]TiO2-NP fate was quantified in organs, tissues, carcass, lavage and body fluids, including excretions. After fast mucociliary airway clearance (fractional range 0.16-0.31), long-term macrophage-mediated clearance (LT-MC) from the alveolar region is 2.6-fold higher after 28d (integral fraction 0.40 ± 0.04) than translocation across the air-blood-barrier (integral fraction 0.15 ± 0.01). A high NP fraction remains in the alveoli (0.44 ± 0.05 after 28d), half of these on the alveolar epithelium and half in interstitial spaces. There is clearance from both retention sites at fractional rates (0.02-0.03 d- 1) by LT-MC. Prior to LT-MC, [48V]TiO2-NP are re-entrained to the epithelium as reported earlier for 20 nm inhaled gold-NP (AuNP) and iridium-NP (IrNP). CONCLUSION: Comparing the 28-day biokinetics patterns of three different inhaled NP materials TiO2-NP, AuNP and IrNP, the long-term kinetics of interstitial relocation and subsequent re-entrainment onto the lung-epithelium is similar for AuNP and Ir-NP but slower than for TiO2-NP. We discuss mechanisms and pathways of NP relocation and re-entrainment versus translocation. Additionally, after 28 days the integral translocated fractions of TiO2-NP and IrNP across the air-blood-barrier (ABB) are similar and become 0.15 while the translocated AuNP fraction is only 0.04. While NP dissolution proved negligible, translocated TiO2-NP and IrNP are predominantly excreted in urine (~ 0.1) while the urinary AuNP excretion amounts to a fraction of only 0.01. Urinary AuNP excretion is below 0.0001 during the first week but rises tenfold thereafter suggesting delayed disagglomeration. Of note, all three NP dissolve minimally, since no ionic radio-label release was detectable. These biokinetics data of inhaled, same-sized NP suggest significant time-dependent differences of the ABB translocation and subsequent fate in the organism.

A transcription factor network represses CFTR gene expression in airway epithelial cells.

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause the inherited disorder cystic fibrosis (CF). Lung disease is the major cause of CF morbidity, though CFTR expression levels are substantially lower in the airway epithelium than in pancreatic duct and intestinal epithelia, which also show compromised function in CF. Recently developed small molecule therapeutics for CF are highly successful for one specific CFTR mutation and have a positive impact on others. However, the low abundance of CFTR transcripts in the airway limits the opportunity for drugs to correct the defective substrate. Elucidation of the transcriptional mechanisms for the CFTR locus has largely focused on intragenic and intergenic tissue-specific enhancers and their activating trans-factors. Here, we investigate whether the low CFTR levels in the airway epithelium result from the recruitment of repressive proteins directly to the locus. Using an siRNA screen to deplete ∼1500 transcription factors (TFs) and associated regulatory proteins in Calu-3 lung epithelial cells, we identified nearly 40 factors that upon depletion elevated CFTR mRNA levels more than 2-fold. A subset of these TFs was validated in primary human bronchial epithelial cells. Among the strongest repressors of airway expression of CFTR were Krüppel-like factor 5 and Ets homologous factor, both of which have pivotal roles in the airway epithelium. Depletion of these factors, which are both recruited to an airway-selective cis-regulatory element at -35 kb from the CFTR promoter, improved CFTR production and function, thus defining novel therapeutic targets for enhancement of CFTR.

Cigarette smoke dampens antiviral signaling in small airway epithelial cells by disrupting TLR3 cleavage.

Cigarette smokers and people exposed to second-hand smoke are at an increased risk for pulmonary viral infections, and yet the mechanism responsible for this heightened susceptibility is not understood. To understand the effect of cigarette smoke on susceptibility to viral infection, we used an air-liquid interface culture system and exposed primary human small airway epithelial cells (SAEC) to whole cigarette smoke, followed by treatment with the viral mimetic polyinosinic polycytidylic acid (poly I:C) or influenza A virus (IAV). We found that prior smoke exposure strongly inhibited production of proinflammatory (interleukin-6 and interleukin-8) and antiviral [interferon-γ-induced protein 10 (IP-10) and interferons] mediators in SAECs in response to poly I:C and IAV infection. Impaired antiviral responses corresponded to increased infection with IAV. This was associated with a decrease in phosphorylation of the key antiviral transcription factor interferon response factor 3 (IRF3). Here, we found that cigarette smoke exposure inhibited activation of Toll-like receptor 3 (TLR3) by impairing TLR3 cleavage, which was required for downstream phosphorylation of IRF3 and production of IP-10. These results identify a novel mechanism by which cigarette smoke exposure impairs antiviral responses in lung epithelial cells, which may contribute to increased susceptibility to respiratory infections.

Peroxisome proliferator-activated receptor-γ agonists attenuate biofilm formation by Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a significant contributor to recalcitrant multidrug-resistant infections, especially in immunocompromised and hospitalized patients. The pathogenic profile of P. aeruginosa is related to its ability to secrete a variety of virulence factors and to promote biofilm formation. Quorum sensing (QS) is a mechanism wherein P. aeruginosa secretes small diffusible molecules, specifically acyl homo serine lactones, such as N-(3-oxo-dodecanoyl)-l-homoserine lactone (3O-C12-HSL), that promote biofilm formation and virulence via interbacterial communication. Strategies that strengthen the host's ability to inhibit bacterial virulence would enhance host defenses and improve the treatment of resistant infections. We have recently shown that peroxisome proliferator-activated receptor γ (PPARγ) agonists are potent immunostimulators that play a pivotal role in host response to virulent P. aeruginosa Here, we show that QS genes in P. aeruginosa (strain PAO1) and 3O-C12-HSL attenuate PPARγ expression in bronchial epithelial cells. PAO1 and 3O-C12-HSL induce barrier derangements in bronchial epithelial cells by lowering the expression of junctional proteins, such as zonula occludens-1, occludin, and claudin-4. Expression of these proteins was restored in cells that were treated with pioglitazone, a PPARγ agonist, before infection with PAO1 and 3O-C12-HSL. Barrier function and bacterial permeation studies that have been performed in primary human epithelial cells showed that PPARγ agonists are able to restore barrier integrity and function that are disrupted by PAO1 and 3O-C12-HSL. Mechanistically, we show that these effects are dependent on the induction of paraoxonase-2, a QS hydrolyzing enzyme, that mitigates the effects of QS molecules. Importantly, our data show that pioglitazone, a PPARγ agonist, significantly inhibits biofilm formation on epithelial cells by a mechanism that is mediated via paraoxonase-2. These findings elucidate a novel role for PPARγ in host defense against P. aeruginosa Strategies that activate PPARγ can provide a therapeutic complement for treatment of resistant P. aeruginosa infections.-Bedi, B., Maurice, N. M., Ciavatta, V. T., Lynn, K. S., Yuan, Z., Molina, S. A., Joo, M., Tyor, W. R., Goldberg, J. B., Koval, M., Hart, C. M., Sadikot, R. T. Peroxisome proliferator-activated receptor-γ agonists attenuate biofilm formation by Pseudomonas aeruginosa.

Glucocorticoid Receptor Binding Inhibits an Intronic IL33 Enhancer and is Disrupted by rs4742170 (T) Allele Associated with Specific Wheezing Phenotype in Early Childhood.

Interleukin 33 (IL-33) is a cytokine constitutively expressed by various cells of barrier tissues that contribute to the development of inflammatory immune responses. According to its function as an alarmin secreted by lung and airway epithelium, IL-33 plays a significant role in pathogenesis of allergic disorders. IL-33 is strongly involved in the pathogenesis of asthma, anaphylaxis, allergy and dermatitis, and genetic variations in IL33 locus are associated with increased susceptibility to asthma. Genome-wide association studies have identified risk "T" allele of the single-nucleotide polymorphism rs4742170 located in putative IL33 enhancer area as susceptible variant for development of specific wheezing phenotype in early childhood. Here, we demonstrate that risk "T" rs4742170 allele disrupts binding of glucocorticoid receptor (GR) transcription factor to IL33 putative enhancer. The IL33 promoter/enhancer constructs containing either 4742170 (T) allele or point mutations in the GR-binding site, were significantly more active and did not respond to cortisol in a pulmonary epithelial cell line. At the same time, the constructs containing rs4742170 (C) allele with a functional GR-binding site were less active and further inhibitable by cortisol. The latter effect was GR-dependent as it was completely abolished by GR-specific siRNA. This mechanism may explain the negative effect of the rs4742170 (T) risk allele on the development of wheezing phenotype that strongly correlates with allergic sensitization in childhood.

The Risk G Allele of the Single-Nucleotide Polymorphism rs928413 Creates a CREB1-Binding Site That Activates IL33 Promoter in Lung Epithelial Cells.

Cytokine interleukin 33 (IL-33) is constitutively expressed by epithelial barrier cells, and promotes the development of humoral immune responses. Along with other proinflammatory mediators released by the epithelium of airways and lungs, it plays an important role in a number of respiratory pathologies. In particular, IL-33 significantly contributes to pathogenesis of allergy and asthma; genetic variations in the IL33 locus are associated with increased susceptibility to asthma. Large-scale genome-wide association studies have identified minor "G" allele of the single-nucleotide polymorphism rs928413, located in the IL33 promoter area, as a susceptible variant for early childhood and atopic asthma development. Here, we demonstrate that the rs928413(G) allele creates a binding site for the cAMP response element-binding protein 1 (CREB1) transcription factor. In a pulmonary epithelial cell line, activation of CREB1, presumably via the p38 mitogen-activated protein kinases (MAPK) cascade, activates the IL33 promoter containing the rs928413(G) allele specifically and in a CREB1-dependent manner. This mechanism may explain the negative effect of the rs928413 minor "G" allele on asthma development.