Efferocytosis of SARS-CoV-2-infected dying cells impairs macrophage anti-inflammatory functions and clearance of apoptotic cells

  1. Ana CG Salina
  2. Douglas dos-Santos
  3. Tamara S Rodrigues
  4. Marlon Fortes-Rocha
  5. Edismauro G Freitas-Filho
  6. Daniel L Alzamora-Terrel
  7. Icaro MS Castro
  8. Thais FC Fraga da Silva
  9. Mikhael HF de Lima
  10. Daniele C Nascimento
  11. Camila M Silva
  12. Juliana E Toller-Kawahisa
  13. Amanda Becerra
  14. Samuel Oliveira
  15. Diego B Caetité
  16. Leticia Almeida
  17. Adriene Y Ishimoto
  18. Thais M Lima
  19. Ronaldo B Martins
  20. Flavio Veras
  21. Natália B do Amaral
  22. Marcela C Giannini
  23. Letícia P Bonjorno
  24. Maria IF Lopes
  25. Maira N Benatti
  26. Sabrina S Batah
  27. Rodrigo C Santana
  28. Fernando C Vilar
  29. Maria A Martins
  30. Rodrigo L Assad
  31. Sergio CL de Almeida
  32. Fabiola R de Oliveira
  33. Eurico Arruda Neto
  34. Thiago M Cunha
  35. José C Alves-Filho
  36. Vania LD Bonato
  37. Fernando Q Cunha
  38. Alexandre T Fabro
  39. Helder I Nakaya
  40. Dario S Zamboni
  41. Paulo Louzada-Junior
  42. Rene DR Oliveira
  43. Larissa D Cunha  Is a corresponding author
  1. Departamento de Biologia Celular e Molecular e Bioagentes Patogênicos, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Brazil
  2. Hospital Israelita Albert Einstein, Brazil
  3. Departamento de Bioquímica e Imunologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, Brazil
  4. Departamento de Farmacologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Brazil
  5. Center of Research in Inflammatory Diseases (CRID), Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Brazil
  6. Divisão de Imunologia Clinica, Emergência, Doenças Infecciosas e Unidade de Terapia Intensiva, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Brazil
  7. Departamento de Patologia e Medicina Legal, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Brazil
  8. Departamento de Cirurgia e Anatomia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Brazil
5 figures, 1 table and 3 additional files

Figures

Figure 1 with 1 supplement
Macrophages engulf apoptotic cells (AC) carrying viable SARS-CoV-2.

(A) Immunoblot of caspase-8 and caspase-3 cleavage in Calu-3 and Vero CCL81 cells in response to infection with SARS-CoV-2 for 48 hr (CoV2, detected with rabbit anti-Spike), UV irradiation (UV), or …

Figure 1—figure supplement 1
Support data for Figure 1.

(A) Immunofluorescence images of tissue samples immunolabeled with secondary antibodies only used for detection of anti-cleaved caspase-3 (red, anti-rabbit IgG conjugated to Alexa 594), anti-dsRNA …

Figure 2 with 1 supplement
Engulfment of SARS-CoV-2-infected dying cells impairs macrophage anti-inflammatory functions.

Macrophages were unstimulated (Cntrl), co-incubated with apoptotic Vero CCL81 cells (AC) isolated from UV-irradiated (UV-AC) or SARS-CoV-2-infected (CoV2-AC) cultures, stimulated with the …

Figure 2—figure supplement 1
Support data for Figure 2.

(A) Representative gating strategy used to determine the CD206 expression on the surface of macrophages in response to the different stimuli by flow cytometry (backgating corresponds to FMO control …

Figure 3 with 1 supplement
Engulfment of SARS-CoV-2-infected dying cells causes inflammatory cytokine production.

Macrophages were left unstimulated (Cntrl), co-incubated with apoptotic Vero CCL81 cells (AC) isolated from UV-irradiated (UV-AC) or SARS-CoV-2-infected (CoV2-AC) cultures, stimulated with the …

Figure 3—figure supplement 1
Support data for Figure 3.

(A) THP-1-derived macrophages were incubated with UV-AC, Cov2-AC, or CoV2-Sup obtained from infected Vero CCL81 cells or infected with CoV2. Expression of IL6 in macrophages showed as fold change …

Figure 4 with 1 supplement
Engulfment of SARS-CoV-2-infected dying cells suppresses continual efferocytosis by macrophages.

(A) Expression of efferocytic receptors in human primary monocyte-derived macrophages. Macrophages were left unstimulated (Cntrl), co-incubated for 24 hr with apoptotic Vero CCL81 cells (AC) …

Figure 4—figure supplement 1
Support data for Figure 4.

(A) THP-1-derived macrophages were incubated with UV-AC or CoV2-AC obtained from infected Vero CCL81 cells or infected with CoV2. Expression of the efferocytic receptors CD36, SRA-I, ITGB5, TIMD4, …

Lung monocytes and macrophages of severe COVID-19 patients express reduced levels of efferocytic receptors.

(A–B) Representative histological findings in post-mortem lung tissue from control patients (Cntrl) and COVID-19 patients, obtained by ultrasound-guided minimally invasive autopsy. Tissue samples …

Tables

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Antibodyanti-Sars-CoV-2 Spike (rabbit polyclonal)AbcamCat: #ab272504 RRID: AB_2847845WB and FACS (1:1000); Immunofluorescence (1:1000)
Antibodyanti-CD11b+CD11 c (mouse monoclonal)AbcamCat: #ab1211 RRID: AB_442947Immunofluorescence (1:300)
Antibodyanti-human CD68 (mouse monoclonal)AbcamCat: #ab955 RRID: AB_307338Immunofluorescence (1:100)
Antibodyanti-CD206-FITC (mouse monoclonal)BD BiosciencesCat: #551135 RRID: AB_394065FACS (1:200)
Antibodyanti-caspase-3 (rabbit polyclonal)Cell SignalingCat: #9662 RRID: AB_331439WB (1:1000)
Antibodyanti-cleaved caspase-3 (rabbit monoclonal)Cell SignalingCat: #9664 RRID: AB_10694088WB and Immunofluorescence (1:1000); FACS (1:200)
Antibodyanti-caspase-8 (rabbit monoclonal)Cell SignalingCat: #4790 RRID: AB_10545768WB (1:1000)
Antibodyanti-cleaved caspase-8 (rabbit monoclonal)Cell SignalingCat: #9496 RRID: AB_561381WB (1:1000)
Antibodyanti-βactin HRP conjugate (mouse monoclonal)Cell SignalingCat: #12262 RRID: AB_2566811WB (1:5000)
Antibodyanti-human CD36 (rabbit polyclonal)Thermo Fisher ScientificCat: # PA5-81996 RRID: AB_2789157Immunofluorescence (1:100)
Antibodyanti-human Mertk (rabbit monoclonal)Thermo Fisher ScientificCat: # MA5-31991 RRID: AB_2809285Immunofluorescence (1:150)
Antibodyanti-human cytokeratin 18 (rabbit polyconal)GeneTexCat: #GTX112977 RRID: AB_10719880Immunofluorescence (1:500)
Antibodyanti-dsRNA J2 (mouse monoclonal)SCICONS English & Scientific Consulting KftCat: #10010200 RRID: AB_2651015Immunofluorescence (1:1000)
Antibodydonkey anti-rabbit IgG HRP (donkey polyclonal)Jackson ImmunoResearchCat: #711-036-152 RRID: AB_2340590WB (1:5000)
AntibodyAlexa-594 goat-anti mouse IgG (goat polyclonal)Thermo Fisher ScientificCat: #A11005 RRID: AB_2534073Immunofluorescence (1:500)
AntibodyAlexa-488 donkey-anti rabbit IgG (donkey polyclonal)Thermo Fisher ScientificCat: #A21206 RRID: AB_2535792FACS (1:2500) and Immunofluorescence (1:500)
AntibodyAlexa-594 goat-anti rabbit IgG (goat polyclonal)Thermo Fisher ScientificCat: #A11012 RRID: AB_141359Immunofluorescence (1:500 or 1:1000)
AntibodyAlexa-488 F(ab)'2 goat-anti mouse IgG (goat polyclonal)Thermo Fisher ScientificCat: #A11017 RRID: AB_2534084Immunofluorescence (1:500)
AntibodyAlexa-647 donkey-anti mouse IgG (donkey polyclonal)Thermo Fisher ScientificCat: #A31571 RRID: AB_162542Immunofluorescence (1:500 or 1:1000)
AntibodyAlexa-647 goat anti-rabbit IgG (goat polyclonal)Thermo Fisher ScientificCat: #A21244 RRID: AB_2535812Immunofluorescence (1:500)
Cell line (Homo sapiens)Calu-3BCRJ #0264ATCC #HTB-55
Cell line (H. sapiens)Jurkat E6-1BCRJ #0125ATCC #TIB-152
Cell line (H. sapiens)THP-1Dario Zamboni’s labATCC: #TIB-202
Cell line (Cercopithecus aethiops)Vero CCL81Dario Zamboni’s labATCC: #CCL-81
Strain, strain background (human)SARS-CoV-2Brazil/SPBR-02/2020
Strain, strain background (human)Coxsackievirus B5Roger M. Loria’s lab
Chemical compound, drugFITC Annexin VBD BiosciencesCat: #556,420FACS (1:200)
Chemical compound, drugPurified Recombinant Annexin VBD BiosciencesCat: #556,416
Chemical compound, drugZombie NIR Fixable ViabilityBiolegendCat: #423,106FACS (1:400)
Chemical compound, drugZombie Violet Fixable Viability KitBiolegendCat: #423,114FACS (1:400)
Chemical compound, drugRemdesivirBOC SciencesCat: #1809249-37-3
Chemical compound, drugphorbol 12-myristate 13-acetate (PMA)InvivoGenCat: #Tlrl-pma
Chemical compound, drugAnnexin V MicroBeadsMiltenyi BiotecCat: #130-090-201
Chemical compound, drugZ-VAD-FMKSelleckchemCat: #S7023
Chemical compound, drugGlycineSigmaCat: #G7126
Chemical compound, drugcomplete, Mini Protease Inhibitor CocktailSigmaCat: #11836153001
Chemical compound, drugCytochalasin DSigmaCat: #C8273
Chemical compound, drugCellTrace CFSE Cell Proliferation KitThermo Fisher ScientificCat: #C34554
Chemical compound, drugCellTrace Far Red Cell Proliferation KitThermo Fisher ScientificCat: #C34564
Chemical compound, drugCellTrace Violet Cell Proliferation KitThermo Fisher ScientificCat: #C34557
Chemical compound, drugpHrodo Red AM Intracellular pH IndicatorThermo Fisher ScientificCat: #P35372
Chemical compound, drugHoechst 33,342Thermo Fisher ScientificCat: #H3570
Commercial assay or kitBD Cytofix/Cytoperm Fixation/Permeabilization KitBD BiosciencesCat: #554,715
Commercial assay or kitCytoTox 96 Non-Radioactive Cytotoxicity AssayBD BiosciencesCat: #G1780
Commercial assay or kitHuman Inflammatory Cytokine Cytometric Bead Array (CBA)BD BiosciencesCat: #551,811
Commercial assay or kitHuman IL-6 DuoSet ELISAR&D SystemsCat: #DY206-05
Commercial assay or kitPowerUp SYBR Green Master MixThermo Fisher ScientificCat: #A25742
Commercial assay or kitZenon Rabbit IgG Labeling KitsThermo Fisher ScientificCat: #Z25306
Commercial assay or kitDirect-zol RNA Miniprep KitsZymo ResearchCat: #R2052
Software, algorithmAdobe PhotoshopAdobe Inc
Software, algorithmFlowJo v10.8.0FlowJo, LLC
Software, algorithmGraphpad PRISM 8.0GraphPad Software, Inc
Software, algorithmImageJ (Fiji)National Institutes of Health
Software, algorithmNIS ElementsNikon Instruments Inc.
Software, algorithmClusterProfilerWu et al., 2021
Software, algorithmfgsea RKorotkevich et al., 2016
Software, algorithmR v4.0.4
Software, algorithmSeurat v4.1.0Stuart et al., 2019
Software, algorithmEnrichrChen et al., 2013

Additional files

Supplementary file 1

Custom gene sets incorporating associated to efferocytic pathway and related to human diseases (related to Figure 5C).

https://cdn.elifesciences.org/articles/74443/elife-74443-supp1-v2.docx
Supplementary file 2

Medical characteristics of COVID-19 patients.

https://cdn.elifesciences.org/articles/74443/elife-74443-supp2-v2.docx
Transparent reporting form
https://cdn.elifesciences.org/articles/74443/elife-74443-transrepform1-v2.pdf

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