Response Of Lung Epithelial Cells Research Articles

Early growth response 1/Krüppel-like factor 5 pathway inhibitor alleviates lipopolysaccharide-induced lung injury by promoting autophagy

Early transcription factors play critical roles in the development of acute lung injury/acute respiratory distress syndrome (ALI/ARDS). Early growth response 1 (EGR1) is a transcription factor essential for various biological processes, including regulation of metabolism, differentiation, and inflammation. However, its role in ALI has been poorly reported. In this study, we aimed to determine the effect of EGR1 on ALI to gain insights into the theoretical basis for further treatment of ALI. By employing concerted molecular biology techniques, we showed that EGR1 protein was upregulated in mice. EGR1 protein was upregulated in mice and human lung epithelial cells in response to lipopolysaccharide (LPS) stimulation. EGR1 knockdown promoted autophagy and reduced LPS-induced pro-inflammatory mediator production. EGR1 was preferentially bound to the GCGTGGGCG motif region and EGR1-binding peak-related genes were mainly enriched in autophagy and injury stress-related pathways. Additionally, EGR1 promoted Krüppel-like factor 5 (KLF5) transcription by binding to the KLF5 promoter region, and KLF5 knockdown significantly decreased inflammatory damage, suggesting that EGR1 promotes ALI progression by regulating KLF5 expression. Furthermore, ML264, an inhibitor of the EGR1/KLF5 pathway axis, displayed a protective role in ALI to reduce inflammation. In conclusion, our findings demonstrate the potential of EGR1 knockdown to inhibit KLF5 and promote autophagy, further reducing the inflammatory response to mitigate ALI/ARDS. The EGR1/KLF5 pathway axis may be a valuable therapeutic target for the treatment of ALI/ARDS.

Particulate matter composition drives differential molecular and morphological responses in lung epithelial cells.

Particulate matter (PM) is a ubiquitous component of air pollution that is epidemiologically linked to human pulmonary diseases. PM chemical composition varies widely, and the development of high-throughput experimental techniques enables direct profiling of cellular effects using compositionally unique PM mixtures. Here, we show that in a human bronchial epithelial cell model, exposure to three chemically distinct PM mixtures drive unique cell viability patterns, transcriptional remodeling, and the emergence of distinct morphological subtypes. Specifically, PM mixtures modulate cell viability, DNA damage responses, and induce the remodeling of gene expression associated with cell morphology, extracellular matrix organization, and cellular motility. Profiling cellular responses showed that cell morphologies change in a PM composition-dependent manner. Finally, we observed that PM mixtures with higher cadmium content induced increased DNA damage and drove redistribution among morphological subtypes. Our results demonstrate that quantitative measurement of individual cellular morphologies provides a robust, high-throughput approach to gauge the effects of environmental stressors on biological systems and score cellular susceptibilities to pollution.

Isolation and Quantification of Bacterial Membrane Vesicles for Quantitative Metabolic Studies Using Mammalian Cell Cultures.

Bacterial membrane vesicles (BMVs) are produced by most bacteria and participate in various cellular processes, such as intercellular communication, nutrient exchange, and pathogenesis. Notably, these vesicles can contain virulence factors, including toxic proteins, DNA, and RNA. Such factors can contribute to the harmful effects of bacterial pathogens on host cells and tissues. Although the general effects of BMVs on host cellular physiology are well known, the underlying molecular mechanisms are less understood. In this study, we introduce a vesicle quantification method, leveraging the membrane dye FM4-64. We utilize a linear regression model to analyze the fluorescence emitted by stained vesicle membranes to ensure consistent and reproducible vesicle-host interaction studies using cultured cells. This method is particularly valuable for identifying host cellular processes impacted by vesicles and their specific cargo. Moreover, it outcompetes unreliable protein concentration-based methods. We (1) show a linear correlation between the number of vesicles and the fluorescence signal emitted from the FM4-64 dye; (2) introduce the "vesicle load" as a new semi-quantitative unit, facilitating more reproducible vesicle-cell culture interaction experiments; (3) show that a stable vesicle load yields consistent host responses when studying vesicles from Pseudomonas aeruginosa mutants; (4) demonstrate that typical vesicle isolation contaminants, such as flagella, do not significantly skew the metabolic response of lung epithelial cells to P. aeruginosa vesicles; and (5) identify inositol monophosphatase 1 (SuhB) as a pivotal regulator in the vesicle-mediated pathogenesis of P. aeruginosa.

Host–device interactions: exposure of lung epithelial cells and fibroblasts to nickel, titanium, or nitinol affect proliferation, reactive oxygen species production, and cellular signaling

Endoscopic implantation of medical devices for the treatment of lung diseases, including airway stents, unidirectional valves and coils, is readily used to treat central airway disease and emphysema. However, granulation and fibrotic tissue formation impairs treatment effectiveness. To date little is known about the interaction between implanted devices, often made from metals, such as nickel, titanium or nitinol, and cells in the airways. Here, we study the response of lung epithelial cells and fibroblasts to implant device materials. The adhesion and proliferation of bronchial epithelial cells and lung fibroblasts upon exposure to 10 × 3 × 1 mm pieces of nickel, titanium or nitinol is examined using light and scanning electron microscopy. Pro-inflammatory cytokine mRNA expression and release, signaling kinase activity and intracellular free radical production are assessed. Nitinol, and to a lesser extent nickel and titanium, surfaces support the attachment and growth of lung epithelial cells. Nitinol induces a rapid and significant alteration of kinase activity. Cells directly exposed to nickel or titanium produce free radicals, but those exposed to nitinol do not. The response of lung epithelial cells and fibroblasts depends on the metal type to which they are exposed. Nitinol induces cellular surface growth and the induction of kinase activity, while exposure of lung epithelial cells to nickel and titanium induces free radical production, but nitinol does not.Graphical

Computational framework to understand the clinical stages of COVID-19 and visualization of time course for various treatment strategies.

Coronavirus disease 2019 is known to be regulated by multiple factors such as delayed immune response, impaired T cell activation, and elevated levels of proinflammatory cytokines. Clinical management of the disease remains challenging due to interplay of various factors as drug candidates may elicit different responses depending on the staging of the disease. In this context, we propose a computational framework which provides insights into the interaction between viral infection and immune response in lung epithelial cells, with an aim of predicting optimal treatment strategies based on infection severity. First, we formulate the model for visualizing the nonlinear dynamics during the disease progression considering the role of T cells, macrophages and proinflammatory cytokines. Here, we show that the model is capable of emulating the dynamic and static data trends of viral load, T cell, macrophage levels, interleukin (IL)-6 and TNF-α levels. Second, we demonstrate the ability of the framework to capture the dynamics corresponding to mild, moderate, severe, and critical condition. Our result shows that, at late phase (>15 days), severity of disease is directly proportional to pro-inflammatory cytokine IL6 and tumor necrosis factor (TNF)-α levels and inversely proportional to the number of T cells. Finally, the simulation framework was used to assess the effect of drug administration time as well as efficacy of single or multiple drugs on patients. The major contribution of the proposed framework is to utilize the infection progression model for clinical management and administration of drugs inhibiting virus replication and cytokine levels as well as immunosuppressant drugs at various stages of the disease.

Pulmonary infection is associated with an increased IL-6 in acute exacerbation chronic obstructive pulmonary disease

Objective Acute Exacerbation Chronic Obstructive Pulmonary Disease (AECOPD) is associated with an acute worsening of respiratory symptoms that have effects on lung function, quality of life and health economic burden. In addition, the development of pulmonary infections is a common complication of Chronic Obstructive Pulmonary Disease (COPD). In the pathophysiology of AECOPD, interleukin (IL)-6 is a pleiotropic cytokine that can be produced by inflammatory and primary lung epithelial cells in response to a variety of different stimuli. We aim to investigate the correlation between serum cytokine levels and AECOPD with pulmonary infection. Methods 37 AECOPD patients diagnosed with pulmonary infection and 33 patients diagnosed with AECOPD only were selected. All COPD patients were diagnosed according to the Global Initiative for Chronic Obstructive Lung Disease (GOLD) criteria. Serum samples for C-reactive protein (CRP) and cytokines were obtained from the patients immediately after admission. Serum concentrations of cytokines were measured using a fluorescent bead immunoassay on a flow cytometer. Logistic regression was used to identify risk factors for AECOPD co-infection of the lungs. Results Serum characterization of our cohort showed patients with AECOPD and pulmonary infection had higher levels of IL-6 and IL-10 compared with the AECOPD group, and IL-6 was independently associated with AECOPD with pulmonary infection. ROC curve analysis showed that IL-6 was a useful predictor of the incidence of pulmonary infection in AECOPD patients. Conclusions Our findings highlight the role of IL-6 in the pathogenesis of AECOPD with pulmonary infection.

Nsp16 shields SARS-CoV-2 from efficient MDA5 sensing and IFIT1-mediated restriction.

Methylation of the mRNA 5' cap by cellular methyltransferases enables efficient translation and avoids recognition by innate immune factors. Coronaviruses encode viral 2'-O-methyltransferases to shield their RNA from host factors. Here, we generate recombinant SARS-CoV-2 harboring a catalytically inactive 2'-O-methyltransferase Nsp16, Nsp16mut, and analyze viral replication in human lung epithelial cells. Although replication is only slightly attenuated, we find SARS-CoV-2 Nsp16mut to be highly immunogenic, resulting in a strongly enhanced release of type I interferon upon infection. The elevated immunogenicity of Nsp16mut is absent in cells lacking the RNA sensor MDA5. In addition, we report that Nsp16mut is highly sensitive to type I IFN treatment and demonstrate that this strong antiviral effect of type I IFN is mediated by the restriction factor IFIT1. Together, we describe a dual role for the 2'-O-methyltransferase Nsp16 during SARS-CoV-2 replication in avoiding efficient recognition by MDA5 and in shielding its RNA from interferon-induced antiviral responses, thereby identifying Nsp16 as a promising target for generating attenuated and highly immunogenic SARS-CoV-2 strains and as a potential candidate for therapeutic intervention.

Insights into functional connectivity in mammalian signal transduction pathways by pairwise comparison of protein interaction partners of critical signaling hubs.

Growth factors and cytokines activate signal transduction pathways and regulate gene expression in eukaryotes. Intracellular domains of activated receptors recruit several protein kinases as well as transcription factors that serve as platforms or hubs for the assembly of multi-protein complexes. The signaling hubs involved in a related biologic function often share common interaction proteins and target genes. This functional connectivity suggests that a pairwise comparison of protein interaction partners of signaling hubs and network analysis of common partners and their expression analysis might lead to the identification of critical nodes in cellular signaling. A pairwise comparison of signaling hubs across several related pathways might reveal novel signaling modules. Analysis of protein interaction connectome by Venn (PIC-Venn) of transcription factors STAT1, STAT3, NFKB1, RELA, FOS, and JUN, and their common interaction network suggested that BRCA1 and TSC22D3 function as critical nodes in immune responses by connecting the signaling hubs into signaling modules. Transcriptional regulation of critical hubs may play a major role in the lung epithelial cells in response to SARS-CoV-2 and in COVID-19 patients. Mutations and differential expression levels of these critical nodes and modules in pathological conditions might deregulate signaling pathways and their target genes involved in inflammation. Biological connectivity emerges from the structural connectivity of interaction networks across several signaling hubs in related pathways. The main objectives of this study are to identify critical hubs, critical nodes, and modules involved in the signal transduction pathways of innate and adaptive immunity. Application of PIC-Venn to several signaling hubs might reveal novel nodes and modules that can be targeted by small regulatory molecules to simultaneously activate or inhibit cell signaling in health and disease.

Inflammatory response in human lung cells stimulated with plasma from COPD patients.

BackgroundChronic obstructive pulmonary disease (COPD) is a condition resulting from a persistent inflammatory state in the airways even after smoking cessation. Intriguingly, the reasons behind this persistence of the inflammatory influx without smoking exposure have not been fully unraveled. We aimed to explore the hypothesis that systemic inflammation in COPD patients influences lung cell inflammatory response.MethodsWe cultured human lung fibroblast and human airway epithelial cell lines with plasma from COPD patients (four emphysematous-COPD, four asthma-COPD overlap, four chronic bronchitis-COPD, and four bronchiectasis- COPD), and four smokers or ex-smokers without COPD as controls. Non-stimulated cells were used as controls. We measured Interleukine-8 (IL-8), C-reactive protein (CRP) and matrix metalloproteinase-9 (MMP-9) in plasma and culture supernatants by ELISA.ResultsCells stimulated with plasma from COPD patients and non-COPD smoker subjects produced higher CRP, IL- 8 and MMP-9 levels, an increase for COPD in CRP (p=0.029) in epithelial cells and IL-8 (p=0.039) in fibroblasts and decrease for MMP-9 (p=0.039) in fibroblasts, compared with non-stimulated cells. The response was higher in epithelial cells for IL-8 (p=0.003) and in fibroblasts for MMP-9 (p=0.063). The plasma from chronic bronchitis and bronchiectasis phenotypes induced higher IL-8 in fibroblasts.ConclusionsPlasma from COPD patients increases the inflammatory response in lung epithelial cells and lung fibroblasts, with a different response depending on the cell type and clinical phenotype.

Streptococcus pneumoniae and Influenza A Virus Co-Infection Induces Altered Polyubiquitination in A549 Cells.

Epithelial cells are an important line of defense within the lung. Disruption of the epithelial barrier by pathogens enables the systemic dissemination of bacteria or viruses within the host leading to severe diseases with fatal outcomes. Thus, the lung epithelium can be damaged by seasonal and pandemic influenza A viruses. Influenza A virus infection induced dysregulation of the immune system is beneficial for the dissemination of bacteria to the lower respiratory tract, causing bacterial and viral co-infection. Host cells regulate protein homeostasis and the response to different perturbances, for instance provoked by infections, by post translational modification of proteins. Aside from protein phosphorylation, ubiquitination of proteins is an essential regulatory tool in virtually every cellular process such as protein homeostasis, host immune response, cell morphology, and in clearing of cytosolic pathogens. Here, we analyzed the proteome and ubiquitinome of A549 alveolar lung epithelial cells in response to infection by either Streptococcus pneumoniae D39Δcps or influenza A virus H1N1 as well as bacterial and viral co-infection. Pneumococcal infection induced alterations in the ubiquitination of proteins involved in the organization of the actin cytoskeleton and Rho GTPases, but had minor effects on the abundance of host proteins. H1N1 infection results in an anti-viral state of A549 cells. Finally, co-infection resembled the imprints of both infecting pathogens with a minor increase in the observed alterations in protein and ubiquitination abundance.

Protein Disulfide Isomerase A3 Regulates Influenza Neuraminidase Activity and Influenza Burden in the Lung.

Influenza (IAV) neuraminidase (NA) is a glycoprotein required for the viral exit from the cell. NA requires disulfide bonds for proper function. We have recently demonstrated that protein disulfide isomerase (PDI)A3 is required for oxidative folding of IAV hemagglutinin (HA), and viral propagation. However, it not known whether PDIs are required for NA maturation or if these interactions represent a putative target for the treatment of influenza infection. We sought to determine whether PDIA3 is required for disulfide bonds of NA, its activity, and propagation of the virus. Requirement of disulfides for NA oligomerization and activity were determined using biotin switch and redox assays in WT and PDIA3−/− in A549 cells. A PDI specific inhibitor (LOC14) was utilized to determine the requirement of PDIs in NA activity, IAV burden, and inflammatory response in A549 and primary mouse tracheal epithelial cells. Mice were treated with the inhibitor LOC14 and subsequently examined for IAV burden, NA activity, cytokine, and immune response. IAV-NA interacts with PDIA3 and this interaction is required for NA activity. PDIA3 ablation or inhibition decreased NA activity, viral burden, and inflammatory response in lung epithelial cells. LOC14 treatment significantly attenuated the influenza-induced inflammatory response in mice including the overall viral burden. These results provide evidence for PDIA3 inhibition suppressing NA activity, potentially providing a novel platform for host-targeted antiviral therapies.

Ubiquitin specific peptidase 25 alleviates acute lung injury and suppresses the inflammatory response in lung epithelial cells.

As COVID-19 spreads over the world, the treatment of acute lung injury (ALI) has attracted much attention. Considering ubiquitin-specific protease (USP) 25 has been relevant to inflammation, this article focused on its role in ALI and its regulatory mechanism. Lipopolysaccharide (LPS) was applied to separately stimulate mice and human lung epithelial cells to establish in vivo and in vitro ALI models. To discover the effects of USP25 overexpression on mouse, lung pathology, inflammatory factor levels, edema, number of inflammatory cells, and downstream protein levels were evaluated. USP25 overexpression in mice could alleviate LPS-induced lung tissue lesions and edema, and reduce inflammatory factors and inflammatory cells. It also inhibited the levels of downstream TRAF6, MAPK pathway-related proteins, and Fos Proto-Oncogene (FOS) in vivo. Furthermore, BEAS-2B cells were transfected with TNF receptor-associated factor 6 (TRAF6) plasmids to study the role of TRAF6 in the regulatory mechanism of USP25. TRAF6 overexpression was found to reverse the functions of USP25 overexpression on cells. In conclusion, USP25 reduced ALI and inhibited inflammation in lung epithelial cells via regulating TRAF6/MAPK/FOS signaling.

OLFM4 Regulates Lung Epithelial Cell Function in Sepsis-Associated ARDS/ALI via LDHA-Mediated NF-κB Signaling.

BackgroundAcute respiratory distress syndrome (ARDS) is one of the leading causes of death in patients with sepsis. As such, early and accurate identification of sepsis-related ARDS is critical.MethodsBioinformatic analysis was used to explore the GEO datasets. ELISA method was used to detect the plasma or cellular supernatant of relevant proteins. Quantitative real-time PCR was used for mRNA measurements and Western blot was applied for protein measurements. Immunohistochemistry staining and Immunofluorescence staining were used to identify the localization of OLFM4. Cecal ligation and puncture (CLP) model was used to establish sepsis model.ResultsThe bioinformatic analysis results identified ten genes (CAMP, LTF, RETN, LCN2, ELANE, PGLYRP1, BPI, DEFA4, MPO, and OLFM4) as critical in sepsis and sepsis-related ARDS. OLFM4, LCN2, and BPI were further demonstrated to have diagnostic values in sepsis-related ARDS. Plasma expression of OLFM4 and LCN2 was also upregulated in sepsis-related ARDS patients compared to septic patients alone. OLFM4 expression was significantly increased in the lung tissues of septic mice and was co-localized with Ly6G+ neutrophils, F4/80+ macrophages and pro-surfactant C+ lung epithelial cells. In vitro data showed that OLFM4 expression in lung epithelial cells was downregulated upon LPS stimulation, whereas neutrophil media induced OLFM4 expression in lung epithelial cells. Overexpression of OLFM4 and treatment with recombinant OLFM4 effectively suppressed LPS-induced pro-inflammatory responses in lung epithelial cells. Furthermore, the increased levels of LDHA phosphorylation and the downstream NF-κB activation induced by LPS in epithelial cells were effectively diminished by OLFM4 overexpression and recombinant OLFM4 treatment via a reduction in ROS production and HIF1α expression.ConclusionOLFM4 may regulate the pro-inflammatory response of lung epithelial cells in sepsis-related ARDS by modulating metabolic disorders; this result could provide new insights into the treatment of sepsis-induced ARDS.

Exposure to glyphosate and tetrachlorvinphos induces cytotoxicity and global DNA methylation in human cells.

Two organophosphate pesticides-glyphosate and tetrachlorvinphos-have been announced as carcinogens to humans by various authorities, including the European Chemical Agency and the Environmental Protection Agency. We aimed to investigate molecular mechanisms associated with carcinogenicity and to examine changes in global m5C DNA methylation and cytotoxic potential in A549 lung epithelial cells in response to glyphosate and tetrachlorvinphos, and differential gene expression of m5C DNA methyltransferase genes in Sprague Dawley rats to Roundup (commercial formulation of glyphosate). Global m5C level significantly increased after 1500μM glyphosate exposure for 24h. We determined that exposure to tetrachlorvinphos did not significantly increase the m5C level in A549 cells for 24h. Additionally, we did not observe significant DNA methylation alteration for both pesticides after 12h exposure. In the animal study, we observed that DNA methyltransferase genes (DNMT3b and DNMT3a) showed significantly higher expression in Roundup-exposed rats than the control group in the liver and kidney. We also observed that a significant cytotoxic effect was determined after the treatment of the cells with higher concentrations of glyphosate and tetrachlorvinphos. Our results revealed that DNA methylation could be modified by exposure to glyphosate and that exposure to Roundup was associated with the differential expression level of m5C DNA methylation methyltransferase. Finally, exposure to both pesticides increased cytotoxicity.

Acetylation, Methylation and Allysine Modification Profile of Viral and Host Proteins during Influenza A Virus Infection.

Protein modifications dynamically occur and regulate biological processes in all organisms. Towards understanding the significance of protein modifications in influenza virus infection, we performed a global mass spectrometry screen followed by bioinformatics analyses of acetylation, methylation and allysine modification in human lung epithelial cells in response to influenza A virus infection. We discovered 8 out of 10 major viral proteins and 245 out of 2280 host proteins detected to be differentially modified by three modifications in infected cells. Some of the identified proteins were modified on multiple amino acids residues and by more than one modification; the latter occurred either on different or same residues. Most of the modified residues in viral proteins were conserved across >40 subtypes of influenza A virus, and influenza B or C viruses and located on the protein surface. Importantly, many of those residues have already been determined to be critical for the influenza A virus. Similarly, many modified residues in host proteins were conserved across influenza A virus hosts like humans, birds, and pigs. Finally, host proteins undergoing the three modifications clustered in common functional networks of metabolic, cytoskeletal, and RNA processes, all of which are known to be exploited by the influenza A virus.

Loss of Mitochondrial Adenine Nucleotide Translocase (ANT1) Regulates Cellular Senescence and Protects against Chronic Obstructive Pulmonary Disease

Chronic obstructive pulmonary disease (COPD) is a destructive lung disease resulting in alveolar tissue loss, airway remodeling and inflammation with cigarette smoke (CS) being a major causative factor. Mitochondrial dysfunction can lead to changes in cell fate implicated in COPD. Cellular senescence, a state of cell cycle arrest, results in abnormal responses in lung epithelial cells. An important protein in mitochondrial metabolism and cell fate is adenine nucleotide translocase (ANT), a mitochondrial ATP transporter. Our studies suggest that levels of ANT are altered in COPD. We hypothesize that loss of ANTs results in increased cellular senescence thus altering COPD pathogenesis and airway dysfunction. Methods Utilizing siRNA knockdown of SLC25A4 (ANT1) and SLC25A5 (ANT2) in human bronchial epithelial cells, Beas-2bs, we assessed cellular senescence (p53, p21, p16) via western blot, qt-PCR and B-galactocidase analysis. Cells were also exposed to CS extract. Wildtype (WT) and ant1-null mice were exposed to CS for 2 and 6mo or air control with lung histology and morphometry analysis. We performed single cell RNA sequencing on lungs from 2mo air and CS-exposed mice. Single-cell library preparation was performed using 10X Genomics 5prime v1 reagents to analyze 5000 cells/mouse (n=3 mice/group). Alignment was performed using Cell Ranger and standard workflow analysis using R package Seurat v3.2. Cell clusters were generated using unsupervised UMAP. Pathway analysis on differential gene expression lists by cell type was performed using Ingenuity Pathway Analysis. Results Knockdown of ANT1 or ANT2 in Beas-2b cells results in an increase in cell senescence proteins p16, p21, p53 and beta-galactosidase staining. Surprisingly, ant1-null mice exposed to 6months of CS exposure are protected from alveolar destruction (fig.1), airway remodeling and chronic inflammation with fewer macrophages. Single cell RNA sequencing of WT lung cells demonstrates that CS (2 mo) causes a significant increase in genes associated with senescence in alveolar type 1 epithelial cells (AT1, p-value 1.0x10-6) with dysregulation of mitochondrial-related gene pathways when compared to air controls: mitochondrial dysfunction, upregulation of Eif2 signaling and NRF2-mediated oxidative stress, and downregulation in sirtuin signaling. Alveolar type 2 cells (AT2) from WT-smoked mice show downregulation of Eif2 signaling, unfolded protein response, NRF2-mediated oxidative stress, and sirtuin signaling. Expression of slc25a4 (ant1) is upregulated in AT2 cells in WT-smoked mice (p-value 1.7x10-17). Mitochondrial and cell fate pathways were significantly altered in AT1 and AT2 cells from WT-smoked mice but not from ant1-null mice, suggesting a key role in lung epithelial responses in COPD. Conclusions ANTs alter cell senescence in alveolar epithelial cells. An In vivo mouse model shows that ant1 is upregulated in AT2 cells after CS and loss of ant1 is protective against CS lung injury. Ant1 regulates epithelial cell fate, mitochondrial function and CS-related cell injury, providing a potential therapeutic avenue for COPD.

Toxicological assessment of e‐cigarette or vaping product use associated lung injury (EVALI) cartridges and constituents

Rationale Definitive causative agents of the outbreak associated with the use of e-cigarette, or vaping product use associated lung injury (EVALI) are unknown. Patients diagnosed with EVALI have used vape products containing tetrahydrocannabinol (THC) and vitamin E acetate (VEA). Hypothesis We hypothesized that inhalation of these EVALI vape cartridges and their constituents induce pulmonary toxicity, mediated by oxidative damage and inflammatory responses, leading to acute lung injury, and alters SARS-CoV-2 related proteins. Methods Vaping products were recovered from hospitalized patients at the University of Rochester Medical Center. Cartridge constituents were characterized by gas chromatography and mass spectrometry. Comparative toxicity of exposure to common vape cartridge components such as medium-chain triglyceride (MCT) oil, VEA, and patient-derived cartridge aerosols was assessed by in vitro and in vivo models. Lung epithelial cells (BEAS-2B) and monocytes (Mono-Mac-6). Cells were exposed to MCT, VEA, and cartridge aerosols and the elicited inflammatory response, barrier function, and lipid-laden index were determined. C57BL/6 mice were exposed to MCT, VEA, and vape cartridge aerosols for three days (1 hr/day). The immune response was determined by differential cell counts, cytokine milieu, and lipidomics analysis in bronchoalveolar lavage fluid (BALF). Lung surfactant protein (SP-A), SARS-CoV-2 proteins (ACE2 and TMPRSS2) were quantified in the lung homogenates. Results Constituents identified patient-derived vaping products included reactive hydrocarbons and various forms of cannabinoids. VEA and cartridge aerosols induced significantly increased IL-6 and IL-8 responses in lung epithelial cells and monocytes. MCT, VEA, and cartridge aerosols induced barrier dysfunction. Increased lipid-laden indices were observed in MM6 cells. In mice, acute exposure to cartridge aerosols caused a significant infiltration of leukocytes in BALF and increased IL-6, eotaxin, and G-CSF in the BALF. Lipidomics analysis showed significantly increased eicosanoid inflammatory mediators and leukotrienes. Reduced levels of SP-A levels were seen in lung homogenates. Exposure to cartridge aerosols showed the most significant effects and toxicity compared to MCT and VEA. SARS-CoV-2 related proteins were not affected. Conclusion Acute exposure to vape cartridges induces cytotoxicity, barrier dysfunction, and elicit an inflammatory response in lung cells and mice, which are potential predictive toxicological markers of EVALI. Toxicity of vape cartridges was independent of SARS-CoV-2 proteins. This study provides important identification of potential chemical constituents, toxicological responses, and potential biomarkers of EVALI.

MDA5 Governs the Innate Immune Response to SARS-CoV-2 in Lung Epithelial Cells

Recent studies profiling the innate immune signatures in patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) suggest that cellular responses to viral challenge impact disease severity. Yet, the molecular events that underlie cellular recognition and response to SARS-CoV-2 infection remains to be elucidated. Here, we find that SARS-CoV-2 replication induces a delayed interferon (IFN) response in lung epithelial cells. Through a survey of putative sensors involved in detection of RNA virus infection, we found that MDA5 and LGP2 primarily regulate IFN induction in response to SARS-CoV-2 infection. Additionally, we find that IRF-3, -5, and NF-kB/p65 are the key transcription factors regulating the IFN response during SARS-CoV-2 infection. In summary, these findings provide critical insights into the molecular basis of the innate immune recognition and signaling response to SARS-CoV-2.Funding: This work was supported by the following grants to the Sanford Burnham Prebys Medical Discovery Institute: DoD: W81XWH-20-1-0270; DHIPC: U19 AI118610; Fluomics/NOSI: U19 AI135972, as well as generous philanthropic donations from Dinah Ruch and Susan & James Blair. This work was additionally supported by the following grants to Northwestern University Feinberg School of Medicine: a CTSA supplement to NCATS: UL1 TR002389; a CTSA supplement to NUCATS with the generous support of the Dixon family: UL1 TR001422; and a Cancer Center supplement: P30 CA060553. Development and implementation of iPS cell technology for production of airway epithelial cells was supported by Incubation Program from Office of Society Academia Collaboration for Innovation, Kyoto University. Conflict of Interest: The authors declare no competing interests.

MDA5 Governs the Innate Immune Response to SARS-CoV-2 in Lung Epithelial Cells

Recent studies have profiled the innate immune signatures in patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and suggest that cellular responses to viral challenge may affect disease severity. Yet the molecular events that underlie cellular recognition and response to SARS-CoV-2 infection remain to be elucidated. Here, we find that SARS-CoV-2 replication induces a delayed interferon (IFN) response in lung epithelial cells. By screening 16 putative sensors involved in sensing of RNA virus infection, we found that MDA5 and LGP2 primarily regulate IFN induction in response to SARS-CoV-2 infection. Further analyses revealed that viral intermediates specifically activate the IFN response through MDA5-mediated sensing. Additionally, we find that IRF3, IRF5, and NF-κB/p65 are the key transcription factors regulating the IFN response during SARS-CoV-2 infection. In summary, these findings provide critical insights into the molecular basis of the innate immune recognition and signaling response to SARS-CoV-2.

Gene expression profiling of SARS-CoV-2 infections reveal distinct primary lung cell and systemic immune infection responses that identify pathways relevant in COVID-19 disease.

To identify key gene expression pathways altered with infection of the novel coronavirus SARS-CoV-2, we performed the largest comparative genomic and transcriptomic analysis to date. We compared the novel pandemic coronavirus SARS-CoV-2 with SARS-CoV and MERS-CoV, as well as influenza A strains H1N1, H3N2 and H5N1. Phylogenetic analysis confirms that SARS-CoV-2 is closely related to SARS-CoV at the level of the viral genome. RNAseq analyses demonstrate that human lung epithelial cell responses to SARS-CoV-2 infection are distinct. Extensive Gene Expression Omnibus literature screening and drug predictive analyses show that SARS-CoV-2 infection response pathways are closely related to those of SARS-CoV and respiratory syncytial virus infections. We validated SARS-CoV-2 infection response genes as disease-associated using Kaplan–Meier survival estimates in lung disease patient data. We also analysed COVID-19 patient peripheral blood samples, which identified signalling pathway concordance between the primary lung cell and blood cell infection responses.