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1.
Front Immunol ; 13: 1020624, 2022.
Article in English | MEDLINE | ID: covidwho-2119501

ABSTRACT

Background: SARS-CoV-2 infects through the respiratory route and triggers inflammatory response by affecting multiple cell types including type II alveolar epithelial cells. SARS-CoV-2 triggers signals via its Spike (S) protein, which have been shown to participate in the pathogenesis of COVID19. Aim: Aim of the present study was to investigate the effect of SARS-CoV2 on type II alveolar epithelial cells, focusing on signals initiated by its S protein and their impact on the expression of inflammatory mediators. Results: For this purpose A549 alveolar type II epithelial cells were exposed to SARS CoV2 S recombinant protein and the expression of inflammatory mediators was measured. The results showed that SARS-CoV-2 S protein decreased the expression and secretion of IL8, IL6 and TNFα, 6 hours following stimulation, while it had no effect on IFNα, CXCL5 and PAI-1 expression. We further examined whether SARS-CoV-2 S protein, when combined with TLR2 signals, which are also triggered by SARS-CoV2 and its envelope protein, exerts a different effect in type II alveolar epithelial cells. Simultaneous treatment of A549 cells with SARS-CoV-2 S protein and the TLR2 ligand PAM3csk4 decreased secretion of IL8, IL6 and TNFα, while it significantly increased IFNα, CXCL5 and PAI-1 mRNA expression. To investigate the molecular pathway through which SARS-CoV-2 S protein exerted this immunomodulatory action in alveolar epithelial cells, we measured the induction of MAPK/ERK and PI3K/AKT pathways and found that SARS-CoV-2 S protein induced the activation of the serine threonine kinase AKT. Treatment with the Akt inhibitor MK-2206, abolished the inhibitory effect of SARS-CoV-2 S protein on IL8, IL6 and TNFα expression, suggesting that SARS-CoV-2 S protein mediated its action via AKT kinases. Conclusion: The findings of our study, showed that SARS-CoV-2 S protein suppressed inflammatory responses in alveolar epithelial type II cells at early stages of infection through activation of the PI3K/AKT pathway. Thus, our results suggest that at early stages SARS-CoV-2 S protein signals inhibit immune responses to the virus allowing it to propagate the infection while in combination with TLR2 signals enhances PAI-1 expression, potentially affecting the local coagulation cascade.


Subject(s)
Alveolar Epithelial Cells , COVID-19 , Humans , Alveolar Epithelial Cells/metabolism , SARS-CoV-2 , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt , Tumor Necrosis Factor-alpha , RNA, Viral , Plasminogen Activator Inhibitor 1 , Interleukin-6 , Interleukin-8 , Toll-Like Receptor 2
2.
Am J Respir Cell Mol Biol ; 67(3): 389-401, 2022 09.
Article in English | MEDLINE | ID: covidwho-2020635

ABSTRACT

The lung epithelium forms the first barrier against respiratory pathogens and noxious chemicals; however, little is known about how more than 90% of this barrier, made of AT1 (alveolar type 1) cells, responds to injury. Using the Sendai virus to model natural infection in mice, we find evidence that AT1 cells have an intermediary role by persisting in areas depleted of AT2 cells, upregulating IFN responsive genes, and receding from invading airway cells. Sendai virus infection mobilizes airway cells to form alveolar SOX2+ (Sry-box 2+) clusters without differentiating into AT1 or AT2 cells. Large AT2 cell-depleted areas remain covered by AT1 cells, which we name "AT2-less regions", and are replaced by SOX2+ clusters spreading both basally and luminally. AT2 cell proliferation and differentiation are largely confined to topologically distal regions and form de novo alveolar surface, with limited contribution to in situ repairs of AT2-less regions. Time-course single-cell RNA sequencing profiling and RNAscope validation suggest enhanced immune responses and altered growth signals in AT1 cells. Our comprehensive spatiotemporal and genomewide study highlights the hitherto unappreciated role of AT1 cells in lung injury-repair.


Subject(s)
Alveolar Epithelial Cells , Respirovirus Infections , Alveolar Epithelial Cells/metabolism , Animals , Cell Differentiation/physiology , Cells, Cultured , Lung , Mice
3.
Stem Cell Res Ther ; 13(1): 170, 2022 04 27.
Article in English | MEDLINE | ID: covidwho-1817268

ABSTRACT

Alveoli are the functional units of blood-gas exchange in the lung and thus are constantly exposed to outside environments and frequently encounter pathogens, particles and other harmful substances. For example, the alveolar epithelium is one of the primary targets of the SARS-CoV-2 virus that causes COVID-19 lung disease. Therefore, it is essential to understand the cellular and molecular mechanisms by which the integrity of alveoli epithelial barrier is maintained. Alveolar epithelium comprises two cell types: alveolar type I cells (AT1) and alveolar type II cells (AT2). AT2s have been shown to function as tissue stem cells that repair the injured alveoli epithelium. Recent studies indicate that AT1s and subgroups of proximal airway epithelial cells can also participate alveolar repair process through their intrinsic plasticity. This review discussed the potential mechanisms that drive the reparative behaviors of AT2, AT1 and some proximal cells in responses to injury and how an abnormal repair contributes to some pathological conditions.


Subject(s)
COVID-19 , SARS-CoV-2 , Alveolar Epithelial Cells/metabolism , Humans , Pulmonary Alveoli/metabolism , Stem Cells/metabolism
4.
Int J Mol Sci ; 23(5)2022 Feb 26.
Article in English | MEDLINE | ID: covidwho-1736945

ABSTRACT

Disruption of the alveolar-endothelial barrier caused by inflammation leads to the progression of septic acute lung injury (ALI). In the present study, we investigated the beneficial effects of simvastatin on the endotoxin lipopolysaccharide (LPS)-induced ALI and its related mechanisms. A model of ALI was induced within experimental sepsis developed by intraperitoneal injection of a single non-lethal LPS dose after short-term simvastatin pretreatment (10-40 mg/kg orally). The severity of the lung tissue inflammatory injury was expressed as pulmonary damage scores (PDS). Alveolar epithelial cell apoptosis was confirmed by TUNEL assay (DNA fragmentation) and expressed as an apoptotic index (AI), and immunohistochemically for cleaved caspase-3, cytochrome C, and anti-apoptotic Bcl-xL, an inhibitor of apoptosis, survivin, and transcriptional factor, NF-kB/p65. Severe inflammatory injury of pulmonary parenchyma (PDS 3.33 ± 0.48) was developed after the LPS challenge, whereas simvastatin significantly and dose-dependently protected lung histology after LPS (p < 0.01). Simvastatin in a dose of 40 mg/kg showed the most significant effects in amelioration alveolar epithelial cells apoptosis, demonstrating this as a marked decrease of AI (p < 0.01 vs. LPS), cytochrome C, and cleaved caspase-3 expression. Furthermore, simvastatin significantly enhanced the expression of Bcl-xL and survivin. Finally, the expression of survivin and its regulator NF-kB/p65 in the alveolar epithelium was in strong positive correlation across the groups. Simvastatin could play a protective role against LPS-induced ALI and apoptosis of the alveolar-endothelial barrier. Taken together, these effects were seemingly mediated by inhibition of caspase 3 and cytochrome C, a finding that might be associated with the up-regulation of cell-survival survivin/NF-kB/p65 pathway and Bcl-xL.


Subject(s)
Acute Lung Injury , NF-kappa B , Acute Lung Injury/chemically induced , Acute Lung Injury/drug therapy , Acute Lung Injury/metabolism , Alveolar Epithelial Cells/metabolism , Apoptosis , Caspase 3/genetics , Caspase 3/metabolism , Cytochromes c/metabolism , Endotoxins/adverse effects , Humans , Lipopolysaccharides/toxicity , Lung/pathology , NF-kappa B/metabolism , Simvastatin/adverse effects , Survivin/genetics , Up-Regulation
5.
Med Microbiol Immunol ; 211(1): 49-69, 2022 Feb.
Article in English | MEDLINE | ID: covidwho-1704341

ABSTRACT

Metabolic pathways drive cellular behavior. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection causes lung tissue damage directly by targeting cells or indirectly by producing inflammatory cytokines. However, whether functional alterations are related to metabolic changes in lung cells after SARS-CoV-2 infection remains unknown. Here, we analyzed the lung single-nucleus RNA-sequencing (snRNA-seq) data of several deceased COVID-19 patients and focused on changes in transcripts associated with cellular metabolism. We observed upregulated glycolysis and oxidative phosphorylation in alveolar type 2 progenitor cells, which may block alveolar epithelial differentiation and surfactant secretion. Elevated inositol phosphate metabolism in airway progenitor cells may promote neutrophil infiltration and damage the lung barrier. Further, multiple metabolic alterations in the airway goblet cells are associated with impaired muco-ciliary clearance. Increased glycolysis, oxidative phosphorylation, and inositol phosphate metabolism not only enhance macrophage activation but also contribute to SARS-CoV-2 induced lung injury. The cytotoxicity of natural killer cells and CD8+ T cells may be enhanced by glycerolipid and inositol phosphate metabolism. Glycolytic activation in fibroblasts is related to myofibroblast differentiation and fibrogenesis. Glycolysis, oxidative phosphorylation, and glutathione metabolism may also boost the aging, apoptosis and proliferation of vascular smooth muscle cells, resulting in pulmonary arterial hypertension. In conclusion, this preliminary study revealed a possible cellular metabolic basis for the altered innate immunity, adaptive immunity, and niche cell function in the lung after SARS-CoV-2 infection. Therefore, patients with COVID-19 may benefit from therapeutic strategies targeting cellular metabolism in future.


Subject(s)
COVID-19 , Alveolar Epithelial Cells/metabolism , CD8-Positive T-Lymphocytes , Humans , Immunity, Innate , Lung , SARS-CoV-2
6.
PLoS One ; 17(1): e0262832, 2022.
Article in English | MEDLINE | ID: covidwho-1643286

ABSTRACT

Tumor progression locus 2 (Tpl2) is a serine/threonine kinase that regulates the expression of inflammatory mediators in response to Toll-like receptors (TLR) and cytokine receptors. Global ablation of Tpl2 leads to severe disease in response to influenza A virus (IAV) infection, characterized by respiratory distress, and studies in bone marrow chimeric mice implicated Tpl2 in non-hematopoietic cells. Lung epithelial cells are primary targets and replicative niches of influenza viruses; however, the specific regulation of antiviral responses by Tpl2 within lung epithelial cells has not been investigated. Herein, we show that Tpl2 is basally expressed in primary airway epithelial cells and that its expression increases in both type I and type II airway epithelial cells (AECI and AECII) in response to influenza infection. We used Nkx2.1-cre to drive Tpl2 deletion within pulmonary epithelial cells to delineate epithelial cell-specific functions of Tpl2 during influenza infection in mice. Although modest increases in morbidity and mortality were attributed to cre-dependent deletion in lung epithelial cells, no alterations in host cytokine production or lung pathology were observed. In vitro, Tpl2 inhibition within the type I airway epithelial cell line, LET1, as well as genetic ablation in primary airway epithelial cells did not alter cytokine production. Overall, these findings establish that Tpl2-dependent defects in cells other than AECs are primarily responsible for the morbidity and mortality seen in influenza-infected mice with global Tpl2 ablation.


Subject(s)
Alveolar Epithelial Cells/metabolism , Host Microbial Interactions/genetics , Influenza A virus , MAP Kinase Kinase Kinases/metabolism , Orthomyxoviridae Infections/blood , Orthomyxoviridae Infections/immunology , Proto-Oncogene Proteins/metabolism , Animals , Cytokines/metabolism , Disease Models, Animal , Dogs , Female , MAP Kinase Kinase Kinases/genetics , Madin Darby Canine Kidney Cells , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Orthomyxoviridae Infections/mortality , Orthomyxoviridae Infections/virology , Proto-Oncogene Proteins/genetics
7.
Life Sci ; 293: 120324, 2022 Mar 15.
Article in English | MEDLINE | ID: covidwho-1616648

ABSTRACT

AIMS: Angiotensin-converting enzyme (ACE) 2 is the receptor for severe acute respiratory syndrome coronavirus 2 which causes coronavirus disease 2019 (COVID-19). Viral cellular entry requires ACE2 and transmembrane protease serine 2 (TMPRSS2). ACE inhibitors (ACEIs) or angiotensin (Ang) receptor blockers (ARBs) influence ACE2 in animals, though evidence in human lungs is lacking. We investigated ACE2 and TMPRSS2 in type II pneumocytes, the key cells that maintain lung homeostasis, in lung parenchymal of ACEI/ARB-treated subjects compared to untreated control subjects. MAIN METHODS: Ang II and Ang-(1-7) levels and ACE2 and TMPRSS2 protein expression were measured by radioimmunoassay and immunohistochemistry, respectively. KEY FINDINGS: We found that the ratio Ang-(1-7)/Ang II, a surrogate marker of ACE2 activity, as well as the amount of ACE2-expressing type II pneumocytes were not different between ACEI/ARB-treated and untreated subjects. ACE2 protein content correlated positively with smoking habit and age. The percentage of TMPRSS2-expressing type II pneumocytes was higher in males than females and in subjects under 60 years of age but it was not different between ACEI/ARB-treated and untreated subjects. However, there was a positive association of TMPRSS2 protein content with age and smoking in ACEI/ARB-treated subjects, with high TMPRSS2 protein levels most evident in ACEI/ARB-treated older adults and smokers. SIGNIFICANCE: ACEI/ARB treatment influences human lung TMPRSS2 but not ACE2 protein content and this effect is dependent on age and smoking habit. This finding may help explain the increased susceptibility to COVID-19 seen in smokers and older patients with treated cardiovascular-related pathologies.


Subject(s)
Alveolar Epithelial Cells/metabolism , Angiotensin Receptor Antagonists/pharmacology , Angiotensin-Converting Enzyme 2/metabolism , Angiotensin-Converting Enzyme Inhibitors/pharmacology , Renin-Angiotensin System/physiology , Serine Endopeptidases/metabolism , Adult , Age Factors , Aged , Alveolar Epithelial Cells/chemistry , Alveolar Epithelial Cells/drug effects , Angiotensin I/metabolism , Angiotensin II/metabolism , Angiotensin-Converting Enzyme 2/analysis , Angiotensin-Converting Enzyme 2/antagonists & inhibitors , Female , Humans , Lung/chemistry , Lung/drug effects , Lung/metabolism , Male , Middle Aged , Peptide Fragments/metabolism , Renin-Angiotensin System/drug effects , Retrospective Studies , Serine Endopeptidases/analysis , Smoking/metabolism , Smoking/pathology
8.
Microbiol Spectr ; 9(3): e0073521, 2021 12 22.
Article in English | MEDLINE | ID: covidwho-1596765

ABSTRACT

SARS-CoV-2 infection can cause compromised respiratory function and thrombotic events. SARS-CoV-2 binds to and mediates downregulation of angiotensin converting enzyme 2 (ACE2) on cells that it infects. Theoretically, diminished enzymatic activity of ACE2 may result in increased concentrations of pro-inflammatory molecules, angiotensin II, and Bradykinin, contributing to SARS-CoV-2 pathology. Using immunofluorescence microscopy of lung tissues from uninfected, and SARS-CoV-2 infected individuals, we find evidence that ACE2 is highly expressed in human pulmonary alveolar epithelial cells and significantly reduced along the alveolar lining of SARS-CoV-2 infected lungs. Ex vivo analyses of primary human cells, indicated that ACE2 is readily detected in pulmonary alveolar epithelial and aortic endothelial cells. Exposure of these cells to spike protein of SARS-CoV-2 was sufficient to reduce ACE2 expression. Moreover, exposure of endothelial cells to spike protein-induced dysfunction, caspase activation, and apoptosis. Exposure of endothelial cells to bradykinin caused calcium signaling and endothelial dysfunction (increased expression of von Willibrand Factor and decreased expression of Krüppel-like Factor 2) but did not adversely affect viability in primary human aortic endothelial cells. Computer-assisted analyses of molecules with potential to bind bradykinin receptor B2 (BKRB2), suggested a potential role for aspirin as a BK antagonist. When tested in our in vitro model, we found evidence that aspirin can blunt cell signaling and endothelial dysfunction caused by bradykinin in these cells. Interference with interactions of spike protein or bradykinin with endothelial cells may serve as an important strategy to stabilize microvascular homeostasis in COVID-19 disease. IMPORTANCE SARS-CoV-2 causes complex effects on microvascular homeostasis that potentially contribute to organ dysfunction and coagulopathies. SARS-CoV-2 binds to, and causes downregulation of angiotensin converting enzyme 2 (ACE2) on cells that it infects. It is thought that reduced ACE2 enzymatic activity can contribute to inflammation and pathology in the lung. Our studies add to this understanding by providing evidence that spike protein alone can mediate adverse effects on vascular cells. Understanding these mechanisms of pathogenesis may provide rationale for interventions that could limit microvascular events associated with SARS-CoV-2 infection.


Subject(s)
COVID-19/physiopathology , Endothelial Cells/virology , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Alveolar Epithelial Cells/cytology , Alveolar Epithelial Cells/metabolism , Alveolar Epithelial Cells/virology , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/metabolism , Aorta/cytology , Aorta/metabolism , Aorta/virology , Apoptosis , Bradykinin/chemistry , Bradykinin/metabolism , COVID-19/genetics , COVID-19/metabolism , COVID-19/virology , Endothelial Cells/cytology , Endothelial Cells/metabolism , Homeostasis , Humans , Lung/blood supply , Lung/metabolism , Lung/virology , Microcirculation , Receptors, Bradykinin/chemistry , Receptors, Bradykinin/genetics , Receptors, Bradykinin/metabolism , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics
9.
Nat Cell Biol ; 23(12): 1314-1328, 2021 12.
Article in English | MEDLINE | ID: covidwho-1559292

ABSTRACT

The lung is the primary organ targeted by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), making respiratory failure a leading coronavirus disease 2019 (COVID-19)-related mortality. However, our cellular and molecular understanding of how SARS-CoV-2 infection drives lung pathology is limited. Here we constructed multi-omics and single-nucleus transcriptomic atlases of the lungs of patients with COVID-19, which integrate histological, transcriptomic and proteomic analyses. Our work reveals the molecular basis of pathological hallmarks associated with SARS-CoV-2 infection in different lung and infiltrating immune cell populations. We report molecular fingerprints of hyperinflammation, alveolar epithelial cell exhaustion, vascular changes and fibrosis, and identify parenchymal lung senescence as a molecular state of COVID-19 pathology. Moreover, our data suggest that FOXO3A suppression is a potential mechanism underlying the fibroblast-to-myofibroblast transition associated with COVID-19 pulmonary fibrosis. Our work depicts a comprehensive cellular and molecular atlas of the lungs of patients with COVID-19 and provides insights into SARS-CoV-2-related pulmonary injury, facilitating the identification of biomarkers and development of symptomatic treatments.


Subject(s)
COVID-19/genetics , Lung/metabolism , Transcriptome/genetics , Alveolar Epithelial Cells/metabolism , Alveolar Epithelial Cells/pathology , Alveolar Epithelial Cells/virology , COVID-19/metabolism , Fibrosis/metabolism , Fibrosis/pathology , Fibrosis/virology , Humans , Lung/pathology , Lung/virology , Proteomics/methods , SARS-CoV-2/pathogenicity
10.
Am J Respir Crit Care Med ; 204(9): 1024-1034, 2021 11 01.
Article in English | MEDLINE | ID: covidwho-1495777

ABSTRACT

Rationale: ACE2 (angiotensin-converting enzyme 2), the entry receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is expressed in type 2 alveolar epithelial cells (AT2) that may play key roles in postinjury repair. An imbalance between ACE2 and ACE has also been hypothesized to contribute to lung injury. Objectives: To characterize the expression and distribution of ACE2 and ACE and to compare AT2 with endothelial cell expression in coronavirus disease (COVID-19)-related or -unrelated acute respiratory distress syndrome (ARDS) and controls. Methods: Lung tissue stainings (using multiplex immunofluorescence) and serum concentrations of ACEs were determined retrospectively in two different cohorts of patients. AT2 and endothelial cells were stained in lung tissue for ProSPC (pro-surfactant protein C) and CD31, respectively. Measurements and Main Results: Pulmonary ACE2 expression was increased in patients with COVID-19-related and -unrelated ARDS (0.06% of tissue area and 0.12% vs. 0.006% for control subjects; P = 0.013 and P < 0.0001, respectively). ACE2 was upregulated in endothelial cells (0.32% and 0.53% vs. 0.01%; P = 0.009 and P < 0.0001) but not in AT2 cells (0.13% and 0.08% vs. 0.03%; P = 0.94 and P = 0.44). Pulmonary expression of ACE was decreased in both COVID-19-related and -unrelated ARDS (P = 0.057 and P = 0.032). Similar increases in ACE2 and decreases in ACE were observed in sera of COVID-19 (P = 0.0054 and P < 0.0001) and non-COVID-19 ARDS (P < 0.0001 and P = 0.016). In addition, AT2 cells were decreased in patients with COVID-19-related ARDS compared with COVID-19-unrelated ARDS (1.395% vs. 2.94%, P = 0.0033). Conclusions: ACE2 is upregulated in lung tissue and serum of both COVID-19-related and -unrelated ARDS, whereas a loss of AT2 cells is selectively observed in COVID-19-related ARDS.


Subject(s)
Alveolar Epithelial Cells/metabolism , Angiotensin-Converting Enzyme 2/metabolism , COVID-19/metabolism , Peptidyl-Dipeptidase A/metabolism , Respiratory Distress Syndrome/metabolism , Adult , Aged , Biomarkers/metabolism , COVID-19/diagnosis , COVID-19/physiopathology , Case-Control Studies , Female , Humans , Immunohistochemistry , Logistic Models , Male , Middle Aged , Proportional Hazards Models , Respiratory Distress Syndrome/diagnosis , Respiratory Distress Syndrome/virology , Retrospective Studies , Severity of Illness Index , Up-Regulation
11.
Mol Med Rep ; 24(4)2021 Oct.
Article in English | MEDLINE | ID: covidwho-1395036

ABSTRACT

Chronic alcohol abuse increases the risk of mortality and poor outcomes in patients with acute respiratory distress syndrome. However, the underlying mechanisms remain to be elucidated. The present study aimed to investigate the effects of chronic alcohol consumption on lung injury and clarify the signaling pathways involved in the inhibition of alveolar fluid clearance (AFC). In order to produce rodent models with chronic alcohol consumption, wild­type C57BL/6 mice were treated with alcohol. A2a adenosine receptor (AR) small interfering (si)RNA or A2bAR siRNA were transfected into the lung tissue of mice and primary rat alveolar type II (ATII) cells. The rate of AFC in lung tissue was measured during exposure to lipopolysaccharide (LPS). Epithelial sodium channel (ENaC) expression was determined to investigate the mechanisms underlying alcohol­induced regulation of AFC. In the present study, exposure to alcohol reduced AFC, exacerbated pulmonary edema and worsened LPS­induced lung injury. Alcohol caused a decrease in cyclic adenosine monophosphate (cAMP) levels and inhibited α­ENaC, ß­ENaC and γ­ENaC expression levels in the lung tissue of mice and ATII cells. Furthermore, alcohol decreased α­ENaC, ß­ENaC and γ­ENaC expression levels via the A2aAR or A2bAR­cAMP signaling pathways in vitro. In conclusion, the results of the present study demonstrated that chronic alcohol consumption worsened lung injury by aggravating pulmonary edema and impairing AFC. An alcohol­induced decrease of α­ENaC, ß­ENaC and γ­ENaC expression levels by the A2AR­mediated cAMP pathway may be responsible for the exacerbated effects of chronic alcohol consumption in lung injury.


Subject(s)
Acute Lung Injury/metabolism , Alveolar Epithelial Cells/metabolism , Epithelial Sodium Channels/drug effects , Epithelial Sodium Channels/metabolism , Ethanol/pharmacology , Receptors, Adenosine A2/metabolism , Acute Lung Injury/chemically induced , Acute Lung Injury/pathology , Alveolar Epithelial Cells/pathology , Animals , Cyclic AMP/metabolism , Cytokines , Lipopolysaccharides/adverse effects , Lung/metabolism , Lung Injury/chemically induced , Lung Injury/metabolism , Lung Injury/pathology , Mice , Mice, Inbred C57BL , Pulmonary Alveoli/metabolism , Pulmonary Edema/chemically induced , Pulmonary Edema/metabolism , Pulmonary Edema/pathology , RNA Splicing Factors/genetics , RNA Splicing Factors/metabolism , Rats , Receptor, Adenosine A2A/genetics , Receptor, Adenosine A2A/metabolism , Signal Transduction
12.
J Toxicol Sci ; 46(9): 425-435, 2021.
Article in English | MEDLINE | ID: covidwho-1389030

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19). SARS-CoV-2 enters host cells by binding with the receptor angiotensin-converting enzyme 2 (ACE2). While ACE2 is expressed in multiple cell types, it has been implicated in the clinical progression of COVID-19 as an entry point for SARS-CoV-2 into respiratory cells. Human respiratory cells, such as airway and alveolar epithelial type II (ATII) cells, are considered essential for COVID-19 research; however, primary human respiratory cells are difficult to obtain. In the present study, we generated ATII and club cells from human induced pluripotent stem cells (hiPSCs) for SARS-CoV-2 infection and drug testing. The differentiated cells expressed ATII markers (SFTPB, SFTPC, ABCA3, SLC34A2) or club cell markers (SCGB1A1 and SCGB3A2). Differentiated cells, which express ACE2 and TMPRSS2, were infected with SARS-CoV-2. Remdesivir treatment decreased intracellular SARS-CoV-2 viral replication and, furthermore, treatment with bleomycin showed cytotoxicity in a concentration-dependent manner. These data suggest that hiPSC-derived AT2 and club cells provide a useful in vitro model for drug development.


Subject(s)
Adenosine Monophosphate/analogs & derivatives , Alanine/analogs & derivatives , Alveolar Epithelial Cells/drug effects , Antiviral Agents/pharmacology , Bleomycin/toxicity , Cell Differentiation , Induced Pluripotent Stem Cells/drug effects , SARS-CoV-2/drug effects , Toxicity Tests , Adenosine Monophosphate/pharmacology , Alanine/pharmacology , Alveolar Epithelial Cells/metabolism , Alveolar Epithelial Cells/pathology , Alveolar Epithelial Cells/virology , COVID-19/drug therapy , Cell Line , Cell Survival/drug effects , Host-Pathogen Interactions , Humans , Induced Pluripotent Stem Cells/metabolism , Induced Pluripotent Stem Cells/pathology , Induced Pluripotent Stem Cells/virology , Phenotype , SARS-CoV-2/growth & development , SARS-CoV-2/pathogenicity , Virus Replication/drug effects
15.
Cell Stress Chaperones ; 26(5): 859-868, 2021 09.
Article in English | MEDLINE | ID: covidwho-1353732

ABSTRACT

Vaccinations are widely credited with reducing death rates from COVID-19, but the underlying host-viral mechanisms/interactions for morbidity and mortality of SARS-CoV-2 infection remain poorly understood. Acute respiratory distress syndrome (ARDS) describes the severe lung injury, which is pathologically associated with alveolar damage, inflammation, non-cardiogenic edema, and hyaline membrane formation. Because proteostatic pathways play central roles in cellular protection, immune modulation, protein degradation, and tissue repair, we examined the pathological features for the unfolded protein response (UPR) using the surrogate biomarker glucose-regulated protein 78 (GRP78) and co-receptor for SARS-CoV-2. At autopsy, immunostaining of COVID-19 lungs showed highly elevated expression of GRP78 in both pneumocytes and macrophages compared with that of non-COVID control lungs. GRP78 expression was detected in both SARS-CoV-2-infected and un-infected pneumocytes as determined by multiplexed immunostaining for nucleocapsid protein. In macrophages, immunohistochemical staining for GRP78 from deceased COVID-19 patients was increased but overlapped with GRP78 expression taken from surgical resections of non-COVID-19 controls. In contrast, the robust in situ GRP78 immunostaining of pneumocytes from COVID-19 autopsies exhibited no overlap and was independent of age, race/ethnicity, and gender compared with that from non-COVID-19 controls. Our findings bring new insights for stress-response pathways involving the proteostatic network implicated for host resilience and suggest that targeting of GRP78 expression with existing therapeutics might afford an alternative therapeutic strategy to modulate host-viral interactions during SARS-CoV-2 infections.


Subject(s)
Alveolar Epithelial Cells/metabolism , COVID-19/metabolism , Endoplasmic Reticulum Stress , Heat-Shock Proteins/analysis , Receptors, Coronavirus/analysis , SARS-CoV-2/pathogenicity , Adult , Aged , Aged, 80 and over , Alveolar Epithelial Cells/pathology , Alveolar Epithelial Cells/virology , Autopsy , COVID-19/mortality , COVID-19/pathology , COVID-19/virology , Case-Control Studies , Female , Host-Pathogen Interactions , Humans , Macrophages, Alveolar/metabolism , Macrophages, Alveolar/virology , Male , Middle Aged , Proteostasis , Up-Regulation , Young Adult
16.
Sci Rep ; 11(1): 16212, 2021 08 10.
Article in English | MEDLINE | ID: covidwho-1351976

ABSTRACT

During 2020, understanding the molecular mechanism of SARS-CoV-2 infection (the cause of COVID-19) became a scientific priority due to the devastating effects of the COVID-19. Many researchers have studied the effect of this viral infection on lung epithelial transcriptomes and deposited data in public repositories. Comprehensive analysis of such data could pave the way for development of efficient vaccines and effective drugs. In the current study, we obtained high-throughput gene expression data associated with human lung epithelial cells infected with respiratory viruses such as SARS-CoV-2, SARS, H1N1, avian influenza, rhinovirus and Dhori, then performed comparative transcriptome analysis to identify SARS-CoV-2 exclusive genes. The analysis yielded seven SARS-CoV-2 specific genes including CSF2 [GM-CSF] (colony-stimulating factor 2) and calcium-binding proteins (such as S100A8 and S100A9), which are known to be involved in respiratory diseases. The analyses showed that genes involved in inflammation are commonly altered by infection of SARS-CoV-2 and influenza viruses. Furthermore, results of protein-protein interaction analyses were consistent with a functional role of CSF2 and S100A9 in COVID-19 disease. In conclusion, our analysis revealed cellular genes associated with SARS-CoV-2 infection of the human lung epithelium; these are potential therapeutic targets.


Subject(s)
Alveolar Epithelial Cells/metabolism , COVID-19/genetics , Transcriptome , Alveolar Epithelial Cells/virology , COVID-19/metabolism , COVID-19/virology , Calgranulin A/genetics , Calgranulin A/metabolism , Calgranulin B/genetics , Calgranulin B/metabolism , Granulocyte-Macrophage Colony-Stimulating Factor/genetics , Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , Humans , SARS-CoV-2/pathogenicity
17.
Theranostics ; 11(16): 7970-7983, 2021.
Article in English | MEDLINE | ID: covidwho-1337804

ABSTRACT

The novel ß-coronavirus, SARS-CoV-2, the causative agent of coronavirus disease 2019 (COVID-19), has infected more than 177 million people and resulted in 3.84 million death worldwide. Recent epidemiological studies suggested that some environmental factors, such as air pollution, might be the important contributors to the mortality of COVID-19. However, how environmental exposure enhances the severity of COVID-19 remains to be fully understood. In the present report, we provided evidence showing that mdig, a previously reported environmentally-induced oncogene that antagonizes repressive trimethylation of histone proteins, is an important regulator for SARS-CoV-2 receptors neuropilin-1 (NRP1) and NRP2, cathepsins, glycan metabolism and inflammation, key determinants for viral infection and cytokine storm of the patients. Depletion of mdig in bronchial epithelial cells by CRISPR-Cas-9 gene editing resulted in a decreased expression of NRP1, NRP2, cathepsins, and genes involved in protein glycosylation and inflammation, largely due to a substantial enrichment of lysine 9 and/or lysine 27 trimethylation of histone H3 (H3K9me3/H3K27me3) on these genes as determined by ChIP-seq. Meanwhile, we also validated that environmental factor arsenic is able to induce mdig, NRP1 and NRP2, and genetic disruption of mdig lowered expression of NRP1 and NRP2. Furthermore, mdig may coordinate with the Neanderthal variants linked to an elevated mortality of COVID-19. These data, thus, suggest that mdig is a key mediator for the severity of COVID-19 in response to environmental exposure and targeting mdig may be the one of the effective strategies in ameliorating the symptom and reducing the mortality of COVID-19.


Subject(s)
COVID-19/metabolism , COVID-19/virology , Dioxygenases/metabolism , Histone Demethylases/metabolism , Neuropilin-1/metabolism , Nuclear Proteins/metabolism , Polysaccharides/metabolism , SARS-CoV-2/metabolism , Alveolar Epithelial Cells/metabolism , Animals , COVID-19/epidemiology , Cathepsins/metabolism , Cell Line , Cells, Cultured , Dioxygenases/biosynthesis , Dioxygenases/genetics , Environmental Exposure , Histone Demethylases/biosynthesis , Histone Demethylases/genetics , Histones/metabolism , Humans , Nuclear Proteins/biosynthesis , Nuclear Proteins/genetics , Pandemics , Rats , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/metabolism
18.
Front Immunol ; 12: 660632, 2021.
Article in English | MEDLINE | ID: covidwho-1325522

ABSTRACT

The novel SARS-CoV-2virus that caused the disease COVID-19 is currently a pandemic worldwide. The virus requires an alveolar type-2 pneumocyte in the host to initiate its life cycle. The viral S1 spike protein helps in the attachment of the virus on toACE-2 receptors present on type-2 pneumocytes, and the S2 spike protein helps in the fusion of the viral membrane with the host membrane. Fusion of the SARS-CoV-2virus and host membrane is followed by entry of viral RNA into the host cells which is directly translated into the replicase-transcriptase complex (RTC) following viral RNA and structural protein syntheses. As the virus replicates within type-2 pneumocytes, the host immune system is activated and alveolar macrophages start secreting cytokines and chemokines, acting as an inflammatory mediator, and chemotactic neutrophils, monocytes, natural NK cells, and CD8+ T cells initiate the local phagocytosis of infected cells. It is not the virus that kills COVID-19 patients; instead, the aberrant host immune response kills them. Modifying the response from the host immune system could reduce the high mortality due to SARS-CoV-2 infection. The present study examines the viral life cycle intype-2 pneumocytes and resultant host immune response along with possible therapeutic targets.


Subject(s)
COVID-19/immunology , COVID-19/therapy , Immunomodulation , SARS-CoV-2/pathogenicity , Alveolar Epithelial Cells/metabolism , Alveolar Epithelial Cells/pathology , Alveolar Epithelial Cells/virology , Angiotensin-Converting Enzyme 2/metabolism , COVID-19/pathology , COVID-19/virology , Cytokines/immunology , Cytokines/metabolism , Host-Pathogen Interactions/immunology , Humans , Immunity , Macrophages, Alveolar/immunology , Macrophages, Alveolar/metabolism , SARS-CoV-2/physiology
19.
Front Immunol ; 12: 648815, 2021.
Article in English | MEDLINE | ID: covidwho-1325521

ABSTRACT

Multiple lines of evidence have demonstrated that cigarette smoke or Chronic Obstructive Pulmonary Disease upregulates angiotensin-converting enzyme 2, the cellular receptor for the entry of the severe acute respiratory syndrome coronavirus 2, which predisposes individuals to develop severe Coronavirus disease 2019. The reason for this observation is unknown. We recently reported that the loss of function of Miz1 in the lung epithelium in mice leads to a spontaneous COPD-like phenotype, associated with upregulation of angiotensin-converting enzyme 2. We also reported that cigarette smoke exposure downregulates Miz1 in lung epithelial cells and in mice, and Miz1 is also downregulated in the lungs of COPD patients. Here, we provide further evidence that Miz1 directly binds to and represses the promoter of angiotensin-converting enzyme 2 in mouse and human lung epithelial cells. Our data provide a potential molecular mechanism for the upregulation of angiotensin-converting enzyme 2 observed in smokers and COPD patients, with implication in severe Coronavirus disease 2019.


Subject(s)
Angiotensin-Converting Enzyme 2/genetics , Kruppel-Like Transcription Factors/metabolism , Receptors, Virus/genetics , Transcription, Genetic , Alveolar Epithelial Cells/metabolism , Angiotensin-Converting Enzyme 2/metabolism , Animals , BTB-POZ Domain , Cell Line , Cigarette Smoking/adverse effects , Kruppel-Like Transcription Factors/chemistry , Kruppel-Like Transcription Factors/genetics , Mice , Promoter Regions, Genetic , Protein Binding , Receptors, Virus/metabolism , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/metabolism , Transcription, Genetic/drug effects , Tumor Necrosis Factors/pharmacology , Virus Internalization
20.
Int J Mol Sci ; 22(14)2021 Jul 16.
Article in English | MEDLINE | ID: covidwho-1323266

ABSTRACT

Smoking is a major risk factor for chronic obstructive pulmonary disease (COPD) and causes remodeling of the small airways. However, the exact smoke-induced effects on the different types of small airway epithelial cells (SAECs) are poorly understood. Here, using air-liquid interface (ALI) cultures, single-cell RNA-sequencing reveals previously unrecognized transcriptional heterogeneity within the small airway epithelium and cell type-specific effects upon acute and chronic cigarette smoke exposure. Smoke triggers detoxification and inflammatory responses and aberrantly activates and alters basal cell differentiation. This results in an increase of inflammatory basal-to-secretory cell intermediates and, particularly after chronic smoke exposure, a massive expansion of a rare inflammatory and squamous metaplasia associated KRT6A+ basal cell state and an altered secretory cell landscape. ALI cultures originating from healthy non-smokers and COPD smokers show similar responses to cigarette smoke exposure, although an increased pro-inflammatory profile is conserved in the latter. Taken together, the in vitro models provide high-resolution insights into the smoke-induced remodeling of the small airways resembling the pathological processes in COPD airways. The data may also help to better understand other lung diseases including COVID-19, as the data reflect the smoke-dependent variable induction of SARS-CoV-2 entry factors across SAEC populations.


Subject(s)
Airway Remodeling/drug effects , Alveolar Epithelial Cells/drug effects , Cigarette Smoking/adverse effects , Epithelial Cells/metabolism , Alveolar Epithelial Cells/metabolism , Alveolar Epithelial Cells/pathology , Cell Differentiation/drug effects , Cells, Cultured , Cigarette Smoking/metabolism , Epithelial Cells/drug effects , Humans , Neoplasms, Basal Cell/metabolism , Primary Cell Culture , Pulmonary Disease, Chronic Obstructive/etiology , Pulmonary Disease, Chronic Obstructive/metabolism , Pulmonary Disease, Chronic Obstructive/pathology , Respiratory Mucosa/metabolism , Respiratory Mucosa/pathology , Smoke , Smoking/adverse effects , Smoking/metabolism
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