Visualizing the Intracellular Immune Response to SARS CoV 2 with Fast, High Content Imaging
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1. Researchers used high-content imaging to visualize the intracellular immune response to SARS-CoV-2 infection in lung epithelial cells. 2. They found that SARS-CoV-2 replicates rapidly in Calu-3 cells but triggers a robust but delayed innate immune response. 3. Nuclear translocation of transcription factors like IRF3 and NF-kB occurred in SARS-CoV-2 infected cells, coinciding with expression of viral proteins.
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Visualizing the Intracellular Immune Response to SARS CoV 2 with Fast, High Content Imaging
- 1. Welcome! Jason Otterstrom, PhD IDEA Bio-Medical Application Scientist Matthew Whelan, PhD HCS Microscopy and Image Analysis University College London Research Fellow Visualizing the Intracellular Immune Response to SARS-CoV-2 with Fast, High-Content Imaging Ann-Kathrin (AK) Reuschl, PhD Division of Infection & Immunity University College London Postdoctoral Research Associate
- 2. Copyright 2022. All Rights Reserved. Contact Presenter for Permission Visualizing the Intracellular Immune Response to SARS-CoV-2 with Fast, High-Content Imaging Matthew Whelan, PhD HCS Microscopy and Image Analysis University College London Research Fellow
- 3. Imaging slides Dr. Matthew V.X. Whelan UCL
- 4. Looking to the future
- 5. Visualising the SARS-CoV-2 Pandemic
- 6. Aim: How can we implement High Content Screening Microscopy and single cell analysis to investigate: • Kinetics of viral replication • Spatial information about cell-cell Spread • Kinetics of Innate Immune activation in Infected Cells
- 7. Selecting a Fluorescent Marker for SARS-CoV-2 Infection
- 8. Developing a High Content Screening Approach in Calu-3 Epithelial Cell Model 10x/0.4NA 40x/0.75NA Fixation and staining: • Viral infection/spread: • Nuclei (Dapi), • Nucleocapsid (IF), • dsRNA (IF), • CellMask (segmentation) • Innate immune activation • Nuclei (Dapi), • Nucleocapsid (IF), • IRF3/NFκB (IF) • Innate Response (RNA- FISH) • Nuclei (Dapi), • Nucleocapsid (IF), • IL6 mRNA (RNA-FISH), IFIT1 mRNA (RNA-FISH), GAPDH mRNA (RNA-FISH) 2h, 6h, 10h, 24h, 48h, 72h infection 2 TCID50/cell 0.4 TCID50/cell 0.04 TCID50/cell 0.004 TCID50/cell Automated Image Analysis
- 9. 6hpi 10hpi 24hpi 48hpi 72hpi Nuclei Nucleocapsid CellMask Visualising Viral Protein Expression: SARS-CoV2 Infection Occurs Rapidly
- 10. 6hpi 10hpi 24hpi 48hpi 72hpi Nuclei Nucleocapsid CellMask Cells analysed: 484,557 Visualising Viral Protein Expression: SARS-CoV2 Infection Occurs Rapidly
- 11. Visualising Viral Replication: Spatial replication differs between first wave and later SARS-CoV2 isolates Nuclei dsRNA Mock VIC IC19 Alpha VIC Infected Cells = ( ) IC19 Infected Cells = ( ) Alpha Infected Cells = ( )
- 12. Approach to Quantifying Innate immune Responses: Proinflammatory Transcription Factors Transcriptional Response - Interferons - ISGs - Proinflammatory Mediators
- 13. Implementing a High Content Screening Approach: Nuclear Intensity in Infected Cells (First Approach) Nuclear Nucleocapsid Merge Nuclei Nucleocapsid NF-κB
- 14. Nuclear Localisation of IRF3 and NF-κB Coincides with Cells Becoming N-positive (Expressing Viral Proteins) Uninfected Cells = ( ) Nucleocapsid + Cells = ( ) IRF3 Nucleocapsid Mock (24hpi) +SARS-CoV-2 (24hpi) NF-κB Nucleocapsid Mock (24hpi) +SARS-CoV-2 (24hpi) Uninfected Cells = ( ) Nucleocapsid + Cells = ( ) IRF3 IRF3 NF-κB NF-KB Cells analysed: 26,400 Cells analysed: 27, 773
- 15. IRF3 Nucleocapsid Mock (24hpi) +SARS-CoV-2 (24hpi) NF-κB Nucleocapsid Mock (24hpi) +SARS-CoV-2 (24hpi) IRF3 IRF3 NF-κB NF-KB Cells analysed: 26,400 Cells analysed: 27, 773 Innate activation is limited to N-positive cells in Early Infection Bystander Cells = ( ) Bystander Cells = ( )
- 16. Visualising Response to Innate Sensing: IL-6 (NFκB regulated Cytokine) mRNA Transcripts Upregulated Uninfected Cells = ( ) Nucleocapsid + Cells = ( ) Bystander Cells = ( ) Mock (24hpi) +SARS-CoV-2 (24hpi) Nuclei IL-6 mRNA Nucleocapsid
- 17. GAPDH mRNA Expression Unaf in N-positive Cells Uninfected Cells = ( ) Nucleocapsid + Cells = ( ) Bystander Cells = ( ) Mock (24hpi) +SARS-CoV-2 (24hpi) Nuclei Nucleocapsid GAPDH mRNA
- 18. Furthering the pipeline: Nuclear / Cytoplasmic Ratio Reveals Alpha Variant Antagonises Innate Immunity 24hpi Cells analysed: 3,000 per condition Infected? (N-positive) No Yes OFF ON Innate Immune Sensing? How Much? Ratio New analysis pipeline: (CellProfiler/Python) Nucleo- capsid IRF3 VIC Alpha
- 19. Looking to the Future! Rapid Translocation analysis (Athena) Mock Viral Protein Cells analysed: 10,000 per condition + = Interferon • Developed with IDEA-Bio • Fast Efficient Nuclear translocation analysis • Optimised for 10x magnification to allow for large single cell populations • Discussed later in Webinar Super Resolution Imaging • SRRF Microscopy (Super-Resolution Radial Fluctuations • Algorithm processes: 100 frame Widefield Images • Resolution of <100nm
- 20. Investigating Viral Spread 1. Make Montage of Well 10x 64 sub-positions. 2. Segment cell population (CellProfiler/Python 3. Quantify object neighbours 1px distance 4. Output: 5. (A) Number of neighbours 6. (B) Percentage of surface touching Viral Cell-Cell Spread (CellProfiler → Athena) 1. Nucleocapsid 1. Merge 2. 3. (A) 3. (B)
- 27. Looking to the future
- 28. Acknowledgements Greg Towers (UCL) Lucy Thorne Lorena Zuliani-Alvarez Jane Turner The rest of the lab THANK YOU FOR REAGENTS: Laura McCoy (UCL): N antibody NIBSC: Virus and reagents Dalan Bailey (Pirbright): cells Wendy Barclay (Imperial) Imperial ATACCC Study Team Ajit Lalvani (Imperial) Jake Dunning (Imperial) Maria Zambon (PHE) Clare Jolly (UCL) Ann-Kathrin Reuschl Maddy Noursadeghi (UCL) The rest of lab UCL Medicinal Chemists David Selwood Ben Graham • UCL COVID19 RAPID RESPONSE FUND • UKRI funding G2P Consortium Thank you for listening! Joe Grove (UCL) Yasu Takeuchi (UCL) Our paper:
- 29. Copyright 2022. All Rights Reserved. Contact Presenter for Permission Visualizing the Intracellular Immune Response to SARS-CoV-2 with Fast, High-Content Imaging Jason Otterstrom, PhD IDEA Bio-Medical Application Scientist
- 30. 1. IDEA Bio-Medical 2. WiScan® Hermes Microscope 3. WiSoft® Athena Analysis Software For Virology Summary
- 31. 25 years experience designing electro-optics & precision motion systems Experts in Biotechnology & Pharmaceutical Sciences Company
- 33. X, Y, Z Motion ✓ Unique stable-plate design: Stationary plate, Mobile objective Precision Objective Motion
- 34. X, Y, Z Motion 10x ✓ Unique stable-plate design: Stationary plate, Mobile objective ✓ Rapid plate scanning Colors: 4 Fields: 1 50 ms Exposure Plate Format Scan Time (mm:ss) 96 01:39 384 05:25 Round-bottom plates supported! Figure from: Liu, H. et al. (2016) PLoS ONE 11(10): e0164645 Precision Objective Motion
- 35. X, Y, Z Motion ✓ Unique stable-plate design: Stationary plate, Mobile objective ✓ Rapid plate scanning ✓ Automatic 3 objective switching Precision Objective Motion
- 36. X, Y, Z Motion 2X 4X 10X 20X 40X 60X ✓ Unique stable-plate design: Stationary plate, Mobile objective ✓ Rapid plate scanning ✓ Automatic 3 objective switching ✓ Magnifications 2X – 60X: air, oil, water Precision Objective Motion
- 37. ✓ Unique stable-plate design: Stationary plate, Mobile objective ✓ Rapid plate scanning ✓ Automatic 3 objective switching ✓ Magnifications 2X – 60X: air, oil, water ✓ Up to 7 fluorescence colors: 405nm – 694nm 10x Fluorescence Channel Standard Configuration DAPI ✔ CFP FITC ✔ YFP TRITC ✔ mCherry CY5 ✔ Transmission White LED ✔ Laser Diode ✔ Flexible illumination
- 38. 60x/1.42 Oil 60x/0.90 Air Yeast 60% shorter exposure time with oil Same illumination & contrast Automated Scanning with Oil Objectives Automated immersion oil dispensing 60x/1.42
- 39. Automated Scanning with Oil Objectives Automated immersion oil dispensing Nuclear Pore Complex 100 nm resolution Multi-color Super-Resolution Imaging via SRRF 60x/1.42
- 40. Automated Scanning with Oil Objectives Automated immersion oil dispensing 60x/1.42 Multi-color Super-Resolution Imaging via SRRF
- 41. WiSoft: Athena Image Analysis Software for Life Scientists
- 42. Athena: Easy to Use Simple parameter definition Dynamic segmentation readout
- 43. Athena: Informative Data Visualization Heat Maps Time-Evolution Plots Dose Curves Scatter Plots
- 44. CELL MORPHOLOGY PROTEIN EXPRESSION CELL COUNT CELL CYCLE SPHEROIDS SUPER-RESOLUTION IMAGING VIRAL PLAQUE ASSAY TRANSLOCATION + INTRACELLULAR GRANULES VIRAL SPREAD QUANTIFICATION INTRANUCLEAR FOCI RNA & ORGANELLE CO-LOCALIZATION MITOCHONDRIA QUANTIFICATION SCRATCH ASSAY LIVE-DEAD TOXICOLOGY ASSAYS ZEBRAFISH FLUORESCENCE QUANTIFICATION Athena: Ready-Made Applications
- 45. Translocation: Compartmentalized Intensity & Spot Count Quantify • Nuclear Intensity • Cytoplasmic Intensity • Spot Counting & Morphology • Nuclear : Cytoplasm Ratios Labels: Nucleus Cytoplasm Masks: Nucleus Cell Nuclear Puncta Cytoplasmic Puncta
- 46. Viral Plaque Assay Quantify • Plaque Count & Morphology • Plaque Density • Cell Count / Plaque • Multi-channel Fluorescence Intensity Labels: Nucleus Infected Cell Masks: Infected Nucleus Plaque
- 47. Viral Spread: Quantify • Infected Cell Count • Neighbor-cell Statistics • Total vs. Infected • Contact Ratio • Cell Morphology • Multi-channel Fluorescence Intensity Single-Cell Infection Measurement Labels: Nucleus Cytoplasm Infected Cell Masks: Nucleus Healthy Cell Border Infected Cell Border
- 48. idea-bio.com Contact: Jason Otterstrom jason-o@idea-bio.com Application Scientist, IDEA Bio Automated Imaging Solutions
- 49. Copyright 2022. All Rights Reserved. Contact Presenter for Permission Visualizing the Intracellular Immune Response to SARS-CoV-2 with Fast, High-Content Imaging Ann-Kathrin Reuschl, PhD Division of Infection & Immunity University College London Postdoctoral Research Associate
- 50. Evolution of SARS-CoV-2 innate antagonism a Dr. Ann-Kathrin Reuschl Division of Infection and Immunity University College London PIs: Prof. Clare Jolly/Prof. Greg Towers
- 51. g b o d a First wave virus
- 52. g b o d a
- 53. The host innate immune response determines the outcome of infection Viral infection Airway epithelium IRF3 NFκB IFN I ISGs Proinflammatory genes Signaling Adaptors Pattern Recognition Receptors (PRR) Pathogen Associated Molecular Patters (PAMPs) Innate immunity is the first line of defense against infections Gene expression
- 54. The host innate immune response determines the outcome of infection Viral infection Airway epithelium IRF3 NFκB IFN I ISGs Proinflammatory genes Interferon Antiviral responses Inflammation Signaling Adaptors Pattern Recognition Receptors (PRR) Pathogen Associated Molecular Patters (PAMPs) Innate immunity is the first line of defense against infections Gene expression Defensive responses
- 55. The host innate immune response determines the outcome of infection • How does SARS-CoV-2 infection trigger innate immune responses in human lung epithelial cells? • What is the effect of SARS-CoV-2 infection on immune cells and the local inflammatory environment? • Have Variants Of Concern evolved improved innate antagonism?
- 56. SARS-CoV-2 replicates rapidly in Calu-3 lung epithelial cells First wave VIC SARS-CoV-2 Lineage B: BetaCoV/Australia/VIC01/2020 Viral replication Infection levels Virus released Thorne, Reuschl, Zuliani-Alvarez, Whelan et al. EMBO J. 2021
- 57. SARS-CoV-2 replicates rapidly in Calu-3 lung epithelial cells First wave VIC SARS-CoV-2 Lineage B: BetaCoV/Australia/VIC01/2020 Thorne, Reuschl, Zuliani-Alvarez, Whelan et al. EMBO J. 2021 MOI 0.4 TCID50/cell N protein Cell Mask DAPI 6h 10h 48h 24h Quantification SARS-CoV-2 infection of Calu-3 cells
- 58. Does SARS-CoV-2 trigger and innate immune response? Viral replication ↑ gRNA and sgRNA MDA5 RIG-I MAVS RNA sensors TBK1 IKKs IRF3 NFκB IFN I ISGs Proinflammatory genes
- 59. SARS-CoV-2 triggers a robust delayed innate immune response in epithelial cells Viral RNA replication 0 10 20 30 40 50 100 102 104 102 104 106 108 1010 Hours Post Infection E copies/mg RNA E RNA SARS-CoV-2 Lineage B: BetaCoV/Australia/VIC01/2020 Thorne, Reuschl, Zuliani-Alvarez, Whelan et al. EMBO J. 2021 Nuclear translocation of innate immune transcription factors MOI 0.4 TCID50/cell N protein Transcription factor NFkB IRF3
- 60. SARS-CoV-2 triggers a robust delayed innate immune response in epithelial cells SARS-CoV-2 Lineage B: BetaCoV/Australia/VIC01/2020 Thorne, Reuschl, Zuliani-Alvarez, Whelan et al. EMBO J. 2021 Viral RNA replication 0 10 20 30 40 50 100 102 104 102 104 106 108 1010 Hours Post Infection E copies/mg RNA E RNA Nuclear translocation of innate immune transcription factors NFkB IRF3
- 61. SARS-CoV-2 triggers a robust delayed innate immune response in epithelial cells Thorne, Reuschl, Zuliani-Alvarez, Whelan et al. EMBO J. 2021 ISGs 0 10 20 30 40 50 100 102 104 102 104 106 108 1010 Hours Post Infection Gene Fold Induction E copies/mg RNA E RNA CXCL10 IFIT2 Interferons 0 10 20 30 40 50 100 102 104 102 104 106 108 1010 Hours Post Infection Gene Fold Induction E copies/mg RNA E RNA IFNb IFNl1 IFNl3 Pro-inflammatory mediators 0 10 20 30 40 50 100 102 104 102 104 106 108 1010 Hours Post Infection Gene Fold Induction E copies/mg RNA Viral replication overlaid with host gene expression Interferons 10 20 30 40 50 102 104 106 108 1010 Hours Post Infection E copies/mg RNA E RNA IFNb IFNl1 IFNl3 ISGs 0 10 20 30 40 50 100 102 104 102 104 106 108 1010 Hours Post Infection Gene Fold Induction E copies/mg RNA E RNA CXCL10 IFIT2 Pro-inflammatory mediators 0 10 20 30 40 50 100 102 104 102 104 106 108 1010 Hours Post Infection Gene Fold Induction E copies/mg RNA E RNA IL-6 CCL5
- 62. SARS-CoV-2 triggers a robust delayed innate immune response in epithelial cells Thorne, Reuschl, Zuliani-Alvarez, Whelan et al. EMBO J. 2021 Single-cell fluorescence in situ hybridisation (FISH) to detect gene expression:
- 63. s i C t r l s i M A V S s i R I G - I s i M D A 5 0 1000 2000 3000 4000 5000 Fold induction **** *** *** IFNb s i C t r l s i M A V S s i R I G - I s i M D A 5 0 1000 2000 3000 4000 Fold induction *** ** *** CXCL10 Innate immune activation during SARS-CoV-2 infection MAVS signaling governs the epithelial inflammatory response during infection Thorne, Reuschl, Zuliani-Alvarez, Whelan et al. EMBO J. 2021 Viral replication ↑ gRNA and sgRNA MDA5 RIG-I MAVS RNA sensors TBK1 IKKs IRF3 NFκB IFN I ISGs Proinflammatory genes
- 64. MAVS signaling governs the epithelial inflammatory response during infection Thorne, Reuschl, Zuliani-Alvarez, Whelan et al. EMBO J. 2021 Viral replication ↑ gRNA and sgRNA MDA5 RIG-I MAVS RNA sensors TBK1 IKKs IRF3 NFκB IFN I ISGs Proinflammatory genes siRNA knockdown s i C t r l s i M A V S s i R I G - I s i M D A 5 0 1000 2000 3000 4000 Fold induction *** ** *** CXCL10 s i C t r l s i M A V S s i R I G - I s i M D A 5 0 1000 2000 3000 4000 5000 Fold induction **** *** *** IFNb Innate immune activation during SARS-CoV-2 infection
- 65. Does epithelial infection drive local inflammation? Thorne, Reuschl, Zuliani-Alvarez, Whelan et al. EMBO J. 2021 SARS-CoV-2 infected Calu-3 cells Conditioned media transfer MDM Soluble mediators
- 66. Epithelial responses to SARS-CoV-2 drive macrophage activation Thorne, Reuschl, Zuliani-Alvarez, Whelan et al. EMBO J. 2021 SARS-CoV-2 infected Calu-3 cells Conditioned media transfer MDM Epithelial CoM: Macrophage activation Mock SARS-CoV-2 Mock+ MAVS k/d SARS-CoV-2+MAVS k/d Soluble mediators
- 67. Epithelial responses to SARS-CoV-2 drive macrophage activation Thorne, Reuschl, Zuliani-Alvarez, Whelan et al. EMBO J. 2021 SARS-CoV-2 infected Calu-3 cells Conditioned media transfer MDM Epithelial CoM: Macrophage activation Mock SARS-CoV-2 Mock+ MAVS k/d SARS-CoV-2+MAVS k/d Soluble mediators +/- MAVS
- 68. Thorne, Reuschl, Zuliani-Alvarez, Whelan et al. EMBO J. 2021 MAVS-activation of SARS-CoV-2 links epithelial infection to macrophage inflammation
- 69. a First wave VIC/IC19 Does SARS-CoV-2 evolve improved innate antagonism? vs.
- 70. Does SARS-CoV-2 evolve improved innate antagonism? Thorne, Bouhaddou, Reuschl, Zuliani-Alvarez et al. Nature. 2021 Alpha has acquired coding changes outside of spike:
- 71. Alpha replicates efficiently in primary epithelial cultures vs. Thorne, Bouhaddou, Reuschl, Zuliani-Alvarez et al. Nature. 2021 IC19 Primary bronchial HAEs
- 72. Alpha has evolved improved innate antagonism vs. Thorne, Bouhaddou, Reuschl, Zuliani-Alvarez et al. Nature. 2021
- 73. Taking a global view Thorne, Bouhaddou, Reuschl, Zuliani-Alvarez et al. Nature. 2021 Interferon-stimulated genes (ISG) RNASeq 10h 24h
- 74. Alpha induces less antiviral responses than first wave lineages RNASeq Proteomics Interferon stimulated genes: RT-PCR Thorne, Bouhaddou, Reuschl, Zuliani-Alvarez et al. Nature. 2021
- 75. Alpha enhances expression of innate antagonists Orf6, Orf9b and N Mock Alpha VIC IC19 RNASeq Proteomics 250kDa 100kDa 55kDa 55kDa 15kDa S N Orf6b Tubulin M o c k I C 1 9 V I C B . 1 . 1 . 7 Upregulation of viral gene and protein expression by Alpha compared to first wave variants: Thorne, Bouhaddou, Reuschl, Zuliani-Alvarez et al. Nature. 2021 Viral replication ↑ gRNA and sgRNA
- 76. Alpha has evolved improved innate antagonism Thorne, Bouhaddou, Reuschl, Zuliani-Alvarez et al. Nature. 2021
- 77. Evolution of SARS-CoV-2 innate antagonism • SARS-CoV-2 replicates rapidly in lung epithelial cells and triggers a delayed proinflammatory and ISG immune response mediated by MAVS (RIG-I/MDA-5) • Epithelial RNA-sensing of infection drives pro-inflammatory macrophage activation • Spike and non-spike mutations act in concert to mediate efficient infection and evasion of host immune responses • Alpha has evolved to efficiently antagonise the host innate immune response by upregulation of innate antagonists Orf6 Orf9b and N • Improved innate antagonism might contribute to efficient transmission of SARS- CoV-2
- 78. Thank you for your attention! Towers Lab (UCL) Greg Towers Lucy Thorne Giulia Dowgier Jane Turner The rest of the lab Jolly Lab (UCL) Clare Jolly Matt Whelan Dejan Mesner Taylor Bronzovich Noursadeghi Lab (UCL) UCL COVID19 Rapid Response Fund UKRI funding G2P Consortium Krogan Lab (UCSF) Nevan Krogan Lorena Zuliani-Alvarez Mehdi Bouhaddou Goodfellow Lab (Cambridge) Bonfanti Lab (Crick) Barclay Lab (Imperial College) Lucy Thorne Ann-Kathrin Reuschl Lorena Zuliani-Alvarez Matt Whelan McCoy Lab (UCL)