Human Small Airway Epithelial Cells Research Articles

Epidermal growth factor receptor signaling governs the host inflammatory response to invasive aspergillosis.

The epidermal growth factor receptor (EGFR) has been identified as an epithelial cell receptor for Mucorales fungi and Candida albicans. Blocking EGFR with small molecule inhibitors reduces disease severity in mouse models of mucormycosis and oropharyngeal candidiasis. In contrast, cases of invasive aspergillosis have been reported in cancer patients who were treated with EGFR inhibitors, suggesting that EGFR signaling may play a protective role in the host defense against this infection. Here, we analyzed transcriptomic data from the lungs of mice with invasive aspergillosis and found evidence that Aspergillus fumigatus infection activates multiple genes that are predicted to function in the EGFR signaling pathway. We also found that A. fumigatus infection activates EGFR in both a human small-airway epithelial (HSAE) cell line and in the lungs of immunosuppressed mice. EGFR signaling in HSAE cells is required for maximal endocytosis of A. fumigatus and for fungal-induced proinflammatory cytokine and chemokine production. In a corticosteroid immunosuppressed mouse model of invasive pulmonary aspergillosis, inhibition of EGFR with gefitinib decreased whole-lung cytokine and chemokine levels and reduced accumulation of phagocytes in the lung, leading to a decrease in fungal killing, an increase in pulmonary fungal burden, and accelerated mortality. Thus, EGFR signaling is required for pulmonary epithelial cells to orchestrate the host innate immune defense against invasive aspergillosis in immunosuppressed hosts.IMPORTANCEWhen A. fumigatus infects the lungs, it invades epithelial cells that line the airways. During this process, the fungus interacts with epithelial cell receptors. This interaction stimulates epithelial cells to endocytose the fungus. It also induces these cells to secrete proinflammatory cytokines and chemokines that recruit phagocytes to the site of infection where they can kill the fungus. Here, we show that in small-airway epithelial cells, the EGFR acts as a sensor for A. fumigatus that triggers the production of chemokines in response to fungal infection. In corticosteroid-immunosuppressed mice, blocking EGFR with the kinase inhibitor gefitinib reduces chemokine production in the lungs. This leads to decreased accumulation of neutrophils and dendritic cells in the lungs, reduced A. fumigatus killing, and increased mortality. These results provide a potential explanation as to why some cancer patients who are treated with EGFR inhibitors develop invasive aspergillosis.

Epidermal Growth Factor Receptor Signaling Governs the Host Inflammatory Response to Invasive Aspergillosis.

The epidermal growth factor receptor (EGFR) has been identified as an epithelial cell receptor for Mucorales fungi and Candida albicans. Blocking EGFR with small molecule inhibitors reduces disease severity in mouse models of mucormycosis and oropharyngeal candidiasis. In contrast, cases of invasive aspergillosis have been reported in cancer patients who were treated with EGFR inhibitors, suggesting that EGFR signaling may play a protective role in the host defense against this infection. Here, we analyzed transcriptomic data from the lungs of mice with invasive aspergillosis and found evidence that Aspergillus fumigatus infection activates multiple genes that are predicted to function in the EGFR signaling pathway. We also found that A. fumigatus infection activates EGFR in both a human small airway epithelial (HSAE) cell line and in the lungs of immunosuppressed mice. EGFR signaling in HSAE cells is required for maximal endocytosis of A. fumigatus and for fungal-induced proinflammatory cytokine and chemokine production. In a corticosteroid immunosuppressed mouse model of invasive pulmonary aspergillosis, inhibition of EGFR with gefitinib decreased whole lung chemokine levels and reduced accumulation of phagocytes in the lung, leading to a decrease in fungal killing, an increase in pulmonary fungal burden, and accelerated mortality. Thus, EGFR signaling is required for pulmonary epithelial cells to orchestrate the host innate immune defense against invasive aspergillosis in immunosuppressed hosts.

Magnetoactive, Kirigami-Inspired Hammocks to Probe Lung Epithelial Cell Function

IntroductionMechanical forces provide critical biological signals to cells. Within the distal lung, tensile forces act across the basement membrane and epithelial cells atop. Stretching devices have supported studies of mechanical forces in distal lung epithelium to gain mechanistic insights into pulmonary diseases. However, the integration of curvature into devices applying mechanical forces onto lung epithelial cell monolayers has remained challenging. To address this, we developed a hammock-shaped platform that offers desired curvature and mechanical forces to lung epithelial monolayers.MethodsWe developed hammocks using polyethylene terephthalate (PET)-based membranes and magnetic-particle modified silicone elastomer films within a 48-well plate that mimic the alveolar curvature and tensile forces during breathing. These hammocks were engineered and characterized for mechanical and cell-adhesive properties to facilitate cell culture. Using human small airway epithelial cells (SAECs), we measured monolayer formation and mechanosensing using F-Actin staining and immunofluorescence for cytokeratin to visualize intermediate filaments.ResultsWe demonstrate a multi-functional design that facilitates a range of curvatures along with the incorporation of magnetic elements for dynamic actuation to induce mechanical forces. Using this system, we then showed that SAECs remain viable, proliferate, and form an epithelial cell monolayer across the entire hammock. By further applying mechanical stimulation via magnetic actuation, we observed an increase in proliferation and strengthening of the cytoskeleton, suggesting an increase in mechanosensing.ConclusionThis hammock strategy provides an easily accessible and tunable cell culture platform for mimicking distal lung mechanical forces in vitro. We anticipate the promise of this culture platform for mechanistic studies, multi-modal stimulation, and drug or small molecule testing, extendable to other cell types and organ systems.

Blockade of endolysosomal acidification suppresses TLR3-mediated proinflammatory signaling in airway epithelial cells

BackgroundEndolysosomal compartments are acidic and contain low pH-dependent proteases, and these conditions are exploited by respiratory viruses, such as SARS-CoV-2 and influenza virus, for escaping into the cytosol. Moreover, endolysosomes contain various pattern recognition receptors (PRRs), which respond to virus-derived pathogen-associated molecular patterns (PAMPs) by production of pro-inflammatory cytokines/chemokines. However, excessive pro-inflammatory responses can lead to a potentially lethal cytokine storm. ObjectivesHere we investigated the endosomal PRR expression profile in primary human small airway epithelial cells (HSAECs), and whether blockade of endolysosomal acidification affects their cytokine/chemokine production after challenge with virus-derived stimulants. MethodsHSAECs were exposed to stimulants mimicking virus-derived PAMPs, either in the absence or presence of compounds causing blockade of endolysosomal acidification, followed by measurement of cytokine expression and release. ResultsWe show that toll-like receptor 3 (TLR3) is the major endosomal PRR expressed by HSAECs, and that TLR3 expression is strongly induced by TLR3 agonists, but not by a range of other PRR agonists. We also demonstrate that TLR3 engagement with its agonists elicits a robust pro-inflammatory cytokine/chemokine response, which is profoundly suppressed through blockade of endolysosomal acidification, by bafilomycin A1, monensin, or niclosamide. Using TLR3 reporter cells, it was confirmed that TLR3 signaling is strongly induced by Poly(I:C) and that blockade of endolysosomal acidification efficiently blocked TLR3 signaling. Finally, we show that blockade of endolysosomal acidification causes a reduction in the levels of TLR3 mRNA and protein. ConclusionThese findings show that blockade of endolysosomal acidification suppresses TLR3-dependent cytokine and chemokine production in HSAECs. Clinical implicationThese findings may be exploited for therapeutic strategies aiming to ameliorate the cytokine storm in response to respiratory virus infection.

Epigenetic Regulation of Carbonic Anhydrase 9 Expression by Nitric Oxide in Human Small Airway Epithelial Cells.

DNA methylation is a crucial epigenetic modification that regulates gene expression and determines cell fate; however, the triggers that alter DNA methylation levels remain unclear. Recently, we showed that S-nitrosylation of DNA methyltransferase (DNMT) induces DNA hypomethylation and alters gene expression. Furthermore, we identified DBIC, a specific inhibitor of S-nitrosylation of DNMT3B, to suppress nitric oxide (NO)-induced gene alterations. However, it remains unclear how NO-induced DNA hypomethylation regulates gene expression and whether this mechanism is maintained in normal cells and triggers disease-related changes. To address these issues, we focused on carbonic anhydrase 9 (CA9), which is upregulated under nitrosative stress in cancer cells. We pharmacologically evaluated its regulatory mechanisms using human small airway epithelial cells (SAECs) and DBIC. We demonstrated that nitrosative stress promotes the recruitment of hypoxia-inducible factor 1 alpha to the CA9 promoter region and epigenetically induces CA9 expression in SAECs. Our results suggest that nitrosative stress is a key epigenetic regulator that may cause diseases by altering normal cell function.

Induction of CXCL3 in airway epithelial cells by a novel IL-9 receptor complex

Abstract Cytokine signaling is critical for cell-to-cell communication in development, homeostasis and disease. Interleukin (IL)-9 signaling promotes allergic diseases by acting on both immune and structural cells. In hematopoietic cells, IL-9 receptor (IL-9R) consists of two subunits – the α-chain (IL-9Rα) and a common γ (γc) chain. However, IL-9-responsive epithelial cells express IL-9Rα but not the γc chain raising a question on how these cells respond to IL-9 in the absence of the established IL-9R complex. Here, we discovered that in airway epithelial cells, IL-9Rα pairs with IL-13Rα1, forming a novel type II IL-9R. Using proximity ligation assay, we show that the IL-9Rα /IL-13Rα1 chains are in IL-9-dependent proximity in both mouse tracheal epithelial cells (mTEC) and human small airway epithelial cells (SAEC). IL-9-induced STAT3 activation in SAEC was blocked by anti-IL-13Rα1 but not by anti-γc. Using RNA-seq, in vitro IL-9 treatment of SAEC induced the expression of genes including CXCL3, a key chemokine implicated in asthma. Importantly, intranasal administration of IL-9 increased Cxcl3 expression in mTECs and CXCL3 protein in the lung, that corresponds with an expansion in the number of CXCR2+ immune cells in the lung. Thus, we identify a novel type II IL-9R which is composed of IL-9Rα and IL-13Rα1 and show that IL-9 signaling via type II IL-9R induces CXCL3 expression in epithelial cells to contribute to airway inflammation.

Induction of TNFα by flavin adenine dinucleotide (FADH) protects neonatal C57Bl6 lungs from high oxygen induced lung injury

Background: Bronchopulmonary dysplasia (BPD) is a serious lung disease caused by oxidative stress, predominately affecting premature infants that require prolonged oxygen support. The co-factor flavin adenine dinucleotide (FADH) facilitates glutathione reductase (GR) enzymatic activity and increases the bioavailability of the antioxidant glutathione (GSH). As such, we hypothesized that intranasal delivery of FADH can improve redox homeostasis and attenuate lung injury following chronic high oxygen supplementation (0.85% FiO2) by altering pro-inflammatory signal transduction pathways in a C57Bl6 newborn mouse model for BPD. Methods: We used a prolonged hyperoxia-induced lung injury rodent model to recapitulate the clinical and histological changes present in BPD. Newborn mice were housed in FiO2 85% (hyperoxic) or 21% O2 (normoxic) from day of birth until post-natal day (PN) 14. FADH (7 μM) or saline (control) treatments were administered daily via intranasal insufflation on PN 14-21 in a final volume of 1.5μL/g body weight. BALF and lung tissue samples were collected and evaluated for redox stress, lung injury/development, and inflammation using standard immunohistological techniques, morphometric analysis, and standard molecular assays. BALF cytokines were measured using a flow cytometric approach (BioLegend). Two-dimensional cell migration assays were tracked via microscopy daily until 100% confluence reached; the ½ maximal peak values were evaluated to compare rate of wound closure following very low (2.5 ng/mL) TNFα or IL12 treatment. Transepithelial resistance and potential differences were measured using an EVOM device (World Precision Instrument) across human small airway epithelial cells, and equivalent short circuit currents (Isc) calculated in accordance with Ohm’s Law, where V=IR. Single data comparisons were performed using paired Student’s t-tests. Multiple comparisons were performed using ANOVA followed by post-hoc testing in Sigma Plot. Results: FADH protected neonatal lungs from high oxygen induced oxidative stress: GSH/GSSG Eh (a measure of oxidative stress) improved from -168.77 mV ± 3.64 mV to -179.10 mV ± 1.85 mV (n=5 BALF measurements). FADH also improved lung injury scores from 0.187±0.038 to 0.03±0.014 (p=0.005), decreased neutrophil migration (p<0.001), and increased macrophages (p<0.001) when compared to age-matched untreated pups. FADH-mediated increase in TNFα (and not IL12) significantly increased the rate of lung epithelial cell wound closure from [2.27±0.07 to 1.97±0.1 average days for ½ complete wound closure, n=6, p=0.01] and increased Isc from 6.5±.21 to 9.2±1.0 μA/cm2, n=3, p=0.03. Conclusions: Our findings indicate that FADH protects the preterm lung from hyperoxic injury by 1) increasing the bioavailability of GSH and 2) via TNFα signaling to increase the rate of wound closure and repair of epithelial cell function. Significances: The mechanisms identified through this study could be targeted as a potential adjunct therapy for BPD patients to attenuate high oxygen induced lung injury. Funding: Supported by R01HL137033 (MH). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Polycyclic aromatic hydrocarbon (PAH) activation of AhR inhibits glutathione S-transferase (GST) activity in primary human small airway epithelial cells (hSAECs)

Background: PAH represents the organic phase of particulate matter (PM) in air pollution emitted via the incomplete combustion of various compounds (i.e. diesel fuel, wood, and plastics). It is widely regarded that PAH activation of aryl hydrocarbon receptor (AhR) plays an important role in pro-inflammatory signaling, however the mechanisms remain unknown. In contrast- endogenous AhR agonists, such as 6-Formylindolo[3,2-b]carbazole (FICZ), exert pleiotropic protective effects against inflammation, fibrosis, and oxidative stress. The objective of our research program is to delineate the differences between activation of AhR by PAH (with adverse effects) compared to its beneficial homeostatic activation by endogenous FICZ. PM may induce oxidative stress in part through inhibition of antioxidant defense mechanisms. GSTs are a superfamily of enzymes that plays an important role in protecting cells from oxidative damage (by conjugating reduced glutathione to substrates under pro-oxidizing conditions). We hypothesize that PAH activation of AhR contributes to oxidative stress via inhibition of GST activity in primary hSAECs. Methods: Primary hSAECs were subjected to varying dosages of PAH [0, 250, 500, 1000, 5000 ng/mL] to model airborne PM exposure levels, and to increasing doses of FICZ [0, 0.50, 10, 100, 1000 nM] to model normal and excessively high endogenous ligand activation of AhR. We determined the relative potencies of PAH and FICZ by measuring CYP1A1 and CYP1B1 gene expression. Oxidative stress was measured using DHE-labeling and GST activity was measured with enzymatic kits (Sigma Aldrich). AhR activity was pharmacologically inhibited using 10μM CH223191. Statistical analysis was conducted using SigmaPlot 14.0 (Systat Software). Results: PAH and FICZ maximally stimulate hSAEC CYP1A1 and CYP1B1 expression at 1000ng/mL and 100nM, respectively, and are therefore defined as equipotent concentrations used in our studies. In a representative experiment in hSAECs, PAH (1000ng/mL) significantly increased hSAEC oxidative stress vs FICZ (100nM) n=6, p<0.01), while significantly decreasing GST specific activity vs equipotent FICZ activation of AhR in primary hSAECs from a 66-year-old female without lung disease (26.6±0.874 vs 28.3±.611 nmol/mL/min); n=6 from 2 independent assays; p=0.032. Similar differential effects of PAH vs FICZ activation of AhR on GST were seen in additional experiments with male hSAECs and Calu-3 cells (male). Importantly, CH223191 inhibition of GST enzymatic activity suggests that AhR plays an important role in protecting cells from oxidative stress. Conclusions: Our studies indicate that airborne PAH exposure can have detrimental health effects on the lung via aberrant AhR signaling. Significance: Understanding activity of AhR signaling can leads to novel therapeutic options for redox disorders in the lung. Funding: VA 5I01BX001777-05 (RP) and R01HL137033 (MH). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Nanoplastics Penetrate Human Bronchial Smooth Muscle and Small Airway Epithelial Cells and Affect Mitochondrial Metabolism.

Micro- and nanoplastic particles, including common forms like polyethylene and polystyrene, have been identified as relevant pollutants, potentially causing health problems in living organisms. The mechanisms at the cellular level largely remain to be elucidated. This study aims to visualize nanoplastics in bronchial smooth muscle (BSMC) and small airway epithelial cells (SAEC), and to assess the impact on mitochondrial metabolism. Healthy and asthmatic human BSMC and SAEC in vitro cultures were stimulated with polystyrene nanoplastics (PS-NPs) of 25 or 50 nm size, for 1 or 24 h. Live cell, label-free imaging by holotomography microscopy and mitochondrial respiration and glycolysis assessment were performed. Furthermore, 25 and 50 nm NPs were shown to penetrate SAEC, along with healthy and diseased BSMC, and they impaired bioenergetics and induce mitochondrial dysfunction compared to cells not treated with NPs, including changes in oxygen consumption rate and extracellular acidification rate. NPs pose a serious threat to human health by penetrating airway tissues and cells, and affecting both oxidative and glycolytic metabolism.

Genome-Wide Analysis Identifies Nuclear Factor 1C as a Novel Transcription Factor and Potential Therapeutic Target in SCLC

IntroductionRecent insights regarding mechanisms mediating stemness, heterogeneity, and metastatic potential of lung cancers have yet to be fully translated to effective regimens for the treatment of these malignancies. This study sought to identify novel targets for lung cancer therapy. MethodsTranscriptomes and DNA methylomes of 14 SCLC and 10 NSCLC lines were compared with normal human small airway epithelial cells (SAECs) and induced pluripotent stem cell (iPSC) clones derived from SAEC. SCLC lines, lung iPSC (Lu-iPSC), and SAEC were further evaluated by DNase I hypersensitive site sequencing (DHS-seq). Changes in chromatin accessibility and depths of transcription factor (TF) footprints were quantified using Bivariate analysis of Genomic Footprint. Standard techniques were used to evaluate growth, tumorigenicity, and changes in transcriptomes and glucose metabolism of SCLC cells after NFIC knockdown and to evaluate NFIC expression in SCLC cells after exposure to BET inhibitors. ResultsConsiderable commonality of transcriptomes and DNA methylomes was observed between Lu-iPSC and SCLC; however, this analysis was uninformative regarding pathways unique to lung cancer. Linking results of DHS-seq to RNA sequencing enabled identification of networks not previously associated with SCLC. When combined with footprint depth, NFIC, a transcription factor not previously associated with SCLC, had the highest score of occupancy at open chromatin sites. Knockdown of NFIC impaired glucose metabolism, decreased stemness, and inhibited growth of SCLC cells in vitro and in vivo. ChIP-seq analysis identified numerous sites occupied by BRD4 in the NFIC promoter region. Knockdown of BRD4 or treatment with Bromodomain and extra-terminal domain (BET) inhibitors (BETis) markedly reduced NFIC expression in SCLC cells and SCLC PDX models. Approximately 8% of genes down-regulated by BETi treatment were repressed by NFIC knockdown in SCLC, whereas 34% of genes repressed after NFIC knockdown were also down-regulated in SCLC cells after BETi treatment. ConclusionsNFIC is a key TF and possible mediator of transcriptional regulation by BET family proteins in SCLC. Our findings highlight the potential of genome-wide chromatin accessibility analysis for elucidating mechanisms of pulmonary carcinogenesis and identifying novel targets for lung cancer therapy.

Cooperative interaction of interferon regulatory factor -1 and bromodomain-containing protein 4 on RNA polymerase activation for intrinsic innate immunity.

The human orthopneumovirus, Respiratory Syncytial Virus (RSV), is the causative agent of severe lower respiratory tract infections (LRTI) and exacerbations of chronic lung diseases. In immune competent hosts, RSV productively infects highly differentiated epithelial cells, where it elicits robust anti-viral, cytokine and remodeling programs. By contrast, basal cells are relatively resistant to RSV infection, in part, because of constitutive expression of an intrinsic innate immune response (IIR) consisting of a subgroup of interferon (IFN) responsive genes. The mechanisms controlling the intrinsic IIR are not known. Here, we use human small airway epithelial cell hSAECs as a multipotent airway stem cell model to examine regulatory control of an intrinsic IIR pathway. We find hSAECs express patterns of intrinsic IIRs, highly conserved with pluri- and multi-potent stem cells. We demonstrate a core intrinsic IIR network consisting of Bone Marrow Stromal Cell Antigen 2 (Bst2), Interferon Induced Transmembrane Protein 1 (IFITM1) and Toll-like receptor (TLR3) expression are directly under IRF1 control. Moreover, expression of this intrinsic core is rate-limited by ambient IRF1• phospho-Ser 2 CTD RNA Polymerase II (pSer2 Pol II) complexes binding to their proximal promoters. In response to RSV infection, the abundance of IRF1 and pSer2 Pol II binding is dramatically increased, with IRF1 complexing to the BRD4 chromatin remodeling complex (CRC). Using chromatin immunoprecipitation in IRF1 KD cells, we find that the binding of BRD4 is IRF1 independent. Using a small molecule inhibitor of the BRD4 acetyl lysine binding bromodomain (BRD4i), we further find that BRD4 bromodomain interactions are required for stable BRD4 promoter binding to the intrinsic IIR core promoters, as well as for RSV-inducible pSer2 Pol II recruitment. Surprisingly, BRD4i does not disrupt IRF1-BRD4 interactions, but disrupts both RSV-induced BRD4 and IRF1 interactions with pSer2 Pol II. We conclude that the IRF1 functions in two modes- in absence of infection, ambient IRF1 mediates constitutive expression of the intrinsic IIR, whereas in response to RSV infection, the BRD4 CRC independently activates pSer2 Pol II to mediates robust expression of the intrinsic IIR. These data provide insight into molecular control of anti-viral defenses of airway basal cells.

Abstract 5445: Unravelling the molecular mechanisms of stemness in small cell lung cancer using an iPSC-based approach

Abstract Introduction: The cancer stem cell hypothesis explains the heterogeneity, drug resistance, and metastatic potential of Small Cell Lung Cancer (SCLC). To address limited surgical tissue availability, we developed an induced pluripotent stem cell (iPSC) model for investigating SCLC stemness mechanisms and potential novel therapies. Cigarette smoke condensates (CSC) have previously been linked to stem cell activation in lung epithelial cells. We aimed to transform normal lung iPSCs (Lu-iPSC) through prolonged CSC exposure to identify candidate genes in the transition to cancer stem cells. Methods: Four Lu-iPSC clones derived from normal human small airway epithelial cells (SAEC) of two healthy donors were exposed to CSC at different concentrations for short- (7 days) and long-term (6 months) periods. RNA-seq was performed to identify differentially expressed genes (DEGs) relative to respective SAEC. Gene expression data of 23 SCLC cell lines and 38 NSCLC from the public databases was extracted and analyzed relative to SAEC. In vitro tri-lineage differentiation studies, drug sensitivity assays, and phenotypic characterization by flow cytometry was performed in this study. Results: Gene expression profiles of Lu-iPSCs closely resembled those of SCLC rather than NSCLC. Short-term CSC exposure reduced Lu-iPSC proliferation while increasing the expression of key stemness markers such as SALL1 in a dose-dependent manner. Concurrently, CSC exposure downregulated the expression of nodal and its inhibitors, LEFTY1 and LEFTY2, which can be considered as the earliest signs of differentiation events in Lu-iPSC. Long term CSC exposure induced expression of epithelial mesenchymal transition markers including ZEB1 and ZEB2. In-vitro trilineage differentiation studies revealed that CSC particularly decreased expression of endodermal lineage specific markers (GATA4, SOX17, and FOXA2) without affecting mesodermal and ectodermal markers. Importantly, CSC induced the phenotypic expression of several lung cancer stem cell markers (CD44, CD133, and CD338) along with resistance to cisplatin and topotecan. RNA seq analysis of long-term CSC exposed Lu-iPSCs identified 92 common DEGs (53 up and 39 down) relative to their controls. Gene set enrichment analysis on DEGs indicated upregulation of Notch signaling, IGFBPs, TGF beta receptor signaling, and NCAM1 interactions while GABA receptor activation and GPCR signaling were downregulated. Comprehensive RNA seq analysis of CSC exposed and unexposed Lu-iPSCs relative to SCLC and NSCLC, identified potential candidate genes associated with SCLC stemness. Conclusion: Our findings suggest that this iPSC model shows promise for investigating mechanisms mediating stemness in SCLC and identifying candidate genes and their associated pathways as potential targets for preclinical development of novel treatment regimens for these highly lethal neoplasms. Citation Format: Sudheer Kumar Gara, Haitao Wang, Anand Singh, Tuana Tolunay, Vivek Shukla, Ruihong Wang, Mary Zhang, Stephanie Shiffka, David S. Schrump. Unravelling the molecular mechanisms of stemness in small cell lung cancer using an iPSC-based approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5445.

SARS-CoV-2 spike protein-ACE2 interaction increases carbohydrate sulfotransferases and reduces N-acetylgalactosamine-4-sulfatase by p38 MAPK

Immunostaining in lungs of patients who died with COVID-19 infection showed increased intensity and distribution of chondroitin sulfate and decline in N-acetylgalactostamine-4-sulfatase (Arylsulfatase B; ARSB). To explain these findings, human small airway epithelial cells were exposed to the SARS-CoV-2 spike protein receptor binding domain (SPRBD) and transcriptional mechanisms were investigated. Phospho-p38 MAPK and phospho-SMAD3 increased following exposure to the SPRBD, and their inhibition suppressed the promoter activation of the carbohydrate sulfotransferases CHST15 and CHST11, which contributed to chondroitin sulfate biosynthesis. Decline in ARSB was mediated by phospho-38 MAPK-induced N-terminal Rb phosphorylation and an associated increase in Rb-E2F1 binding and decline in E2F1 binding to the ARSB promoter. The increases in chondroitin sulfotransferases were inhibited when treated with phospho-p38-MAPK inhibitors, SMAD3 (SIS3) inhibitors, as well as antihistamine desloratadine and antibiotic monensin. In the mouse model of carrageenan-induced systemic inflammation, increases in phospho-p38 MAPK and expression of CHST15 and CHST11 and declines in DNA-E2F binding and ARSB expression occurred in the lung, similar to the observed effects in this SPRBD model of COVID-19 infection. Since accumulation of chondroitin sulfates is associated with fibrotic lung conditions and diffuse alveolar damage, increased attention to p38-MAPK inhibition may be beneficial in ameliorating Covid-19 infections.

Dysfunctional neutrophil type 1 interferon responses in preschool children with recurrent wheezing and IL-4–mediated aeroallergen sensitization

BackgroundThe innate mechanisms associated with viral exacerbations in preschool children with recurrent wheezing are not understood. ObjectiveWe assessed differential gene expression in blood neutrophils from preschool children with recurrent wheezing, stratified by aeroallergen sensitization, at baseline and after exposure to polyinosinic-polycytidylic acid (poly(I:C)). We also examined whether poly(I:C)-stimulated blood neutrophils influenced airway epithelial gene expression. MethodsBlood neutrophils were purified and cultured overnight with poly(I:C) and underwent next-generation sequencing with Reactome pathway analysis. Primary human small airway epithelial cells were treated with poly(I:C)-treated neutrophil culture supernatants and were analyzed for Type 1 interferon gene expression with a targeted array. Symptoms and exacerbations were assessed in participants over 12 months. Results436 genes were differently expressed in neutrophils from children with versus without aeroallergen sensitization at baseline, with significant downregulation of Type 1 interferons. These Type 1 interferons were significantly upregulated in sensitized children after poly(I:C) stimulation. Confirmatory experiments demonstrated similar upregulation of Type 1 interferons in IL-4 treated neutrophils stimulated with poly(I:C). Poly(I:C)-treated neutrophil supernatants from children with aeroallergen sensitization also induced a Type 1 interferon response in epithelial cells. Children with aeroallergen sensitization also had higher symptom scores during exacerbations and these symptom differences persisted for three days after prednisolone treatment. ConclusionsType 1 interferon responses are dysregulated in preschool children with aeroallergen sensitization, which is in turn associated with exacerbation severity. Given the importance of Type 1 interferon signaling in viral resolution, additional studies of neutrophil Type 1 interferon responses are needed in this population.

Real-Time Exposure to 3D-Printing Emissions Elicits Metabolic and Pro-Inflammatory Responses in Human Airway Epithelial Cells.

Three-dimensional (3D) printer usage in household and school settings has raised health concerns regarding chemical and particle emission exposures during operation. Although the composition of 3D printer emissions varies depending on printer settings and materials, little is known about the impact that emissions from different filament types may have on respiratory health and underlying cellular mechanisms. In this study, we used an in vitro exposure chamber system to deliver emissions from two popular 3D-printing filament types, acrylonitrile butadiene styrene (ABS) and polylactic acid (PLA), directly to human small airway epithelial cells (SAEC) cultured in an air-liquid interface during 3D printer operation. Using a scanning mobility particle sizer (SMPS) and an optical particle sizer (OPS), we monitored 3D printer particulate matter (PM) emissions in terms of their particle size distribution, concentrations, and calculated deposited doses. Elemental composition of ABS and PLA emissions was assessed using scanning electron microscopy coupled with energy dispersive X-ray spectroscopy (SEM-EDX). Finally, we compared the effects of emission exposure on cell viability, inflammation, and metabolism in SAEC. Our results reveal that, although ABS filaments emitted a higher total concentration of particles and PLA filaments emitted a higher concentration of smaller particles, SAEC were exposed to similar deposited doses of particles for each filament type. Conversely, ABS and PLA emissions had distinct elemental compositions, which were likely responsible for differential effects on SAEC viability, oxidative stress, release of inflammatory mediators, and changes in cellular metabolism. Specifically, while ABS- and PLA-emitted particles both reduced cellular viability and total glutathione levels in SAEC, ABS emissions had a significantly greater effect on glutathione relative to PLA emissions. Additionally, pro-inflammatory cytokines including IL-1β, MMP-9, and RANTES were significantly increased due to ABS emissions exposure. While IL-6 and IL-8 were stimulated in both exposure scenarios, VEGF was exclusively increased due to PLA emissions exposures. Notably, ABS emissions induced metabolic perturbation on amino acids and energy metabolism, as well as redox-regulated pathways including arginine, methionine, cysteine, and vitamin B3 metabolism, whereas PLA emissions exposures caused fatty acid and carnitine dysregulation. Taken together, these results advance our mechanistic understanding of 3D-printer-emissions-induced respiratory toxicity and highlight the role that filament emission properties may play in mediating different respiratory outcomes.

Liquid plug propagation in computer-controlled microfluidic airway-on-a-chip with semi-circular microchannels.

This paper introduces a two-inlet, one-outlet lung-on-a-chip device with semi-circular cross-section microchannels and computer-controlled fluidic switching that enables a broader systematic investigation of liquid plug dynamics in a manner relevant to the distal airways. A leak-proof bonding protocol for micro-milled devices facilitates channel bonding and culture of confluent primary small airway epithelial cells. Production of liquid plugs with computer-controlled inlet channel valving and just one outlet allows more stable long-term plug generation and propagation compared to previous designs. The system also captures both plug speed and length as well as pressure drop concurrently. In one demonstration, the system reproducibly generates surfactant-containing liquid plugs, a challenging process due to lower surface tension that makes the plug formation less stable. The addition of surfactant decreases the pressure required to initiate plug propagation, a potentially significant effect in diseases where surfactant in the airways is absent or dysfunctional. Next, the device recapitulates the effect of increasing fluid viscosity, a challenging analysis due to higher resistance of viscous fluids that makes plug formation and propagation more difficult particularly in airway-relevant length scales. Experimental results show that increased fluid viscosity decreases plug propagation speed for a given air flow rate. These findings are supplemented by computational modeling of viscous plug propagation that demonstrates increased plug propagation time, increased maximum wall shear stress, and greater pressure differentials in more viscous conditions of plug propagation. These results match physiology as mucus viscosity is increased in various obstructive lung diseases where it is known that respiratory mechanics can be compromised due to mucus plugging of the distal airways. Finally, experiments evaluate the effect of channel geometry on primary human small airway epithelial cell injury in this lung-on-a-chip. There is more injury in the middle of the channel relative to the edges highlighting the role of channel shape, a physiologically relevant parameter as airway cross-sectional geometry can also be non-circular. In sum, this paper describes a system that pushes the device limits with regards to the types of liquid plugs that can be stably generated for studies of distal airway fluid mechanical injury.

Brilacidin as a Broad-Spectrum Inhibitor of Enveloped, Acutely Infectious Viruses.

Alphaviruses, belonging to the Togaviridae family, and bunyaviruses, belonging to the Paramyxoviridae family, are globally distributed and lack FDA-approved vaccines and therapeutics. The alphaviruses Venezuelan equine encephalitis virus (VEEV) and eastern equine encephalitis virus (EEEV) are known to cause severe encephalitis, whereas Sindbis virus (SINV) causes arthralgia potentially persisting for years after initial infection. The bunyavirus Rift Valley Fever virus (RVFV) can lead to blindness, liver failure, and hemorrhagic fever. Brilacidin, a small molecule that was designed de novo based on naturally occurring host defensins, was investigated for its antiviral activity against these viruses in human small airway epithelial cells (HSAECs) and African green monkey kidney cells (Veros). This testing was further expanded into a non-enveloped Echovirus, a Picornavirus, to further demonstrate brilacidin's effect on early steps of the viral infectious cycle that leads to inhibition of viral load. Brilacidin demonstrated antiviral activity against alphaviruses VEEV TC-83, VEEV TrD, SINV, EEEV, and bunyavirus RVFV. The inhibitory potential of brilacidin against the viruses tested in this study was dependent on the dosing strategy which necessitated compound addition pre- and post-infection, with addition only at the post-infection stage not eliciting a robust inhibitory response. The inhibitory activity of brilacidin was only modest in the context of the non-enveloped Picornavirus Echovirus, suggesting brilacidin may be less potent against non-enveloped viruses.

Climate Change and New Challenges for Rural Communities: Particulate Matter Matters.

Climate change presents multiple challenges to rural communities. Here, we investigated the toxicological potential of the six types of particulate matter most common to rural Arkansas: soil, road, and agricultural dusts, pollen, traffic exhaust, and particles from biomass burning in human small airway epithelial cells (SAECs). Biomass burning and agricultural dust demonstrated the most potent toxicological responses, exhibited as significant (p < 0.05) up-regulation of HMOX1 (oxidative stress) and TNFα (inflammatory response) genes as well as epigenetic alterations (altered expression of DNA methyltransferases DNMT1, DNMT3A, and DNMT3B, enzymatic activity, and DNA methylation of alpha satellite elements) that were evident at both 24 h and 72 h of exposure. We further demonstrate evidence of aridification in the state of Arkansas and the presence of winds capable of transporting agricultural dust- and biomass burning-associated particles far beyond their origination. Partnerships in the form of citizen science projects may provide important solutions to prevent and mitigate the negative effects of the rapidly evolving climate and improve the well-being of rural communities. Furthermore, the identification of the most toxic types of particulate matter could inform local policies related to agriculture, biomass burning, and dust control.

Transcriptome analysis reveals the impact of NETs activation on airway epithelial cell EMT and inflammation in bronchiolitis obliterans

Bronchiolitis obliterans (BO) is a chronic airway disease that was often indicated by the pathological presentation of narrowed and irreversible airways. However, the molecular mechanisms of BO pathogenesis remain unknown. Although neutrophil extracellular traps (NETs) can contribute to inflammatory disorders, their involvement in BO is unclear. This study aims to identify potential signaling pathways in BO by exploring the correlations between NETs and BO. GSE52761 and GSE137169 datasets were downloaded from gene expression omnibus (GEO) database. A series of bioinformatics analyses such as differential expression analysis, gene ontology (GO), Kyoto encyclopedia of genes and genomes (KEGG), and gene set enrichment analysis (GSEA) were performed on GSE52761 and GSE137169 datasets to identify BO potential signaling pathways. Two different types of BO mouse models were constructed to verify NETs involvements in BO. Additional experiments and bioinformatics analysis using human small airway epithelial cells (SAECs) were also performed to further elucidate differential genes enrichment with their respective signaling pathways in BO. Our study identified 115 differentially expressed genes (DEGs) that were found up-regulated in BO. Pathway enrichment analysis revealed that these genes were primarily involved in inflammatory signaling processes. Besides, we found that neutrophil extracellular traps (NETs) were formed and activated during BO. Our western blot analysis on lung tissue from BO mice further confirmed NETs activation in BO, where neutrophil elastase (NE) and myeloperoxidase (MPO) expression were found significantly elevated. Transcriptomic and bioinformatics analysis of NETs treated-SAECs also revealed that NETs-DEGs were primarily associated through inflammatory and epithelial-to-mesenchymal transition (EMT) -related pathways. Our study provides novel clues towards the understanding of BO pathogenesis, in which NETs contribute to BO pathogenesis through the activation of inflammatory and EMT associated pathways. The completion of our study will provide the basis for potential novel therapeutic targets in BO treatment.

Mechanisms of antiviral activity of the new hDHODH inhibitor MEDS433 against respiratory syncytial virus replication

Human respiratory syncytial virus (RSV) is an important cause of acute lower respiratory infections, for which no effective drugs are currently available. The development of new effective anti-RSV agents is therefore an urgent priority, and Host-Targeting Antivirals (HTAs) can be considered to target RSV infections. As a contribution to this antiviral avenue, we have characterized the molecular mechanisms of the anti-RSV activity of MEDS433, a new inhibitor of human dihydroorotate dehydrogenase (hDHODH), a key cellular enzyme of de novo pyrimidine biosynthesis. MEDS433 was found to exert a potent antiviral activity against RSV-A and RSV-B in the one-digit nanomolar range. Analysis of the RSV replication cycle in MEDS433-treated cells, revealed that the hDHODH inhibitor suppressed the synthesis of viral genome, consistently with its ability to specifically target hDHODH enzymatic activity. Then, the capability of MEDS433 to induce the expression of antiviral proteins encoded by Interferon-Stimulated Genes (ISGs) was identified as a second mechanism of its antiviral activity against RSV. Indeed, MEDS433 stimulated secretion of IFN-β and IFN-λ1 that, in turn, induced the expression of some ISG antiviral proteins, such as IFI6, IFITM1 and IRF7. Singly expression of these ISG proteins reduced RSV-A replication, thus likely contributing to the overall anti-RSV activity of MEDS433. Lastly, MEDS433 proved to be effective against RSV-A replication even in a primary human small airway epithelial cell model. Taken as a whole, these observations provide new insights for further development of MEDS433, as a promising candidate to develop new strategies for treatment of RSV infections.