TLR3 and TLR7 RNA Sensor Activation during SARS-CoV-2 Infection
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cell Cultures
2.2. 3D Cultures:Multicellular Spheroids Formation
2.3. MTT Assay for Cell Viability
2.4. SARS-CoV-2 Propagation and Infection
2.5. Viral RNA Detection
2.6. MTCSs Treatment for RNA Sensor Pathways Analysis
2.7. Gene Expression Analysis
2.8. Immunofluorescence Analysis
2.9. Wesern Blot Analysis
2.10. IRF3 and NF-κB Expression and Phosphorilation Analysis
2.11. Soluble Factors Quantification by ELISA Assay
2.12. Statistical Analysis
3. Results
3.1. Calu-3/MRC-5 Multicellular Spheroids Are Efficiently Infected by SARS-CoV-2
3.2. Calu-3 and MRC-5 Permissivity to SARS-CoV-2 Infection
3.3. Calu-3/MRC-5 MTCS Response to SARS-CoV-2 Infection
3.4. SARS-CoV-2 Infection Induced an Increase in Cytokines and Interferon Secretion
3.5. TLR3 and TLR7 Activation Followed Different Signal Pathways after SARS-CoV-2 Infection
4. Discussion
Supplementary Materials
Author Contributions
Funding
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Target Gene | PrimeTime qPCR Primer Assay * |
---|---|
RIG-I | Hs.PT.58.4273674 |
MDA5 | Hs.PT.58.1224165 |
TLR3 | Hs.PT.58.25887499.g |
TLR4 | Hs.PT.58.38700156.g |
TLR7 | Hs.PT.58.39183219.g |
TLR8 | Hs.PT.58.15023918.g |
IRF3 | Hs.PT.58.27933933.g |
NF-κB | Hs.PT.58.20344216 |
GAPDH | Hs.PT.58.25887499.g |
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Bortolotti, D.; Gentili, V.; Rizzo, S.; Schiuma, G.; Beltrami, S.; Strazzabosco, G.; Fernandez, M.; Caccuri, F.; Caruso, A.; Rizzo, R. TLR3 and TLR7 RNA Sensor Activation during SARS-CoV-2 Infection. Microorganisms 2021, 9, 1820. https://doi.org/10.3390/microorganisms9091820
Bortolotti D, Gentili V, Rizzo S, Schiuma G, Beltrami S, Strazzabosco G, Fernandez M, Caccuri F, Caruso A, Rizzo R. TLR3 and TLR7 RNA Sensor Activation during SARS-CoV-2 Infection. Microorganisms. 2021; 9(9):1820. https://doi.org/10.3390/microorganisms9091820
Chicago/Turabian StyleBortolotti, Daria, Valentina Gentili, Sabrina Rizzo, Giovanna Schiuma, Silvia Beltrami, Giovanni Strazzabosco, Mercedes Fernandez, Francesca Caccuri, Arnaldo Caruso, and Roberta Rizzo. 2021. "TLR3 and TLR7 RNA Sensor Activation during SARS-CoV-2 Infection" Microorganisms 9, no. 9: 1820. https://doi.org/10.3390/microorganisms9091820