Macrophage innate training induced by IL-4 and IL-13 activation enhances OXPHOS driven anti-mycobacterial responses

  1. Mimmi LE Lundahl  Is a corresponding author
  2. Morgane Mitermite
  3. Dylan Gerard Ryan
  4. Sarah Case
  5. Niamh C Williams
  6. Ming Yang
  7. Roisin I Lynch
  8. Eimear Lagan
  9. Filipa M Lebre
  10. Aoife L Gorman
  11. Bojan Stojkovic
  12. Adrian P Bracken
  13. Christian Frezza
  14. Frederick J Sheedy
  15. Eoin M Scanlan
  16. Luke AJ O'Neill
  17. Stephen V Gordon
  18. Ed C Lavelle  Is a corresponding author
  1. School of Biochemistry and Immunology, Adjuvant Research Group, Trinity Biomedical Sciences Institute, Trinity College Dublin, Ireland
  2. School of Chemistry, Scanlan Research Group, Trinity Biomedical Sciences Institute, Trinity College Dublin, Ireland
  3. School of Veterinary Medicine, UCD Veterinary Sciences Centre, University College Dublin, Ireland
  4. School of Biochemistry and Immunology, Inflammation Research Group, Trinity Biomedical Sciences Institute, Trinity College Dublin, Ireland
  5. Hutchison/MRC Research centre, MRC Cancer Unit, University of Cambridge, United Kingdom
  6. School of Biochemistry and Immunology, Macrophage Homeostasis Group, Trinity Biomedical Sciences Institute, Trinity College Dublin, Ireland
  7. School of Genetics and Microbiology, Department of Genetics, Trinity College Dublin, Ireland
13 figures, 1 table and 2 additional files

Figures

Figure 1 with 1 supplement
Training with IL-4 and IL-13 enhances BMDM mycobacterial killing capacity.

(A) Schematic of protocol for BCG infection following acute activation (Day 0) or training (Day 6) with IL-4 and IL-13. (B–C) BCG Denmark uptake (B) and killing after h as indicated (C) measured by …

Figure 1—figure supplement 1
CFU counts and cytokine secretion from BCG killing experiments.

(A–B) BCG Denmark CFU counts (A) and cytokine secretion (B) on Day 0 or Day 6 after h following BMDM BCG infection. BMDMs were previously incubated with media control, IL-4 with IL-13 or IFNγ with …

Figure 2 with 3 supplements
Innate training of BMDMs with IL-4 and IL-13 enhances pro-inflammatory responses.

(A–B) Cytokine secretion following BMDM 24 h incubation with irradiated M. tuberculosis (Mtb) (A) TLR agonists (B) or media on Day 6 as indicated. BMDMs were previously incubated with media, IL-4 …

Figure 2—figure supplement 1
BMDMs either classically activated with IFNγ and LPS or alternatively activated with IL-4 and IL-13.

(A) Gating strategy for flow cytometry analysis, gating on cells, live cells and CD11b+F480+BMDMs. (B) Surface marker expression of CD80, CD206 and MHC II on BMDMs incubated with media as a control, …

Figure 2—figure supplement 2
IL-4 and IL-13 innate training enhances inflammatory and bactericidal responses following TLR 1/2 stimulation.

(A–B) Cytokine secretion from BMDMs following incubation with media control, IL-4 with IL-13 or IFNγ with LPS for 24 hr on Day –1 and incubated with media or PAM3CSK4 on Day 0 (A) or Day 6 (B) for …

Figure 2—figure supplement 3
Methylation contributes to IL-4 and IL-13 innate training.

(A) Cytokine secretion following BMDM incubation with media control or IL-4 with IL-13 for 24 hr on Day –1, with or without 1 hr pre-incubation with methylation inhibitor MTA, and incubation with …

Figure 2—figure supplement 3—source code 1

Unedited and labelled western blots.

Unedited western blots for histone H3, H3K4me3, H3K27me3 and H3K9me2, accompanying ponceau stains and figure where size and wells are labelled.

https://cdn.elifesciences.org/articles/74690/elife-74690-fig2-figsupp3-code1-v2.zip
Figure 3 with 1 supplement
BMDMs trained with IL-4 and IL-13 retain M2-typical metabolism upon mycobacterial challenge.

(A) Extracellular flux analysis of BMDMs, following 24 hr incubation with IL-4 and IL-13, IFNγ and LPS or media (n=3). Mitochondrial stress test inhibitors: oligomycin (OM; blocks ATP synthase), …

Figure 3—source data 1

MetaboAnalyst R-history.

Statistical analysis carried out by MetaboAnalyst. Raw data can be found in Source data 1.

https://cdn.elifesciences.org/articles/74690/elife-74690-fig3-data1-v2.txt
Figure 3—figure supplement 1
Challenged BMDMs previously trained with IL-4 and IL-13 retain M2-typical metabolism.

(A–B) qPCR of indicated mRNA in BMDMs following incubation with media control, IL-4 with IL-13 or IFNγ with LPS on for 24 h Day –1 and stimulated with media (A) or PAM3CSK4 (B) on Day 6 for 6 hr (Pkm…

Figure 4 with 1 supplement
OXPHOS drives inflammatory cytokine responses in IL-4/13 trained BMDMs.

(A) Cytokine secretion from BMDMs following incubation with media, IL-4 with IL-13 or IFNγ with LPS for 24 hr on Day –1 and incubation with irradiated M. tuberculosis (Mtb) on Day 6 for 24 hr, with …

Figure 4—figure supplement 1
Innate training with IL-4 and IL-13 enhancement of pro-inflammatory responses is not dependent upon glucose metabolism.

(A–B) Cytokine secretion from BMDMs following incubation with media control, IL-4 with IL-13 or IFNγ with LPS for 24 hr on Day –1 and incubation with irradiated M. tuberculosis H37Rv (Mtb) (A) or …

BMDMs trained with IL-4 and IL-13 retain M2-typical metabolism: schematic summary of results.

Pathways have been simplified. Key: metabolic pathways strongly upregulated by M1/M2 macrophage activation are highlighted by red/blue colored boxes, respectively. Inhibitors are indicated by green …

Figure 6 with 2 supplements
IL-10 inhibits bactericidal and pro-inflammatory training induced by IL-4 and IL-13 and alters yeast β-glucan training.

(A) Cytokine secretion following BMDM incubation with media or IL-4 and IL-13, with or without IL-10, for 24 hr on Day –1 and incubated for 24 hr with media or irradiated M. tuberculosis (Mtb) on …

Figure 6—figure supplement 1
Alternative macrophage activation with or without IL-10.

(A) Cytokine secretion from BMDMs incubated with media as a control or IL-4 and IL-13, with or without IL-10 for 24 hr on Day-1, then incubated with media or Mtb for 24 hr on Day 0 (n=4). (B) …

Figure 6—figure supplement 2
CFU counts and cytokine secretion from BCG killing experiments.

(A–B) BCG Denmark CFU counts (A) and cytokine secretion (B) on Day 0 or Day 6 after h following BMDM BCG infection. BMDMs were previously incubated with media control or IL-4 and IL-13, with or …

Figure 7 with 1 supplement
IL-4 and IL-13 innate training enhances MoDM secreted TNFα following Mtb stimulation.

Cytokine secretion following MoDM 24 hr incubation with irradiated M. tuberculosis (Mtb) or media on Day 0 or on Day 6 as indicated. MoDM were previously incubated with media, IL-4 with IL-13 – with …

Figure 7—figure supplement 1
IL-4 and IL-13 innate training alters MoDM responses following PAM3CSK4 stimulation.

Cytokine secretion following MoDM 24 hr incubation with PAM3CSK4 or media on Day 0 or on Day 6 as indicated. MoDM were previously incubated with media, IL-4 with IL-13 – with or without the addition …

Author response image 1
Fig 1 and Fig 1-figure supplement 1 (n = 2 on Day 0, n = 1 on Day 6).
Author response image 2
Fig 6 and Fig 6-figure supplement 2 (n = 2 on Day 0, n = 1 on Day 6).
Author response image 3
Following BCG infection on Day 6, where BMDMs were incubated with media control or IL-4/13 on Day -1 for 24 hours.
Author response image 4
100 ng/ml IFNγ, 50 ng/ml LPS, 50 ng/ml each.

Nos2 expression after 24 hours.

Author response image 5
Author response image 6

Tables

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Strain, strain background (Escherichia coli, serotype R515)Lipopolysaccharide, LPSEnzoCat# ALX-581–007 L002
Strain, strain background (H. sapiens)Primary cell isolationBuffy packs from Irish Blood Transfusion ServiceN/A
Strain, strain background (M. musculus, C57BL/6JOlaHsd)Primary cell isolationIn-house coloniesC57BL/6JOlaHsdBoth sexes employed
Strain, strain background (Mycobacterium bovis)Bacille Calmette-Guérin (BCG) Denmark 1331Gift to Prof. Gordon from Prof. Behr, McGill University, CanadaN/A
Strain, strain background (Mycobacterium tuberculosis, strain H37Rv)Irradiated whole cells of M. tuberculosisBEI resourcesNR-49098Non-viable bacteria
Cell line (M. musculus)L929gift of Prof. Muñoz-Wolf, Trinity College, Dublin
gift of Prof. Sheedy, Trinity College, Dublin
N/ACell lines maintained in E. Lavelle and F. Sheedy labs.
AntibodyAnti-CD11b-APC-eFluor 780 (Rat monoclonal)Thermo Fisher ScientificCat# 47-0112-82, Clone M1/70FACS (0.1 μl per test)
AntibodyAnti-CD206-PE (Rat monoclonal)BioLegendCat# 141706, Clone C068C2FACS (0.4 μl per test)
AntibodyAnti-CD80-FITC (Hamster monoclonal)BD BiosciencesCat# 561954, Clone 16–10 A1FACS (0.15 μl per test)
AntibodyAnti-F4/80-PerCP-Cy5.5 (Rat monoclonal)Thermo Fisher ScientificCat# 45-4801-82, Clone BM8FACS (0.25 μl per test)
AntibodyAnti-Histone H3 (Mouse monoclonal)Active MotifCat# 39763WB (1/3,000)
AntibodyAnti-H3K4me3 (Rabbit polyclonal)AbcamCat# ab8580WB (1/1000)
AntibodyAnti- H3K9me2 (Mouse monoclonal)AbcamCat# ab1220WB (1/1000)
AntibodyAnti-H3K27me3 (Mouse monoclonal)Active MotifCat# 61017WB (1/1000)
AntibodyAnti-MHC class II-eFlour 450 (Rat monoclonal)Thermo Fisher ScientificCat# 48-5321-82, Clone M5/114.15.2FACS (0.2 μl per test)
AntibodyAnti-mouse IgG (Goat monoclonal)LI-CORCat# 925–32210WB (1/5000 dilution)
AntibodyAnti-Rabbit IgG (Goat monoclonal)LI-CORCat# 926–32211WB (1/2500)
AntibodyFc block: Anti-CD16/CD32 (Rat monoclonal)BD BiosciencesCat# 553142, Clone 2.4G2FACS (0.5 μl per test)
Sequence-based reagentActb _FPrimer BLASTInvitrogen Custom DNA OligosGCTTCTTTGCAGCTCCTTCGT
Sequence-based reagentActb _RPrimer BLASTInvitrogen Custom DNA OligosCGTCATCCATGGCGAACTG
Sequence-based reagentArg1 _FPrimer BLASTInvitrogen Custom DNA OligosTACAAGACAGGGCTCCTTTCAG
Sequence-based reagentArg1 _RPrimer BLASTInvitrogen Custom DNA OligosTGAGTTCCGAAGCAAGCCAA
Sequence-based reagentChitl3 _FPrimer BLASTInvitrogen Custom DNA OligosAAGCTCTCCAGAAGCAATCC
Sequence-based reagentChitl3 _RPrimer BLASTInvitrogen Custom DNA OligosAGAAGAATTGCCAGACCTGTGA
Sequence-based reagentLdha _FPrimer BLASTEurofins genomics (MWG)GAGACTTGGCTGAGAGCATAA
Sequence-based reagentLdha _RPrimer BLASTEurofins genomics (MWG)GATACATGGGACACTGAGGAA
Sequence-based reagentMyc _FPrimer BLASTInvitrogen Custom DNA OligosCAGCGACTCTGAAGAAGAGCA
Sequence-based reagentMyc _RPrimer BLASTInvitrogen Custom DNA OligosGACCTCTTGGCAGGGGTTTG
Sequence-based reagentNos2 _FPrimer BLASTInvitrogen Custom DNA OligosTCCTGGACATTACGACCCCT
Sequence-based reagentNos2 _RPrimer BLASTInvitrogen Custom DNA OligosCTCTGAGGGCTGACACAAGG
Sequence-based reagentPkm2 _FPrimer BLASTEurofins genomics (MWG)TGTCTGGAGAAACAGCCAAG
Sequence-based reagentPkm2_RPrimer BLASTEurofins genomics (MWG)CGAATAGCTGCAAGTGGTAGA
Sequence-based reagentRetnla _FPrimer BLASTInvitrogen Custom DNA OligosCAGCTGATGGTCCCAGTGAAT
Sequence-based reagentRetnla _RPrimer BLASTInvitrogen Custom DNA OligosAGTGGAGGGATAGTTAGCTGG
Sequence-based reagentSdha _FPrimer BLASTEurofins genomics (MWG)GGAACACTCCAAAAACAGACC
Sequence-based reagentSdha _RPrimer BLASTEurofins genomics (MWG)CCACCACTGGGTATTGAGTAGAA
Sequence-based reagentTbp _FPrimer BLASTInvitrogen Custom DNA OligosCAGGAGCCAAGAGTGAAGAACA
Sequence-based reagentTbp _RPrimer BLASTInvitrogen Custom DNA OligosAAGAACTTAGCTGGGAAGCCC
Peptide, recombinant proteinHeat-Shocked Bovine Serum Albumin (BSA)Thermo Fisher ScientificCat# 12881630
Peptide, recombinant proteinM-MLV reverse transcriptasePromegaCat# M3683
Peptide, recombinant proteinRecombinant human IFNγPeprotechCat# 300–02
Peptide, recombinant proteinRecombinant human IL-10PeprotechCat# 200–10
Peptide, recombinant proteinRecombinant human IL-13PeprotechCat# 200–13
Peptide, recombinant proteinRecombinant human IL-4PeprotechCat# 200–04
Peptide, recombinant proteinRecombinant human M-CSFProspec Protein SpecialistsCat# CYT-308
Peptide, recombinant proteinRecombinant murine IFNγPeprotechCat# 315–05
Peptide, recombinant proteinRecombinant murine IL-10PeprotechCat# 210–10
Peptide, recombinant proteinRecombinant murine IL-13PeprotechCat# 210–13
Peptide, recombinant proteinRecombinant murine IL-4PeprotechCat# 214–14
Commercial assay or kitBCA Protein Assay Kit (Pierce)Thermo Fisher ScientificCat# 23225
Commercial assay or kitGriess Reagent System kitPromegaCat# G2930
Commercial assay or kitHigh Pure RNA Isolation KitRocheCat# 11828665001
Commercial assay or kitHuman IL-10 ELISA Kit uncoatedInvitrogenCat# 88710688
Commercial assay or kitHuman IL-6 ELISA Kit uncoatedInvitrogenCat# 88706688
Commercial assay or kitHuman TNFα ELISA Kit uncoatedInvitrogenCat# 88734688
Commercial assay or kitMouse IL-10 ELISA MAXBioLegendCat# 431411
Commercial assay or kitMouse IL-6 ELISA MAXBioLegendCat# 431301
Commercial assay or kitMouse TNFα DuoSet ELISAR&D SystemsCat# DY410
Chemical compound, drug2-deoxyglucose, 2-DGSigma AldrichCat# D8375
Chemical compound, drugAntimycin-ASigma AldrichCat# A8674
Chemical compound, drugBPTESSigma AldrichCat# SML0601-5mg
Chemical compound, drugCpGInvivoGenCat# ODN M362
Chemical compound, drugFCCPSigma AldrichCat# C2920
Chemical compound, drugGlucoseSigma AldrichCat# G8270
Chemical compound, drugL-GlutamineGibcoCat# 25030–024
Chemical compound, drugGlycerolSigma AldrichCat# G2025
Chemical compound, drugMTASigma AldrichCat# D5011-25MG
Chemical compound, drugOligomycinSigma AldrichCat# 75351
Chemical compound, drugPAM3CSK4InvivoGenCat# tlrl-pms
Chemical compound, drugPoly I:CInvivoGenCat# tlrl-pic
Chemical compound, drugPyruvateSigma AldrichCat# P5280
Chemical compound, drugResiquimod/R848InvivoGenCat# tlrl-r848
Chemical compound, drugRotenoneSigma AldrichCat# R8875
Chemical compound, drugSodium ChlorideSigma AldrichCat# S9888
Chemical compound, drugValine-d8CK isotopesCat# DLM-488
Chemical compound, drugWGP DispersibleInvivoGenCat# tlrl-wgp
Software, algorithmFlowJo 7FlowJo LLC, Franklin Lakes, New Jerseyhttps://www.flowjo.com/solutions/flowjo
Software, algorithmImageJNational Institutes of Health and the Laboratory for Optical and Computational Instrumentationhttps://imagej.nih.gov/ij/
Software, algorithmMetaboAnalyst 5.0Xia Lab @ McGillhttps://www.metaboanalyst.ca/
Software, algorithmMicrosoft Office ExcelMicrosoft, Redmond, Washingtonhttps://products.office.com/en-au/excel
Software, algorithmPrism 8.2GraphPad Software, San Diego, Californiahttps://www.graphpad.com/scientific-software/prism/
Software, algorithmTracefinder 5.0Thermo Fisher Scientific, Waltham, Massachusettshttps://www.thermofisher.com/ie/en/home/industrial/mass-spectrometry/liquid-chromatography-mass-spectrometry-lc-ms/lc-ms-software/lc-ms-data-acquisition-software/tracefinder-software.html
Other4% PFA in PBSSanta Cruz BiotechnologyCat# NC0238527Fixing buffer for flow cytometry and confocal microscopy
OtherAcetonitrileThermo Fisher ScientificCat# 10001334Extraction buffer for Metabolomics
OtherDMEM (high glucose)Sigma AldrichCat# D5671Cell culture media (BMDMs)
OtherDMEM (no glucose)GibcoCat# 11966025Cell culture media (BMDMs)
OtherdNTP MixMeridian BioscienceCat# BIO-39028Nucleotides for cDNA synthesis
OtherFBSBioseraBatch# 015BS551Serum for cell culture media
OtherFixable Viability Stain 510InvitrogenCat# 564406Viability stain used for FACS
OtherHoechst 33342Thermo Fisher ScientificCat# 10150888DNA dye used for confocal microscopy
OtherKAPA SYBR FAST Rox low qPCR Kit Master MixSigma AldrichCat# KK4622Nucleic acid stain for qPCR
OtherLymphoprepStemcell TechnologiesCat# 07851Density gradient medium for isolating PBMCs
OtherMethanolThermo Fisher ScientificCat# 10284580Extraction buffer for Metabolomics
OtherMiddlebrook 7H11 powderSigma AldrichCat# M0428For making Mycobacterial culture media (agar plates)
OtherMiddlebrook 7H9 powderSigma AldrichCat# M0178For making Mycobacterial culture media
OtherModified Auramine-O stain and quencherScientific Device LaboratoryCat# 345–04 LMycobacterial stain for confocal micrsocopy
OtherPBS, sterileGibcoCat# 14190094Cell wash buffer
OtherPenicillin-StreptomycinGibcoCat# 15-070-063Antibiotics for cell culture
OtherPhalloidin-Alexa Fluor 647InvitrogenCat# A22287Stain actin for confocal microscopy
OtherRadio-Immunoprecipitation Assay (RIPA) bufferSigma AldrichCat# R0278-50MLCell lysis buffer
OtherRandom Hexamer Primer MixMeridian BioscienceCat# BIO-38028Primers for cDNA synthesis
OtherReverse Transcriptase BufferPromegaCat# A3561Buffer for cDNA synthesis
OtherRNAseOUTInvitrogenCat# 10777019RNAse inhibitor for cDNA synthesis
OtherRPMI 1640 GlutamaxGibcoCat# 21875034Cell culture media (MoDMs)
OtherSeahorse Calibration Fluid pH 7.4AgilentPart# 100840–000Extracellular flux calibration fluid
OtherSeahorse XF DMEM MediumAgilentCat# 103575–100Extracellular flux culture media
OtherTween20Sigma AldrichCat# P1379-1LDetergent for cell lysis and ELISA wash buffer
OtherVectashield mounting mediaVWRCat# 101098–042Mounting media for confocal microscopy
OtherWater, sterileBaxterCat# UKF7114Solvent

Additional files

Transparent reporting form
https://cdn.elifesciences.org/articles/74690/elife-74690-transrepform1-v2.docx
Source data 1

Macrophage Innate Training Induced by IL-4 and IL-13 Activation Enhances OXPHOS Driven Anti-Mycobacterial Responses Dataset.

Raw data, calculations and results of statistical analyses for all included figures.

https://cdn.elifesciences.org/articles/74690/elife-74690-data1-v2.xlsx

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