Long-Term Culture Captures Injury-Repair Cycles of Colonic Stem Cells.

The colonic epithelium can undergo multiple rounds of damage and repair, often in response to excessive inflammation. The responsive stem cell that mediates this process is unclear, in part because of a lack of in vitro models that recapitulate key epithelial changes that occur in vivo during damage and repair. Here, we identify a Hopx+ colitis-associated regenerative stem cell (CARSC) population that functionally contributes to mucosal repair in mouse models of colitis. Hopx+ CARSCs, enriched for fetal-like markers, transiently arose from hypertrophic crypts known to facilitate regeneration. Importantly, we established a long-term, self-organizing two-dimensional (2D) epithelial monolayer system to model the regenerative properties and responses of Hopx+ CARSCs. This system can reenact the "homeostasis-injury-regeneration" cycles of epithelial alterations that occur in vivo. Using this system, we found that hypoxia and endoplasmic reticulum stress, insults commonly present in inflammatory bowel diseases, mediated the cyclic switch of cellular status in this process.

Cell Culture Differentiation and Proliferation Conditions Influence the In Vitro Regeneration of the Human Airway Epithelium.

The human airway mucociliary epithelium can be recapitulated in vitro using primary cells cultured in an air-liquid interface (ALI), a reliable surrogate to perform pathophysiological studies. As tremendous variations exist among media used for ALI-cultured human airway epithelial cells, the aim of our study was to evaluate the impact of several media (BEGM, PneumaCult, Half & Half, and Clancy) on cell type distribution using single-cell RNA sequencing and imaging. Our work revealed the impact of these media on cell composition, gene expression profile, cell signaling, and epithelial morphology. We found higher proportions of multiciliated cells in PneumaCult-ALI and Half & Half, stronger EGF signaling from basal cells in BEGM-ALI, differential expression of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) entry factor ACE2, and distinct secretome transcripts depending on the media used. We also established that proliferation in PneumaCult-Ex Plus favored secretory cell fate, showing the key influence of proliferation media on late differentiation epithelial characteristics. Altogether, our data offer a comprehensive repertoire for evaluating the effects of culture conditions on airway epithelial differentiation and will aid in choosing the most relevant medium according to the processes to be investigated, such as cilia, mucus biology, or viral infection. We detail useful parameters that should be explored to document airway epithelial cell fate and morphology.

Investigation of CFTR Function in Human Nasal Epithelial Cells Informs Personalized Medicine.

We broaden the clinical versatility of human nasal epithelial (HNE) cells. HNEs were isolated from 10 participants harboring CFTR variants: nine with rare variants (Q359R [n=2], G480S, R334W [n=5], and R560T) and one person harboring R117H;7T;TG10/5T;TG12. Cultures were differentiated at air-liquid interface. CFTR function was measured in Ussing chambers at three conditions - baseline, ivacaftor, and elexacaftor+tezacaftor+ivacaftor (ETI). Four participants initiated modulators. Q359R HNEs had 5.4% (%WT) baseline CFTR function and 25.5% with ivacaftor. With therapy, sweat [Cl-] decreased and symptoms resolved. G480S HNEs had 4.1% baseline and 32.1% CFTR function with ETI. Clinically, FEV1 increased and sweat [Cl-] decreased (119 to 46mmol/L) with ETI. In vitro cultures derived from five individuals harboring R334W showed a moderate increase in CFTR function with exposure to modulators. For one of these participants, ETI was begun in vivo; symptoms and FEV1 improved. c.1679G>C (R560T) HNEs had <4% baseline CFTR function and no modulator response. RNA analysis confirmed that c.1679G>C completely mis-splices. A symptomatic patient harboring R117H;7T;TG10/5T;TG12 exhibited reduced CFTR function (17.5%) in HNEs, facilitating mild CF diagnosis. HNEs responded to modulators (ivacaftor: 32.8%, ETI: 55.5%) and, since beginning therapy, lung function improved. While reaffirming HNE use for guiding therapeutic approaches, we inform predictions on modulator response (e.g. R334W) and closely assess variants affecting splicing (e.g. c.1679G>C). Notably, functional studies in HNEs harboring R117H;7T;TG10/5T;TG12 facilitated mild CF diagnosis, suggesting use for HNE functional studies as a clinical diagnostic test.

A gel-coated air-liquid-interface culture system with tunable substrate stiffness matching healthy and diseased lung tissues.

Since its invention in the late 1980s, the air-liquid-interface (ALI) culture system has been the standard in vitro model for studying human airway biology and pulmonary diseases. However, in a conventional ALI system, cells are cultured on a porous plastic membrane that is much stiffer than human airway tissues. Here, we develop a gel-ALI culture system by simply coating the plastic membrane with a thin layer of hydrogel with tunable stiffness matching that of healthy and fibrotic airway tissues. We determine the optimum gel thickness that does not impair the transport of nutrients and biomolecules essential to cell growth. We show that the gel-ALI system allows human bronchial epithelial cells (HBECs) to proliferate and differentiate into pseudostratified epithelium. Furthermore, we discover that HBECs migrate significantly faster on hydrogel substrates with stiffness matching that of fibrotic lung tissues, highlighting the importance of mechanical cues in human airway remodeling. The developed gel-ALI system provides a facile approach to studying the effects of mechanical cues in human airway biology and in modeling pulmonary diseases.NEW & NOTEWORTHY In a conventional ALI system, cells are cultured on a plastic membrane that is much stiffer than human airway tissues. We develop a gel-ALI system by coating the plastic membrane with a thin layer of hydrogel with tunable stiffness matching that of healthy and fibrotic airway tissues. We discover that human bronchial epithelial cells migrate significantly faster on hydrogel substrates with pathological stiffness, highlighting the importance of mechanical cues in human airway remodeling.

Pan-antiviral effects of a PIKfyve inhibitor on respiratory virus infection in human nasal epithelium and mice.

Endocytosis, or internalization through endosomes, is a major cell entry mechanism used by respiratory viruses. Phosphoinositide 5-kinase (PIKfyve) is a critical enzyme for the synthesis of phosphatidylinositol (3, 5)biphosphate (PtdIns (3, 5)P2) and has been implicated in virus trafficking via the endocytic pathway. In fact, antiviral effects of PIKfyve inhibitors against SARS-CoV-2 and Ebola have been reported, but there is little evidence regarding other respiratory viruses. In this study, we demonstrated the antiviral effects of PIKfyve inhibitors on influenza virus and respiratory syncytial virus in vitro and in vivo. PIKfyve inhibitors Apilimod mesylate (AM) and YM201636 concentration-dependently inhibited several influenza strains in an MDCK cell-cytopathic assay. AM also reduced the viral load and cytokine release, while improving the cell integrity of human nasal air-liquid interface cultured epithelium infected with influenza PR8. In PR8-infected mice, AM (2 mg/mL), when intranasally treated, exhibited a significant reduction of viral load and inflammation and inhibited weight loss caused by influenza infection, with effects being similar to oral oseltamivir (10 mg/kg). In addition, AM demonstrated antiviral effects in RSV A2-infected human nasal epithelium in vitro and mouse in vivo, with an equivalent effect to that of ribavirin. AM also showed antiviral effects against human rhinovirus and seasonal coronavirus in vitro. Thus, PIKfyve is found to be involved in influenza and RSV infection, and PIKfyve inhibitor is a promising molecule for a pan-viral approach against respiratory viruses.

H5N1 clade 2.3.4.4b avian influenza viruses replicate in differentiated bovine airway epithelial cells cultured at air-liquid interface.

Highly pathogenic avian influenza (HPAI) H5N1 viruses are responsible for disease outbreaks in wild birds and poultry, resulting in devastating losses to the poultry sector. Since 2020, an increasing number of outbreaks of HPAI H5N1 was seen in wild birds. Infections in mammals have become more common, in most cases in carnivores after direct contact with infected birds. Although ruminants were previously not considered a host species for HPAI viruses, in March 2024 multiple outbreaks of HPAI H5N1 were detected in goats and cattle in the United States. Here, we have used primary bronchus-derived well-differentiated bovine airway epithelial cells (WD-AECs) cultured at air-liquid interface to assess the susceptibility and permissiveness of bovine epithelial cells to infection with European H5N1 virus isolates. We inoculated bovine WD-AECs with three low-passage HPAI clade 2.3.4.4b H5N1 virus isolates and detected rapid increases in viral genome loads and infectious virus during the first 24 h post-inoculation, without substantial cytopathogenic effects. Three days post-inoculation infected cells were still detectable by immunofluorescent staining. These data indicate that multiple lineages of HPAI H5N1 may have the propensity to infect the respiratory tract of cattle and support extension of avian influenza surveillance efforts to ruminants. Furthermore, this study underscores the benefit of WD-AEC cultures for pandemic preparedness by providing a rapid and animal-free assessment of the host range of an emerging pathogen.

Novel air-liquid interface culture model to investigate stiffness-dependent behaviors of alveolar epithelial cells.

Pulmonary alveoli are functional units in gas exchange in the lung, and their dysfunctions in lung diseases such as interstitial pneumonia are accompanied by fibrotic changes in structure, elevating the stiffness of extracellular matrix components. The present study aimed to test the hypothesis that such changes in alveoli stiffness induce functional alteration of epithelial cell functions, exacerbating lung diseases. For this, we have developed a novel method of culturing alveolar epithelial cells on polyacrylamide gel with different elastic modulus at an air-liquid interface. It was demonstrated that A549 cells on soft gels, mimicking the modulus of a healthy lung, upregulated mRNA expression and protein synthesis of surfactant protein C (SFTPC). By contrast, the cells on stiff gels, mimicking the modulus of the fibrotic lung, exhibited upregulation of SFTPC gene expression but not at the protein level. Cell morphology, as well as cell nucleus volume, were also different between the two types of gels.

Boron Nitride Nanosheets Induce Lipid Accumulation and Autophagy in Human Alveolar Lung Epithelial Cells Cultivated at Air-Liquid Interface.

Hexagonal boron nitride (hBN) is an emerging 2D material attracting significant attention due to its superior electrical, chemical, and therapeutic properties. However, inhalation toxicity mechanisms of hBN in human lung cells are poorly understood. Here, cellular interaction and effects of hBN nanosheets is investigated in alveolar epithelial cells cultured on porous inserts and exposed under air-liquid interface conditions for 24 h. hBN is taken up by the cells as determined in a label-free manner via RAMAN-confocal microscopy, ICP-MS, TEM, and SEM-EDX. No significant (p > 0.05) effects are observed on cell membrane integrity (LDH release), epithelial barrier integrity (TEER), interleukin-8 cytokine production or reactive oxygen production at tested dose ranges (1, 5, and 10 µg cm-2). However, it is observed that an enhanced accumulation of lipid granules in cells indicating the effect of hBN on lipid metabolism. In addition, it is observed that a significant (p < 0.05) and dose-dependent (5 and 10 µg cm-2) induction of autophagy in cells after exposure to hBN, potentially associated with the downstream processing and breakdown of excess lipid granules to maintain lipid homeostasis. Indeed, lysosomal co-localization of lipid granules supporting this argument is observed. Overall, the results suggest that the continuous presence of excess intracellular lipids may provoke adverse outcomes in the lungs.

Collagen Tubular Airway-on-Chip for Extended Epithelial Culture and Investigation of Ventilation Dynamics.

The lower respiratory tract is a hierarchical network of compliant tubular structures that are made from extracellular matrix proteins with a wall lined by an epithelium. While microfluidic airway-on-a-chip models incorporate the effects of shear and stretch on the epithelium, week-long air-liquid-interface culture at physiological shear stresses, the circular cross-section, and compliance of native airway walls have yet to be recapitulated. To overcome these limitations, a collagen tube-based airway model is presented. The lumen is lined with a confluent epithelium during two-week continuous perfusion with warm, humid air while presenting culture medium from the outside and compensating for evaporation. The model recapitulates human small airways in extracellular matrix composition and mechanical microenvironment, allowing for the first time dynamic studies of elastocapillary phenomena associated with regular breathing and mechanical ventilation, as well as their impacts on the epithelium. A case study reveales increasing damage to the epithelium during repetitive collapse and reopening cycles as opposed to overdistension, suggesting expiratory flow resistance to reduce atelectasis. The model is expected to promote systematic comparisons between different clinically used ventilation strategies and, more broadly, to enhance human organ-on-a-chip platforms for a variety of tubular tissues.

Oxygen levels affect oviduct epithelium functions in air-liquid interface culture.

Key reproductive events such as fertilization and early embryonic development occur in the lumen of the oviduct. Since investigating these processes in vivo is both technically challenging and ethically sensitive, cell culture models have been established to reproduce the oviductal microenvironment. Compartmentalized culture systems, particularly air-liquid interface cultures (ALI; cells access the culture medium only from the basolateral cell side), result in highly differentiated oviduct epithelial cell cultures. The oxygen (O2) tension within the oviduct is 4-10% across species, and its reduced O2 content is presumed to be important for early reproductive processes. However, cell culture models of the oviduct are typically cultivated without O2 regulation and therefore at about 18% O2. To investigate the impact of O2 levels on oviduct epithelium functions in vitro, we cultured porcine oviduct epithelial cells (POEC) at the ALI using both physiological (5%) and supraphysiological (18%) O2 levels and two different media regimes. Epithelium architecture, barrier function, secretion of oviduct fluid surrogate (OFS), and marker gene expression were comparatively assessed. Under all culture conditions, ALI-POEC formed polarized, ciliated monolayers with appropriate barrier function. Exposure to 18% O2 accelerated epithelial differentiation and significantly increased the apical OFS volume and total protein content. Expression of oviduct genes and the abundance of OVGP1 (oviduct-specific glycoprotein 1) in the OFS were influenced by both O2 tension and medium choice. In conclusion, oviduct epithelial cells can adapt to a supraphysiological O2 environment. This adaptation, however, may alter their capability to replicate in vivo tissue characteristics.

Revealing the pathogenesis of gastric intestinal metaplasia based on the mucosoid air-liquid interface.

BACKGROUND: Gastric intestinal metaplasia (GIM) is an essential precancerous lesion. Although the reversal of GIM is challenging, it potentially brings a state-to-art strategy for gastric cancer therapeutics (GC). The lack of the appropriate in vitro model limits studies of GIM pathogenesis, which is the issue this work aims to address for further studies. METHOD: The air-liquid interface (ALI) model was adopted for the long-term culture of GIM cells in the present work. This study conducted Immunofluorescence (IF), quantitative real-time polymerase chain reaction (qRT-PCR), transcriptomic sequencing, and mucoproteomic sequencing (MS) techniques to identify the pathways for differential expressed genes (DEGs) enrichment among different groups, furthermore, to verify novel biomarkers of GIM cells. RESULT: Our study suggests that GIM-ALI model is analog to the innate GIM cells, which thus can be used for mucus collection and drug screening. We found genes MUC17, CDA, TRIM15, TBX3, FLVCR2, ONECUT2, ACY3, NMUR2, and MAL2 were highly expressed in GIM cells, while GLDN, SLC5A5, MAL, and MALAT1 showed down-regulated, which can be used as potential biomarkers for GIM cells. In parallel, these genes that highly expressed in GIM samples were mainly involved in cancer-related pathways, such as the MAPK signal pathway and oxidative phosphorylation signal pathway. CONCLUSION: The ALI model is validated for the first time for the in vitro study of GIM. GIM-ALI model is a novel in vitro model that can mimic the tissue micro-environment in GIM patients and further provide an avenue for studying the characteristics of GIM mucus. Our study identified new markers of GIM as well as pathways associated with GIM, which provides outstanding insight for exploring GIM pathogenesis and potentially other related conditions.

In vitro toxicological evaluation of aerosols generated by a 4th generation vaping device using nicotine salts in an air-liquid interface system.

BACKGROUND: Electronic cigarettes (EC) have gained popularity, especially among young people, with the introduction of fourth-generation devices based on e-liquids containing nicotine salts that promise a smoother vaping experience than freebase nicotine. However, the toxicological effects of nicotine salts are still largely unknown, and the chemical diversity of e-liquids limits the comparison between different studies to determine the contribution of each compound to the cytotoxicity of EC aerosols. Therefore, the aim of this study was to evaluate the toxicological profile of controlled composition e-liquid aerosols to accurately determine the effects of each ingredient based on exposure at the air-liquid interface. METHODS: Human lung epithelial cells (A549) were exposed to undiluted aerosols of controlled composition e-liquids containing various ratios of propylene glycol (PG)/vegetable glycerin (VG) solvents, freebase nicotine, organic acids, nicotine salts, and flavoured commercial e-liquids. Exposure of 20 puffs was performed at the air-liquid interface following a standard vaping regimen. Toxicological outcomes, including cytotoxicity, inflammation, and oxidative stress, were assessed 24 h after exposure. RESULTS: PG/VG aerosols elicited a strong cytotoxic response characterised by a 50% decrease in cell viability and a 200% increase in lactate dehydrogenase (LDH) production, but had no effects on inflammation and oxidative stress. These effects occurred only at a ratio of 70/30 PG/VG, suggesting that PG is the major contributor to aerosol cytotoxicity. Both freebase nicotine and organic acids had no greater effect on cell viability and LDH release than at a 70/30 PG/VG ratio, but significantly increased inflammation and oxidative stress. Interestingly, the protonated form of nicotine in salt showed a stronger proinflammatory effect than the freebase nicotine form, while benzoic acid-based nicotine salts also induced significant oxidative stress. Flavoured commercial e-liquids was found to be cytotoxic at a threshold dose of ≈ 330 µg/cm². CONCLUSION: Our results showed that aerosols of e-liquids consisting only of PG/VG solvents can cause severe cytotoxicity depending on the concentration of PG, while nicotine salts elicit a stronger pro-inflammatory response than freebase nicotine. Overall, aerosols from fourth-generation devices can cause different toxicological effects, the nature of which depends on the chemical composition of the e-liquid.

An experimental and modeling approach to describe the deactivation of cellulases at the air-liquid interface.

Understanding the reaction mechanisms involved in the enzymatic hydrolysis of cellulose is important because it is kinetically the most limiting step of the bioethanol production process. The present work focuses on the enzymatic deactivation at the air-liquid interface, which is one of the aspects contributing to this global deactivation. This phenomenon has already been experimentally proven, but this is the first time that a model has been proposed to describe it. Experiments were performed by incubating Celluclast cocktail solutions on an orbital stirring system at different enzyme concentrations and different surface-to-volume ratios. A 5-day follow-up was carried out by measuring the global FPase activity of cellulases for each condition tested. The activity loss was proven to depend on both the air-liquid surface area and the enzyme concentration. Both observations suggest that the loss of activity takes place at the air-liquid surface, the total amount of enzymes varying with volume or enzyme concentration. Furthermore, tests performed using five individual enzymes purified from a Trichoderma reesei cocktail showed that the only cellulase that is deactivated at the air-liquid interface is cellobiohydrolase II. From the experimental data collected by varying the initial enzyme concentration and the ratio surface to volume, it was possible to develop, for the first time, a model that describes the loss of activity at the air-liquid interface for this configuration.

A novel approach to investigate severe asthma and COPD: the 3d ex vivo respiratory mucosa model.

Purpose: This article illustrates the replication of asthma and COPD conditions in a laboratory setting and the potential applications of this methodology.Introduction: Biologic drugs have been shown to enhance the treatment of severe asthma and COPD. Monoclonal antibodies against specific targets have dramatically changed the management of these conditions. Although the inflammatory pathways of asthma and COPD have already been clearly outlined, alternative mechanisms of action remain mostly unexplored. They could provide additional insights into these diseases and their clinical management.Aims: In vivo or in vitro models have thus been developed to test alternative hypotheses. This study describes sophisticated ex vivo models that mimic the response of human respiratory mucosa to disease triggers, aiming to narrow the gap between laboratory studies and clinical practice.Results: These models successfully replicate crucial aspects of these diseases, such as inflammatory cell presence, cytokine production, and changes in tissue structure, offering a dynamic platform for investigating disease processes and evaluating potential treatments, such as monoclonal antibodies. The proposed models have the potential to enhance personalized medicine approaches and patient-specific treatments, helping to advance the understanding and management of respiratory diseases.

Acute exposure to seasonal PM2.5 induces toxicological responses in A549 cells cultured at the air-liquid interface mediated by oxidative stress and endoplasmic reticulum stress.

Atmospheric fine particulate matter (PM2.5) enters the human body through respiration and poses a threat to human health. This is not only dependent on its mass concentration in the atmosphere, but also related to seasonal variations in its chemical components, which makes it important to study the cytotoxicity of PM2.5 in different seasons. Traditional immersion exposure cannot simulate the living environment of human epithelial cells in the human body, making this method unsuitable for evaluating the inhalation toxicity of PM2.5. In this study, a novel air-liquid interface (ALI) particulate matter exposure device (VITROCELL Cloud 12 system) was used to evaluate the toxic effects and potential mechanisms of human lung epithelial cells (A549) after exposure to seasonal PM2.5. PM2.5 samples from four seasons were collected and analyzed for chemical components. After 6 h of exposure to seasonal PM2.5, winter PM2.5 exhibited the highest cytotoxicity among most toxicity indicators, especially apoptosis rate, reactive oxygen species (ROS), inflammatory responses and DNA damage (γ-H2AX). The effect of autumn PM2.5 on apoptosis rate was significantly higher than that in spring, and there was no significant difference in other toxicity indicators between spring and autumn. The cytotoxicity of summer PM2.5 was the lowest among the four seasons. It should be noted that even exposure to low doses of summer PM2.5 leads to significant DNA damage in A459 cells. Correlation analysis results showed that water-soluble ions, metallic elements, and polycyclic aromatic hydrocarbons (PAHs) were associated with most toxicological endpoints. Inhibitors of oxidative stress and endoplasmic reticulum (ER) stress significantly inhibited cellular damage, indicating that PM2.5-induced cytotoxicity may be related to the generation of ROS and ER stress. In addition, PM2.5 can induce ER stress through oxidative stress, which ultimately leads to apoptosis.

Modular air-liquid interface aerosol exposure system (MALIES) to study toxicity of nanoparticle aerosols in 3D-cultured A549 cells in vitro.

We present a novel lung aerosol exposure system named MALIES (modular air-liquid interface exposure system), which allows three-dimensional cultivation of lung epithelial cells in alveolar-like scaffolds (MatriGrids®) and exposure to nanoparticle aerosols. MALIES consists of multiple modular units for aerosol generation, and can be rapidly assembled and commissioned. The MALIES system was proven for its ability to reliably produce a dose-dependent toxicity in A549 cells using CuSO4 aerosol. Cytotoxic effects of BaSO4- and TiO2-nanoparticles were investigated using MALIES with the human lung tumor cell line A549 cultured at the air-liquid interface. Experiments with concentrations of up to 5.93 × 105 (BaSO4) and 1.49 × 106 (TiO2) particles/cm3, resulting in deposited masses of up to 26.6 and 74.0 µg/cm2 were performed using two identical aerosol exposure systems in two different laboratories. LDH, resazurin reduction and total glutathione were measured. A549 cells grown on MatriGrids® form a ZO-1- and E-Cadherin-positive epithelial barrier and produce mucin and surfactant protein. BaSO4-NP in a deposited mass of up to 26.6 µg/cm2 resulted in mild, reversible damage (~ 10% decrease in viability) to lung epithelium 24 h after exposure. TiO2-NP in a deposited mass of up to 74.0 µg/cm2 did not induce any cytotoxicity in A549 cells 24 h and 72 h after exposure, with the exception of a 1.7 fold increase in the low exposure group in laboratory 1. These results are consistent with previous studies showing no significant damage to lung epithelium by short-term treatment with low concentrations of nanoscale BaSO4 and TiO2 in in vitro experiments.

Diesel exhaust particle exposure exacerbates ciliary and epithelial barrier dysfunction in the multiciliated bronchial epithelium models.

Airway epithelium, the first defense barrier of the respiratory system, facilitates mucociliary clearance against inflammatory stimuli, such as pathogens and particulates inhaled into the airway and lung. Inhaled particulate matter 2.5 (PM2.5) can penetrate the alveolar region of the lung, and it can develop and exacerbate respiratory diseases. Although the pathophysiological effects of PM2.5 in the respiratory system are well known, its impact on mucociliary clearance of airway epithelium has yet to be clearly defined. In this study, we used two different 3D in vitro airway models, namely the EpiAirway-full-thickness (FT) model and a normal human bronchial epithelial cell (NHBE)-based air-liquid interface (ALI) system, to investigate the effect of diesel exhaust particles (DEPs) belonging to PM2.5 on mucociliary clearance. RNA-sequencing (RNA-Seq) analyses of EpiAirway-FT exposed to DEPs indicated that DEP-induced differentially expressed genes (DEGs) are related to ciliary and microtubule function and inflammatory-related pathways. The exposure to DEPs significantly decreased the number of ciliated cells and shortened ciliary length. It reduced the expression of cilium-related genes such as acetylated α-tubulin, ARL13B, DNAH5, and DNAL1 in the NHBEs cultured in the ALI system. Furthermore, DEPs significantly increased the expression of MUC5AC, whereas they decreased the expression of epithelial junction proteins, namely, ZO1, Occludin, and E-cadherin. Impairment of mucociliary clearance by DEPs significantly improved the release of epithelial-derived inflammatory and fibrotic mediators such as IL-1ß, IL-6, IL-8, GM-CSF, MMP-1, VEGF, and S100A9. Taken together, it can be speculated that DEPs can cause ciliary dysfunction, hyperplasia of goblet cells, and the disruption of the epithelial barrier, resulting in the hyperproduction of lung injury mediators. Our data strongly suggest that PM2.5 exposure is directly associated with ciliary and epithelial barrier dysfunction and may exacerbate lung injury.

In vitro evaluation of physicochemical-dependent effects of polymeric nanoparticles on their cellular uptake and co-localization using pulmonary calu-3 cell lines.

OBJECTIVE: The study evaluated physicochemical properties of eight different polymeric nanoparticles (NPs) and their interaction with lung barrier and their suitability for pulmonary drug delivery. METHODS: Eight physiochemically different NPs were fabricated from Poly lactic-co-glycolic acid (PLGA, PL) and Poly glycerol adipate-co-ω-pentadecalactone (PGA-co-PDL, PG) via emulsification-solvent evaporation. Pulmonary barrier integrity was investigated in vitro using Calu-3 under air-liquid interface. NPs internalization was investigated using a group of pharmacological inhibitors with subsequent microscopic visual confirmation. RESULTS: Eight NPs were successfully formulated from two polymers using emulsion-solvent evaporation; 200, 500 and 800 nm, negatively-charged and positively-charged. All different NPs did not alter tight junctions and PG NPs showed similar behavior to PL NPs, indicating its suitability for pulmonary drug delivery. Active endocytosis uptake mechanisms with physicochemical dependent manner were observed. In addition, NPs internalization and co-localization with lysosomes were visually confirmed indicating their vesicular transport. CONCLUSION: PG and PL NPs had shown no or low harmful effects on the barrier integrity, and with effective internalization and vesicular transport, thus, prospectively can be designed for pulmonary delivery applications.

Interleukin-13 Treatment of Living Lung Tissue Model Alters the Metabolome and Proteome-A Nano-DESI MS Metabolomics and Shotgun Proteomics Study.

Asthma is a chronic respiratory disease with one of the largest numbers of cases in the world; thus, constant investigation and technical development are needed to unravel the underlying biochemical mechanisms. In this study, we aimed to develop a nano-DESI MS method for the in vivo characterization of the cellular metabolome. Using air-liquid interface (ALI) cell layers, we studied the role of Interleukin-13 (IL-13) on differentiated lung epithelial cells acting as a lung tissue model. We demonstrate the feasibility of nano-DESI MS for the in vivo monitoring of basal-apical molecular transport, and the subsequent endogenous metabolic response, for the first time. Conserving the integrity of the ALI lung-cell layer enabled us to perform temporally resolved metabolomic characterization followed by "bottom-up" proteomics on the same population of cells. Metabolic remodeling was observed upon histamine and corticosteroid treatment of the IL-13-exposed lung cell monolayers, in correlation with alterations in the proteomic profile. This proof of principle study demonstrates the utility of in vivo nano-DESI MS for characterizing ALI tissue layers, and the new markers identified in our study provide a good starting point for future, larger-scale studies.

Preterm ovine respiratory epithelial cell responses to mechanical ventilation, lipopolysaccharide, and interleukin-13.

Mechanical ventilation causes airway injury, respiratory epithelial cell proliferation, and lung inflammation in preterm sheep. Whether preterm epithelial cells respond similarly to adult epithelial cells or are altered by mechanical ventilation is unknown. We test the hypothesis that mechanical ventilation alters the responses of preterm airway epithelium to stimulation in culture. Respiratory epithelial cells from the trachea, left mainstem bronchi (LMSB), and distal bronchioles were harvested from unventilated preterm lambs, ventilated preterm lambs, and adult ewes. Epithelial cells were grown in culture or on air-liquid interface (ALI) and challenged with combinations of either media only, lipopolysaccharide (LPS; 10 ng/mL), bronchoalveolar fluid (BALF), or interleukin-13 (IL-13). Cell lysates were evaluated for mRNA changes in cytokine, cell type markers, Notch pathway, and acute phase markers. Mechanical ventilation altered preterm respiratory epithelium cell types. Preterm respiratory epithelial cells responded to LPS in culture with larger IL-8 induction than adults, and mechanical ventilation further increased cytokines IL-1ß and IL-8 mRNA induction at 2 h. IL-8 protein is detected in cell media after LPS stimulation. The addition of BALF from ventilated preterm animals increased IL-1ß mRNA to LPS (fivefold) in both preterm and adult cells and suppressed IL-8 mRNA (twofold) in adults. Preterm respiratory epithelial cells, when grown on ALI, responded to IL-13 with an increase in goblet cell mRNA. Preterm respiratory epithelial cells responded to LPS and IL-13 with responses similar to adults. Mechanical ventilation or exposure to BALF from mechanically ventilated animals alters the responses to LPS.NEW & NOTEWORTHY Preterm lamb respiratory epithelial cells can be extracted from the trachea and bronchi and frozen, and the preterm cells can respond in culture to stimulation with LPS or IL-13. Brief mechanical ventilation changes the distribution and cell type of preterm respiratory cells toward an adult phenotype, and mechanical ventilation alters the response to LPS in culture. Bronchoalveolar lavage fluid from preterm lambs receiving mechanical ventilation also alters unventilated preterm and adult responses to LPS.