Direct exposure to SARS-CoV-2 and cigarette smoke increases infection severity and alters the stem cell-derived airway repair response
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Abstract
Most demographic studies are now associating current smoking status with increased risk of severe COVID-19 and mortality from the disease but there remain many questions about how direct cigarette smoke exposure affects SARS-CoV-2 airway cell infection. We directly exposed mucociliary air-liquid interface (ALI) cultures derived from primary human nonsmoker airway basal stem cells (ABSCs) to short term cigarette smoke and infected them with live SARS-CoV-2. We found an increase in the number of infected airway cells after cigarette smoke exposure as well as an increased number of apoptotic cells. Cigarette smoke exposure alone caused airway injury that resulted in an increased number of ABSCs, which proliferate to repair the airway. But we found that acute SARS-CoV-2 infection or the combination of exposure to cigarette smoke and SARS-CoV-2 did not induce ABSC proliferation. We set out to examine the underlying mechanism governing the increased susceptibility of cigarette smoke exposed ALI to SARS-CoV-2 infection. Single cell profiling of the cultures showed that infected airway cells displayed a global reduction in gene expression across all airway cell types. Interestingly, interferon response genes were induced in SARS-CoV-2 infected airway epithelial cells in the ALI cultures but smoking exposure together with SARS-CoV-2 infection reduced the interferon response. Treatment of cigarette smoke-exposed ALI cultures with Interferon β-1 abrogated the viral infection, suggesting that the lack of interferon response in the cigarette smoke-exposed ALI cultures allows for more severe viral infection and cell death. In summary, our data show that acute smoke exposure allows for more severe proximal airway epithelial disease from SARS-CoV-2 by reducing the mucosal innate immune response and ABSC proliferation and has implications for disease spread and severity in people exposed to cigarette smoke.
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SciScore for 10.1101/2020.07.28.226092: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Table 2: Resources
No key resources detected.
Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar …
SciScore for 10.1101/2020.07.28.226092: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Table 2: Resources
No key resources detected.
Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
- No protocol registration statement was detected.
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SciScore for 10.1101/2020.07.28.226092: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement Tissues were procured under Institutional Review Board-approved protocols at the David Geffen School of Medicine at UCLA. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources After several washes of Tris-Buffered Saline and Tween-20 (TBST), secondary antibodies were incubated on samples for 1 hour in darkness, washed, and mounted using Vectashield hardest mounting medium with DAPI (Vector Labs H-1500). Vectashield hardest mounting medium with DAPIsuggested: None…SciScore for 10.1101/2020.07.28.226092: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement Tissues were procured under Institutional Review Board-approved protocols at the David Geffen School of Medicine at UCLA. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources After several washes of Tris-Buffered Saline and Tween-20 (TBST), secondary antibodies were incubated on samples for 1 hour in darkness, washed, and mounted using Vectashield hardest mounting medium with DAPI (Vector Labs H-1500). Vectashield hardest mounting medium with DAPIsuggested: NoneExperimental Models: Cell Lines Sentences Resources Virus titer was measured in Vero-E6 cells by TCID50 assay. Vero-E6suggested: NoneSoftware and Algorithms Sentences Resources Data and Code Availability This study generated a single cell RNA sequencing dataset of ALI cultures that were exposed to cigarette smoke and/or SARS-CoV-2 or neither. SARS-CoV-2suggested: (Active Motif Cat# 91345, AB_2847847)IF images were obtained using an LSM700 or LSM880 Zeiss confocal microscope and composite images generated using ImageJ. ImageJsuggested: (ImageJ, SCR_003070)Cellular suspensions were loaded on a Chromium Controller instrument (10X Genomics) to generate single-cell Gel Bead-In-EMulsions (GEMs) GEMssuggested: (GEMS, SCR_009188)Raw sequencing data were filtered by read quality, adapter- and polyA-trimmed, and reads were aligned to a hybrid human hg38-Sars Covid2 transcriptome using the Cell Range software (10X Genomics) and the STAR aligner. STARsuggested: (STAR, SCR_015899)Gene ontology enrichments were determined using the Metascape tool (Zhou et al., 2019). Metascapesuggested: (Metascape, SCR_016620)Data from additional tools added to each annotation on a weekly basis.
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