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Airway epithelial cells arrayed in the inner lining of the airways of the lung are believed to be the major source for the development of malignancy of the lung. The advent of in vitro cell culture model made it easy to understand the molecular mechanism of carcinogenesis at a cellular level, where the airway epithelial cells are cultured on a 2D surface submerged in the culture media. However, this method of culturing airway epithelial cells does not reflect their true nature, and thus results obtained have their limitations. Further, they exhibit dissimilar morphology, transcriptome, and secretome when compared to the cells in vivo. Thus, the experimental data obtained from 2D culture models are inconclusive and, in most cases, could not be validated further in in vivo settings. These limitations can be addressed by culturing the airway epithelial cells on air-liquid interface (ALI), where they develop ciliated morphology similar to that of the lung. Experiments performed with this 3D model provide reliable data that are more realistic, and, in many cases, could replace the requirement of further in vivo validation. Here, we provide the detailed protocol of a 3D culture system called ALI culture for growing human-derived primary small airway epithelial cells to study the cellular and molecular changes associated with lung cancer., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

Objective: Cysteinyl leukotrienes (CysLTs), a group of inflammatory lipid mediators, are found elevated in obese-asthmatic patients. Leukotriene D 4 (LTD 4 ), a representative CysLT, is implicated in promoting lung inflammation and remodelling in allergic asthma, but its role in non-allergic asthma, especially in obese-asthmatic patients, is not known. Here, using primary human small airway epithelial cells (SAECs) we have investigated the mechanism of LTD 4 -induced inflammation and remodelling and assessed high proneness of obese mice to develop asthma upon challenge with allergen ovalbumin (OVA)., Methods: Primary human small airway epithelial cells (SAECs) were stimulated with different concentrations of LTD 4 for different time intervals and various inflammatory markers were measured through cytokine array, membrane-based ELISA and Western blotting. An air-liquid interface (ALI) model of SAECs was used to study the effects of LTD 4 -induced remodelling in SAECs using Western blotting, H&E staining and PAS staining. Further, OVA-based murine model was used to examine the propensity of high-fat diet (HFD)-fed obese mice to develop asthma symptoms by studying the infiltration of inflammatory cells (assessed by bronchioalveolar lavage (BAL) cytology) and airway remodelling (assessed by histopathology) upon allergen exposure., Results: The human primary small airway epithelial cells (SAECs) treated with LTD 4 showed significant alterations in the levels of inflammatory markers such as GM-CSF, TNF-α, IL-1β, EGF and eotaxin in dose- and time-dependent manner. Further, LTD 4 enhanced the activation of inflammasomes as evidenced by increased levels of NALP3, cleaved caspase-1 and IL-1β. LTD 4 also enhanced inflammation by increasing the expression of COX-2 in SAECs. The airway remodelling markers Vimentin and Muc5AC were found elevated in ALI culture of SAECs when stimulated with LTD 4 , as it also increased TGF-β levels and activation of Smad2/3 phosphorylation in SAECs. Last, sensitization and challenge of HFD-fed obese mice with OVA showed increased infiltration of inflammatory cells in BAL and enhanced levels of remodeling phenotypes like loss of cilia, mucus cell metaplasia and collagen deposition in mice lung tissues., Conclusion: The results suggest that LTD 4 could induce inflammatory response in human airway epithelial cell by activating NALP3 inflammasome. LTD 4 could further promote airway epithelial cells' remodelling through TGF-β/smad2/3-mediated pathway. Our in vivo results suggested that obesity predisposed the OVA challenged mice to develop lung inflammation and remodelling akin to asthma-like phenotypes during obesity.

Background: Cysteinyl leukotrienes (CysLTs), the potent lipid inflammatory mediators, are elevated in many pathological conditions and implicated in various inflammatory diseases including asthma, however their role in airway epithelial cells modulation is not clearly understood. We have investigated the effects of a CysLT, Leukotriene D 4 (LTD 4 ) on human airway epithelial cells, and assessed its role and mode of action in these cells., Methodology: Human small airway epithelial cells (SAECs) and A549 cells were incubated with different concentrations of LTD 4 for different time intervals. Subsequently trypan blue dye exclusion assay, MTT assay, Western blotting, RT-PCR and immunofluorescence experiments were performed to examine the effects of LTD 4 on proliferation and related molecular changes in the airway epithelial cells., Results: The treatment of human airway epithelial cells with LTD 4 resulted in a significant increase in cell proliferation and modulation in the expression of receptors, CysLT 1 R and CysLT 2 R in SAECs as well as A549 cells. In both types of cells, LTD 4 increased the expression levels of PCNA and c-myc, and trans-activated EGF receptor and increased the activation of ERK1/2. When treated along with epidermal growth factor (EGF), LTD 4 showed a marginal additive effect in ERK1/2 and EGFR phosphorylation compared to LTD 4 alone in both types of airway epithelial cells., Conclusion: In conclusion, these results suggest that sustained presence of lipid inflammatory mediator LTD 4 could induce human airway epithelial cell proliferation through ERK1/2 phosphorylation, either directly via CysLT 1 receptor or by transactivating EGFR., (Copyright © 2018 Elsevier Inc. All rights reserved.)