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Air-liquid interphase culture confers SARS-CoV-2 susceptibility to A549 alveolar epithelial cells.

ABSTRACT: The human lung cell A549 is susceptible to infection with a number of respiratory viruses. However, A549 cells are resistant to Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) infection in conventional submerged culture, and this would appear to be due to low expression levels of the SARS-CoV-2 entry receptor: angiotensin-converting enzyme-2 (ACE2). Here, we examined SARS-CoV-2 susceptibility to A549 cells after adaptation to air-liquid interface (ALI) culture. A549 cells in ALI culture yielded a layer of mucus on their apical surface, exhibited decreased expression levels of the proliferation marker KI-67 and intriguingly became susceptible to SARS-CoV-2 infection. We found that A549 cells increased the endogenous expression levels of ACE2 and TMPRSS2 following adaptation to ALI culture conditions. Camostat, a TMPRSS2 inhibitor, reduced SARS-CoV-2 infection in ALI-cultured A549 cells. These findings indicate that ALI culture switches the phenotype of A549 cells from resistance to susceptibility to SARS-CoV-2 infection through upregulation of ACE2 and TMPRSS2.

SUBMITTER: Sasaki M

PROVIDER: S-EPMC8423671 | biostudies-literature | 2021 Nov

REPOSITORIES: biostudies-literature

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Air-liquid interphase culture confers SARS-CoV-2 susceptibility to A549 alveolar epithelial cells.

Sasaki Michihito M Kishimoto Mai M Itakura Yukari Y Tabata Koshiro K Intaruck Kittiya K Uemura Kentaro K Toba Shinsuke S Sanaki Takao T Sato Akihiko A Hall William W WW Orba Yasuko Y Sawa Hirofumi H

Biochemical and biophysical research communications 20210910

The human lung cell A549 is susceptible to infection with a number of respiratory viruses. However, A549 cells are resistant to Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) infection in conventional submerged culture, and this would appear to be due to low expression levels of the SARS-CoV-2 entry receptor: angiotensin-converting enzyme-2 (ACE2). Here, we examined SARS-CoV-2 susceptibility to A549 cells after adaptation to air-liquid interface (ALI) culture. A549 cells in ALI cul ...[more]

PMID: 34517212

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Project description:BackgroundExposure to indoor air pollution from solid fuel combustion is associated with lung diseases and cancer. This study investigated the cytotoxicity and molecular mechanisms of biomass combustion-derived particles in human pulmonary alveolar epithelial cells (HPAEpiC) using a platform that combines air-liquid interface (ALI) and dynamic culture (DC) systems.MethodsHPAEpiC were cultured on the surface of polycarbonate (PC) membranes on the ALI-DC platform. The cells were sprayed with an aerosolized solution of biomass combustion soluble constituents (BCSCs) and simultaneously nourished with culture medium flowing beneath the permeable PC membranes. The ALI-DC method was compared with the traditional submerged culture approach. BCSC particle morphology and dosages deposited on the chip were determined for particle characterization. Flow cytometry, scanning electron microscopy, and transmission electron microscopy were used to investigate the apoptosis rate of HPAEpiC and changes in the cell ultrastructure induced by BCSCs. Additionally, the underlying apoptotic pathway was examined by determining the protein expression levels by western blotting.ResultsScanning electron microscope images demonstrated that the sample processing and delivering approach of the ALI-DC platform were suitable for pollutant exposure. Compared with the submerged culture method, a significant decline in cell viability and increase in apoptosis rate was observed after BCSC exposure on the ALI-DC platform, indicating that the ALI-DC platform is a more sensitive system for investigating cytotoxicity of indoor air pollutants in lung cells. The morphology and ultrastructure of the cells were damaged after exposure to BCSCs, and the p53 pathway was activated. The Bcl-2/Bax ratio was reduced, upregulating caspase-9 and caspase-3 expression and subsequently inducing apoptosis of HPAEpiC. The addition of N-acetyl cysteine antioxidant significantly alleviated the cytotoxicity induced by BCSCs.ConclusionA novel ALI-DC platform was developed to study the cytotoxicity of air pollutants on lung cells. Using the platform, we demonstrated that BCSCs could damage the mitochondria, produce reactive oxygen species, and activate p53 in HPAEpiC, ultimately inducing apoptosis.

Project description:BackgroundEpidemiological studies have related desert dust events to increased respiratory morbidity and mortality. Although the Sahara is the largest source of desert dust, Saharan dust (SD) has been barely examined in toxicological studies. Here, we aimed to assess the NLRP3 inflammasome-caspase-1-pathway-dependent pro-inflammatory potency of SD in comparison to crystalline silica (DQ12 quartz) in an advanced air-liquid interface (ALI) co-culture model. Therefore, we exposed ALI co-cultures of alveolar epithelial A549 cells and macrophage-like differentiated THP-1 cells to 10, 21, and 31 µg/cm² SD and DQ12 for 24 h using a Vitrocell Cloud system. Additionally, we exposed ALI co-cultures containing caspase (CASP)1-/- and NLRP3-/- THP-1 cells to SD.ResultsCharacterization of nebulized DQ12 and SD revealed that over 90% of agglomerates of both dusts were smaller than 2.5 μm. Characterization of the ALI co-culture model revealed that it produced surfactant protein C and that THP-1 cells remained viable at the ALI. Moreover, wild type, CASP1-/-, and NLRP3-/- THP-1 cells had comparable levels of the surface receptors cluster of differentiation 14 (CD14), toll-like receptor 2 (TLR2), and TLR4. Exposing ALI co-cultures to non-cytotoxic doses of DQ12 and SD did not induce oxidative stress marker gene expression. SD but not DQ12 upregulated gene expressions of interleukin 1 Beta (IL1B), IL6, and IL8 as well as releases of IL-1β, IL-6, IL-8, and tumor necrosis factor α (TNFα). Exposing wild type, CASP1-/-, and NLRP3-/- co-cultures to SD induced IL1B gene expression in all co-cultures whereas IL-1β release was only induced in wild type co-cultures. In CASP1-/- and NLRP3-/- co-cultures, IL-6, IL-8, and TNFα releases were also reduced.ConclusionsSince surfactants can decrease the toxicity of poorly soluble particles, the higher potency of SD than DQ12 in this surfactant-producing ALI model emphasizes the importance of readily soluble SD components such as microbial compounds. The higher potency of SD than DQ12 also renders SD a potential alternative particulate positive control for studies addressing acute inflammatory effects. The high pro-inflammatory potency depending on NLRP3, CASP-1, and IL-1β suggests that SD causes acute lung injury which may explain desert dust event-related increased respiratory morbidity and mortality.

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Air-liquid interphase culture confers SARS-CoV-2 susceptibility to A549 alveolar epithelial cells.

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Air-liquid interphase culture confers SARS-CoV-2 susceptibility to A549 alveolar epithelial cells.

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