scholarly journals Effect of ORF7 of SARS-CoV-2 on the Chemotaxis of Monocytes and Neutrophils In Vitro

2021 ◽  
Vol 2021 ◽  
pp. 1-9
Author(s):  
Gang Wang ◽  
Jun Guan ◽  
Guojun Li ◽  
Fengtian Wu ◽  
Qin Yang ◽  
...  
Keyword(s):  
A549 Cells ◽  
Type Ii ◽  
Local Inflammation ◽  
Transwell Assay ◽  
Reading Frame ◽  
Type Ii Pneumocyte ◽  
Specific Effects ◽  
Significant Public Health ◽  
Immunological Changes

Coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is currently the most significant public health threat worldwide. Patients with severe COVID-19 usually have pneumonia concomitant with local inflammation and sometimes a cytokine storm. Specific components of the SARS-CoV-2 virus trigger lung inflammation, and recruitment of immune cells to the lungs exacerbates this process, although much remains unknown about the pathogenesis of COVID-19. Our study of lung type II pneumocyte cells (A549) demonstrated that ORF7, an open reading frame (ORF) in the genome of SARS-CoV-2, induced the production of CCL2, a chemokine that promotes the chemotaxis of monocytes, and decreased the expression of IL-8, a chemokine that recruits neutrophils. A549 cells also had an increased level of IL-6. The results of our chemotaxis Transwell assay suggested that ORF7 augmented monocyte infiltration and reduced the number of neutrophils. We conclude that the ORF7 of SARS-CoV-2 may have specific effects on the immunological changes in tissues after infection. These results suggest that the functions of other ORFs of SARS-CoV-2 should also be comprehensively examined.

scholarly journals Effect of ORF7 of SARS-CoV-2 on the chemotaxis of monocytes and neutrophils in vitro

2021 ◽  
Author(s):  
Gang Wang ◽  
Jun Guan ◽  
Guojun Li ◽  
Fengtian Wu ◽  
Qin Yang ◽  
...  
Keyword(s):  
A549 Cells ◽  
Type Ii ◽  
Local Inflammation ◽  
Transwell Assay ◽  
Reading Frame ◽  
Type Ii Pneumocyte ◽  
Specific Effects ◽  
Significant Public Health ◽  
Immunological Changes

AbstractCoronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is currently the most significant public health threats in worldwide. Patients with severe COVID-19 usually have pneumonia concomitant with local inflammation and sometimes a cytokine storm. Specific components of the SARS-CoV-2 virus trigger lung inflammation, and recruitment of immune cells to the lungs exacerbates this process, although much remains unknown about the pathogenesis of COVID-19. Our study of lung type II pneumocyte cells (A549) demonstrated that ORF7, an open reading frame (ORF) in the genome of SARS-CoV-2, induced the production of CCL2, a chemokine that promotes the chemotaxis of monocytes, and decreased the expression of IL-8, a chemokine that recruits neutrophils. A549 cells also had an increased level of IL-6. The results of our chemotaxis transwell assay suggested that ORF7 augmented monocyte infiltration and reduced the number of neutrophils. We conclude that the ORF7 of SARS-CoV-2 may have specific effects on the immunological changes in tissues after infection. These results suggest that the functions of other ORFs of SARS-CoV-2 should also be comprehensively examined.

Rv3351c, aMycobacterium tuberculosisgene that affects bacterial growth and alveolar epithelial cell viability

2015 ◽  
Vol 61 (12) ◽  
pp. 938-947 ◽  
Author(s):  
Rebecca L. Pavlicek ◽  
Kari Fine-Coulson ◽  
Tuhina Gupta ◽  
Frederick D. Quinn ◽  
James E. Posey ◽  
...  
Keyword(s):  
Alveolar Epithelial Cell ◽  
Alveolar Epithelial Cells ◽  
Host Cells ◽  
Gene Products ◽  
Type Ii ◽  
Intracellular Replication ◽  
Cytotoxic Effects ◽  
Alveolar Epithelial ◽  
Type Ii Pneumocyte

Despite the interactions known to occur between various lower respiratory tract pathogens and alveolar epithelial cells (AECs), few reports examine factors influencing the interplay between Mycobacterium tuberculosis bacilli and AECs during infection. Importantly, in vitro studies have demonstrated that the M. tuberculosis hbha and esxA gene products HBHA and ESAT6 directly or indirectly influence AEC survival. In this report, we identify Rv3351c as another M. tuberculosis gene that impacts the fate of both the pathogen and AEC host. Intracellular replication of an Rv3351c mutant in the human AEC type II pneumocyte cell line A549 was markedly reduced relative to the complemented mutant and parent strain. Deletion of Rv3351c diminished the release of lactate dehydrogenase and decreased uptake of trypan blue vital stain by host cells infected with M. tuberculosis bacilli, suggesting attenuated cytotoxic effects. Interestingly, an isogenic hbha mutant displayed reductions in AEC killing similar to those observed for the Rv3351c mutant. This opens the possibility that multiple M. tuberculosis gene products interact with AECs. We also observed that Rv3351c aids intracellular replication and survival of M. tuberculosis in macrophages. This places Rv3351c in the same standing as HBHA and ESAT6, which are important factors in AECs and macrophages. Defining the mechanism(s) by which Rv3351c functions to aid pathogen survival within the host may lead to new drug or vaccine targets.

Type II pneumocyte hypertrophy without activation of surfactant biosynthesis after partial pneumonectomy

1993 ◽  
Vol 264 (2) ◽  
pp. L153-L159 ◽  
Author(s):  
B. D. Uhal ◽  
M. D. Etter
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Tritiated Thymidine ◽  
Cell Cycle Distribution ◽  
Type Ii ◽  
Adult Rats ◽  
Type Ii Pneumocyte ◽  
Significant Difference

Hypertrophic and normotrophic type II pneumocytes were isolated from pneumonectomized adult rats by unit gravity (1 g) sedimentation or by fluorescence-activated cell sorting (FACS). In vivo or in vitro, hypertrophic cells incorporated significantly more 5-bromo-2'-deoxyuridine or tritiated thymidine into acid-insoluble material than did normotrophic cells. By FACS analysis of cell subpopulations isolated by 1 g, > 97% of normotrophic cells had G0-phase DNA content. In contrast, the cell cycle distribution of hypertrophic cells was 75% G1, 5% S, and 20% G2/M phases. Rates of incorporation of tritiated choline into total cellular phosphatidylcholine (PC) were identical in type II cells isolated from normal or pneumonectomized rats. The intracellular contents of disaturated phosphatidylcholine (DSPC) and total PC, as well as the ratio of these two lipids, were the same in hypertrophic and normotrophic cells from pneumonectomized rats and in cells isolated from normal rats. No significant difference was observed in the rate at which hypertrophic or normotrophic cells incorporated choline into DSPC. These results demonstrate that type II pneumocyte hypertrophy after pneumonectomy reflects balanced cell growth secondary to cell cycle progression in vivo. The data also indicate that epithelial cell hypertrophy after pneumonectomy, in contrast to that which develops after more acute lung injury, occurs without activation of surfactant biosynthesis or storage.

Type II pneumocyte proliferation in vitro: problems and future directions

1991 ◽  
Vol 261 (4) ◽  
pp. L110-L117 ◽  
Author(s):  
Bruce D. Uhal ◽  
Kevin M. Flowers ◽  
D. Eugene Rannels
Keyword(s):  
Cell Cycle ◽  
Primary Culture ◽  
Alveolar Epithelium ◽  
Preliminary Evaluation ◽  
Type I ◽  
Type Ii Cells ◽  
Type Ii ◽  
Type Ii Pneumocyte ◽  
Cell Cycle Block

In adult animals, the type II pneumocyte is progenitor of both the type I and type II alveolar epithelium. In primary culture, however, the fate of this cell is uncertain. Type II cells in culture lose their differentiated properties and eventually resemble type I cells, but a lack of specific markers has complicated the characterization of the phenotype acquired in vitro. Furthermore, limited proliferation of these cells in vitro has precluded definition of the relationship between type II cell proliferation and differentiation. Recent work in this laboratory has involved the correlation of flow cytometric cell cycle analysis with phenotype markers. Initial results indicate that isolation of type II cells induces cell cycle block similar to that sustained by other cell types in response to stress. In addition, preliminary evaluation of phenotype suggests that traditional markers become ambiguous beyond the 1st day of primary culture. These results raise concern related to the interpretation of experiments conducted in vitro. This report discusses the implications of these findings and directions for future work. alveolar epithelium; bromodeoxyuridine; cell cycle block; flow cytometry; lung injury

Microfluidic shear stress-regulated surfactant secretion in alveolar epithelial type II cells in vitro

2014 ◽  
Vol 306 (7) ◽  
pp. L672-L683 ◽  
Author(s):  
Sanjeev Kumar Mahto ◽  
Janna Tenenbaum-Katan ◽  
Ayala Greenblum ◽  
Barbara Rothen-Rutishauser ◽  
Josué Sznitman
Keyword(s):  
Shear Stress ◽  
Actin Cytoskeleton ◽  
Intracellular Transport ◽  
A549 Cells ◽  
Cell Periphery ◽  
Type Ii ◽  
Alveolar Epithelial ◽  
Surfactant Secretion ◽  
Flow Stimulation

We investigated the role of flow-induced shear stress on the mechanisms regulating surfactant secretion in type II alveolar epithelial cells (ATII) using microfluidic models. Following flow stimulation spanning a range of wall shear stress (WSS) magnitudes, monolayers of ATII (MLE-12 and A549) cells were examined for surfactant secretion by evaluating essential steps of the process, including relative changes in the number of fusion events of lamellar bodies (LBs) with the plasma membrane (PM) and intracellular redistribution of LBs. F-actin cytoskeleton and calcium levels were analyzed in A549 cells subjected to WSS spanning 4–20 dyn/cm2. Results reveal an enhancement in LB fusion events with the PM in MLE-12 cells upon flow stimulation, whereas A549 cells exhibit no foreseeable changes in the monitored number of fusion events for WSS levels ranging up to a threshold of ∼8 dyn/cm2; above this threshold, we witness instead a decrease in LB fusion events in A549 cells. However, patterns of LB redistribution suggest that WSS can potentially serve as a stimulus for A549 cells to trigger the intracellular transport of LBs toward the cell periphery. This observation is accompanied by a fragmentation of F-actin, indicating that disorganization of the F-actin cytoskeleton might act as a limiting factor for LB fusion events. Moreover, we note a rise in cytosolic calcium ([Ca2+]c) levels following stimulation of A549 cells with WSS magnitudes ranging near or above the experimental threshold. Overall, WSS stimulation can influence key components of molecular machinery for regulated surfactant secretion in ATII cells in vitro.

scholarly journals Overexpression of 14-3-3γ Induces the Migration and Invasion of Human Lung Adenocarcinoma A549 Cells

2021 ◽  
Author(s):  
Pritsana Raungrut ◽  
Nidanut Champoochana ◽  
Paramee Thongsuksai ◽  
Kamontip Promnares
Keyword(s):  
Lung Cancer ◽  
Cancer Progression ◽  
A549 Cells ◽  
Migration And Invasion ◽  
P Value ◽  
Transwell Assay ◽  
Scratch Assay ◽  
Invasion And Migration ◽  
Tumor Cell Migration

Objective: 14-3-3 gamma (γ) is known to modulate the development and progression of many cancers. However, the evidence in lung cancer is still unclear. In this study, effects of 14-3-3γ on tumor cell migration and invasion were investigated. Material and Methods: A 14-3-3γ expression vector was made and transfected into A549 cells. In-vitro scratch assay and transwell assay were applied to assess migration and invasion, respectively. Western blotting was used to detect expression of proteins related to epithelial–mesenchymal transition.Results: Closing rate of scratch wounds, both in classical and non-classical scratch assay, was significantly increased in 14-3-3γ-overexpressing cells in comparison to the controls. Similarly, by transwell assay, a significant increase in the invasion and migration was shown in the 14-3-3γ-overexpressing cells in comparison to the null vector cells, by approximately 79.2% (p-value=0.002) and 131.2% (p-value<0.001), respectively. In addition, increased 14-3-3γ expression resulted in a significant increase of β-catenin and Snail but not for E-cadherin and vimentin. Conclusion: The study demonstrates the role of 14-3-3γ protein on lung cancer progression via migration and invasion processes, possibly providing a new targeted therapy for non-small cell lung cancer.

Exosurf enhances adenovirus-mediated gene transfer to alveolar type II cells

1997 ◽  
Vol 273 (4) ◽  
pp. L741-L748 ◽  
Author(s):  
S. Machelle Manuel ◽  
Yi Guo ◽  
Sadis Matalon
Keyword(s):  
Gene Transfer ◽  
Luciferase Activity ◽  
A549 Cells ◽  
Luciferase Reporter ◽  
Luciferase Expression ◽  
Alveolar Type ◽  
Type Ii ◽  
Luciferase Reporter Gene ◽  
Pulmonary Epithelium

We assessed the role of surfactant replacement mixtures in the enhancement of adenovirus-mediated gene transfer to pulmonary epithelial cells both in vitro and in vivo. A549 cells, a pulmonary epithelium-derived adenocarcinoma cell line, were incubated with either media alone or media containing 10 μg phospholipid/ml Exosurf or Infasurf for 50 min followed by addition of a replication-deficient adenovirus (E1-deleted) expressing the luciferase reporter gene [AdCMV-Luc; 10 plaque-forming units (PFU)/cell] for 4 h. Pretreatment with Exosurf, but not Infasurf, at 37°C, but not at 4°C, enhanced luciferase activity in A549 cells 24 h later by 156% ( P < 0.01). Intratracheal instillation of AdCMV-Luc (2 × 109PFU) into rats resulted in luciferase expression mainly in alveolar macrophages and to a smaller extent in alveolar type II (ATII) cells 24 h later. However, when the AdCMV-Luc instillation was preceded by Exosurf (250 μl; 25 mg/ml), a 10-fold increase in ATII cell luciferase activity was noted. Preincubation of cultured ATII cells with Exosurf also enhanced their transfection by AdCMV-Luc by 515% ( P < 0.001). The results of these studies provide a new strategy for targeting ATII cells for gene delivery.

Cell surface carbohydrates modulate neutrophil adherence to alveolar type II cells in vitro

1993 ◽  
Vol 264 (4) ◽  
pp. L391-L400 ◽  
Author(s):  
B. Crestani ◽  
C. Rolland ◽  
A. Petiet ◽  
N. Colas-Linhart ◽  
M. Aubier
Keyword(s):  
Cell Surface ◽  
A549 Cells ◽  
Sialic Acids ◽  
Alveolar Type ◽  
Type Ii ◽  
Adhesive Interaction ◽  
Human Polymorphonuclear Leukocyte ◽  
Type Ii Cell ◽  
Surface Carbohydrates

We characterized the influence of phosphorylated sugars and cell surface sialic acids on the adherence of human polymorphonuclear leukocyte (PMN) to rat alveolar type II cell (ATII cells) and human-derived A549 cell monolayers in vitro. Percent adherence of radiolabeled polymorphonuclear leukocytes was assessed after incubating cells with the carbohydrates, enzymes, or lectins to be tested. Lactose-1-phosphate (Lact1P) and maltose-1-phosphate (Malt1P) (10 mM) inhibited adherence of PMN to ATII cells and A549 cells. Maximal inhibition followed treatment of both PMN and rat ATII cells and amounted to 85 +/- 7% with Lact1P and 92 +/- 3% with Malt1P. Inhibition was concentration dependent. Incubation of PMN with mannose-6-phosphate reduced adherence to rat ATII cells and A549 cells by 36 +/- 11 and 39 +/- 8%, respectively. Maximal concentrations of sugars did not alter cellular viability. Neuraminidase-induced desialilation of ATII cells increased adherence of PMN by 36 +/- 7% to rat ATII cells and by 86 +/- 18% to A549 cells. Masking of terminal sialic acids on rat ATH cells with Limulus polyphemus agglutinin (100 micrograms/ml) increased adherence by 50 +/- 2%. These results indicate that cell surface carbohydrates are involved in the regulation of the adhesive interaction between PMN and ATII cells in vitro.

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