Human Small Airway Epithelial Cells Research Articles

Chlorine inhalation toxicology‐on‐a‐chip

Accidental or environmental exposures to chlorine (Cl2 ) gas represent a major health threat. In particular, inhalation toxicology of Cl2 is an area of active research that focuses on investigating the deleterious effects of Cl2 on the human respiratory system and developing effective countermeasures to mitigate the risk of Cl2 -induced lung injury. While traditional cell culture models have gained widespread use as a simple and convenient research tool for in vitro investigation of Cl2 toxicity, they have limited capacity to recapitulate the complexity of native human lung tissues and thus largely fail to generate accurate predictions on pulmonary responses to Cl2 . Here we present two distinct microengineered biomimetic models that reconstruct the small airway and alveolar regions of the human lung for experimental studies of respiratory toxicity of Cl2 in the distal lung, which remains an outstanding question in inhalation toxicology of Cl2 . Our microengineered small airway model is composed of two microfabricated 3D chambers separated by a semipermeable membrane. The design of this microdevice makes it possible to mimic tissue compartmentalization in native airways by permitting long-term co-culture of primary human small airway epithelial cells, primary human pulmonary microvascular endothelial cells, and lung fibroblasts in a physiological 3D microenvironment to engineer fully differentiated airway epithelium directly exposed to air and supported by vascularized, perfusable 3D stromal tissues. The alveolar model, which is constructed in the same device, uses primary human type II pneumocytes harvested from iPSC-derived human alveolar organoids, some of which are transdifferentiated into type I alveolar epithelial cells during the course of controlled microfluidic culture in our device. In this study, these models were capable of producing human lung tissues that exhibited differentiated phenotype and functional capacity that could be maintained for extended periods (over 1 month). Notably, our data revealed the potential of the underlying pulmonary vasculature to accelerate and promote differentiation and maturation of lung epithelial cells during air-liquid interface culture. Using the lung-on-a-chip systems in conjunction with an in-house Cl2 exposure system, we conducted a preliminary study in which the devices were exposed to various concentrations of Cl2 gas (10, 50, 100 and 300 ppm) for 15 min. Our preliminary data showed the deleterious potential of Cl2 to induce acute injury of both small airway and alveolar tissues in a dose-dependent manner. Studies are underway for more extensive screening of Cl2 toxicity and in-depth analysis of lung injury in our models at the molecular, cellular, and tissue levels. We believe that the microengineered systems developed in this work represent an important advance in our ability to model acute respiratory responses to toxic gases and may provide a potentially powerful in vitro platform to study biochemical threats and develop medical countermeasures against them.

Reversal of NADPH Oxidase-Dependent Early Oxidative and Inflammatory Responses in Chronic Obstructive Pulmonary Disease by Puerarin.

In the present study, we investigated effects of Puerarin on the early oxidative and inflammatory responses in the lung triggered by acute cigarette smoking (ACS). C57BL/6 mice were exposed to ACS for 1 hr in the presence or absence of Puerarin and harvested at 2, 6, and 24 hours. ACS induced significant increases in superoxide production in mouse lungs at 2 and 6 hours; and superoxide production was also elevated in a time and concentration dependent manner in cigarette smoke extract (CSE) stimulated human small airway epithelial cells (HSAECs), which was dose-dependently abrogated by Puerarin. ACS exposure upregulated NOX1, NOX2, and NOX4 protein expression in mouse lungs. Likewise, NOX1 and NOX4 were upregulated in CSE-stimulated HSAECs. These responses were significantly or completely attenuated by Puerarin. ACS induced significant infiltrations of neutrophils and macrophages in mouse lung parenchyma and BAL fluid, which were completely or significantly abrogated by Puerarin, so was the activation of the NF-кB pathway and the upregulation in inflammatory mediators including TNF-α, KC (murine homolog of IL-8), COX-2, IL-6 and MCP-1. Nuclear translocation of p65, IL-8 secretion, and upregulation of COX-2 in CSE stimulated HSAECs were also markedly attenuated by Puerarin. Moreover, ACS or CSE stimulated upregulation in reactive oxygen species (ROS) production and expression of inflammatory mediators were alleviated by ROS scavenger TEMPO in vivo and vitro, with no synergy combining with Puerarin, indicating that the effects of Puerarin are redox-sensitive following activation of NOX. In summary, our data for the first time demonstrate that Puerarin robustly attenuates NOX isoform-dependent ROS production and inflammatory activation in ACS exposed mice and CSE treated HSAECs, indicating that Puerarin might be used as a robust therapeutic agent for early or early stage COPD.

Junin Virus Activates p38 MAPK and HSP27 Upon Entry.

Junín virus (JUNV), a New World arenavirus, is a rodent-borne virus and the causative agent of Argentine hemorrhagic fever. Humans become infected through exposure to rodent host secreta and excreta and the resulting infection can lead to an acute inflammatory disease with significant morbidity and mortality. Little is understood about the molecular pathogenesis of arenavirus hemorrhagic fever infections. We utilized Reverse Phase Protein Microarrays (RPPA) to compare global alterations in the host proteome following infection with an attenuated vaccine strain, Candid#1 (CD1), and the most parental virulent strain, XJ13, of JUNV in a human cell culture line. Human small airway epithelial cells were infected with CD1 or XJ13 at an MOI of 10, or mock infected. To determine proteomic changes at early timepoints (T = 1, 3, 8 and 24 h), the JUNV infected or mock infected cells were lysed in compatible buffers for RPPA. Out of 113 proteins that were examined by RPPA, 14 proteins were significantly altered following JUNV infection. Several proteins were commonly phosphorylated between the two strains and these correspond to entry and early replication events, to include p38 mitogen-activated protein kinase (MAPK), heat shock protein 27 (HSP27), and nuclear factor kappa B (NFκB). We qualitatively confirmed the alterations of these three proteins following infection by western blot analysis. We also determined that the inhibition of either p38 MAPK, with the small molecule inhibitor SB 203580 or siRNA knockdown, or HSP27, by siRNA knockdown, significantly decreases JUNV replication. Our data suggests that HSP27 phosphorylation at S82 upon virus infection is dependent on p38 MAPK activity. This work sheds light on the nuances of arenavirus replication.

Silencing of long non-coding RNA ZFAS1 alleviates LPS-induced acute lung injury by mediating the miR-96-5p/OXSR1 axis in sepsis

BackgroundExtensive studies have revealed that long non-coding RNAs (lncRNAs) are associated with sepsis-induced acute lung injury (ALI). This study focused on the function and potential mechanisms of lncRNA zinc finger antisense 1 (ZFAS1) in a cell model of sepsis-induced ALI. MethodsTo induce sepsis-induced ALI in vitro and in vivo, mice were subjected to cecal ligation and puncture (CLP) operation, and human small airway epithelial cells (HSAECs) were stimulated with lipopolysaccharide (LPS) (10 μg/mL). Relative expression of oxidative stress-responsive 1 (OXSR1), lncRNA ZFAS1, and microRNA (miR)-96-5p was detected by quantitative real-time polymerase chain reaction (qRT-PCR). Relative protein expression of Bax, Bcl-2, and OXSR1 was determined by western blotting. Moreover, enzyme-linked immunosorbent assay was used to measure the levels of IL-6, IL-1β, and TNF-α. A dual-luciferase reporter assay was conducted to test the targeting interplay between ZFAS1/OXSR1 and miR-96-5p. ResultsUp-regulation of lncRNA ZFAS1 and OXSR1 and down-regulation of miR-96-5p was observed in lung tissues of CLP-induced mice and LPS-treated HSAECs. Decreased ZFAS1 expression or increased miR-96-5p expression repressed inflammation and apoptosis and promoted cell viability in LPS-treated HSAECs. The lncRNA ZFAS1 competitively binds to miR-96-5p and inversely modulates miR-96-5p expression. MiR-96-5p directly targets OXSR1 and inversely regulates OXSR1 expression. In addition, the protective effects of ZFAS1 knockdown on LPS-induced HSAECs were reversed by miR-96-5p inhibition or OXSR1 overexpression. ConclusionsDown-regulation of lncRNA ZFAS1 attenuated LPS-induced ALI in HSAECs by regulating the miR-96-5p/OXSR1 axis.

IDF21-0230 Anti-inflammatory activity of glyoxalase-1 inducer supports further evaluation for prevention of COVID-19

Background: Pre-clinical and clinical studies suggest an essential role of proteases, plasma membrane-associated transmembrane protease serine 2 (TMPRSS2) and furin, in virion fusion with lung alveolar epithelial cells for viral infectivity of coronavirus SARS-CoV-2. Angiotensin converting enzyme-2 (ACE2) and glucose regulated protein-78 (GRP78) are considered to be receptors for cell surface binding of the spike protein of SARS-CoV-2. Risk of progression of associated COVID-19 disease to severe symptoms and risk of mortality is linked to increased levels of inflammatory mediators, including monocyte chemoattract protein-1 (MCP-1) and interleukin-8 (IL8), which are also likely risk factors for severe, life-threatening disease. Increased MCP-1 and IL8 in obese subjects and patients with diabetes may contribute to poor clinical outcomes when infected with coronavirus. In a double blind placebo-controlled clinical crossover study in overweight and obese subjects, treatment with Glo1 inducer, trans-resveratrol and hesperetin combination (tRES-HESP), once daily by oral capsule dietary supplement for 8 weeks, decreased expression of MCP-1, IL8, the receptor for advanced glycation endproducts (RAGE) and cyclo-oxygenase-2 (COX-2) in peripheral blood mononuclear cells (PBMCs). Placebo had no effect. We hypothesized that tRES-HESP may decrease protease and inflammatory gene expression linked to SARS-CoV-2 infection and COVID-19 progression. If so, the anti-inflammatory activity of Glo1 inducer in PBMCs found in clinical trial may also be evidenced in human small airway alveolar epithelial cells in primary culture. Aim: The aim of this project was to assess the anti-inflammatory effect of tRES-HESP on human alveolar epithelial cells in primary culture, assessing effects on protease and inflammatory gene expression considered essential for SARS-CoV-2 infectivity and progression of COVID-19 severity. Method: Human small airway epithelial cells (SAECs) were purchased from Lonza. SAECs were cultured in Small Airway Growth Media with supplements, according to the supplier’s instructions. SAECs (10,000 cells/cm2) were cultured with and without 5 µM tRES-HESP and the effect of basal gene expression studied after 72 h for mRNA (qRT-PCR) and protein (Western blotting); and significance of effect assessed by Student’s t-test, n = 3 or 4. The concentration of glucose in the medium was 6 mM. Results: In SAEC primary cultures, tRES-HESP decreased basal protein levels of TMPRSS2 (- 58%; P<0.001) and furin protease (- 43%; P<0.01) after 72 h. tRES-HESP also decreased mRNA of TMPRSS2 (- 62%, P<0.01), GRP78 (- 67%, P<0.001), COX-2 (-34%, P<0.001) and RAGE mRNA (- 56%, P<.001) and decreased basal secretion of MCP-1 and IL8 (-29% and - 25%, P<0.001). Discussion: tRES-HESP decreased basal expression of proteases essential for SARS-CoV-2 infection, GRP78 and secretion of inflammatory mediator risk predictors of severe COVID-19. These effects are likely due to activation of Nrf2 by tRES-HESP and decrease of unfolded protein response activation by suppression of dicarbonyl stress. These outcomes suggest previous anti-inflammatory response of tRES-HESP in overweight and obese subjects found in PBMCs may also occur concomitantly in the lung. TMPRSS2 is an antioxidant response element-linked gene down regulated by activation of Nrf2. These anti-proteolytic and anti-inflammatory responses of tRES-HESP suggest it is suitable for further evaluation for prevention of COVID-19, particularly in obesity and diabetes.

A Model of Human Small Airway on a Chip for Studies of Subacute Effects of Inhalation Toxicants.

Testing for acute inhalation hazards is conducted in animals; however, a number of robust in vitro human cell-based alternatives have been developed and tested. These models range in complexity from cultures of cell lines or primary cells in air-liquid interface on Transwells, to more complex and physiologically relevant flow- and mechanical stimulation-enabled tissue chips. Although the former models are relatively straightforward to establish and can be tested in medium/high throughput, the latter require specialized equipment and lack in throughput. In this study, we developed a device that can be easily manufactured while allowing for the production of a differentiated lung tissue. This multilayered microfluidic device enables coculture of primary human small airway epithelial cells and lung microvascular endothelial cells under physiological conditions for up to 18 days and recreates the parenchymal-vascular interface in the distal lung. To explore the potential of this airway on a chip for applications in inhalation toxicology, we also devised a system that allows for direct gas/aerosol exposures of the engineered airway epithelium to noxious stimuli known to cause adverse respiratory effects, including dry flowing air, lipopolysaccharide, particulate matter, and iodomethane. This study generated quantitative, high-content data that were indicative of aberrant changes in biochemical (lactate dehydrogenase), barrier (dextran permeability), functional (ciliary beating), and molecular (imaging for various markers) phenotypes of the small airway epithelium due to inhalational exposures. This study is significant because it established an in vitro model of human small airway on a chip that can be used in medium/high-throughput studies of subacute effects of inhalation toxicants.

HnRNP C modulates MERS-CoV and SARS-CoV-2 replication by governing the expression of a subset of circRNAs and cognitive mRNAs

ABSTRACT Host circular RNAs (circRNAs) play critical roles in the pathogenesis of viral infections. However, how viruses modulate the biogenesis of host proviral circRNAs to facilitate their replication remains unclear. We have recently shown that Middle East respiratory syndrome coronavirus (MERS-CoV) infection increases co-expression of circRNAs and their cognate messenger RNAs (mRNAs), possibly by hijacking specific host RNA binding proteins (RBPs). In this study, we systemically analysed the interactions between the representative circRNA–mRNA pairs upregulated upon MERS-CoV infection and host RBPs. Our analysis identified heterogeneous nuclear ribonucleoprotein C (hnRNP C) as a key host factor that governed the expression of numerous MERS-CoV-perturbed circRNAs, including hsa_circ_0002846, hsa_circ_0002061, and hsa_circ_0004445. RNA immunoprecipitation assay showed that hnRNP C could bind physically to these circRNAs. Specific knockdown of hnRNP C by small interfering RNA significantly (P < 0.05 to P < 0.0001) suppressed MERS-CoV replication in human lung adenocarcinoma (Calu-3) and human small airway epithelial (HSAEC) cells. Both MERS-CoV and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection increased the total and phosphorylated forms of hnRNP C to activate the downstream CRK-mTOR pathway. Treatment of MERS-CoV- (IC50: 0.618 µM) or SARS-CoV-2-infected (IC50: 1.233 µM) Calu-3 cells with the mTOR inhibitor OSI-027 resulted in significantly reduced viral loads. Collectively, our study identified hnRNP C as a key regulator of MERS-CoV-perturbed circRNAs and their cognate mRNAs, and the potential of targeting hnRNP C-related signalling pathways as an anticoronaviral strategy.

Genome-scale CRISPR screens identify host factors that promote human coronavirus infection

BackgroundThe COVID-19 pandemic has resulted in 275 million infections and 5.4 million deaths as of December 2021. While effective vaccines are being administered globally, there is still a great need for antiviral therapies as antigenically novel SARS-CoV-2 variants continue to emerge across the globe. Viruses require host factors at every step in their life cycle, representing a rich pool of candidate targets for antiviral drug design.MethodsTo identify host factors that promote SARS-CoV-2 infection with potential for broad-spectrum activity across the coronavirus family, we performed genome-scale CRISPR knockout screens in two cell lines (Vero E6 and HEK293T ectopically expressing ACE2) with SARS-CoV-2 and the common cold-causing human coronavirus OC43. Gene knockdown, CRISPR knockout, and small molecule testing in Vero, HEK293, and human small airway epithelial cells were used to verify our findings.ResultsWhile we identified multiple genes and functional pathways that have been previously reported to promote human coronavirus replication, we also identified a substantial number of novel genes and pathways. The website https://sarscrisprscreens.epi.ufl.edu/ was created to allow visualization and comparison of SARS-CoV2 CRISPR screens in a uniformly analyzed way. Of note, host factors involved in cell cycle regulation were enriched in our screens as were several key components of the programmed mRNA decay pathway. The role of EDC4 and XRN1 in coronavirus replication in human small airway epithelial cells was verified. Finally, we identified novel candidate antiviral compounds targeting a number of factors revealed by our screens.ConclusionsOverall, our studies substantiate and expand the growing body of literature focused on understanding key human coronavirus-host cell interactions and exploit that knowledge for rational antiviral drug development.

Propylene glycol, a component of electronic cigarette liquid, damages epithelial cells in human small airways

BackgroundElectronic cigarettes (e-cigarettes) are used worldwide as a substitute for conventional cigarettes. Although they are primarily intended to support smoking cessation, e-cigarettes have been identified as a gateway to smoking habits for young people. Multiple recent reports have described the health effects of inhaling e-cigarettes. E-cigarette liquid (e-liquid) is mainly composed of propylene glycol (PG) and glycerol (Gly), and the aerosol generated by these devices primarily contains these two components. Thus, this study aimed to evaluate the effects of PG and Gly on human small airway epithelial cells (SAECs).MethodsSAECs were exposed to PG or Gly, and cell proliferation, cell viability, lactate dehydrogenase (LDH) release, DNA damage, cell cycle, and apoptosis were evaluated. Additionally, SAECs derived from chronic obstructive pulmonary disease (COPD) patients (COPD-SAECs) were investigated.ResultsExposure of SAECs to PG significantly inhibited proliferation (1%, PG, p = 0.021; 2–4% PG, p < 0.0001) and decreased cell viability (1–4% PG, p < 0.0001) in a concentration-dependent manner. Gly elicited similar effects but to a reduced degree as compared to the same concentration of PG. PG also increased LDH release in a concentration-dependent manner (3% PG, p = 0.0055; 4% PG, p < 0.0001), whereas Gly did not show a significant effect on LDH release. SAECs exposed to 4% PG contained more cells that were positive for phosphorylated histone H2AX (p < 0.0001), a marker of DNA damage, and an increased proportion of cells in the G1 phase (p < 0.0001) and increased p21 expression (p = 0.0005). Moreover, caspase 3/7-activated cells and cleaved poly (ADP-ribose) polymerase 1 expression were increased in SAECs exposed to 4% PG (p = 0.0054). Furthermore, comparing COPD-SAECs to SAECs without COPD in PG exposure, cell proliferation, cell viability, DNA damage and apoptosis were significantly greater in COPD-SAECs.ConclusionPG damaged SAECs more than Gly. In addition, COPD-SAECs were more susceptible to PG than SAECs without COPD. Usage of e-cigarettes may be harmful to the respiratory system, especially in patients with COPD.

Investigation of the role of the autophagic protein LC3B in the regulation of human airway epithelium cell differentiation in COPD using a biomimetic model.

Chronic obstructive pulmonary disease (COPD) is one of the most lethal chronic disease worldwide; however, the establishment of reliable in vitro models for exploring the biological mechanisms of COPD remains challenging. Here, we determined the differences in the expression and characteristics of the autophagic protein LC3B in normal and COPD human small airway epithelial cells and found that the nucleus of COPD cells obviously accumulated LC3B. We next established 3D human small airway tissues with distinct disease characteristics by regulating the biological microenvironment, extracellular matrix, and air-liquid interface culture methods. Using this biomimetic model, we found that LC3B affects the differentiation of COPD cells into basal, secretory, mucous, and ciliated cells. Moreover, although chloroquine and ivermectin effectively inhibited the expression of LC3B in the nucleus, chloroquine specifically maintained the performance of LC3B in cytoplasm, thereby contributing to the differentiation of ciliated cells and subsequent improvement in the beating functions of the cilia, whereas ivermectin only facilitated differentiation of goblet cells. We demonstrated that the autophagic mechanism of LC3B in the nucleus is one factor regulating the ciliary differentiation and function of COPD cells. Our innovative model can be used to further analyze the physiological mechanisms in the in vitro airway environment.

Broad-Spectrum In Vitro Antiviral Activity of ODBG-P-RVn: An Orally-Available, Lipid-Modified Monophosphate Prodrug of Remdesivir Parent Nucleoside (GS-441524).

ABSTRACTThe necessity for intravenous administration of remdesivir confines its utility for treatment of coronavirus disease 2019 (COVID-19) to hospitalized patients. We evaluated the broad-spectrum antiviral activity of ODBG-P-RVn, an orally available, lipid-modified monophosphate prodrug of the remdesivir parent nucleoside (GS-441524), against viruses that cause diseases of human public health concern, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). ODBG-P-RVn showed 20-fold greater antiviral activity than GS-441524 and had activity nearly equivalent to that of remdesivir in primary-like human small airway epithelial cells. Our results warrant in vivo efficacy evaluation of ODBG-P-RVn.IMPORTANCE While remdesivir remains one of the few drugs approved by the FDA to treat coronavirus disease 2019 (COVID-19), its intravenous route of administration limits its use to hospital settings. Optimizing the stability and absorption of remdesivir may lead to a more accessible and clinically potent therapeutic. Here, we describe an orally available lipid-modified version of remdesivir with activity nearly equivalent to that of remdesivir against emerging viruses that cause significant disease, including Ebola and Nipah viruses. Our work highlights the importance of such modifications to optimize drug delivery to relevant and appropriate human tissues that are most affected by such diseases.

Interaction of Ectodomain of Respiratory Syncytial Virus G Protein with TLR2/ TLR6 Heterodimer: An In vitro and In silico Approach to Decipher the Role of RSV G Protein in Pro-inflammatory Response against the Virus.

Human respiratory syncytial virus (RSV) has been shown to be linked with various forms of respiratory diseases, such as common cold and lower respiratory tract illnesses like pneumonia and bronchiolitis. TLRs play critical role in generating host immune response against RSV. TLRs are expressed not only on leukocytes but also on many other cell types and can recognize RSV. Previous studies have established that RSV can interact with TLR4 and initiate inflammatory cascade of cytokines. The data from a recent study indicated that TLR2/TLR6 is involved in RSV recognition and subsequent innate immune activation. However, the nature of binding and the envelope protein of RSV involved in this interaction with TLRs are not studied yet. We hypothesized that RSV G protein can bind to TLRs and mediate the inflammatory immune response against the virus infection. Therefore, we investigated whether RSV G protein could activate innate immune response through TLR signaling. Different TLR antagonists were used to assess the effect of the exposure of RSV and RSV G ectodomain in human primary small airway epithelial cells (HSAECs). Various inflammatory cytokines, chemokines and type I IFNs were measured by ELISA along with their mRNA expression by qPCR. In silico interaction of RSV G protein with TLR2/TLR6 was also analyzed. Results of ELISA and qPCR analysis have shown that TLR2/TLR6 signaling is activated in HSAECs upon RSV and RSV G protein exposure which initiates innate immune response against RSV. Moreover, RSV envelope protein G plays a crucial role in binding and activation of TLR2/TLR6 signaling. In summary, our study shows that TLR2/TLR6 play important role in the activation of innate immune response upon RSV recognition which could be helpful in promoting RSV clearance and preventing RSV-induced disease.

Toxicity of stainless and mild steel particles generated from gas\u2013metal arc welding in primary human small airway epithelial cells

Welding fumes induce lung toxicity and are carcinogenic to humans but the molecular mechanisms have yet to be clarified. The aim of this study was to evaluate the toxicity of stainless and mild steel particles generated via gas–metal arc welding using primary human small airway epithelial cells (hSAEC) and ToxTracker reporter murine stem cells, which track activation of six cancer-related pathways. Metal content (Fe, Mn, Ni, Cr) of the particles was relatively homogenous across particle size. The particles were not cytotoxic in reporter stem cells but stainless steel particles activated the Nrf2-dependent oxidative stress pathway. In hSAEC, both particle types induced time- and dose-dependent cytotoxicity, and stainless steel particles also increased generation of reactive oxygen species. The cellular metal content was higher for hSAEC compared to the reporter stem cells exposed to the same nominal dose. This was, in part, related to differences in particle agglomeration/sedimentation in the different cell media. Overall, our study showed differences in cytotoxicity and activation of cancer-related pathways between stainless and mild steel welding particles. Moreover, our data emphasizes the need for careful assessment of the cellular dose when comparing studies using different in vitro models.

Bromodomain Containing Protein 4 (BRD4) Regulates Expression of its Interacting Coactivators in the Innate Response to Respiratory Syncytial Virus.

Bromodomain-containing protein 4 plays a central role in coordinating the complex epigenetic component of the innate immune response. Previous studies implicated BRD4 as a component of a chromatin-modifying complex that is dynamically recruited to a network of protective cytokines by binding activated transcription factors, polymerases, and histones to trigger their rapid expression via transcriptional elongation. Our previous study extended our understanding of the airway epithelial BRD4 interactome by identifying over 100 functionally important coactivators and transcription factors, whose association is induced by respiratory syncytial virus (RSV) infection. RSV is an etiological agent of recurrent respiratory tract infections associated with exacerbations of chronic obstructive pulmonary disease. Using a highly selective small-molecule BRD4 inhibitor (ZL0454) developed by us, we extend these findings to identify the gene regulatory network dependent on BRD4 bromodomain (BD) interactions. Human small airway epithelial cells were infected in the absence or presence of ZL0454, and gene expression profiling was performed. A highly reproducible dataset was obtained which indicated that BRD4 mediates both activation and repression of RSV-inducible gene regulatory networks controlling cytokine expression, interferon (IFN) production, and extracellular matrix remodeling. Index genes of functionally significant clusters were validated independently. We discover that BRD4 regulates the expression of its own gene during the innate immune response. Interestingly, BRD4 activates the expression of NFκB/RelA, a coactivator that binds to BRD4 in a BD-dependent manner. We extend this finding to show that BRD4 also regulates other components of its functional interactome, including the Mediator (Med) coactivator complex and the SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin (SMARC) subunits. To provide further insight into mechanisms for BRD4 in RSV expression, we mapped 7,845 RSV-inducible Tn5 transposase peaks onto the BRD4-dependent gene bodies. These were located in promoters and introns of cytostructural and extracellular matrix (ECM) formation genes. These data indicate that BRD4 mediates the dynamic response of airway epithelial cells to RNA infection by modulating the expression of its coactivators, controlling the expression of host defense mechanisms and remodeling genes through changes in promoter accessibility.

Paramyxovirus replication induces the hexosamine biosynthetic pathway and mesenchymal transition via the IRE1α-XBP1s arm of the unfolded protein response.

The paramyxoviridae, respiratory syncytial virus (RSV), and murine respirovirus are enveloped, negative-sense RNA viruses that are the etiological agents of vertebrate lower respiratory tract infections (LRTIs). We observed that RSV infection in human small airway epithelial cells induced accumulation of glycosylated proteins within the endoplasmic reticulum (ER), increased glutamine-fructose-6-phosphate transaminases (GFPT1/2) and accumulation of uridine diphosphate (UDP)-N-acetylglucosamine, indicating activation of the hexosamine biosynthetic pathway (HBP). RSV infection induces rapid formation of spliced X-box binding protein 1 (XBP1s) and processing of activating transcription factor 6 (ATF6). Using pathway selective inhibitors and shRNA silencing, we find that the inositol-requiring enzyme (IRE1α)-XBP1 arm of the unfolded protein response (UPR) is required not only for activation of the HBP, but also for expression of mesenchymal transition (EMT) through the Snail family transcriptional repressor 1 (SNAI1), extracellular matrix (ECM)-remodeling proteins fibronectin (FN1), and matrix metalloproteinase 9 (MMP9). Probing RSV-induced open chromatin domains by ChIP, we find XBP1 binds and recruits RNA polymerase II to the IL6, SNAI1, and MMP9 promoters and the intragenic superenhancer of glutamine-fructose-6-phosphate transaminase 2 (GFPT2). The UPR is sustained through RSV by an autoregulatory loop where XBP1 enhances Pol II binding to its own promoter. Similarly, we investigated the effects of murine respirovirus infection on its natural host (mouse). Murine respirovirus induces mucosal growth factor response, EMT, and the indicators of ECM remodeling in an IRE1α-dependent manner, which persists after viral clearance. These data suggest that IRE1α-XBP1s arm of the UPR pathway is responsible for paramyxovirus-induced metabolic adaptation and mucosal remodeling via EMT and ECM secretion.

Abstract 2299: An unbiased in vivo screen for activating EGFR mutations

Abstract Cancer tissues harbor thousands of mutations, and a given oncogene may be mutated at hundreds of sites across different samples. Most of these somatic mutations are expected to be inconsequential passenger mutations that reflect the general instability of the tumors. The discovery of most of the currently known driver mutations has been facilitated by their accumulation in mutation hot-spots within their respective genes. However, a vast majority of mutations in cancer tissues are rare and their functional significance remains unknown. Several lines of in vitro and clinical evidence also indicate that there is a significant number of, as yet unidentified, activating driver mutations which could serve as predictive markers in oncology. We are developing our in vitro Screen for Activating Mutations (“iSCREAM”)[1] further and advancing it to better recapitulate natural intratumor heterogeneity by taking the system in vivo. To establish the in vivo model for screening activating mutations we utilize the SALE-Y cells which are immortalized human small airway epithelial cells harboring an activating YAP1 variant[2]. In immunocompromised mice, SALE-Y cells fail to form tumors up to 120 days, but they become tumorigenic after transduction of mutations activating the EGFR-MAPK signaling pathway[2]. We will use the SALE-Y cell background for transduction of an expression library encoding thousands of different randomly mutated variants of the kinase to be tested. EGFR will be used to set up the model because: i) EGFR mutants are known to of transforming the SALE-Y cells[2] and ii) we have previously used EGFR as a model to set up the in vitro screen[1]. The SALE-Y expressing the random collection of EGFR variants will be inoculated subcutaneously into immunocompromised mice, and the activating mutations will be given an opportunity to outcompete passenger mutations during subsequent expansion and suvival of the xenograft. Genomic DNA will be harvested from the tumor tissue and subjected to targeted next-generation sequencing allowing unbiased identification of enriched activating mutations promoting tumor growth.

A robust SARS-CoV-2 replication model in primary human epithelial cells at the air liquid interface to assess antiviral agents

There are, besides remdesivir, no approved antivirals for the treatment of SARS-CoV-2 infections. To aid in the search for antivirals against this virus, we explored the use of human tracheal airway epithelial cells (HtAEC) and human small airway epithelial cells (HsAEC) grown at the air-liquid interface (ALI). These cultures were infected at the apical side with one of two different SARS-CoV-2 isolates. Each virus was shown to replicate to high titers for extended periods of time (at least 8 days) and, in particular an isolate with the D614G in the spike (S) protein did so more efficiently at 35 °C than 37 °C. The effect of a selected panel of reference drugs that were added to the culture medium at the basolateral side of the system was explored. Remdesivir, GS-441524 (the parent nucleoside of remdesivir), EIDD-1931 (the parent nucleoside of molnupiravir) and IFN (β1 and λ1) all resulted in dose-dependent inhibition of viral RNA and infectious virus titers collected at the apical side. However, AT-511 (the free base form of AT-527 currently in clinical testing) failed to inhibit viral replication in these in vitro primary cell models. Together, these results provide a reference for further studies aimed at selecting SARS-CoV-2 inhibitors for further preclinical and clinical development.

The bioactivity of soluble Fas ligand is modulated by key amino acids of its stalk region.

We have previously reported that the 26-amino acid N-terminus stalk region of soluble Fas ligand (sFasL), which is separate from its binding site, is required for its biological function. Here we investigate the mechanisms that link the structure of the sFasL stalk region with its function. Using site-directed mutagenesis we cloned a mutant form of sFasL in which all the charged amino acids of the stalk region were changed to neutral alanines (mut-sFasL). We used the Fas-sensitive Jurkat T-cell line and mouse and human alveolar epithelial cells to test the bioactivity of sFasL complexes, using caspase-3 activity and Annexin-V externalization as readouts. Finally, we tested the effects of mut-sFasL on lipopolysaccharide-induced lung injury in mice. We found that mutation of all the 8 charged amino acids of the stalk region into the non-charged amino acid alanine (mut-sFasL) resulted in reduced apoptotic activity compared to wild type sFasL (WT-sFasL). The mut-sFasL attenuated WT-sFasL function on the Fas-sensitive human T-cell line Jurkat and on primary human small airway epithelial cells. The inhibitory mechanism was associated with the formation of complexes of mut-sFasL with the WT protein. Intratracheal administration of the mut-sFasL to mice 24 hours after intratracheal Escherichia coli lipopolysaccharide resulted in attenuation of the inflammatory response 24 hours later. Therefore, the stalk region of sFasL has a critical role on bioactivity, and changes in the structure of the stalk region can result in mutant variants that interfere with the wild type protein function in vitro and in vivo.

ROR\u03b3t inhibitors block both IL-17 and IL-22 conferring a potential advantage over anti-IL-17 alone to treat severe asthma

BackgroundRORγt is a transcription factor that enables elaboration of Th17-associated cytokines (including IL-17 and IL-22) and is proposed as a pharmacological target for severe asthma.MethodsIL-17 immunohistochemistry was performed in severe asthma bronchial biopsies (specificity confirmed with in situ hybridization). Primary human small airway epithelial cells in air liquid interface and primary bronchial smooth muscle cells were stimulated with recombinant human IL-17 and/or IL-22 and pro-inflammatory cytokines measured. Balb/c mice were challenged intratracheally with IL-17 and/or IL-22 and airway hyperreactivity, pro-inflammatory cytokines and airway neutrophilia measured. Balb/c mice were sensitized intraperitoneally and challenged intratracheally with house dust mite extract and the effect of either a RORγt inhibitor (BIX119) or an anti-IL-11 antibody assessed on airway hyperreactivity, pro-inflammatory cytokines and airway neutrophilia measured.ResultsWe confirmed in severe asthma bronchial biopsies both the presence of IL-17-positive lymphocytes and that an IL-17 transcriptome profile in a severe asthma patient sub-population. Both IL-17 and IL-22 stimulated the release of pro-inflammatory cytokine and chemokine release from primary human lung cells and in mice. Furthermore, IL-22 in combination with IL-17, but neither alone, elicits airway hyperresponsiveness (AHR) in naïve mice. A RORγt inhibitor specifically blocked both IL-17 and IL-22, AHR and neutrophilia in a mouse house dust mite model unlike other registered or advanced pipeline modes of action. Full efficacy versus these parameters was associated with 90% inhibition of IL-17 and 50% inhibition of IL-22. In contrast, anti-IL-17 also blocked IL-17, but not IL-22, AHR or neutrophilia. Moreover, the deregulated genes in the lungs from these mice correlated well with deregulated genes from severe asthma biopsies suggesting that this model recapitulates significant severe asthma-relevant biology. Furthermore, these genes were reversed upon RORγt inhibition in the HDM model. Cell deconvolution suggested that the responsible cells were corticosteroid insensitive γδ-T-cells.ConclusionThese data strongly suggest that both IL-17 and IL-22 are required for Th2-low endotype associated biology and that a RORγt inhibitor may provide improved clinical benefit in a severe asthma sub-population of patients by blocking both IL-17 and IL-22 biology compared with blocking IL-17 alone.

Hookah Smoke Mediates Cancer-Associated Epigenomic and Transcriptomic Signatures in Human Respiratory Epithelial Cells

IntroductionAlthough communal smoking of hookah by means of water pipes is perceived to be a safe alternative to cigarette smoking, the effects of hookah smoke in respiratory epithelia have not been well characterized. This study evaluated epigenomic and transcriptomic effects of hookah smoke relative to cigarette smoke in human respiratory epithelial cells. MethodsPrimary normal human small airway epithelial cells from three donors and cdk4 and hTERT-immortalized small airway epithelial cells and human bronchial epithelial cells were cultured for 5 days in normal media with or without cigarette smoke condensates (CSCs) or water pipe condensates (WPCs). Cell count, immunoblot, RNA sequencing, quantitative real-time reverse-transcriptase polymerase chain reaction, methylation-specific polymerase chain reaction, and quantitative chromatin immunoprecipitation techniques were used to compare effects of hookah and cigarette smoke on cell proliferation, global histone marks, gene expression, and promoter-related chromatin structure. ResultsCSC and WPC decreased global H4K16ac and H4K20me3 histone marks and mediated distinct and overlapping cancer-associated transcriptome signatures and pathway modulations that were cell line dependent and stratified across lung cancer cells in a histology-specific manner. Epiregulin encoding a master regulator of EGFR signaling that is overexpressed in lung cancers was up-regulated, whereas FILIP1L and ABI3BP encoding mediators of senescence that are repressed in lung cancers were down-regulated by CSC and WPC. Induction of epiregulin and repression of FILIP1L and ABI3BP by these condensates coincided with unique epigenetic alterations within the respective promoters. ConclusionsThese findings support translational studies to ascertain if hookah-mediated epigenomic and transcriptomic alterations in cultured respiratory epithelia are detectable and clinically relevant in hookah smokers.