PI3Kg inhibition circumvents inflammation and mortality in SARS-CoV-2 and other infections
Data files
Mar 27, 2024 version files 6.52 MB
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2024_Main_Figure_1.csv
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2024_Main_Figure_2.csv
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2024_Main_Figure_3.csv
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2024_Main_Figure_4.csv
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2024_Main_Figure_5.csv
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2024_Main_figure_6.csv
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2024_Main_Figure_7.csv
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2024_Supplement_Figure_10.csv
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2024_Supplement_Figure_11.csv
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2024_Supplement_Figure_12.csv
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2024_Supplement_Figure_13.csv
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2024_Supplement_Figure_2.csv
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2024_Supplement_Figure_3.csv
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2024_Supplement_Figure_4.csv
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2024_Supplement_Figure_5.csv
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2024_Supplement_Figure_7.csv
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2024_Supplement_Figure_8.csv
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2024_Supplement_Figure_9.csv
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README.md
Abstract
Virulent infectious agents such as SARS-CoV-2 and Methicillin Resistant Staphylococcus Aureus (MRSA) induce tissue damage that recruits neutrophils and monocyte/macrophages that promote T cell exhaustion, fibrosis, vascular leak, epithelial cell depletion, and fatal organ damage. Neutrophils and macrophages recruited to pathogen infected lungs, including SARS-CoV-2 infected lungs, express phosphatidylinositol 3-kinase gamma (PI3Kg), a signaling protein that coordinately controls granulocyte and monocyte trafficking to diseased tissues and immune suppressive, pro-fibrotic transcription in myeloid cells. PI3Kg deletion and inhibition with the clinical PI3Kg inhibitor eganelisib promoted survival in models of infectious diseases, including SARS-CoV-2 and MRSA, by suppressing inflammation, vascular leak, organ damage and cytokine storm. These results demonstrate essential roles for PI3Kg in inflammatory lung disease and support the potential use of PI3Kg inhibitors to suppress inflammation in severe infectious diseases.
README: PI3Kg inhibition circumvents inflammation and mortality in SARS-CoV-2 and other infections
File name: README_Dataset-PI3KgCOVIDproject.txt
Authors: Judith A. Varner
Other contributors: Ryan M. Shepard, Anghesom Ghebremedhin, Isa Pratumchai, Sally Robinson, Courtney Betts, Jingjing Hu, Roman Sasik, Kathleen M. Fisch, Jaroslav Zak, Hui Chen, Marc Paradise, Mohammad Amjad, Jason Rivera, Satoshi Uchiyama, Hideya Seo, Alex Campos, Denise Ann Dayao, Saul Tzipori, Cesar Piedra-Mora, Soumita Das, Farnaz Hasteh, Hana Russo, Xin Sun, Le Xu, Laura Crotty Alexander, Jason M. Duran, Mazen Odish, Victor Pretorius, Nell C. Kirchberger, Shao-ming Chin, Tami Von Schalscha, David Cheresh, John D. Morrey, Rossitza Alargova, Brenda O’Connell, Theodore A. Martinot, Sandip P. Patel, Victor Nizet, Amanda J. Martinot, Lisa M. Coussens, John Teijaro
Date created: 2024-03-14
Dataset Version and Release History
Current Version:
- Number: 1.0.0
- Date: 2024-03-14
- Persistent identifier: DOI:10.6076/D1DK5D
- Summary of changes: n/a
Embargo Provenance: n/a
- Scope of embargo: n/a
- Embargo period: n/a
Dataset Attribution and Usage
Dataset Title: Data for the article " PI3Kgamma inhibition circumvents inflammation and mortality in SARS-CoV-2 and other infections"
Persistent Identifier: https://doi.org/10.6076/D1DK5D
Dataset Contributors:
- Creators: Judith A. Varner
Date of Issue: 2024-03-14
Publisher: University of California, San Diego
License: Use of these data is covered by the following license:
- Title: CC0 1.0 Universal (CC0 1.0)
- Specification: https://creativecommons.org/publicdomain/zero/1.0/; the authors respectfully request to be contacted by researchers interested in the re-use of these data so that the possibility of collaboration can be discussed.
Suggested Citations:
- Dataset citation:
- Corresponding publication: Shepard, Ghebremedhin, et al PI3Kgamma inhibition circumvents inflammation and mortality in SARS-CoV-2 and other infections
Contact Information
- Name: Judith A. Varner
- Affiliations: Department of Pathology, Moores Cancer Center, University of California, San Diego
- ORCID ID: https://orcid.org/0000-0002-9251-0600
- Email: jvarner@health.ucsd.edu
Additional Dataset Metadata
Acknowledgements
- Funding sources: National Institutes of Health grant R01CA226909 (JAV) National Institutes of Health grant R01CA167426 (JAV) National Institutes of Health grant R01DE027325 (JAV) California Office of the President Emergency COVID-19 Research Seed Funding R00RG2398 (JAV) Cowlin Family Foundation (SPP) Fast Grants, Emergent Ventures, Mercatus Center at George Mason University (AJM) NIH-ACTIV program (BO’C, TM) National Institutes of Health, Department of Health and Human Services Contract No. HHSN272201700041I/75N93021F00002 (JM) 1R01AI176554-01 (VN)
Dates and Locations
Dates of data collection: Data collected between 2020 and 2023
Geographic locations of data collection: Lab work was performed at University of California, San Diego; Tufts University; Utah State University; The Scripps Research Institute
Methodological Information
- Methods of data collection/generation: see manuscript for details
Data and File Overview
Summary Metrics
- File count: 16
- Total file size: 2.6 MB
- Range of individual file sizes: 2.8 kB-1.5 MB
- File formats: .csv
Naming Conventions
- File naming scheme: Files correspond to article figure number and part. Main Figure supporting data files are labeled: “Supporting Data Main Figure”. Supplementary Figure supporting data files are labeled “Supporting Data Supplementary Figure”. Figure subparts are labeled A, B, C…
Table of Contents
- Main Figure 1.csv
- Main Figure 2.csv
- Main Figure 3.csv
- Main Figure 4.csv
- Main Figure 5.csv
- Main Figure 6.csv
- Main Figure 7.csv
- Supplementary Figure 1.csv
- Supplementary Figure 2.csv
- Supplementary Figure 3.csv
- Supplementary Figure 4.csv
- Supplementary Figure 5.csv
- Supplementary Figure 7.csv
- Supplementary Figure 8.csv
- Supplementary Figure 9.csv
- Supplementary Figure 10.csv
- Supplementary Figure 11.csv
- Supplementary Figure 12.csv
- Supplementary Figure 13.csv
Setup
Unpacking instructions: n/a
Recommended software/tools: Spreadsheet software such as excel
File/Folder Details
Details for: Main Figure 1.csv
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Description: csv file with data for figure subparts (Fig1D-Fig1Q)containing immunohistochemistry data for normal versus COVID-19 patient lungs and bronchoalveolar lavage. "Null" indicates blank fields.
Format(s): .csv
Size(s): 7 KB
Dimensions: 24 rows by 48 columns with figure parts from 1D-1Q
Variables:
Figure 1D variables
*Columns A through B: CD68+ cells/mm2 in normal (n=4) lungs and COVID-19 (n=7) lungs.
Figure 1E variables
*Columns C through D: Myeloperoxidase+ cells/mm2 in normal (n=7) lungs and COVID-19 (n=14) lungs.
Fig 1G variables
*Columns E through F: Trichrome+ pixels/mm2 in normal (n=6) lung and COVID-19 lung(n=8) lung.
Fig 1I variables
*Columns G through I, CD68+ cells/mm2 and Myeloperoxidase+ cells/mm2 in normal (n=3) and COVID bronchoalveolar lavage samples (n=7).
Fig 1J variables
*Columns J through P: Density (cells/mm2) of CD3, Macrophages, monocytes, granulocytes, myeloid other; and immune other in normal (n=4) and COVID-19 (n=5) in bronchoalveolar lavage measured by iterative multiplex immunohistochemistry.
Fig 1L variables
*Columns Q-V: Density (cells/mm2) of non-immune cells, Other nucleated cells, PanCK+sKRT5+ SPB+in normal (n=3) and COVID-19 (n=14) lung tissue measured by iterative multiplex immunohistochemistry.
Fig 1M variables
*Columns W-AF: Table of values of CD8+ T cells, CD4+ T cells, CD20+ B cells, Total macrophages, Total DCs, Monocytes, Granulocytes, Myeloid other, and Immune other cells in normal (n=3) and COVID-19 (n=14) lung tissue measured by iterative multiplex immunohistochemistry.
Fig 1N variables
*Columns AG-AI: CD163-CD68+ cells, CD163+CD68+ cells in normal (n=3) and COVID-19 (n=14) lung tissue measured by iterative multiplex immunohistochemistry.
Fig 1O variables
*Columns AJ-AM: Tregs, Th1 T cells, Th2, Th17, Th9, Th0 T cells in normal (n=3) and COVID-19 (n=14) lung tissue measured by iterative multiplex immunohistochemistry.
Fig 1P variables
*Columns AN-AQ: PD1+ Tregs, Th1 T cells, Th other cells in normal (n=3) and COVID-19 (n=14) lung tissue measured by iterative multiplex immunohistochemistry.
Fig 1Q variables
*Columns AR-AV: PD1 and EOMES subpopulations of CD3+CD8 T cells in normal (n=3) and COVID-19 (n=14) lung tissue measured by iterative multiplex immunohistochemistry.
Details for: Main Figure 2.csv
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Description: an excel workbook file containing RNA sequencing analyses for normal versus COVID-19 patient lungs and bronchoalveolar lavage. "Null" indicates blank fields.
Format(s): .csv
Size(s): 925 KB
Dimensions: 2519 rows X 75 columns containing figure subparts Fig2A-Fig2I.
*Variables:
Fig 2A-B Variables
*Gene names and symbols (columns A-B), Log Fold Change (log FC) values (Column C) and statistics (Columns D-G, rows 3-639 for Differential mRNA expression in uninfected “normal” (n=5) and COVID-19 infected (n=8) bronchoalveolar lavage.
Fig 2C Variables
*Table of GSEA Hallmark pathways up- or down-regulated in BAL from COVID-19 infected versus uninfected patients. Column H-I: Hallmark pathways, Columns J-Q, Pathway size, enrichment scores, statistical significance metrics
Fig 2D-E Variables
*Table of Gene names and symbols (columns R-U), Log Fold Change (log FC) values (Column V) and statistics (Columns W-Z), rows 3-2519 for Differential mRNA expression in uninfected “normal” and COVID-19 infected lungs.
Fig 2F Variables
*Table of GSEA Hallmark pathways up- or down-regulated in lungs from COVID-19 infected versus uninfected patients. (Column AA )Hallmark pathways, Columns AB-AI, Pathway size, enrichment scores, statistical significance metrics.
Fig 2G Variables
*Table of normalized gene expression values in terms of mRNA read counts for CD163 (Column AJ-AL), SFTPB (Column AM-AN) SFTPC (Column AO-AP),C1R (Column AQ-AR), in COVID19 (n=12) versus normal (n=5) patient lungs, rows 3-15.
Fig 2H Variables:
*Table of deconvoluted normalized cell types in Columns AS-BE of BAL from normal (n=5) and COVID-19 (n=12) patients performed using log-normal regression algorithm of Danaher et al and its implementation in Bioconductor from normalized expression values (counts per million, cpm).
Fig 2I Variables:
*Table of deconvoluted normalized cell types in Column BF-BW versus individual lung tissue from normal (n=5) and COVID-19 (n=12) patients performed using log-normal regression algorithm of Danaher et al and its implementation in Bioconductor from normalized expression values (counts per million, cpm).
Details for: Main Figure 3.csv
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Description: an excel workbook file containing K18-hACE2Tg mouse SARS-CoV-2 animal model data."Null" indicates blank fields.
Format(s): .csv
Size(s): 3 KB
Dimensions: 21 rows X 33 columns containing figure subparts Fig3A-Fig3O.
Variables:
Figure 3A variables
*Table of values of PI3KCG gene transcripts in lung tissue from normal and COVID19 patients (Columns A-C)
Figure 3E variables
*Table of values of IBA1+ macrophages/mm2, MPO+ granulocytes/mm2 and PI3K mRNA+ cells/mm2 in lung tissue from uninfected and SARS-CoV-2 infected Syrian golden hamsters (Columns D-F).
Figure 3I variables
*Table of values of myeloperoxidase+ neutrophils/mm2 and IBA1+ macrophages/mm2 in hamster lungs at 0, 2, 4, 7 10 and 14 days after infection and treatment with vehicle (Columns G-K) or IPI-549 (Columns L-P)
Figure 3K variables
*Table of values of weight loss over time of mice that were infected with SARS-CoV-2 and treated with vehicle or IPI-549 (Columns Q-AA)
Figure 3L variables
*Table of values of viral load (pfu virus/gm lung tissue) in SARS-CoV-2 infected mice that were treated with vehicle or IPI-549(Columns AB-AC)
Figure 3N variables
*Table of values of F4/80+ macrophages/mm2 in lung tissue from mice infected with SARS-CoV-2 and treated with vehicle or IPI-549 (Column AD-AE)
Figure 3O variables
*Table of values of MPO+ granulocytes/mm2 in lung tissue from mice infected with SARS-CoV-2 and treated with vehicle or IPI-549 (Columns AF-AG)
Details for: Main Figure 4.csv
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- Description: an excel workbook file containing RNA sequencing analyses for normal versus COVID-19 patient lungs and bronchoalveolar lavage."Null" indicates blank fields.
- Format(s): .csv
- Size(s): 2.8 MB
- Dimensions: 13892 rows X 58 columns containing figure subparts Fig4A-Fig4F
- Variables
Figure 4A variables\
*Table of values of significant, differentially expressed genes in Sars-CoV-2 infected vehicle treated and Sars-CoV-2 infected IPI-549 treated mouse lung (Columns A-T).
Figure 4B variables
*Table of values of Gene enrichment signatures up and down-regulated in SARS-CoV-2 infected vehicle treated vs IPI-549 treated mouse lungs (Columns U-Y).
Figure 4C variables
*Table of values of cell type enrichment signature expressed in SARS-CoV-2 infected vehicle treated vs IPI-549 treated mouse lung (Columns Z-AD)
Figure 4D variables
*Table of values of transcripts per million of myeloid cell genes Pik3cg, Adgre1, Cd68, Ly6c2, and Itgam (Columns AE-AF)
Figure 4E variables
*Table of values of gene set enrichment signatures common to mouse and human SARS-CoV-2 infected lungs that are down-regulated in IPI-549 treated lungs (Columns AG-AL).
Figure 4F variables
*Table of values of differentially expressed genes upregulated in SARS-CoV-2 infected, IPI-549 treated mouse lungs versus SARS-CoV-2 infected human and mouse lung tissues (Columns AM-BF).
Details for: Main Figure 5.csv
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Description: A csv file containing SARS-CoV-2 animal model data."Null" indicates blank fields.
Format(s): .csv
Size(s): 9 KB
Dimensions: 31 rows X 40 columns containing data for figure subparts Fig5B-Fig5J.
*Variables:
Figure 5B variables
*Table of Values: Kaplan-Meier survival analysis of the effect of treatment with vehicle versus IPI-549 at day 0-4 and 2-4 on survival from mouse-adapted Sars-CoV2 infection and sham infection, where 1 indicated death and 0 indicates survival at endpoint (Columns A-E)
Figure 5C variables
*Table of Values: Summary percent weight change of sham infected (n=5) and mouse-adapted Sars-CoV-2 infected mice that were treated with vehicle (n=5)versus IPI-549 (n=10) at day 0-4 (n=10) and 2-4 (n=10)(Columns F-I)
Figure 5D variables
*Table of Values of percent weight change of sham infected (n=5) and mouse-adapted Sars-CoV-2 infected individual mice that were treated with vehicle (n=5)versus IPI-549 (n=10) at day 0-4 (n=10) and 2-4 (n=10)(Columns J-T)
Figure 5E variables
*Table of Values of relative copy number of genomic viral RNA per 300 ng total RNA in tissues from mice that were inoculated with vehicle alone, infected with mouse adapted SARS-CoV-2 and treated with vehicle or IPI-549 from 0-2 days or infected with mouse adapted SARS-CoV-2 and treated with IPI-549 from 2-4 days post inoculation (Columns U-X)
Figure 5F variables
*Table of Values of subgenomic viral RNA per 300 ng total RNA in tissues from mice that were inoculated with vehicle alone, infected with mouse adapted SARS-CoV-2 and treated with vehicle or IPI-549 from 0-2 days or infected with mouse adapted SARS-CoV-2 and treated with IPI-549 from 2-4 days post inoculation (columns Y-AB).
Figure 5H variables
*Table of values of macrophages (CD68+ cells/mm2) in sham infected (n=4) and SARS-CoV-2 infected, vehicle (n=5) or IPI-549 treated from d0-2(n=10)and from d2-4 (n=2)(Columns AC-AF)
Figure 5I variables
*Table of values of MPO+ granulocytes (cells/mm2) in sham infected (n=4) and SARS-CoV-2 infected, vehicle (n=5) or IPI-549 treated from d0-2 (n=9)and from d2-4(n=2)(Columns AG-AJ)
Figure 5J variables
*Table of values of surfactant C+ cells (cells/mm2) in sham infected (n=4) and SARS-CoV-2 infected, vehicle (n=5) or IPI-549 treated from d0-2 (n=10)and from d2-4(n=2)(Columns AK-AN)
Details for: Main Figure 6.csv
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Description: an excel workbook file containing experimental data testing the role of PI3Kgamma in mouse models of acute respiratory distress syndrome."Null" indicates blank fields.
Format(s): .csv
Size(s): 8 KB
Dimensions: 56 rows X 40 columns containing figure subparts Fig6A-Fig6L.
*Variables:
Figure 6A variables
*Columns A-C: Kaplan Meier survival data for MRSA infected WT (n=8)and Pik3cg-/- mice (n=8).
Figure 6B variables
*Columns D-H: Percent macrophages in thioglycolate stimulated WT (n=4) and Pik3cg-/- (n=4)mice and in vehicle (n=4) vs IPI-549 treated (n=4)as determined by flow cytometry.
Figure 6D variables
*Columns I-K: Kaplan-Meier survival for LPS stimulated WT (n=10)and Pik3cg-/- (n=10) female mice. Columns L-N: Kaplan-Meier survival for LPS stimulated WT (n=10)and Pik3cg-/- (n=10)male mice.
Figure 6F variables
*Columns O-T: Macrophages/ml in bronchoalveolar lavage of WT (n=10)and Pik3cg-/- (n=10)and vehicle (n=10)and IPI-549 treated (n=10) mice that were stimulated with chronic LPS (rows 3-8), chronic Poly IC (rows 9-15)or acute LPS (rows 18-22).
Figure 6H variables
*Columns U-X: Macrophages/mm2 in lung tissue from WT vs Pik3cg-/- (11 microscope fields/5 mice each) and vehicle vs IPI-549 (4 microscope fields from 5 mice each)treated Chronic LPS stimulated mice. Columns Y-AB: Macrophages/mm2 in lung tissue from WT vs Pik3cg-/- (19 microscope fields/5 mice each) and vehicle vs IPI-549 (8 microscope fields from 5 mice each) treated Chronic Poly IC stimulated mice. Columns AC-AF: Macrophages/mm2 in lung tissue from WT vs Pik3cg-/- (6 microscope fields/5 mice each)and vehicle vs IPI-549 (8 treated microscope fields/5 mice each) acute LPS stimulated mice.
Figure 6J variables
*Columns AG-AI, rows 3-11: Evans Blue ug/ml in lungs from Naïve (n=7), vehicle (n=4)and IPI-549 (n=5)treated mice that were stimulated with chronic LPS; rows 12-20: Evans Blue ug/ml in lungs from Naïve (n=7), vehicle (n=5)and IPI-549 (n=5)treated mice that were stimulated with chronic Poly IC ; rows 21-32: Evans Blue ug/ml in lungs from Naïve (n=7), vehicle (n=9)and IPI-549 (n=10)treated mice that were stimulated with acute LPS.
Figure 6K variables
*Columns AJ-AL: Serum concentration of TNFalpha(pg/ml) in Naïve (n=5) and LPS stimulated Vehicle (n=5) and IPI-549 (n=5)treated mice
Figure 6L variables
*Columns AM-AN: mRNA expression of Tnfa (rows 4-7), Il6 (rows 9-13), Il1b (rows 15-19), Ccl2 (rows 21-25)in lung myeloid cells isolated from Vehicle (n=3) and IPI-549 (n=3)treated mice
Details for: Main Figure 7.csv
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Description: a csv file containing empirical data from mouse and hamster viral infectivity studies."Null" indicates blank fields.
Format(s): .csv
Size(s): 8 KB
Dimensions: 29 rows X 54 columns containing data for figure subparts Fig7B-Fig7I.
Variables:
Figure 7B variables
*Columns A-F: Mouse weight (as percent of starting weight) on days 1-8 after sham inoculation (PBS, n-3) and MHV inoculation in vehicle (n=5) and IPI-549 (n=5)treated mice.
Figure 7C variables
*Columns G-L: MHV quantification by PCR in lungs of mice at days 2, 5 and 8 after sham inoculation (PBS, n-3) and MHV inoculation in vehicle (n=5) and IPI-549 (n=5)treated mice.
Figure 7D variables
*Columns M-N: Macrophages/ml in BAL from day 5 mice (n=5 mice with 5 fields/mouse)treated with vehicle and n=5 mice with 5 fields/mouse) treated with IPI-549.
Figure 7F variables
*Columns O-U: Macrophages/mm2 in lungs of mice at days 2, 5 and 8 after sham inoculation (PBS, n-3) and MHV inoculation in vehicle (n=5) and IPI-549 (n=5)treated mice.
Figure 7G variables
*Columns V-AA: MPO+ cells/mm2 in lungs of mice at days 2, 5 and 8 after sham inoculation (PBS, n-3) and MHV inoculation in vehicle (n=5) and IPI-549 (n=5)treated mice
Figure 7H variables
*Columns AB-AD: Concentration of TNFa, IL5, IL12 and IFNg (pg/ml) in lungs from sham inoculated mice (PBS, n-3) and MHV inoculated mice that were treated with vehicle (n=5) or IPI-549 (n=5).
Figure 7I variables
*Columns AE-BB: Relative mRNA expression (2-dCT) of Tnfa, Ifng, Il12b, Il10, Gbp3 and Gbp5 in lysates of lungs from sham infected (n=3) and MHV infected, vehicle-treated (n=5 per time point) and IPI-549-treated (n=5 per time point) animals at day 2, 5 and 8 post-inoculation.
Details for: Supplementary Figure 2.csv
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Description: A csv file containing quantification of immune cell types in SARS-CoV-2 patient and normal lungs determined by mIHC."Null" indicates blank fields.
Format(s): .csv
Size(s): 11 MB
Dimensions: 117 rows X 33 columns containing data for Supplementary Figure 2.
Variables:
Supplementary Figure 2A variables
*Columns A-C: Density of surfactant protein B positive (SPB+) cells in normal (n=3), virus- (n=11) and virus- (n=3)lungs.
Supplementary Figure 2B variables
*Column D-F: Proportion of PD-L1+SPB+ cells in virus+ in normal (n=3), virus- (n=11) and virus+ (n=3)lungs.
Supplementary Figure 2C variables
*Columns G-I: Proportion of Ki67+SPB+ cells in normal (n=3), virus- (n=11) and virus+ (n=3)lungs.
Supplementary Figure 2D variables
*Column J-L: Average immune composition per group in normal (n=3), virus- (n=11) and virus+ (n=3)lungs.
Supplementary Figure 2E variables
*Column M-O: Average abundance of CD163+/- macrophages in normal (n=3), virus- (n=11) and virus+ (n=3)lungs.
Supplementary Figure 2F variables
Column P-R: Percent PD-L1 positive, DC-LAMP+ and PD-L1 positive DC-LAMP- dendritic cells in normal (n=3), virus- (n=11) and virus+ (n=3)lungs.
Supplementary Figure 2G variables
*Columns S-U: Percentage of PD-L1 positive granulocytes in normal (n=3), virus- (n=11) and virus+ (n=3) lungs.
Supplementary Figure 2H variables
*Columns V-X: Percent of Treg and Th1 in normal (n=3), virus- (n=11) and virus+(n=3)lung as well as Th2, Th0, Th17, Th9 cells within CD3+CD8- subsets in normal (n=0-1), virus+ (n=2-4) and virus-(n=0-1)lung.
Supplementary Figure 2I variables
*Columns Y-AA: Percent of PD-1+ CD3+CD8- Th1, Treg, Th other (Th2, Th17, Th9, and Th0) cells in normal (n=3), virus- (n=11) and virus+ (n=3)lungs.
Supplementary Figure 2J variables
*Columns AB-AD: Percentage of PD-L1-positive CD20+ B cells in normal (n=3), virus- (n=11) and virus+ (n=3)lungs.
Supplementary Figure 2K variables
*Columns AE-AG: Percentage of PD-1+ CD20+ B cells in normal (n=3), virus - (n=11) and virus+ (n=3) lungs.
Details for: Supplementary Figure 3.csv
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Description: A csv file containing GSEA analyses of BAL from recently hospitalized COVID-19 patients versus normal BAL and gene expression analyses comparing COVID-19 infected lungs and normal lungs."Null" indicates blank fields.
Format(s): .csv
Size(s): 63 KB
Dimensions: 55 rows X 114 columns containing data for Supplementary Figure 3.
*Variables:
Supplementary Figure 3A variables
*Columns A-I: Table of significantly upregulated GO pathways associated with BAL of recently hospitalized COVID-19 patients
Supplementary Figure 3B-D variables
*Columns J-CK: Hallmark pathways and corresponding genes that are up or down regulated in lungs from COVID-19 patients versus normal patients.
Supplementary Figure 3E variables
*Columns CL-CT: Pathways from Reactome, KEGG, GO and WikiPathway gene sets and corresponding genes that are up or down regulated in lungs from COVID-19 patients versus normal patients.
Supplementary Figure 3F variables
*Columns CU-DQ: Expression values of collagen genes in lungs from COVID-19 patients versus normal patients.
Details for: Supplementary Figure 4.csv
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Description: A csv file containing GSEA data for human virus+ lungs vs normal or virus- lungs."Null" indicates blank fields.
Format(s): .csv
Size(s): 154 kB
Dimensions: 43 rows X 68 columns containing data for Supplementary Figure 4.
Variables:
Supplementary Figure 4A-B Variables
*Columns A-I: Differentially expressed genes, with LogFold Change and p values, in lung tissue of COVID-19 patients who were virus positive versus virus negative at the time of death.
Supplementary Figure 4C variables
*Columns J-BG: Hallmark Pathways, statistics and genes that are upregulated or downregulated in virus positive versus virus negative lung tissue of COVID-19 patients.
Supplementary Figure 4D variables
*Columns BH-BP: Pathways from Reactome, KEGG, GO and WikiPathway gene sets and corresponding genes that are up or down regulated in virus+ lungs from COVID-19 patients versus normal patients.
Details for: Supplementary Figure 5.csv
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Description: A csv file containing data on the effect of PI3Kgamma inhibition in models of SARS-CoV-2 infection."Null" indicates blank fields.
Format(s): .csv
Size(s): 1 MB
Dimensions: 1842 rows X 255 columns containing data for Supplementary Figure 5.
*Variables:
Supplementary Figure 5A-B variables
*Table of Gene names and statistics (Columns A-T) up- or down-regulated in macrophages from COVID-19 lungs versus normal lungs.
Supplementary Figure 5C variables
*Columns U-CV: Hallmark Pathways that are up- or down-regulated in macrophages from COVID19 lungs versus normal lungs
Supplementary Figure 5D-E variables
*Table of Gene names and statistics (Columns CW-DP) up- or down-regulated in granulocytes from COVID-19 lungs versus normal lungs
Supplementary Figure 5F variables
*Columns DQ-GF: Hallmark Pathways that are up- or down-regulated in granulocytes from COVID19 lungs versus normal lungs
Supplementary Figure 5G-H variables
*Columns GL-GZ: Genes that are significantly up- or down-regulated in eopithelium from COVID19 lungs versus normal lungs
Supplementary Figure 5I variables
*Columns HA-JA: Hallmark Pathways that are up-regulated in epithelium from COVID19 lungs versus normal lungs
Details for: Supplementary Figure 7.csv
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Description: A csv file containing data on the effect of PI3Kgamma inhibition in models of SARS-CoV-2 infection."Null" indicates blank fields.
Format(s): .csv
Size(s): 5 KB
Dimensions: 58 rows X 28 columns containing data for Supplementary Figure 7.
*Variables:
Supplementary Figure 7A variables
*Columns A-M: Individual percent body weight changes over time in SARS-Co-V2 infected hamsters that were treated with vehicle or IPI-549
Supplementary Figure 7B variables
*Columns N-P: Kaplan Meier survival plots of hACE2-Tg mice infected with SARS-Co-V2 that were treated with vehicle or IPI-549
Supplementary Figure 7E variables
*Columns Q-AB: Percent monocytes of CD45+ cells, percent granulocytes of CD45+ cells, percent macrophages of CD45+ cells and viral load (pfu/gram tissue) in lungs from mice infected with K18-hACE2-Tg mice and treated with vehicle or IPI-549
Details for: Supplementary Figure 8.csv
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Description: A csv file containing empirical data on the effect of PI3Kgamma deletion on MRSA and E.coli effects on mouse cytokine production and bacterial survival."Null" indicates blank fields.
Format(s): .csv
Size(s): 3 KB
Dimensions: 50 rows X 5 columns containing data for Supplementary Figure 8.
*Variables
Supplementary Figure 8 variables
*Table of values of canonical macrophage genes differentially expressed in SARS-CoV-2 infected vehicle treated vs IPI-549 treated mouse lungs (Columns A-E).
Details for: Supplementary Figure 9.csv
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Description: A csv file containing SARS-CoV-2 mouse model data gene expression data."Null" indicates blank fields."Null" indicates blank fields.
Format(s): .csv
Size(s): 1.5 MB
Dimensions: 4909 rows X 159 columns containing data for Supplementary Figure 9.
*Variables
Supplementary Figure 9A variables
*Table of values (A-D) of Cxcl10 mRNA expression (2-delta CT) in sham infected (n=4) and SARS-CoV-2 infected, vehicle (n=5) or IPI-549 treated from d0-2 (n=10)and from d2-4(n=2)(rows 3-14) and Tnfa mRNA expression (2-delta CT) in sham infected (n=4) and SARS-CoV-2 infected, vehicle (n=5) or IPI-549 treated from d0-2 (n=10)and from d2-4(n=2)(rows 16-27)
Supplementary Figure 9B variables
*Table of values of Cxcl10 serum protein expression (pg/ml) in sham infected (n=4) and SARS-CoV-2 infected, vehicle (n=5) or IPI-549 treated from d0-2 (n=10)and from d2-4(n=2)(Columns E-H),
Supplementary Figure 9C variables
*Columns I-R: Table of genes names and values that are significantly (p adj <0.05) differentially expressed in sham (vehicle only, n=4) versus mouse adapted Sars-CoV-2 infected, vehicle-treated (n=5) mouse lungs.
Supplementary Figure 9D Variables
*Columns S-Z Table of GSEA Hallmark pathways and statistics that are significantly up- or down-regulated in lungs from sham (vehicle only) versus mouse adapted Sars-CoV-2 infected, vehicle-treated mice.
Supplementary Figure 9E Variables
*Columns AA-AU: Table of significant (p adj <0.05) differentially expressed genes and values in Sham (vehicle only, n=4) and mouse adapted Sars-CoV-2 infected mouse lungs treated with vehicle (n=5) or with IPI-549 from d0-2 (n=8) or d2-4 (n=2).
Supplementary Figure 9F Variables
*Columns AV-AZ: Tables of Gene Ontogeny (GO, Reactome, KEGG and Wiki Pathways that are down or up regulated in maSARS-CoV-2 infected, IPI-549 d2-4 treated mouse lungs versus maSARS-CoV-2 infected, vehicle treated mouse lungs.
Supplementary Figure 9G Variables
*Columns BA-BU: Table of gene IDs, names and values from Gene Ontogeny GO:0009611 Response to Wounding pathway that are significantly (p adj <0.05) upregulated in mouse adapted Sars-CoV-2 infected, IPI-549 treated d2-4 (n=2) versus mouse adapted Sars-CoV-2 infected, vehicle-treated (n=5) mouse lungs.
Supplementary Figure 9H Variables
*Columns BV-CL: Table of Protein-Protein Interaction (PPI) networks that are upregulated in in mouse adapted Sars-CoV-2 infected, IPI-549 treated d2-4 (n=2) versus mouse adapted Sars-CoV-2 infected, vehicle-treated (n=5) mouse lungs.
Supplementary Figure 9I Variables
*Columns CM-DP: Table of expression values of significant (p adj <0.05) differentially expressed Interferon_Gamma and Viral_Response pathway genes in human and mouse SARS-CoV-2 infected versus mouse adapted Sars-CoV-2 infected IPI-549 treated d2-4 (n=2)lungs.
Supplementary Figure 9J Variables
*Columns DQ-DV: GSEA Gene Ontogeny, Wikipathways, KEGG and Reactome Pathways that are significantly down-regulated in IPI-549 d2-4 treated lungs SARS-CoV-2 infected lungs versus mouse and human SARS-CoV-2 infected lungs.
Supplementary Figure 9K Variables
*Columns DW-ES: Disease Associated Gene Network signatures that are downregulated in IPI-549 d2-4 treated SARS-CoV-2 infected lungs versus mouse and human SARS-CoV-2 infected lungs
Details for: Supplementary Figure 10.csv
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Description: A csv file containing empirical data on the effect of PI3Kgamma deletion on MRSA and E.coli effects on mouse cytokine production and bacterial survival."Null" indicates blank fields.
Format(s): .csv
Size(s): 2 KB
Dimensions: 17 rows X 23 columns containing data for Supplementary Figure 10.
*Variables
Supplementary Figure 10A
*Table of Values: Kaplan-Meier survival analysis of survival of wildtype or PI3Kgamma from infection with multi-drug resistant Escherichia coli mouse-adapted Sars-CoV2 infection and sham infection, where 1 indicated death and 0 indicates survival at endpoint (Columns A-C)
Supplementary Figure 10B
*Columns D-G: Table of values of IL-1b (pg/ml) and IL-6 (pg/ml)in serum from MRSA infected WT and Pik3cg-/- mice.
Supplementary Figure 10C\
*Columns H-S: Quantification of bacteria (cfu/mg tissue) in vivo in WT and Pik3cg-/- mice in liver, spleen and kidney at 4h and 24h determined by ex vivo colony forming assay.
Supplementary Figure 10D\
*Columns T-W: Percent survival of MRSA or E.coli bacteria in in vitro culture with WT and Pik3cg-/- bone marrow derived macrophages (n=3).
Details for: Supplementary Figure 11.csv
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Description: A csv file containing macrophage densities in BAL from acute LPS, chronic LPS and poly I:C stimulated animals."Null" indicates blank fields.
Format(s): .csv
Size(s): 2 KB
Dimensions: 8 rows X 28 columns containing data for Supplementary Figure 11.
Variables
Supplementary Figure 11A
*Columns A-AB: Macrophage density in acute LPS, chronic LPS, chronic poly I:C and acute poly I:C stimulated, vehicle versus IPI-549 treated animals (n=5) at 24, 48 or 72h post stimulation.
Details for: Supplementary Figure 12.csv
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Description: A csv file containing empirical data on the effect of PI3Kgamma inhibition on vascular leak induced by inflammatory stimuli and VEGF."Null" indicates blank fields.
Format(s): .csv
Size(s): 3 KB
Dimensions: 13 rows X 37 columns containing data for Supplementary Figure 12.
*Variables
Supplementary Figure 12A
*Columns A-I: Quantification of vascular leak (mean +/- SEM) in naïve (n=7) and acute LPS, chronic LPS or chronic Poly I:C stimulated WT (n=5) versus Pik3cg-/- (n=4-5) mice.
Supplementary Figure 12B
*Columns J-0: Quantification of vascular leak in hearts of acute systemic LPS, chronic systemic LPS, and chronic systemic Poly I:C stimulated, vehicle (n=4-9) versus IPI-549-treated (n=5-10) animals.
Supplementary Figure 12C
*Columns P-U: Quantification of vascular leak in kidneys of acute systemic LPS, chronic systemic LPS (n=4-5), and chronic systemic Poly I:C stimulated vehicle (n=4-9) versus IPI-549-treated (n=5-10) animals.
Supplementary Figure 12D
*Columns V-AA: Quantification of vascular leak in heart, kidney and lungs of animals treated chronically by intranasal Poly I:C administration and either vehicle (n=5) or IPI-549 (n=5).
Supplementary Figure 12E
*Columns AB-AG: Quantification of CCL2, IL6, IL-10 in serum from WT versus Pik3cg-/- and vehicle versus IPI-549-treated mice chronically stimulated with LPS (n=10).
Supplementary Figure 12F
*Columns AH-AK: Quantification of VEGF-A mediated vascular leak (mean +/- SEM) in WT (n=7) vs Pik3cg-/- and vehicle vs IPI-549-treated WT (n=5) mice.
Details for: Supplementary Figure 13.csv
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Description: A csv file containing cytokine analysis in models of acute respiratory distress."Null" indicates blank fields.
Format(s): .csv
Size(s): 10 KB
Dimensions: 92 rows X 50 columns containing data for Supplementary Figure 13.
*Variables
Supplementary Figure 13A Variables
*Columns A-D: Concentration of cytokines and chemokines in serum from naïve and acute systemic LPS-stimulated WT mice at 0h (Naïve), 24h, 48h and 72h post-stimulation (mean +/- SEM, n=5).
Supplementary Figure 13B Variables
*Columns E-P: Concentration of IL6 and IL1b in serum from naïve and acute systemic LPS-stimulated WT versus PI3Kg-/- mice at 0h (Naïve), 24h and 48h post-stimulation (mean +/- SEM, n=5).
Supplementary Figure 13C Variables
*Columns Q-V: Concentration of TNFalpha in serum from naïve and chronic LPS-stimulated WT versus PI3Kg-/- mice and vehicle versus IPI-549-treated animals (mean +/- SEM, n=5.
Supplementary Figure 13D Variables
*Columns W-Z: Concentration of TNFalpha in bronchoalveolar lavage fluid (BALF) and peritoneal lavage fluid (PLF) from naïve and acute LPS -stimulated, vehicle and IPI-549-treated animals (mean +/- SEM, n=5).
Supplementary Figure 13E Variables
*Columns AA-AX: Mean +/-SEM mRNA expression (2-deltaCT) of cytokines in CD11b+ and CD11b-cells isolated from lungs of chronic LPS stimulated, vehicle versus IPI-549 treated animals (n=3).
Methods
Human subjects
All human tissue analyses were conducted on de-identified tissue under guidelines established by the Institutional Review Board for human subject research of the University of California, San Diego. Postmortem tissue studies received IRB exemption from oversight as research on deceased patients is not classified as human subjects research by the US Department of Health and Human Services or the US Food and Drug Administration. All patients in this study were admitted early in the pandemic during the first 3-6 months after the first cases of COVID-19 were reported in the US when no specific therapeutics had been developed. De-identified lung tissue was obtained upon rapid autopsy of deceased COVID-19 patients formalin-fixed for 48h and processed by the Department of Pathology, UCSD, into paraffin-embedded tissue blocks by the Moores Cancer Center histology shared resource, UCSD, San Diego, CA. Normal human lung tissue was obtained from consented patients during lung cancer surgery at the Moores Cancer Center, UCSD and processed into paraffin embedded tissue blocks by the UCSD Department of Pathology. Bronchoalveolar lavage cells from COVID positive and from COVID-19 negative patients were pelleted, fixed, paraffin embedded and sectioned. All tissue was used for immunohistochemical and bioinformatics analysis.
Animals
8-9-week-old C57BL/6 stock 000664 (RRID:IMSR_JAX:000664) male and female mice were purchased from Jackson Laboratories, Bar Harbor, ME. K18-hACE2 (RRID:IMSR_JAX:034860) 6-8 week old male mice were purchased from Jackson Laboratories. 10-month-old female Balb/c animals (RRID:IMSR_CRL:028) were purchased from Charles River for SARS-CoV-2 infection studies. Golden Syrian Hamsters (Mesocricetus auratus; genotype: HsdHan: AURA) six- to seven-week-old were from Envigo, USA. Pik3cg-/- mice in the C57BL/6 background (RRID:MGI:3619226) were maintained in the Varner lab at the University of California, San Diego. MRSA, MHV studies and ARDS studies were performed at the University of California, San Diego with the approval of the Institutional Animal Care and Use Committees and Institutional Biosafety Committees of the University of California, San Diego. SARS-CoV-2. SARS-CoV-2 infection of K18-hACE2 mice studies were performed at The Scripps Research Institute, La Jolla, CA with the approval of the Institutional Animal Care and Use Committee of The Scripps Research Institute. maSARS-CoV-2 mouse models were performed with support from the NIH-ACTIV program of NIAID, NIH at The Institute for Antiviral Research, Animal, Dairy and Veterinary Science, Utah State University, Logan, UT, with the approval of the Institutional Animal Care and Use Committees and Institutional Biosafety Committees of Utah State University. SARS-CoV-2 hamster studies were performed at the Department of Infectious Diseases and Global Health, Tufts University Cummings School of Veterinary Medicine, North Grafton, MA, USA. All animal experiments were performed with the approval of the Institutional Animal Care and Use Committees and Institutional Biosafety Committees of Tufts University. SARS-CoV-2 hamster experiments were conducted in ABSL3 facilities on the campus of Tufts University.
Reagents
Murine L929 cells (RRID:CVCL_0462, L cell, L-929, derivative of Strain L) were purchased from ATCC (Bethesda, MD) and maintained in completed Dulbecco’s Modified Eagle Medium (DMEM) media supplemented with 10% heat-inactivated fetal bovine serum (FBS) and antibiotics (100 μg/mL penicillin and 100 μg/mL streptomycin). Murine hepatitis virus MHV-A59 infectious clone in L929 cells (BEI NR-43000). SARS-CoV-2, Mouse-Adapted, MA10 variant, infectious clone in Calu-3 cells (BEI NR-55429) were obtained from BEI Resources, NIAID, NIH, Bethesda, Maryland, USA). Formulated IPI-549 and vehicle were provided by Infinity Pharmaceuticals (Cambridge, MA).
Human histopathology
Lung tissue was obtained upon rapid autopsy of recently deceased COVID-19 patients by the Department of Pathology, UCSD, formalin fixed for 48h, and processed into paraffin embedded tissue blocks by the Moores Cancer Center histology shared resource, UCSD, San Diego, CA. Normal human lung tissue was obtained during lung cancer surgery at the Moores Cancer Center, UCSD and processed into paraffin embedded tissue blocks by the UCSD Department of Pathology. Bronchoalveolar lavage cells from COVID-19 positive and from COVID-19 negative patients were pelleted, fixed, paraffin embedded and sectioned. Glass slides containing 4-5µm thick tissue sections were deparaffinized and stained with hematoxylin and eosin or Mason’s Trichrome by the Moores Cancer Center histology shared resource. Alternatively, slides were deparaffinized, rehydrated and treated for 20 minutes with Diva Decloaker (901-DV2004X-071017 Biocare Medical) antigen retrieval solution followed by with endogenous peroxidase blocking, and incubation with 5% normal goat serum for 1h. Slides were then incubated with 1:200 anti-human CD68 (RRID:AB_2074844 clone PG-M1, Dako/Agilent), 1:400 anti-PI3 Kinase-gamma (RRID:AB_1904087 Cell Signaling Technology, Inc., clone D55D5, #5405) or 1:200 anti-Myeloperoxidase (RRID:AB_2864724 ab208670, Abcam). Following primary antibody incubation, slides were washed in Tris buffered saline containing Tween 20 (TBST) and incubated with anti-rabbit horseradish peroxidase (HRP)-conjugated polymer (RRID:AB_2336820 Vectastain Elite ABC-HRP PK-6101, Vector Laboratories) for 30 minutes at room temperature, washed in TBST and counterstained with hematoxylin. Slides were mounted with Cytoseal permanent mounting medium (Richard-Allan Scientific Cat #8310-4). Biomarker quantification was performed using QPath open source digital image analysis software.
Multiplex IHC
Sequential IHC was performed on 5 μm FFPE sections using an adapted protocol based on methodology previously described (14-15). Briefly, slides were deparaffinized and stained with hematoxylin (S3301, Dako, Santa Clara, CA), followed by whole-slide scanning at 20X magnification on an Aperio AT2 (Leica Biosystems, Wetzlar, Germany). Tissues then underwent 20 minutes heat-mediated antigen retrieval in pH 6.0 Citra solution (BioGenex, Fremont, CA), followed by 10 minutes endogenous peroxidase blocking in Dako Dual Endogenous Enzyme Block (S2003, Dako, Santa Clara, CA), then 10 minutes protein blocking with 5% normal goat serum and 2.5% BSA in TBST. Primary antibody conditions are listed in Supplementary Table 1. Following primary antibody incubation, slides were washed in TBST, and incubated with either anti-rat, anti-mouse, or anti-rabbit Histofine Simple Stain MAX PO horseradish peroxidase (HRP)-conjugated polymer (RRID:AB_2811178 Biosciences, Tokyo, Japan) for 30 minutes at room temperature, followed by AEC chromogen (RRID:AB_2336076 Vector Laboratories, Burlingame, CA). Slides were digitally scanned following each chromogen development, then AEC was removed with 100% EtOH. Between rounds, peroxidase blocking with Dako Dual Endogenous Enzyme Block was performed for 10 minutes at RT to inactivate HRP enzyme still present upon the secondary antibody of the previous round. Another primary produced in a distinct species could be utilized within the same staining cycle. Between cycles, citrate antigen retrieval was completed as described above to remove all primary antibodies primary-secondary antibody complexes.
Following iterative staining, computational image processes and analysis was performed. Scanned images were registered in MATLAB version R2018b using the SURF algorithm in the Computer Vision Toolbox (The MathWorks, Inc., Natick, MA). Image processing and cell quantification were performed using FIJI (FIJI Is Just ImageJ) (62). AEC signal was extracted for quantification and visualization in FIJI using a custom macro for color deconvolution. Briefly, the FIJI plugin Color_Deconvolution [H AEC] was used to separate hematoxylin, followed by postprocessing steps for signal cleaning and background elimination. AEC signal was extracted in FIJI using with the NIH plugin RGB_to_CMYK. Color deconvoluted images were processed in CellProfiler Version 3.5.1 (63) to quantify single cell mean intensity signal measurements for every stained marker. FCS Express 6 Image Cytometry RUO (De Novo Software, Glendale, CA) was used to perform hierarchical gating and cell classification based on expression of known markers as shown in Supp. Data. Fig. 1. For visualization, signal-extracted images were overlaid and pseudocolored in FIJI.
TempoSeq FFPE tissue RNA Sequencing
Two five-micron FFPE sections from each of twelve postmortem lung specimens from COVID-19 patients, five normal lung specimens, eight BALF specimens from COVID-19 patients and five BALF specimens from normal patients were used to perform TempoSeq (Templated Oligo assay with Sequencing readout) FFPE human whole transcriptome RNA sequencing at BioSpyder Technologies, Inc, Carlsbad, CA, as previously described (25-26). Of these, one upper lung and one lower lung specimens from each of two patients were included in initial analysis. Two 5 FFPE tissue sections per sample were scraped from glass slides, paraffin removed and at least two 25-nucleotide long oligonucleotides specific for 19,283 genes (21,111 probes) were used to prepare full-length (50-nucleotide-long) probes that were amplified prior to sequencing library preparation. Prepared libraries were sequenced on an Illumina HiSeq2500; mapped reads were generated by TempO-SeqR alignment of demultiplexed FASTQ files using Bowtie, allowing for up to 2 mismatches in the 50-nucleotide target sequence.
RNA sequencing analysis
Cell type deconvolution was performed using the log-normal regression algorithm of Danaher et al. (27) and its implementation in Bioconductor (28). Inputs were the normalized expression values (cpm), the background matrix of background values set to a value of 6, and the training matrix containing log2 expression values in the training set, which is part of the R package and named “Human_Cell_Landscape”. The training matrix originally contained 102 human cell types, but we excluded non-specific cell types not expected to be in these tissues, such as glial cells, kidney cells, etc. A total of 56 cell types for the lung tissue remained.
GeoMx Digital Spatial Profiling (DSP)
nanoString GeoMx digital spatial profiling was applied to COVID and normal lung tissues to acquire spatially resolved, quantitative measurements of gene expression in human lung macrophages, granulocytes and epithelium in FFPE tissue sections. Three cases of post-mortem COVID lung tissues and one case of surgically resected normal lung tissue were sectioned, deparaffinized, rehydrated and treated with low pH retrieval solution. nanoString GeoMX Digital Spatial ImmuneOncology and COVID-19 RNA probes were hybridized to sections overnight. Sections were then stained with a cocktail of immunofluorescent antibodies comprised of Alexa fluor 532-labeled anti-CD68 (RRID:AB_2074852 Clone KP1, 1:100, Novus Biologicals, USA), Alexa fluor 647-labeled Myeloperoxidase (RRID: AB_964678 Clone 2C7, 1:500, Novus Biologicals, USA), Alexa fluor 594 labelled anti-PanCytokeratin (AE-1, AE-3, Novus Biologicals, NBP2-33200AF594) and SYTO13 Nuclear stain (ThermoFisher S7575). Digital scanning of slides was performed on the GeoMX Digital Profiler (nanoString Technologies, Inc.). Regions of Interest (ROI) and cell type with a spatial resolution of approximately 10 mm were selected, exposed to UV light for RNA probe cleavage and oligonucleotide collection. Samples were sequenced on a NovaSeq6000. Counts from barcodes corresponding to RNA probes were normalized with internal positive and negative controls and then normalized to ROI area.
DSP RNA sequence analysis
Hamster SARS-CoV-2 model
Hamster viral infection: Six- to seven-week-old Golden (Syrian) Hamsters (Envigo; weight range 90-110g, equal number of male and females) were inoculated with SARS-CoV-2 intranasally with 1 x 105 TCID50 in 100µl volume as described (71-72). The viral isolate USA-WA1/2020 (BEI Resources) was propagated on Vero E6 cells and clarified by centrifugation. Uninfected hamsters were sham inoculated with phosphate buffered saline intranasally. Hamsters were evaluated daily for changes in bodyweight, temperature, and demeanor or clinical signs of disease. Hamsters were euthanized at 2-, 4-, and 7-days post infection (dpi). Lung tissue was collected at necropsy and fixed in 10% neutral buffered formalin for 24 hours before being transferred to 70% ethanol. For PI3Kg inhibition studies, hamsters were administered 30 mg/kg IPI-549 or vehicle alone by orogastric gavage on days 0, 1, 2, and 3 following challenge and body weight monitored daily. Hamsters were euthanized at 2-, 4-, 7- and 14-days post infection. Lung tissue was collected at necropsy and fixed in 10% neutral buffered formalin for 24 hours before being transferred to 70% ethanol.
Hamster SARS-CoV-2 model histopathology and immunohistochemistry: At time of fixation, lungs were suffused with 10% formalin to expand the alveoli. All tissues were fixed in 10% formalin and blocks sectioned at 5 µm. Slides were baked for 30-60 min at 65 degrees then deparaffinized in xylene and rehydrated through a series of graded ethanol to distilled water. Heat induced epitope retrieval (HIER) was performed using a pressure cooker on steam setting for 25 minutes in citrate buffer (ThermoFisher Scientific, AP-9003-500) followed by treatment with 3% hydrogen peroxide. Slides were then rinsed in distilled water and protein blocked (BioCare, BE965H) for 15 min followed by rinses in 1x phosphate buffered saline. Mouse Anti-SARS-CoV-2 nucleocapsid antibody (RRID:AB_2827977 Sino biological; 40143-MM05) diluted 1:1000, rabbit anti-myeloperoxidase (RRID:AB_2335676 Dako-Agilent; A0398) diluted 1:1000, or rabbit anti-Iba-1 antibody (RRID:AB_839504 Dako; 019-19741) diluted 1:4000 followed by rabbit Mach-2 HRP-Polymer (BioCare; RHRP520L) were incubated for 30 minutes then counterstained with hematoxylin followed by bluing using 0.25% ammonia water. Labeling was performed on a Biocare IntelliPATH autostainer. All antibodies were incubated for 60 min at room temperature. Tissue pathology was assessed independently by two board-certified veterinary pathologists.
Hamster SARS-CoV-2 RNAscope: RNAscope in situ hybridization as directed with the following modifications using a custom probe for hamster PI3Kg (ACD Cat. No. 1071581-C1 and DapB (ACD Cat.No 310043) as a negative control. In brief, after slides were deparaffinized in xylene and rehydrated through a series of graded ethanol to distilled water, retrieval was performed for 30 min in ACD P2 retrieval buffer (ACD Cat. No. 322000) at 95-98°C, followed by treatment with protease III (ACD Cat. No. 322337) diluted 1:10 in PBS for 20 min at 40°C. Slides were then incubated with 3% H2O2 in PBS for 10 minutes at room temperature. All washes were performed in 0.5x kit provided SSC. Slides were developed using the RNAscope® 2.5 HD Detection Reagents-RED (ACD Cat. No.322360).
Hamster SARS-CoV2 Quantitative Image Analysis: Quantitative image analysis was performed using HALO software (v3.0.311.405; Indica Labs) on at least one lung lobe cross-section from each animal as described (71-72). In cases where more than one cross-section was available, all lung lobes were quantified as an individual data point. For PI3Kg, the whole slide digital images were deconvoluted using the Indica Labs – Deconvolution algorithm (v1.1.1). The deconvoluted images were analyzed with Indica Labs – FISH-IF (v2.1.5) algorithm to determine the PI3Kg copies per mm2 total tissue area. For Iba-1, the Indica Labs - Multiplex IHC algorithm (v3.1.4) was used for quantitation. In all instances, manual inspection of all images was performed on each sample to ensure that the annotations were accurate.
Mouse SARS-CoV-2 models
SARS-CoV-2 viral infection in hACE2 transgenic mice: K18-hACE2 male mice (Jackson Laboratories) were intranasally (i.n.) infected with 5000 Plaque Forming Units (PFU) of SARS-CoV-2/human/USA/WA-CDC-WA1/2020 (SARS2-WA, BEI Resources, NIAID, NIH, Bethesda, Maryland, USA). After 0-2 days post-infection (p.i.), mice were treated with either vehicle or 25mg/kg IPI-549 once daily by oral gavage (n=5 or 10 per study). Weight loss was monitored at day 0, 3, 4 and 5 p.i. Lungs were collected at day 5 p.i. and stored in z-Fix for histological analysis or in TRIzol (ThermoFisher Scientific, 15596018) for RNA-sequencing. Some lungs were dissociated and processed for flow cytometry and virus detection. SARS-CoV-2 viral infection in hACE2 transgenic mice were performed 6 times.
Mouse SARS-CoV-2 model flow cytometry
Single cell suspensions were incubated with Ghost Dye™ Violet 510 Live Dead fixable stain BUV510 (Invitrogen), and then with Fc-blocking reagent (BD Bioscences) followed by incubation with fluorescently labeled antibodies at 4°C for 1h. Primary antibodies used for cell surface marker staining were: anti-mouse/human CD11b BUV563 (M1/70, Biolegend), anti-mouse-CD11c APA-Cy7 (N418, Biolegend), anti-mouse CD170/Siglec-F BV421 (S17007L, Biolegend), anti-mouse CD206/MMR (C068C2, Biolegend), anti-mouse F4/80 PerCP-Cy5.5 (BM8, Biolegend) anti-mouse CD90.2/Thy1.2 (30-H12, Biolegend), anti-mouse Ly-6G BV650 (1A8, Biolegend), anti-mouse CD64 BV711 (X54-5/7.1, Biolegend), anti-mouse/human CD44 (IM7, BD Bioscience) anti-mouse Ly-6C APC-A (HK1.4, Biolegend), anti-mouse NK-1.1 (PK136, BD Bioscience), anti-mouse CD45 Alexa Fluor 700 (BD Bioscience, 30-F11), anti-mouse CD19 BUV737 (1D3, BD Bioscience), anti-mouse CD8a BUV395 (53-6.7, BD Bioscience), anti-mouse CD4 (RM4-5, Cytek), anti-TCRB PE-Cy7 (H57-597, Biolegend). FACS was performed on a Cytek Aurora with 5 lasers. Data analysis was performed using FlowJo (Treestar/BD Biosciences).
Mouse SARS-CoV-2 viral infection: Mouse-adapted SARS-CoV-2 (MA10 variant, infectious clone in Calu-3 cells, BEI NR-55429) (73) was prepared based on a stock titer = 1.97 x 104 pfu/ml. Female BALB/c mice 270 to 300 days old were ear-tagged, randomized to groups, and infected with 1 x 103 pfu/mouse in 90µl SARS-CoV-2 or Modified Eagle’s Medium (MEM) sham by intranasal instillation at the Antiviral Institute of Utah State University. Within less than an hour at day 0, mice were administered by oral gavage with 15mg/kg IPI-549 or vehicle in a volume of 0.1 ml/25 g-mouse from either day 0-4 or day 2-4. Individual whole-body weights and survival were monitored for up to 6 days. After morning treatments on day 2 in the afternoon, 5 animals treated with vehicle and 10 animals treated with IPI-549 were necropsied. The remaining animals were monitored until death or day 6. Sera and lung tissues were collected from all mice. Lung homogenates in Trizol were used for RT-PCR analyses and heat-inactivated sera were analyzed for presence of cytokines.
Mouse SARS-CoV2 tissue processing: Sera and lungs were collected from IPI-549 and vehicle treated groups on days 2 and 5, respectively. Blood samples were collected by cardiac puncture, clotted, centrifuged to collect sera, heat-inactivated at 65°C for 45 min to inactivate virus and stored frozen. On day 5, the left lung lobe was perfused with 10% buffered formalin for H&E staining and analysis. A 22-g blunt-ended needle was inserted in the trachea to gently fill the lung with fixative. The tissues were immersed in formalin overnight at refrigeration temperature before removing from the BSL3 lab, embedding, sectioning and scoring pathology. The right lung was weighed, bead-homogenized in 0.3 ml modified Eagle’s Medium (MEM) and two aliquots of 0.1 mL of homogenate added to 1 ml Trizol prepared and frozen until processing for RNA extraction. The remaining homogenized lung volume was used for infectious cell culture SARS virus assay using CCID50 readout. RNA was extracted using the standard Trizol-method. The pellet was dissolved in 0.1 mL of RNase-free water. Total extracted RNA was quantified using a Nanodrop™ spectrophotometer so that 300 ng could be added to each RT-PCR reaction.
Mouse SARS-CoV2 RT-PCR: Two different primer/probe sets were used for purposes of comparison, one to detect genomic viral RNA (gRNA), and another to detect subgenomic viral RNA (sgRNA). The sgPCRs measure any one of viral replication products (S, 3a, E, M, 6, 7a, 7b, 8 and N). Any PCR product amplified from a subgenomic coding sequences is present in both the genomic and subgenomic sequences. Therefore, the approach to distinguish sgRNA from gRNA is to amplify a PCR product in both the 5’ UTR leader sequence and within specific subgenomic sequence. Validated primer/probe sets in the leader and E sequences (74-75) were used: Forward primer (leader sequence): CGATCTCTTGTAGATCTGTTCTC Reverse primer (E sgRNA): ATATTGCAGCAGTACGCACACA Probe: (5'FAM)/ACACTAGCCATCCTTACTGCGCTTCG/(3'BHQ-2).
Mouse SARS-CoV2 gRNA: Genomic viral RNA does not specifically reflect replicating virus. The primer/probe set was developed at Utah State University from a prototypic SARS-CoV-2 sequence, SARS-CoV-2/human/USA/WA-CDC-WA1/2020, in the ORF1ab sequence (accession MN985325.10). This sequence is only present in gRNA, not sgRNA. The samples were run in duplicate using the same program settings above. Forward primer: CACTAGTGCCACAAGAGCAC Reverse primer: TGCGAGCAGAAGGGTAGTAG Probe: (5'FAM)/TCCAGGGACCACCTGGTACTGGT/(3'BHQ-2). Each sample was run in duplicate using the Probe No-ROX One-step kit. The Magnetic Induction Cycler was programmed at 10 min. at 45°C, 2 min. at 95°C, then 40 cycles of 5 sec. at 95°C and 20 sec. at 60°C. A standard curve consisted of 1-log10 serial dilutions of a positive sample. The C(t) values of experimental samples were interpolated from the standard curve to obtain the relative copy number. The values are reported as relative copy number per 300 ng total RNA added to the RT-PCR reaction.
Mouse SARS-CoV-2 infectious virus assay: A published assay to quantify infectious virus was used with some modification (76). Confluent or near-confluent Vero E6 monolayers were prepared in 96-well disposable microplates the day before testing. Cells were maintained in MEM supplemented with FBS as required for each cell line. For antiviral assays, the same medium was used but with FBS reduced to 2% or less and supplemented with 50 µg/ml of gentamicin. The lung tissue of each animal lung was homogenized in MEM supplemented with 10% FBS using a bead-homogenizer, and the tissue fragments were allowed to settle. Growth media was removed from the cells and the tissue homogenate was applied in 0.1 ml volume to wells at 2X concentration. Plates were incubated at 37oC with 5% CO2 until >80% cytopathic effect (CPE) was observed in virus control wells. The plates were then stained with 0.01% neutral red for approximately two hours at 37°C in a 5% CO2 incubator. The neutral red medium was removed by complete aspiration, and the cells were rinsed 1X with phosphate buffered solution (PBS) to remove residual dye. The PBS was completely removed, and the incorporated neutral red was eluted with 50% Sorensen’s citrate buffer/50% ethanol for at least 30 minutes. Neutral red dye penetrates into living cells, thus, the more intense the red color, the larger the number of viable cells present in the wells. The dye content in each well is quantified using a spectrophotometer at 540 nm wavelength. The dye content in each set of wells is converted to a percentage of dye present in untreated control wells using a spreadsheet and normalized based on the virus control. The data are reported as 50% cell-culture infectious doses (CCID50) per gram tissue. The terminology of TCID50 and CCID50 are interchangeable.
Mouse SARS-CoV-2 RNA sequencing and analysis
RNA sequencing libraries were prepared in two batches from ribodepleted RNA extracted from lung tissue of animals enrolled the murine SARS-CoV-2 studies described above. Libraries were sequenced at the University of California San Diego Institute for Genomic Medicine, Genomics Center utilizing an Illumina NovaSeq 6000. Quality trimming of FASTQ files was performed using Trimmomatic (v0.36). Paired-end reads were aligned to the mouse reference genome (GRCm39; annotation file, vM29) using STAR (v2.5.3a), followed by RSEM (v1.3.0) to quantify expression levels of transcripts. The R BioConductor package DESeq2 was used to perform differential gene expression analysis from unnormalized counts. Lowly expressed genes – defined as those genes with mean <10 reads across all samples – were excluded prior to differential expression analysis. Plots represent the regularized log transformed (rlog) read counts. Sample clustering is supervised or unsupervised (Pearson correlation) where indicated. Significance was defined by using an adjusted p-value cut-off of <0.05 after multiple testing correction. Total differentially expressed genes or genes differentially expressed in multiple group comparisons were extracted for gene annotation and functional enrichment analysis using GSEA.
Cell type enrichment scores in mouse RNA sequencing samples were determined from the expression frequency of cell type markers from PanglaoDB https://panglaodb.se/, a single cell sequencing resource for gene expression data collected and integrated from multiple studies. The reference matrix originally contained 178 cell types, but the data were filtered for cells of lung and immune origin. A total of 32 cell types remained. Only genes specific to mouse were included in the analysis. Cell type enrichment scores were calculated using the standard formula (nN/kM), where N is the total number of genes in the library, k is the number of genes in a specific cell type reference list, M is the number of differentially upregulated or downregulated genes, and n is the number of genes in both k and M. Significance was calculated using a Fisher's exact test and plotted as minus log10 p-value.
Murine hepatitis corona virus A59 strain (MHV-A59) infection
MHV-A59 viral infection: 8-week-old male C57BL/6 mice were inoculated intranasally with 7.5×105 plaque-forming units (pfu) in 24μl of MHV-A59 or sham inoculated with 24μl of 0.9% sterile saline. 15mg/kg IPI-549 or 0.1 ml of vehicle was administered p.o. Body weight and survival were monitored daily. At 2-, 5-, and 8-days post-infection, 5 mice from each treatment group and 3 mice from the sham infection group were euthanized, lungs were excised and rinsed in ice-cold 1xPBS. 100mg of lung tissue was homogenized in cold DMEM with 10% FBS for measurement of viral load by plaque assays, 100mg was homogenized in Trizol for RNA extraction, and the remaining tissue was preserved in 5ml of 10% Zinc Formalin for 24-48h, paraffin embedded and used for histological analysis. Blood was collected in 1.5ml Eppendorf tubes, allowed to clot, and serum was collected for cytokine and chemokine analysis.
MHV loads by plaque assay: To quantify MHV viral load in lung tissue, lung homogenates were rapidly frozen and thawed three times. After centrifugation at 770 x g at 4°C for 10 minutes. Supernatants were collected and used to assay virus content using L929 fibroblasts monolayer on 6 well plates. 200µl of the lung homogenate was added to triplicate wells and plates were incubated at 37°C in a 5% CO2 incubator for 1 hr, rocking every 10 mins. At the end of incubation, 2ml of 0.8% agarose was overlaid onto to each well. The plates were incubated for two days until the formation of virus plaques and the plaques of each well were counted.
MHV model RT-PCR: Total RNA was extracted from 100 mg tissue with TRIzol reagent (Invitrogen, Carlsbad, CA), and 1 μg RNA that was pretreated with 1 U of RQ1 RNase-free DNase (ThermoFisher Scientific) to remove DNA contamination at 37 ºC for 30min, were used for reverse transcription with oligo-dT primer (Bio-Rad). PCR reactions were then performed using the primers shown below to detect the MHV-A59 N gene and other associated genes. PCR products (123 bp) were resolved by electrophoresis in 2% agarose gels and visualized on the iBright FL1000 System machine (Invitrogen, Carlsbad, CA). Total RNA was extracted using Trizol (Invitrogen, Carlsbad, CA) from approximately 100mg of lung tissues). Relative levels of each gene were normalized to those of the housekeeping gene GAPDH. Quantitect primer sets used were: Il1b Mm_Il1b_2_SG, Tnfa Mm_Tnf_1_SG, Il6 Mm_Il6_1_SG, Cxcl10 Mm_Cxcl10_1_SG, Il10 Mm_Il10_1_SG, Il12 Mm_Il12b_1_SG, Ifng Mm_Ifng_1_SG, Ifna Mm_Ifna2_1_SG, Ifnb Mm_Ifnb1_1_SG, Gbp5 Mm_Gbp5_1_SG, Gbp3 Mm_Gbp3_1_SG, Stat1 Mm_Stat1_1_SG, Cd8 Mm_Cd8a_1_SG, Cd4 Mm_Cd4_1_SG, Ccl2 Mm_Ccl2_1_SG, Gzmb Mm_Gzmb_1_SG, and Gapdh Mm_Gapdh_3_SG (Qiagen).
MHV Cytokine analysis: Cytokine concentrations in 25µl murine serum and BALF were determined using ProcartaPlex mouse cytokine panels (EPXR260-26088-901, EPX110-20820-901 and PPX-11-MXCE49Z) according to manufacturer’s directions on a Luminex Magpix instrument (ThermoFisher Scientific). Murine cytokine ELISAs were performed using uncoated ELISA kits (Invitrogen/ThermoFisher Scientific): TNF alpha (RRID:AB_2575076 #88-7324-22), IL6 (RRID:AB_2574986 #88-7064-22), and IL1 (RRID:AB_2574942 #88-7013-22) according to manufacturer’s directions.
MRSA and E. coli K1 strain RS218 infection models
Bacterial Strains: Methicillin-resistant Staphylococcus aureus strain USA300/TCH1516 (MRSA) (ATCC BAA-1717) and E. coli K1 strain RS218 were used in the study. MRSA was propagated in Todd Hewitt broth (THB) and E. coli grown in Luria-Bertani broth (LB), shaking at 37°C to mid-log phase. Bacteria were collected by centrifugation at 4000 RPM for 10 min, washed once with phosphate buffered saline (PBS), and resuspended in experimental buffer.
MRSA animals. Animal studies were conducted under protocols approved by the UC San Diego Institutional Animal Care and Use Committee (IACUC). For systemic infection, 8-12 weeks old wild type (WT) and p110γ−/− (p110γ ko) mice in the C57/Bl6 background were injected with 2x108 colony forming units (CFU) MRSA or 1x107 CFU E. coli intraperitoneally (i.p). Survival of infected mice was monitored every 8 h for 6 days. For CFU enumeration experiments, mice were infected with 2.7 x108 CFU MRSA i.p. and euthanized 24 h after infection, Liver, kidney, and spleen were harvested and dilution plated on THB agar plates. Serum was also collected at this timepoint for measurement of cytokines IL-6 and IL-1β using ELISA kits M6000MLB00C (R&D Systems).
L929 conditioning media. To prepare L929 conditioned media, confluent L929 cells were detached, collected, and passaged 1:10. Cells were then cultured for 7 to 8 days until the media was exhausted. The conditioned media, which contains macrophage growth factor M-CSF, was filtered (0.22 μm) and stored at −20 °C until use.
Murine bone marrow derived macrophages (BMDM). BMDM were generated by flushing bone marrow cells from femurs and tibia of wild type (WT) and Pik3cg-/- C57Bl6 mice and resuspending the collected cells in RPMI 1650 supplemented with 20% L929 conditioned media. On day 7 of culture cells were washed, detached with 0.025% trypsin-EDTA and were seeded on 96-well plates for 48 h before used for bacterial killing assays.
Bacterial killing assay. Bacteria were added to cultured macrophages in 96-well plates at a multiplicity of infection (MOI) of 0.1 (10,000 CFU bacteria per 100,000 macrophages). After centrifugation at 1200 rpm for 5 min to initiate contact between macrophages and bacteria, the plate was incubated in 37°C, 5% CO2 for 1 h. Macrophages were then lysed by 0.1% Triton-X and the lysed samples serially diluted and spot-plated onto THB agar plates for CFU enumeration.
Animal models of inflammation
Peritoneal inflammation: WT and Pik3cg-/- mice were Intraperitoneally injected with 100 µl of a 3% thioglycolate (Sigma) solution. Peritoneal cells and fluids were collected 96 hours later from the peritoneal cavity into 10 ml of PBS. Macrophage enrichment was performed by plating cells in RPMI with 10% FBS and 1% penicillin/streptomycin for 2 h at 37 °C and 5% CO2. After 2 h, non-adherent cells were removed with three PBS washes, and cells were analyzed via flow cytometry for expression of CD45 (30-F11, RRID:AB_1210805), CD11b (M1/70, RRID:AB_469901), and F4/80 (BM8, RRID:AB_469653) (ThermoFisher). All data analysis was performed using the flow cytometry analysis program FloJo (Becton Dickinson).
ARDS Animal models: Inflammatory acute respiratory distress syndrome (ARDS) was modeled in animals by systemic or local administration of lipopolysaccharide (LPS) or poly inosine:cytosine (poly I:C). To model acute inflammation, n=5-10 WT or Pik3cg-/- mice were inoculated by intraperitoneal injection with a single dose of 10 mg/kg LPS (L4391, Sigma-Aldrich, Inc., St. Louis, MO, USA) or 12 mg/kg poly I:C (PCL-40-03, Invivogen, Inc., San Diego, CA, USA). In studies evaluating the effect of PI3Kg inhibitor IPI-549, mice were pre-treated once daily for 2 days with 100µl of Vehicle or IPI-549 (15mg/kg) by oral gavage prior to LPS or poly I:C inoculation. Animals were euthanized 24hr later. All studies were performed three or more times with n= 5-10/group. To model chronic inflammation, WT or Pik3cg-/- mice were inoculated with doses of 3 mg/kg LPS or 12 mg/kg Poly I:C by intraperitoneal injection on three consecutive days. Some WT animals were pre-treated with 100µl of Vehicle or IPI-549 (15mg/kg) by oral gavage (n=5-10) for 1-2 days prior to LPS or poly I:C inoculation and once daily after inoculation for 3 days. Animals were euthanized 24hr after the last treatments. All studies were performed three or more times. In some animals, 5 mg/kg Poly I:C or LPS in 20 µl was administered once daily for three days by intranasal inoculation and animals were treated as described above. All studies were performed three or more times with n= 5-10/group.
ARDS Histopathology: To collect lung tissues from mice for histopathology, mice were euthanized, and lungs were immediately inflated by intratracheal administration of up to 1ml 10% zinc formalin, followed by immersion in 10% zinc formalin for 24-48hrs. Lungs were paraffin embedded and sectioned at the UCSD Moores Cancer Center histology shared resource. Glass slides containing 5µm thick tissue sections were deparaffinized and stained with hematoxylin and eosin or Mason’s Trichrome by the Moores Cancer Center histology shared resource. Alternatively, slides were deparaffinized, rehydrated, treated with Diva Decloaker (Biocare Medical) antigen retrieval solution followed by endogenous peroxidase blocking, and then incubated with 5% normal goat serum for 1 hour. Slides were then incubated with 1:250 anti-F4/80 (RRID:AB_467558 14-4801-82, ThermoFischer Scientific) or 1:500 anti-IBA1 (NB100-1028SS, Novus Biologicals) at 4°C overnight. Following primary antibody incubation, slides were washed in TBST, and incubated with goat anti-rat or horse anti-goat (horseradish peroxidase (HRP)-conjugated polymer (Vector Laboratories, Burlingame, CA) for 30 minutes at room temperature, washed in TBST and counterstained with hematoxylin. Images were taken on a Nikon microscope (Eclipse TE2000-U) and analyzed using Metamorph image capture analysis software (Version 6.3r5, Molecular Devices).
ARDS Cytokine analysis: To collect serum for cytokine analysis, blood was collected from the submandibular vein in living animals or from the ocular vein immediately after euthanasia of mice. Blood was allowed to clot for 30-60min at room temperature, then was centrifuged at 2000 x g for 10 min at 4°C. Serum was collected and stored at -80°C until use.
ARDS RNA analysis: To collect lung tissue for RNA analysis, animals were perfused with 5–10 ml of PBS to remove blood prior to excision of intact lungs. RNA was extracted from lungs using Trizol (Invitrogen, Carlsbad, CA).
ARDS CD11b+ cell isolation: Murine lung tissues were minced in a petri dish on ice and then enzymatically dissociated in Hanks Balanced Salt Solution with Ca2+ and Mg2+ containing 0.5 mg/ml Collagenase IV (Sigma), 0.1 mg/ml Hyaluronidase V (Sigma), 0.6 U/ml Dispase II (Roche), 0.005 MU/ml DNAse I(Sigma) and 0.2 mg/ml soybean trypsin inhibitor (Worthington Biochemical) at 37°C for 15 min. Red blood cells were lysed with red blood cell lysis solution (Sigma), and the resulting suspension was filtered through a 70 µm cell strainer to produce a single cell suspension. Cells were centrifuged, at 300×g for 10 minutes, washed 2x in 1x PBS with 2mM EDTA and 0.5% BSA buffer and resuspended in 90 µL of 1x PBS with 2mM EDTA and 0.5% BSA per 107 total cells. CD11b+ cells were isolated by anti-CD11b magnetic bead pull down (130-049-601, Miltenyi Biotech, San Diego, CA) as follows: 10 µl of anti-CD11b MicroBeads was then added per 107 total cells, mixed well and incubated for 15 minutes in ice. Cells were then washed with 1−2 ml of 1x PBS with 2mM EDTA and 0.5% BSA buffer per 107 cells and centrifuged at 300×g for 10 minutes. The pellet was then resuspended at a concentration of 108 cells in 500 µL of buffer, applied to an LS column (Miltenyi, Cat #130-042-401) on a QuadroMACs magnetic separator (Miltenyi, Cat#130-091-051). The flow through containing CD11b- cells was collected for RNA extraction, and CD11b+ cells were eluted using a wash buffer into a separate collection tube for RNA extraction.
ARDS vascular leak assay: To measure vascular leak in mouse models of acute respiratory distress syndrome, LPS or Poly I:C treated mice were injected by tail vein injection with 30mg/kg Evan’s blue dye (100µl of 1% w/v in 0.9% saline) and one hour later, mice were euthanized. Lungs, heart, and kidneys were excised, weighed and dehydrated overnight at 65°C. Evan’s Blue dye was extracted from dried tissue by incubation with formamide at a ratio of 10µl/mg dry weight overnight. Extracted dye was quantified versus a standard curve by measuring absorbance at 620nm.
ARDS bronchoalveolar lavage: To collect cells and fluid from the bronchoalveolar spaces of mouse lungs, mice were euthanized. 1 ml of PBS-EDTA was injected into the lungs via intratracheal cannulation and then aspirated into a 15 mL conical tube; cells were pelleted by centrifugation at 300xg, and the supernatant (bronchoalveolar lavage fluid, BALF) was cryopreserved at -80°C for further analysis. Two additional lavages were performed; cells from all three lavages were pooled, centrifuged 5min at 300 × g at 4 °C, resuspended in 1 mL and centrifuged using a Cytospin centrifuge (ThermoFisher) for morphological analysis by Wright Giemsa staining and counting.
Western blotting to detect PI3Kg: Immortalized murine macrophages were transfected with ribonucleoprotein (RNP) complexes consisting of a one of two guide RNAs targeting the second exon of the Pik3cg gene (sgRNA 1: GCTGGAAGAACTATGAAC and sgRNA 2: GGAGAACTATGAACAAC) and Cas9 nuclease (spCas9 2NLS Nuclease) from Synthego. Each population was Sanger sequenced to confirm gene knockdown. RIPA protein lysates from WT, transfected and mock transfected macrophages were electrophoresed on a 4-12% gradient gel, transferred to PVDF membrane for sequential incubation in rabbit anti-PIK3CG (D55D5) or rabbit anti-GAPDH (14C10) at 1:1000 followed by incubation in anti-rabbit IgG, HRP linked antibody and chemiluminescent substrate.
QUANTIFICATION AND STATISTICAL ANALYSIS
All data were graphed and expressed as the mean +/- standard error of the mean (mean +/- SE) using Graph Pad Prism version 9.1.0. Results were analyzed statistically using one-way Anova with Tukey’s post-hoc test for multiple group analysis and Student’s t-test or Mann Whitney non-parametric t tests for two group analysis using Graph Pad Prism version 9.1.0. Data with p≤0.05 were considered statistically significant and values greater than p>0.05 were considered non-significant. All studies were performed 3 or more times with three or more replicates per group, except for SARS-CoV-2 in vivo assays which were performed one time each with 5-10 replicates per group due to the restricted access to SARS-CoV-2 animal models.
Usage notes
Any spreadsheet program such as Excel.