Mitochondrial respiration contributes to the interferon gamma response in antigen-presenting cells
- Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, United States
- Immunopathogenesis Section, Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, United States
- Department of Microbiology & Molecular Genetics, College of Osteopathic Medicine, Michigan State University, United States
Figures
Figure 1
Forward genetic screen to identify regulators of the IFNγ response.
(A) Representative histograms of the three selected cell surface markers targeted in macrophage CRISPR screens: MHCII, CD40, and PD-L1. Blue histograms indicate expression of each marker in …
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Figure 1—source data 1
Whole-genome profiling of macrophage MHCII, CD40, and PD-L1 expression.
- https://cdn.elifesciences.org/articles/65109/elife-65109-fig1-data1-v2.xlsx
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Figure 1—source data 2
k-means clustering of CRISPR-KO beta scores for regulators of the IFNγ response.
- https://cdn.elifesciences.org/articles/65109/elife-65109-fig1-data2-v2.xlsx
Figure 2
Global analysis of knockout (KO) libraries implicates mitochondrial complex I is a positive regulator of the IFNγ response.
(A) Rank plot of the combined analysis for all genome-wide KO screens. Gene ranks (x-axis) were determined by maximal likelihood estimation (MLE). Known positive (left) and negative (right) …
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Figure 2—source data 1
Whole-genome profiling of the IFNγ response in macrophages.
- https://cdn.elifesciences.org/articles/65109/elife-65109-fig2-data1-v2.xlsx
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Figure 2—source data 2
Gene set enrichment analysis (GSEA) to identify pathways that regulate the IFNγ response in macrophages.
- https://cdn.elifesciences.org/articles/65109/elife-65109-fig2-data2-v2.xlsx
Figure 3 with 1 supplement
Complex I is necessary for IFNγ-induced MHCII and PD-L1 expression.
Metabolic phenotypes in macrophage mutants were confirmed by measuring intracellular (IC) ATP abundance following culture in media containing only (A) glucose or (B) pyruvate. Values are normalized …
Figure 3—figure supplement 1
The complex I dependency of IFNγ signaling is independent of reactive oxygen and nitrogen radicals, and Hif-1α.
(A) sgNTC, sgNdufa1, and sgNdufa2 cells cultured in complete media and treated with or without oligomycin (2.5 μM) for 4 hr. Relative ATP levels were determined as in Figure 2A. (B) Intracellular …
Figure 4
Diminished mitochondrial function specifically limits IFNγ-dependent responses.
(A) TNF and IL-6 production by non-targeting control (NTC) or complex I mutant macrophages stimulated with Pam3CSK4 for 24 hr was determined by ELISA. Statistical testing between mutant and NTC …
Figure 5 with 1 supplement
Complex I is specifically required for IFNγ signaling in human cells.
(A) CD14+ monocytes from healthy human donors were differentiated into macrophages with GM-CSF. Mean fluorescence intensity (MFI) of cell surface markers PD-L1, ICAM1, CD40, and HLA-DR was …
Figure 5—figure supplement 1
Complex I is specifically required for IFNγ signaling in diverse human myeloid cells.
CD14+ monocytes were isolated from healthy human donors and stimulated directly (upper panel) or subsequent to differentiation into macrophages with M-CSF (lower panel). Mean fluorescence intensity …
Figure 6
Complex I inhibition reduces IFNγ receptor activity.
(A) PD-L1 transcript was quantified by qRT-PCR using ΔΔCt relative to β-actin in macrophages of the indicated genotype after stimulation with 10 ng/mL IFNγ. PD-L1 mean fluorescence intensity (MFI) …
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Figure 6—source data 1
Raw blots.
- https://cdn.elifesciences.org/articles/65109/elife-65109-fig6-data1-v2.zip
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Figure 6—source data 2
Labeled raw blots.
- https://cdn.elifesciences.org/articles/65109/elife-65109-fig6-data2-v2.zip
Figure 7 with 1 supplement
Mitochondrial respiration in antigen-presenting cells (APCs) is required for IFNγ-dependent T cell activation.
(A) Cell surface expression of MHCII (mean fluorescence intensity [MFI]) in macrophages (MF) or dendritic cells (DCs) derived from conditionally immortalized progenitor lines. IFNγ was added for 24 …
Figure 7—figure supplement 1
The metabolic dependencies for IFNγ signaling are preserved in macrophages derived from myeloid progenitor lines.
(A–C) Myeloid progenitors cells were conditionally immortalized by transducing murine bone marrow with an estrogen-dependent Hoxb8 transgene that maintains stem-like properties. Following …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (Mus musculus) | L3-Cas9+ | Kiritsy and Ankley et al. (co-submitted) | Primary BMDMs immortalized with J2 virus were transduced with Cas9 and single cell cloned | |
| Cell line (M. musculus) | EGFP-Cas9 iBMDMs | This paper | Primary BMDMs from Jackson Stock 026179 were immortalized with J2 virus | |
| Cell line (M. musculus) | sgNdufa1 EGFP-Cas9 iBMDMs | This paper | Cas9+ iBMDMs were transduced with Ndufa1 sgRNA | |
| Cell line (M. musculus) | sgNdufa2 EGFP-Cas9 iBMDMs | This paper | Cas9+ iBMDMs were transduced with Ndufa2 sgRNA | |
| Cell line (M. musculus) | Cas9+ C57BL/6J estradiol-inducible HoxB8 progenitors | This paper | Myeloid progenitors from Jackson stock 026179 were immortalized with HoxB8 retrovirus and maintained with 10 µM estradiol | |
| Cell line (M. musculus) | sgNdufa1 C57BL/6J estradiol-inducible HoxB8 progenitors | This study | Cas9+ HoxB8 cells were transduced with Ndufa1 sgRNA | |
| Cell line (M. musculus) | Cas9+ C57BL/6J estradiol-inducible HoxB8 progenitors | This paper | Myeloid progenitors from Jackson stock 003288 were immortalized with HoxB8 retrovirus and maintained with 10 µM estradiol | |
| Strain, strain background (M. musculus) | C57BL6J | Jackson Laboratories | Stock 000664 | |
| Strain, strain background (M. musculus) | C7 TCR-transgenic mice (specific for ESAT-6 antigen) | PMID:18779346 | Mice were donated to and maintained by the Behar laboratory | |
| Peptide, recombinant protein | ESAT-6 peptide (MTEQQW NFAGIEAAA) | New England Peptide | ||
| Recombinant DNA reagent | sgOpti | Addgene | RRID:Addgene_ 85681 | |
| Recombinant DNA reagent | sgOpti with blasticidin and zeocyin selection | Kiritsy and Ankley et al. (co- submitted) | sgOpti (RRID 85681) was modified with bacterial selection replaced with zeocyin and mammalian selection replaced with blasticidin | |
| Recombinant DNA reagent | sgOpti with hygromycin and kanamycin selection | Kiritsy and Ankley et al. (co-submitted) | sgOpti (RRID 85681) was modified with bacterial selection replaced with kanamycin and mammalian selection replaced with hygromycin | |
| Recombinant DNA reagent | VSVG | Addgene | RRID:Addgene_8454 | |
| Recombinant DNA reagent | psPax2 | Addgene | RRID:Addgene_12260 | |
| Antibody | MHCII-PE, clone M5/114.15.2 (rat monoclonal) | BioLegend | RRID:AB_313323 | FC (1:800) |
| Recombinant DNA reagent | Mouse CRISPR KO pooled library (BRIE) | Addgene | RRID:Addgene_7363 | |
| Chemical compound, drug | Rotenone | Sigma | Cat# R8875 | |
| Chemical compound, drug | Oligomycin | Cayman | Cat# 11342 | |
| Chemical compound, drug | CCCP | Cayman | Cat# 25458 | |
| Chemical compound, drug | 1400W | Cayman | Cat# 81520 | |
| Chemical compound, drug | N-Acetyl cysteine | Cayman | Cat# 20261 | |
| Chemical compound, drug | Dimethyloxallyl glycine (DMOG) | Cayman | Cat# 71210 | |
| Chemical compound, drug | UK5099 | Cayman | Cat# 16,980 | |
| Chemical compound, drug | 2-Deoxyglucose (2-DG) | Cayman | Cat# 14325 | |
| Chemical compound, drug | MitoTEMPO hydrate | Cayman | Cat# 16621 | |
| Chemical compound, drug | Sodium azide | Sigma | Cat# S2002 | |
| Chemical compound, drug | Antimycin A | Sigma | Cat# A8674 | |
| Chemical compound, drug | Pam3CSK4 | Invivogen | Cat# tlrl-pms | |
| Chemical compound, drug | Linezolid (LZD) | Gift from Clifton Barry (used here PMID:32477361) | ||
| Antibody | Purified anti-STAT1 antibody clone A15158C (mouse monoclonal) | BioLegend | Cat# 603701; RRID:AB_2749867 | WB (1:1000) |
| Antibody | Phospho-Stat1 Tyr701, clone 58D6 (rabbit monoclonal) | Cell Signaling Technology | Cat# 5375; RRID:AB_10860071 | WB (1:1000) |
| Antibody | Purified anti-STAT1 Phospho Ser727 antibody, clone A15158B (mouse monoclonal) | BioLegend | Cat# 686408; RRID:AB_2650782 | WB (1:1000) |
| Antibody | Jak2 XP, clone D2E12 (rabbit monoclonal) | Cell Signaling Technology | Cat# 4040; RRID:AB_10691469 | WB (1:500) |
| Antibody | Phospho-Jak2 (Tyr1007/1008) antibody (unknown) | Cell Signaling Technology | Cat# 3771; RRID:AB_330403 | WB (1:500) |
| Antibody | Biotin anti-mouse CD119, IFNγ Rα chain antibody clone 2E2 (Armenian hamster monoclonal) | BioLegend | Cat# 112803; RRID:AB_2123476 | WB (1:1000) |
| Antibody | Anti-mouse β-actin antibody, clone C4 (mouse monoclonal) | Santa Cruz Biotechnology | Cat# sc-51850; RRID:AB_629337 | WB (1:2000) |
| Antibody | CD274-Bv421 clone 10F.9G2 (rat monoclonal) | BioLegend | RRID:AB_10897097 | FC (1:400) |
| Commercial assay or kit | Zombie Aqua Fixable Viability Kit | BioLegend | Cat# 423101 | FC (1:100) |
| Commercial assay or kit | DNeasy Blood and Tissue Kit | Qiagen | Cat# 69504 | |
| Antibody | CD40 APC anti-mouse CD40 antibody, clone 3/23 (rat monoclonal) | BioLegend | Cat# 124611;RRID:AB_1134081 | FC (1:200) |
| Antibody | Human anti-CD54, clone HCD54 (mouse monoclonal) | BioLegend | Cat# 322718; RRID:AB_2248731 | FC (1:400) |
| Antibody | Human anti-CD40 clone 5C3 (mouse monoclonal) | BioLegend | Cat# 334307; RRID:AB_1186060 | FC (1:400) |
| Antibody | Human anti-CD274, B7-H1, PD-L1, clone 29E (mouse monoclonal) | BioLegend | Cat# 329713; RRID:AB_10901164 | FC (1:400) |
| Antibody | Human anti-HLA-DR antibody, clone L243 (mouse monoclonal) | BioLegend | Cat# 307657; RRID:AB_2572100 | FC (1:400) |
| Antibody | Anti-mouse IFNγ antibody (rat monoclonal) | BioLegend | Cat# 505807; RRID:AB_315401 | FC (1:200) |
| Peptide, recombinant protein | Murine IL-12 | PeproTech | Cat# 210-12 | |
| Peptide, recombinant protein | Human GM-CSF | PeproTech | Cat# 300-03 | |
| Peptide, recombinant protein | Human IFN gamma | PeproTech | Cat# 300-02 | |
| Peptide, recombinant protein | Murine TNF | PeproTech | Cat# 315-01A | |
| Antibody | Anti-IL4 clone: 11B11 (rat monoclonal) | BioLegend | RRID:AB_2750407 | Neutralization (1:500) |
| Antibody | Goat anti-rabbit HRP (goat polyclonal) | Invitrogen | Cat# 31460 | WB (1:1000) |
| Antibody | Goat anti-mouse HRP (goat polyclonal) | Invitrogen | Cat# 31430 | WB (1:1000) |
| Commercial assay or kit | One-Step RT PCR Kit | Qiagen | Cat# 210215 | |
| Commercial assay or kit | Luna Universal One-Step RT-qPCR Kit | NEB | Cat# E3005 | |
| Commercial assay or kit | Trizol | Thermo Fisher Scientific | Cat# 15596026 | |
| Commercial assay or kit | CellTiter-Glo 2.0 Cell Viability Assay | Promega | Cat# G9241 | |
| Commercial assay or kit | Seahorse assay media | Agilent | Cat# 103575-100 | |
| Software, algorithm | MAGECK | PMID:25476604 | ||
| Peptide, recombinant protein | Interferon gamma | BioLegend | Cat# 575308 | |
| Commercial assay or kit | MojoSORT – Mouse CD4 Naïve T cell Isolation Kit | BioLegend | Cat# 480040 | |
| Commercial assay or kit | MojoSort Human CD14 Nanobeads | BioLegend | Cat# BioLegend 480093 | |
| Commercial assay or kit | IL6 ELISA-max | BioLegend | Cat# 431301 | |
| Commercial assay or kit | TNF ELISA-max | BioLegend | Cat# 430901 | |
| Commercial assay or kit | Human IL1b | R&D Systems | Cat# DY201 | |
| Commercial assay or kit | Human TNF-alpha DuoSet ELISA | R&D Systems | Cat# DY210 | |
| Commercial assay or kit | Greiss reagent | Promega | G2930 | |
| Sequence-based reagent | All oligonucleotide sequences are contained in Supplementary file 1. | This paper | All oligonucleotide sequences are contained in Supplementary file 1 |
Additional files
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Supplementary file 1
Oligonucleotides used in this study.
- https://cdn.elifesciences.org/articles/65109/elife-65109-supp1-v2.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/65109/elife-65109-transrepform1-v2.docx
