Identification of Required Host Factors for SARS-CoV-2 Infection in Human Cells.

To better understand host-virus genetic dependencies and find potential therapeutic targets for COVID-19, we performed a genome-scale CRISPR loss-of-function screen to identify host factors required for SARS-CoV-2 viral infection of human alveolar epithelial cells. Top-ranked genes cluster into distinct pathways, including the vacuolar ATPase proton pump, Retromer, and Commander complexes. We validate these gene targets using several orthogonal methods such as CRISPR knockout, RNA interference knockdown, and small-molecule inhibitors. Using single-cell RNA-sequencing, we identify shared transcriptional changes in cholesterol biosynthesis upon loss of top-ranked genes. In addition, given the key role of the ACE2 receptor in the early stages of viral entry, we show that loss of RAB7A reduces viral entry by sequestering the ACE2 receptor inside cells. Overall, this work provides a genome-scale, quantitative resource of the impact of the loss of each host gene on fitness/response to viral infection.

High-resolution transcriptomic and epigenetic profiling identifies novel regulators of COPD.

Patients with chronic obstructive pulmonary disease (COPD) are still waiting for curative treatments. Considering its environmental cause, we hypothesized that COPD will be associated with altered epigenetic signaling in lung cells. We generated genome-wide DNA methylation maps at single CpG resolution of primary human lung fibroblasts (HLFs) across COPD stages. We show that the epigenetic landscape is changed early in COPD, with DNA methylation changes occurring predominantly in regulatory regions. RNA sequencing of matched fibroblasts demonstrated dysregulation of genes involved in proliferation, DNA repair, and extracellular matrix organization. Data integration identified 110 candidate regulators of disease phenotypes that were linked to fibroblast repair processes using phenotypic screens. Our study provides high-resolution multi-omic maps of HLFs across COPD stages. We reveal novel transcriptomic and epigenetic signatures associated with COPD onset and progression and identify new candidate regulators involved in the pathogenesis of chronic lung diseases. The presence of various epigenetic factors among the candidates demonstrates that epigenetic regulation in COPD is an exciting research field that holds promise for novel therapeutic avenues for patients.

Characteristics and anatomic location of PD-1+TCF1+ stem-like CD8 T cells in chronic viral infection and cancer.

CD8 T cells play an essential role in antitumor immunity and chronic viral infections. Recent findings have delineated the differentiation pathway of CD8 T cells in accordance with the progenitor-progeny relationship of TCF1+ stem-like and Tim-3+TCF1- more differentiated T cells. Here, we investigated the characteristics of stem-like and differentiated CD8 T cells isolated from several murine tumor models and human lung cancer samples in terms of phenotypic and transcriptional features as well as their location compared to virus-specific CD8 T cells in the chronically lymphocytic choriomeningitis virus (LCMV)-infected mice. We found that CD8 tumor-infiltrating lymphocytes (TILs) in both murine and human tumors exhibited overall similar phenotypic and transcriptional characteristics compared to corresponding subsets in the spleen of chronically infected mice. Moreover, stem-like CD8 TILs exclusively responded and produced effector-like progeny CD8 T cells in vivo after antigenic restimulation, confirming their lineage relationship and the proliferative potential of stem-like CD8 TILs. Most importantly, similar to the preferential localization of PD-1+ stem-like CD8 T cells in T cell zones of the spleen during chronic LCMV infection, we found that the PD-1+ stem-like CD8 TILs in lung cancer samples are preferentially located not in the tumor parenchyma but in tertiary lymphoid structures (TLSs). The stem-like CD8 T cells are present in TLSs located within and at the periphery of the tumor, as well as in TLSs closely adjacent to the tumor parenchyma. These findings suggest that TLSs provide a protective niche to support the quiescence and maintenance of stem-like CD8 T cells in the tumor.

Intracellular Pseudomonas aeruginosa within the Airway Epithelium of Cystic Fibrosis Lung Tissues.

Rationale: Pseudomonas aeruginosa is the major bacterial pathogen colonizing the airways of adult patients with cystic fibrosis (CF) and causes chronic infections that persist despite antibiotic therapy. Intracellular bacteria may represent an unrecognized reservoir of bacteria that evade the immune system and antibiotic therapy. Although the ability of P. aeruginosa to invade and survive within epithelial cells has been described in vitro in different epithelial cell models, evidence of this intracellular lifestyle in human lung tissues is currently lacking. Objectives: To detect and characterize intracellular P. aeruginosa in CF airway epithelium from human lung explant tissues. Methods: We sampled lung explant tissues from patients with CF undergoing lung transplantation and non-CF lung donor control tissue. We analyzed lung tissue sections for the presence of intracellular P. aeruginosa using quantitative culture and microscopy, in parallel to histopathology and airway morphometry. Measurements and Main Results: P. aeruginosa was isolated from the lungs of seven patients with CF undergoing lung transplantation. Microscopic assessment revealed the presence of intracellular P. aeruginosa within airway epithelial cells in three of the seven patients analyzed at a varying but low frequency. We observed those events occurring in lung regions with high bacterial burden. Conclusions: This is the first study describing the presence of intracellular P. aeruginosa in CF lung tissues. Although intracellular P. aeruginosa in airway epithelial cells is likely relatively rare, our findings highlight the plausible occurrence of this intracellular bacterial reservoir in chronic CF infections.

Transcriptomics Analysis Identifies the Decline in the Alveolar Type II Stem Cell Niche in Aged Human Lungs.

Aging poses a global public health challenge, which is linked to the rise of age-related lung diseases. The precise understanding of the molecular and genetic changes in the aging lung that elevate the risk of acute and chronic lung diseases remains incomplete. Alveolar type II (AT2) cells are stem cells that maintain epithelial homeostasis and repair the lung after injury. AT2 progenitor function decreases with aging. The maintenance of AT2 function requires niche support from other cell types, but little has been done to characterize alveolar alterations with aging in the AT2 niche. To systematically profile the genetic changes associated with age, we present a single-cell transcriptional atlas comprising nearly half a million cells from the healthy lungs of human subjects spanning various ages, sexes, and smoking statuses. Most annotated cell lineages in aged lungs exhibit dysregulated genetic programs. Specifically, the aged AT2 cells demonstrate loss of epithelial identities, heightened inflammaging characterized by increased expression of AP-1 (Activator Protein-1) transcription factor and chemokine genes, and significantly increased cellular senescence. Furthermore, the aged mesenchymal cells display a remarkable decrease in collagen and elastin transcription and a loss of support to epithelial cell stemness. The decline of the AT2 niche is further exacerbated by a dysregulated genetic program in macrophages and dysregulated communications between AT2 and macrophages in aged human lungs. These findings highlight the dysregulations observed in both AT2 stem cells and their supportive niche cells, potentially contributing to the increased susceptibility of aged populations to lung diseases.

The carboxyl-terminal TSP1-homology domain is the biologically active effector peptide of matricellular protein CCN5 that counteracts profibrotic CCN2.

Cellular Communication Network (CCN) proteins have multimodular structures important for their roles in cellular responses associated with organ development and tissue homeostasis. CCN2 has previously been reported to be secreted as a preproprotein that requires proteolytic activation to release its bioactive carboxyl-terminal fragment. Here, our goal was to resolve whether CCN5, a divergent member of the CCN family with converse functions relative to CCN2, releases the TSP1 homology domain as its bioactive signaling entity. The recombinant CCN5 or CCN3 TSP1 homology domains were produced in ExpiCHO-S or DG44 CHO cells as secretory fusion proteins appended to the carboxyl-terminal end of His-Halo-Sumo or amino-terminal end of human albumin and purified from the cell culture medium. We tested these fusion proteins in various phosphokinase signaling pathways or cell physiologic assays. Fusion proteins with the CCN5 TSP1 domain inhibited key signaling pathways previously reported to be stimulated by CCN2, irrespective of fusion partner. The fusion proteins also efficiently inhibited CCN1/2-stimulated cell migration and gap closure following scratch wound of fibroblasts. Fusion protein with the CCN3 TSP1 domain inhibited these functions with similar efficacy and potency as that of the CCN5 TSP1 domain. The CCN5 TSP1 domain also recapitulated a positive regulatory function previously assigned to full-length CCN5, that is, induction of estrogen receptor-α mRNA expression in triple negative MDA-MB-231 mammary adenocarcinoma cells and inhibited epithelial-to-mesenchymal transition and CCN2-induced mammosphere formation of MCF-7 adenocarcinoma cells. In conclusion, the CCN5 TSP1 domain is the bioactive entity that confers the biologic functions of unprocessed CCN5.

Type-II IFN inhibits SARS-CoV-2 replication in human lung epithelial cells and ex vivo human lung tissues through indoleamine 2,3-dioxygenase-mediated pathways.

Interferons (IFNs) are critical for immune defense against pathogens. While type-I and -III IFNs have been reported to inhibit SARS-CoV-2 replication, the antiviral effect and mechanism of type-II IFN against SARS-CoV-2 remain largely unknown. Here, we evaluate the antiviral activity of type-II IFN (IFNγ) using human lung epithelial cells (Calu3) and ex vivo human lung tissues. In this study, we found that IFNγ suppresses SARS-CoV-2 replication in both Calu3 cells and ex vivo human lung tissues. Moreover, IFNγ treatment does not significantly modulate the expression of SARS-CoV-2 entry-related factors and induces a similar level of pro-inflammatory response in human lung tissues when compared with IFNß treatment. Mechanistically, we show that overexpression of indoleamine 2,3-dioxygenase 1 (IDO1), which is most profoundly induced by IFNγ, substantially restricts the replication of ancestral SARS-CoV-2 and the Alpha and Delta variants. Meanwhile, loss-of-function study reveals that IDO1 knockdown restores SARS-CoV-2 replication restricted by IFNγ in Calu3 cells. We further found that the treatment of l-tryptophan, a substrate of IDO1, partially rescues the IFNγ-mediated inhibitory effect on SARS-CoV-2 replication in both Calu3 cells and ex vivo human lung tissues. Collectively, these results suggest that type-II IFN potently inhibits SARS-CoV-2 replication through IDO1-mediated antiviral response.

Thorium Alters Lung Surfactant Protein Expression in Alveolar Epithelial Cells: In Vitro and In Vivo Investigation.

Thorium-232 (Th), the most abundant naturally occurring nuclear fuel, has been identified as a sustainable source of energy. In view of its large-scale utilization and human evidence of lung disorders and carcinogenicity, it is imperative to understand the effect of Th exposure on lung cells. The present study investigated the effect of Th-dioxide (1-100 µg/mL, 24-48 h) on expression of surfactant proteins (SPs) (SP-A, SP-B, SP-C, and SP-D, which are essential to maintain lung's surface tension and host-defense) in human lung cells (WI26 and A549), representative of alveolar cell type-I and type-II, respectively. Results demonstrated the inhibitory effect of Th on transcriptional expression of SP-A, SP-B, and SP-C. However, Th promoted the mRNA expression of SP-D in A549 and reduced its expression in WI26. To a significant extent, the effect of Th on SPs was found to be in accordance with their protein levels. Moreover, Th exposure altered the extracellular release of SP-D/A from A549, which remained unaltered in WI26. Our results suggested the differential role of oxidative stress and ATM and HSP90 signaling in Th-induced alterations of SPs. These effects of Th were found to be consistent in lung tissues of mice exposed to Th aerosols, suggesting a potential role of SPs in Th-associated lung disorders.

In vitro inflammation and toxicity assessment of pre- and post-incinerated organomodified nanoclays to macrophages using high-throughput screening approaches.

BACKGROUND: Organomodified nanoclays (ONC), two-dimensional montmorillonite with organic coatings, are increasingly used to improve nanocomposite properties. However, little is known about pulmonary health risks along the nanoclay life cycle even with increased evidence of airborne particulate exposures in occupational environments. Recently, oropharyngeal aspiration exposure to pre- and post-incinerated ONC in mice caused low grade, persistent lung inflammation with a pro-fibrotic signaling response with unknown mode(s) of action. We hypothesized that the organic coating presence and incineration status of nanoclays determine the inflammatory cytokine secretary profile and cytotoxic response of macrophages. To test this hypothesis differentiated human macrophages (THP-1) were acutely exposed (0-20 µg/cm2) to pristine, uncoated nanoclay (CloisNa), an ONC (Clois30B), their incinerated byproducts (I-CloisNa and I-Clois30B), and crystalline silica (CS) followed by cytotoxicity and inflammatory endpoints. Macrophages were co-exposed to lipopolysaccharide (LPS) or LPS-free medium to assess the role of priming the NF-κB pathway in macrophage response to nanoclay treatment. Data were compared to inflammatory responses in male C57Bl/6J mice following 30 and 300 µg/mouse aspiration exposure to the same particles. RESULTS: In LPS-free media, CloisNa exposure caused mitochondrial depolarization while Clois30B exposure caused reduced macrophage viability, greater cytotoxicity, and significant damage-associated molecular patterns (IL-1α and ATP) release compared to CloisNa and unexposed controls. LPS priming with low CloisNa doses caused elevated cathepsin B/Caspage-1/IL-1ß release while higher doses resulted in apoptosis. Clois30B exposure caused dose-dependent THP-1 cell pyroptosis evidenced by Cathepsin B and IL-1ß release and Gasdermin D cleavage. Incineration ablated the cytotoxic and inflammatory effects of Clois30B while I-CloisNa still retained some mild inflammatory potential. Comparative analyses suggested that in vitro macrophage cell viability, inflammasome endpoints, and pro-inflammatory cytokine profiles significantly correlated to mouse bronchioalveolar lavage inflammation metrics including inflammatory cell recruitment. CONCLUSIONS: Presence of organic coating and incineration status influenced inflammatory and cytotoxic responses following exposure to human macrophages. Clois30B, with a quaternary ammonium tallow coating, induced a robust cell membrane damage and pyroptosis effect which was eliminated after incineration. Conversely, incinerated nanoclay exposure primarily caused elevated inflammatory cytokine release from THP-1 cells. Collectively, pre-incinerated nanoclay displayed interaction with macrophage membrane components (molecular initiating event), increased pro-inflammatory mediators, and increased inflammatory cell recruitment (two key events) in the lung fibrosis adverse outcome pathway.

Screening human lung cancer with predictive models of serum magnetic resonance spectroscopy metabolomics.

The current high mortality of human lung cancer stems largely from the lack of feasible, early disease detection tools. An effective test with serum metabolomics predictive models able to suggest patients harboring disease could expedite triage patient to specialized imaging assessment. Here, using a training-validation-testing-cohort design, we establish our high-resolution magic angle spinning (HRMAS) magnetic resonance spectroscopy (MRS)-based metabolomics predictive models to indicate lung cancer presence and patient survival using serum samples collected prior to their disease diagnoses. Studied serum samples were collected from 79 patients before (within 5.0 y) and at lung cancer diagnosis. Disease predictive models were established by comparing serum metabolomic patterns between our training cohorts: patients with lung cancer at time of diagnosis, and matched healthy controls. These predictive models were then applied to evaluate serum samples of our validation and testing cohorts, all collected from patients before their lung cancer diagnosis. Our study found that the predictive model yielded values for prior-to-detection serum samples to be intermediate between values for patients at time of diagnosis and for healthy controls; these intermediate values significantly differed from both groups, with an F1 score = 0.628 for cancer prediction. Furthermore, values from metabolomics predictive model measured from prior-to-diagnosis sera could significantly predict 5-y survival for patients with localized disease.

Synthesis of selenophene-containing flavonols and 2-styrylchromones: Evaluation of their activities compared with selenophene-containing chalcones as potential anticancer agents.

Previously, we documented the synthesis and assessed the biological effects of chalcones containing selenium against HT-29 human colorectal adenocarcinoma cells, demonstrating their significant potential. As research on selenium-containing flavonoids remains limited, this article outlines our design and synthesis of three selenium-based flavonols and three 2-styrylchromones. We conducted evaluations of these compounds to determine their impact on human lung cancer cells (A549, H1975, CL1-0, and CL1-5) and their influence on normal lung fibroblast MRC5 cells. Additionally, we included selenium-based chalcones in our testing for comparative purposes. Our findings highlight that the simplest compound, designated as compound 1, exhibited the most promising performance among the tested molecules.

The Roles and Regulatory Mechanisms of Tight Junction Protein Cingulin and Transcription Factor Forkhead Box Protein O1 in Human Lung Adenocarcinoma A549 Cells and Normal Lung Epithelial Cells.

Tight junction (TJ) protein cingulin (CGN) and transcription factor forkhead box protein O1 (FOXO1) contribute to the development of various cancers. Histone deacetylase (HDAC) inhibitors have a potential therapeutic role for some cancers. HDAC inhibitors affect the expression of both CGN and FOXO1. However, the roles and regulatory mechanisms of CGN and FOXO1 are unknown in non-small cell lung cancer (NSCLC) and normal human lung epithelial (HLE) cells. In the present study, to investigate the effects of CGN and FOXO1 on the malignancy of NSCLC, we used A549 cells as human lung adenocarcinoma and primary human lung epithelial (HLE) cells as normal lung tissues and performed the knockdown of CGN and FOXO1 by siRNAs. Furthermore, to investigate the detailed mechanisms in the antitumor effects of HDAC inhibitors for NSCLC via CGN and FOXO1, A549 cells and HLE cells were treated with the HDAC inhibitors trichostatin A (TSA) and Quisinostat (JNJ-2648158). In A549 cells, the knockdown of CGN increased bicellular TJ protein claudin-2 (CLDN-2) via mitogen-activated protein kinase/adenosine monophosphate-activated protein kinase (MAPK/AMPK) pathways and induced cell migration, while the knockdown of FOXO1 increased claudin-4 (CLDN-4), decreased CGN, and induced cell proliferation. The knockdown of CGN and FOXO1 induced cell metabolism in A549 cells. TSA and Quisinostat increased CGN and tricellular TJ protein angulin-1/lipolysis-stimulated lipoprotein receptor (LSR) in A549. In normal HLE cells, the knockdown of CGN and FOXO1 increased CLDN-4, while HDAC inhibitors increased CGN and CLDN-4. In conclusion, the knockdown of CGN via FOXO1 contributes to the malignancy of NSCLC. Both HDAC inhibitors, TSA and Quisinostat, may have potential for use in therapy for lung adenocarcinoma via changes in the expression of CGN and FOXO1.

Liposomal Formulation Reduces Transport and Cell Uptake of Colistin in Human Lung Epithelial Calu-3 Cell and 3D Human Lung Primary Tissue Models.

Respiratory tract infections caused by multi-drug-resistant (MDR) bacteria have been a severe risk to human health. Colistin is often used to treat the MDR Gram-negative bacterial infections as a last-line therapy. Inhaled colistin can achieve a high concentration in the lung but none of aerosolized colistin products has been approved in the USA. Liposome has been reported as an advantageous formulation strategy for antibiotics due to its controlled release profile and biocompatibility. We have developed colistin liposomal formulations in our previous study. In the present study, the cellular uptake and transport of colistin in colistin liposomes were examined in two human lung epithelium in vitro models, Calu-3 monolayer and EpiAirway 3D tissue models. In both models, cellular uptake (p < 0.05) and cellular transport (p < 0.01) of colistin were significantly reduced by the colistin liposome compared to the colistin solution. Our findings indicate that inhaled colistin liposomes could be a promising treatment for extracellular bacterial lung infections caused by MDR Pseudomonas aeruginosa (P. aeruginosa).

Global profiling of lysine lactylation in human lungs.

Lactate is closely related to various cellular processes, such as angiogenesis, responses to hypoxia, and macrophage polarization, while regulating natural immune signaling pathways and promoting neurogenesis and cognitive function. Lysine lactylation (Kla) is a novel posttranslational modification, the examination of which may lead to new understanding of the nonmetabolic functions of lactate and the various physiological and pathological processes in which lactate is involved, such as infection, tumorigenesis and tumor development. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), researchers have identified lactylation in human gastric cancer cells and some other species, but no research on lactylation in human lungs has been reported. In this study, we performed global profiling of lactylation in human lungs under normal physiological conditions, and 724 Kla sites in 451 proteins were identified. After comparing the identified proteins with those reported in human lactylation datasets, 141 proteins that undergo lactylation were identified for the first time in this study. Our work expands the database on human lactylation and helps advance the study on lactylation function and regulation under physiological and pathological conditions.

Replication of Novel Zoonotic-Like Influenza A(H3N8) Virus in Ex Vivo Human Bronchus and Lung.

Human infection with avian influenza A(H3N8) virus is uncommon but can lead to acute respiratory distress syndrome. In explant cultures of the human bronchus and lung, novel H3N8 virus showed limited replication efficiency in bronchial and lung tissue but had a higher replication than avian H3N8 virus in lung tissue.

The role of potassium current in the pulmonary response to environmental oxidative stress.

Exposure of human lung epithelial cells (A549 cell line) to the oxidant pollutant ozone (O3) alters cell membrane currents inducing its decrease, when the cell undergoes to a voltage-clamp protocol ranging from -90 to +70mV. The membrane potential of these cells is mainly maintained by the interplay of potassium and chloride currents. Our previous studies indicated the ability of O3 to activate ORCC (Outward Rectifier Chloride Channel) and consequently increases the chloride current. In this paper our aim was to understand the response of potassium current to oxidative stress challenge and to identify the kind potassium channel involved in O3 induced current changes. After measuring the total membrane current using an intracellular solution with or without potassium ions, we obtained the contribution of potassium to the overall membrane current in control condition by a mathematical approach. Repeating these experiments after O3 treatment we observed a significant decrease of Ipotassium. Treatment of the cells with Iberiotoxin (IbTx), a specific inhibitor of BK channel, we were able to verify the presence and the functionality of BK channels. In addition, the administration of 4-Aminopyridine (an inhibitor of voltage dependent K channels but not BK channels) and Tetraethylammonium (TEA) before and after O3 treatment we observed the formation of BK oxidative post-translation modifications. Our data suggest that O3 is able to inhibit potassium current by targeting BK channel. Further studies are needed to better clarify the role of this BK channel and its interplay with the other membrane channels under oxidative stress conditions. These findings can contribute to identify the biomolecular pathway induced by O3 allowing a possible pharmacological intervention against oxidative stress damage in lung tissue.

Anti-fibrotic effects of nintedanib on lung fibroblasts derived from patients with Progressive Fibrosing Interstitial Lung Diseases (PF-ILDs).

The tyrosine kinase inhibitor nintedanib has been recently approved for the treatment of Interstitial Lung Diseases (ILDs) that manifest a progressive fibrosis phenotype other than Idiopathic pulmonary Fibrosis (IPF). Nintedanib reduces the development of lung fibrosis in various animal models resembling features of PF-ILD and in vitro, it inhibits the fibrosing phenotype of human lung fibroblasts (HLFs) isolated from patients with IPF. To get insight on the cellular and molecular mechanisms that drive the clinical efficiency of nintedanib in patients with non-IPF PF-ILD, we investigated its effects on the fibrosing functions of HLFs derived from patients with PF-hypersensitivity pneumonitis (PF-HP, n = 7), PF-sarcoidosis (n = 5) and pleuroparenchymal fibroelastosis (PPFE, n = 4). HLFs were treated with nintedanib (10 nM-1 µM) and then stimulated with PDGF-BB (25-50 ng/ml) or TGF-ß1 (1 ng/ml) for 24-72 h to assess proliferation and migration or differentiation. At nanomolar concentrations, nintedanib reduced the levels of PDGF receptor and ERK1/2 phosphorylation, the proliferation and the migration of PF-HP, PF-sarcoidosis and PPFE HLFs stimulated with PDGF-BB. Moreover, nintedanib also attenuates the myofibroblastic differentiation driven by TGF-ß1 but only when it is used at 1 µM. The drug reduced the phosphorylation of SMAD2/3 and decreased the induction of collagen, fibronectin and α-smooth muscle actin expression induced by TGF-ß1. In conclusion, our results demonstrate that nintedanib counteracts fundamental fibrosing functions of lung fibroblasts derived from patients with PF-HP, PF-sarcoidosis and PPFE, at concentrations previously reported to inhibit control and IPF HLFs. Such effects may contribute to its clinical benefit in patients suffering from these irreversible ILDs.

Tetrahydrobiopterin from cyanide-degrading bacterium Bacillus pumilus strain SVD06 induces apoptosis in human lung adenocarcinoma cell (A549).

Tetrahydrobiopterin (BH4) is an essential biological cofactor and a derivative of pterin which is considered potent anticancer agents. In continuation of our previous study on the identification of BH4 from cyanide-degrading Bacillus pumilus, the present study focuses on evaluating the anticancer properties of BH4 on A549, a human lung adenocarcinoma. Anticancer activity analysis shows that BH4 inhibited A549 cell growth after 24 h of incubation with 0.02 mg/mL. In acridine orange/ethidium bromide staining, BH4-treated A549 cells showed apoptotic morphology. BH4 treatment caused cell cycle arrest at G0/G1 phase compared to control cells. BH4 augmented p53 expression in alveolar cancer cells by downregulating MDM2 levels. There was downregulation of casp-3 and upregulation of iNOS gene in BH4-treated A549 cells. Further, docking studies indicated that BH4 had significant interactions with the above proteins affirming the apoptosis mechanism. Thus, BH4 could be considered a potential anticancer drug.

Inhibition of TRPC4/5 Channel Is Effective in Protecting against Histamine-Induced Hyperpermeability by Blocking Ca2+ Influx in Lung Microvascular Endothelial Cells.

Dysfunction of lung microvascular endothelium is a major feature in the pathobiology of pulmonary edema and hypoxic respiratory failure. Histamine induces lung microvascular endothelial barrier disruption and hyperpermeability upon evoking intracellular Ca2+ ([Ca2+]i) dynamics via binding to its receptors. Transient receptor potential canonical (TRPC) channels are Ca2+-permeable channel and stimulated by the agonists of G-protein-coupled receptors (GPCR). Here, we assessed histamine induced [Ca2+]i increases in human lung microvascular endothelial cells (HLMVEC) by using live cell Ca2+ imaging. We found that histamine increased [Ca2+]i was maintained at a static elevated level after a transient peak. The elevated Ca2+ plateau was vanished when extracellular Ca2+ was removed, indicating Ca2+ influx from extracellular mediated the histamine-induced Ca2+ plateau. TRPC4/5 channels antagonists, ML204 (10 µM) and HC070 (1 µM), significantly inhibited the Ca2+ plateaus, which was not influenced by Pyr3 or larixyl, the antagonists of TRPC3/6. Furthermore, ML204 or HC070 effectively suppressed the permeability response to histamine in HLMVEC. Our results indicated that histamine-induced Ca2+ influx may be mediated by TRPC4/5 channels and the antagonist of the channel significantly improved histamine-induced HLMVEC dysfunction.

A genomic perspective of the aging human and mouse lung with a focus on immune response and cellular senescence.

BACKGROUND: The aging lung is a complex process and influenced by various stressors, especially airborne pathogens and xenobiotics. Additionally, a lifetime exposure to antigens results in structural and functional changes of the lung; yet an understanding of the cell type specific responses remains elusive. To gain insight into age-related changes in lung function and inflammaging, we evaluated 89 mouse and 414 individual human lung genomic data sets with a focus on genes mechanistically linked to extracellular matrix (ECM), cellular senescence, immune response and pulmonary surfactant, and we interrogated single cell RNAseq data to fingerprint cell type specific changes. RESULTS: We identified 117 and 68 mouse and human genes linked to ECM remodeling which accounted for 46% and 27%, respectively of all ECM coding genes. Furthermore, we identified 73 and 31 mouse and human genes linked to cellular senescence, and the majority code for the senescence associated secretory phenotype. These cytokines, chemokines and growth factors are primarily secreted by macrophages and fibroblasts. Single-cell RNAseq data confirmed age-related induced expression of marker genes of macrophages, neutrophil, eosinophil, dendritic, NK-, CD4+, CD8+-T and B cells in the lung of aged mice. This included the highly significant regulation of 20 genes coding for the CD3-T-cell receptor complex. Conversely, for the human lung we primarily observed macrophage and CD4+ and CD8+ marker genes as changed with age. Additionally, we noted an age-related induced expression of marker genes for mouse basal, ciliated, club and goblet cells, while for the human lung, fibroblasts and myofibroblasts marker genes increased with age. Therefore, we infer a change in cellular activity of these cell types with age. Furthermore, we identified predominantly repressed expression of surfactant coding genes, especially the surfactant transporter Abca3, thus highlighting remodeling of surfactant lipids with implications for the production of inflammatory lipids and immune response. CONCLUSION: We report the genomic landscape of the aging lung and provide a rationale for its growing stiffness and age-related inflammation. By comparing the mouse and human pulmonary genome, we identified important differences between the two species and highlight the complex interplay of inflammaging, senescence and the link to ECM remodeling in healthy but aged individuals.