HIF1α is required for NK cell metabolic adaptation during virus infection
- Rheumatology Division, Washington University School of Medicine, United States
- Department of Chemistry, Department of Medicine, Washington University, United States
- Department of Biomedical Research, Center for Genes, Environment and Health, National Jewish Health, United States
Figures
Figure 1 with 1 supplement
Hypoxia-inducible factor-1α (HIF1α) expression in natural killer (NK) cells.
(A) HIF1α protein expression in splenic NK cells (black) ex vivo compared to FMO control (gray) by flow cytometry. Representative experiment of two independent experiments. (B) Comparison of …
Figure 1—figure supplement 1
Expression of hypoxia-inducible factor-1α (HIF1α) in natural killer (NK) cells.
(A) Representative histogram of HIF1α expression in splenic and liver NK cells direct ex vivo. Data are from two independent experiments from five mice per group. (B) Splenocytes from C57BL/6 mice …
Figure 2 with 1 supplement
Natural killer (NK) cells require hypoxia-inducible factor-1α (HIF1α) for an optimal response to virus infection.
(A) 1αKO or FL control mice were infected with 50K plaque forming unit (pfu) then monitored for weight loss over 7 days. Each data represents four mice from two independent experiments. (B) Splenic …
Figure 2—figure supplement 1
Normal development in hypoxia-inducible factor-1α (HIF1α) KO natural killer (NK) cells.
(A) Quantification of NK cells absolute numbers (left) and frequencies (right) of NK cells in Flox controls and 1αKO direct ex vivo. Data are from three independent experiments with 6–9 mice per …
Figure 3 with 1 supplement
Hypoxia-inducible factor-1α (HIF1α) is dispensable for natural killer (NK) cell effector function.
(A) Representative histograms (top) and quantification (bottom) of CD69 MFI on NK cells and frequency of KLRG1+ and IFNγ+ NK cells at day 1.5 post-infection (pi) of 1αKO or FL control mice. Data are …
Figure 3—figure supplement 1
Ly49H + and Ly49H- hypoxia-inducible factor-1α (HIF1α) KO natural killer (NK) cells have similar activation compared to Flox controls.
(A–C) 1αKO or Flox mice infected with murine cytomegalovirus (MCMV) received a dose of 50K plaque forming unit (pfu) and analyzed as indicated. (A) Histogram (left) and quantification of frequency …
Figure 4 with 1 supplement
Cell division in hypoxia-inducible factor-1α (HIF1α) KO natural killer (NK) cells is normal but numbers are reduced.
(A, B) 1αKO or FL control mice infected with murine cytomegalovirus (MCMV) received a dose of 50K plaque forming unit (pfu) and analyzed as indicated. (A) Ki67 expression with representative …
Figure 4—figure supplement 1
Proliferation is normal in hypoxia-inducible factor-1α (HIF1α) KO natural killer (NK) cells.
(A–D) 1αKO or Flox mice infected with murine cytomegalovirus (MCMV) received a dose of 50K plaque forming unit (pfu) and analyzed as indicated. (A) Representative contour plots (left) and …
Figure 5 with 1 supplement
Hypoxia-inducible factor-1α (HIF1α) KO natural killer (NK) cells are predisposed to apoptosis.
(A) mRNA transcripts for levels of apoptosis associated genes Bcl-2, Bim, and cytochrome c was measured in 1αKO or FL control NK cells at day 3 post IL-2 stimulation. Data are from two independent …
Figure 5—figure supplement 1
Hypoxia-inducible factor-1α (HIF1α) KO natural killer (NK) cells are predisposed to cell death.
(A–C) Enriched NK cells stimulated with 2000 units/ml of IL-2 for 3 days then harvested and analyzed as indicated. Data represents four mice per time point from two independent experiments. (A) …
Figure 6 with 1 supplement
Impaired glycolytic activity contributes to impaired survival of hypoxia-inducible factor-1α (HIF1α) KO natural killer (NK) cells.
Enriched NK cells from 1αKO or FL control mice were IL-2 treated for 3 days then collected and analyzed as follows. (A) mRNA transcript levels of HIF1α for both cohort of mice measured with qRT-PCR. …
Figure 6—figure supplement 1
Hypoxia-inducible factor-1α (HIF1α) KO natural killer (NK) cells have dysregulated metabolism.
Enriched NK cells from 1αKO and Flox mice were subjected to a mitochondrial stress test, then, oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured direct ex vivo. …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Mus musculus) | β2m KO; B6.129P2-B2mtm1Unc/J | Jackson Laboratories | IMSR Cat# JAX:002070, RRID:IMSR_JAX:002070 | |
| Strain, strain background (Mus musculus) | HIF1a Flox; B6.129-Hif1atm3Rsjo/J | Jackson Laboratories | IMSR Cat# JAX:007561, RRID:IMSR_JAX:007561 | |
| Strain, strain background (Mus musculus) | NKp46iCre; Ncr1tm1.1(icre)Viv/J | Eric Vivier | MGI Cat# 5309017, RRID:MGI:5309017 | |
| Strain, strain background (Mus musculus) | Wild-type; C57BL/6NCr | Charles River | MGI Cat# 5657970, RRID:MGI:5657970 | |
| Strain, strain background (murine cytomegalovirus) | MCMV | Cheng et al., 2010 | ||
| Antibody | Anti-SQSTM1/p62 Alexa Fluor 647 (rabbit monoclonal) | Abcam | Cat# ab194721 | Flow (1:500) |
| Antibody | Anti-CD49a Brilliant Violet 421 (Armenian hamster monoclonal) | BD Biosciences | Cat# 740046, RRID:AB_2739815 | Flow (1:100) |
| Antibody | Anti-Bcl-2 Alexa Fluor 488 (mouse monoclonal) | BioLegend | Cat# 633506, RRID:AB_2028390 | Flow (1:100) |
| Antibody | Anti-Cytochrome c Alexa Fluor 647 (mouse monoclonal) | BioLegend | Cat# 612310, RRID:AB_2565241 | Flow (1:100) |
| Antibody | Anti-DNAM-1 PE (rat monoclonal) | BioLegend | Cat# 132006, RRID:AB_1279173 | Flow (1:100) |
| Antibody | Anti-HIF1a PE (mouse monoclonal) | BioLegend | Cat# 359704, RRID:AB_2562423 | Flow (5:100) |
| Antibody | Anti-Ly49A PE (rat monoclonal) | BioLegend | Cat# 116808, RRID:AB_313759 | Flow (1:200) |
| Antibody | Anti-NK1.1 BV650 (mouse monoclonal) | BioLegend | Cat# 108736, RRID:AB_2563159 | Flow (1:100) |
| Antibody | Anti-Mcl-1 PE (rabbit monoclonal) | Cell Signaling Technology | Cat# 65617, RRID:AB_2799688 | Flow (1:50) |
| Antibody | APC-AffiniPure F(ab')2 Fragment Donkey Anti-Goat IgG (H + L) (min X Ck,GP,Sy Hms,Hrs,Hu,Ms,Rb,Rat Sr Prot) antibody (polyclonal) | Jackson ImmunoResearch Labs | Cat# 705-136-147, RRID:AB_2340407 | Flow (1:300) |
| Antibody | Anti-Ly49H FITC (mouse monoclonal) | Made in-house FACS | Flow (1:100) | |
| Antibody | Anti-Bim (rabbit monoclonal) | Thermo Fisher Scientific | Cat# MA5-14848, RRID:AB_10981505 | Flow (1:200) |
| Antibody | Anti-CD3e AF700 (Syrian hamster monoclonal) | Thermo Fisher Scientific | Cat# 56-0033-82, RRID:AB_837094 | Flow (2:100) |
| Antibody | Anti-CD19 Alexa Fluor 700 (rat monoclonal) | Thermo Fisher Scientific | Cat# 56-0193-80, RRID:AB_837082 | Flow (1:100) |
| Antibody | Anti-CD11b eFluor 450 (rat monoclonal) | Thermo Fisher Scientific | Cat# 48-0112-82, RRID:AB_1582236 | Flow (1:100) |
| Antibody | Anti-CD27 PE-Cy7 (Armenian hamster monoclonal) | Thermo Fisher Scientific | Cat# 25-0271-82, RRID:AB_1724035 | Flow (1:100) |
| Antibody | Anti-CD49b APC (rat monoclonal) | Thermo Fisher Scientific | Cat# 17-5971-82, RRID:AB_469485 | Flow (1:100) |
| Antibody | Anti-CD69 PE-Cy7 (Armenian hamster monoclonal) | Thermo Fisher Scientific | Cat# 25-0691-82, RRID:AB_469637 | Flow (1:100) |
| Antibody | Anti-Granzyme B APC (mouse monoclonal) | Thermo Fisher Scientific | Cat# MHGB05, RRID:AB_10373420 | Flow (5:100) |
| Antibody | Anti-IFNg eFluor450 (rat monoclonal) | Thermo Fisher Scientific | Cat# 48-7311-82,, RRID:AB_1834366 | Flow (1:100) |
| Antibody | Anti-Ki-67 eFluor 450 (rat monoclonal) | Thermo Fisher Scientific | Cat# 48-5698-80, RRID:AB_1115115 | Flow (1:100) |
| Antibody | Anti-KLRG1 APC (Syrian hamster monoclonal) | Thermo Fisher Scientific | Cat# 17-5893-82, RRID:AB_469469 | Flow (1:100) |
| Antibody | Anti-Ly49G PE (mouse monoclonal) | Thermo Fisher Scientific | Cat# 12-5885-82, RRID:AB_466005 | Flow (2:100) |
| Antibody | Anti-Ly49H APC (mouse monoclonal) | Thermo Fisher Scientific | Cat# 17-5886-82, RRID:AB_10598809 | Flow (1:100) |
| Antibody | Anti-Ly49I FITC (mouse monoclonal) | Thermo Fisher Scientific | Cat# 11-5895-85, RRID:AB_465302 | Flow (1:100) |
| Antibody | Anti-NKG2A PE (mouse monoclonal) | Thermo Fisher Scientific | Cat# 12-5897-82, RRID:AB_46602 | Flow (2:100) |
| Antibody | Anti-NKp46 PerCPeFluor710 (rat monoclonal) | Thermo Fisher Scientific | Cat#: 46-3351-82, RRID:AB_1834441 | Flow (1:100) |
| Other | Viability stain eFluor 506 | Thermo Fisher Scientific | Cat# 65-0866-14 | Flow (1:1000) |
| Other | Viability stain eFluor 780 | Thermo Fisher Scientific | Cat# 65-0865-14 | Flow (1:1000) |
| Sequence-based reagent | Actin b | IDT DNA | TAQman assay | Forward: 5′-AGCTCATTGTAGAAGGTGTGG-3′;reverse: 5′-GGTGGGAATGGGTCAGAAG-3′;probe: 5′-TTCAGGGTCAGGATACCTCTCTTGCT-3′ |
| Sequence-based reagent | Bcl-2 | IDT DNA | PowerSYBR Green PCR Master Mix | Forward: 5'-CCTGTGGATGACTGAGTACCTG-3'; reverse; 5'-AGCCAGGAGAAATCAAACAGAGG-3' |
| Sequence-based reagent | Bim | IDT DNA | PowerSYBR Green PCR Master Mix | Forward: 5'-GGAGATACGGATTGCACAGGAG-3'; reverse: 5'-CTCCATACCAGACGGAAGATAAAG-3' |
| Sequence-based reagent | Cytochrome c | IDT DNA | PowerSYBR Green PCR Master Mix | Forward: 5'-GAGGCAAGCATAAGACTGGACC-3'; reverse: 5'-ACTCCATCAGGGTATCCTCTCC-3' |
| Sequence-based reagent | gapdh | IDT DNA | PowerSYBR Green PCR Master Mix | Forward: 5'-GTTGTCTCCTGCGACTTCA-3'; reverse: 5'-GGTGGTCCAGGGTTTCTTA-3' |
| Sequence-based reagent | glut1 | IDT DNA | PowerSYBR Green PCR Master Mix | Forward: 5'-AAGAAGCTGACGGGTCGCCTCATGC-3'; reverse: 5'-TGAGAGGGACCAGAGCGTGGTG-3' |
| Sequence-based reagent | gpi1 | IDT DNA | PowerSYBR Green PCR Master Mix | Forward: 5'-GTTGCCTGAAGAGGCCAGG-3'; reverse: 5'-GCTGTTGCTTGATGAAGCTGATC-3' |
| Sequence-based reagent | hif1a | IDT DNA | PowerSYBR Green PCR Master Mix | Forward: 5'-CATCAGTTGCCACTTCCCCA-3'; reverse: 5'-GGCATCCAGAAGTTTTCTCACAC-3' |
| Sequence-based reagent | hk2 | IDT DNA | PowerSYBR Green PCR Master Mix | Forward: 5'-GGAGAGCACGTGTGACGAC-3'; reverse: 5'-GATGCGACAGGCCACAGCA-3', |
| Sequence-based reagent | hprt | IDT DNA | PowerSYBR Green PCR Master Mix | Forward: 5'- CTGGTGAAAAGGACCTCTCGAAG-3'; reverse: 5'- CCAGTTTCACTAATGACACAAACG-3' |
| Sequence-based reagent | ldha | IDT DNA | PowerSYBR Green PCR Master Mix | Forward: 5'-CACAAGCAGGTGGTGGACAG-3'; reverse: 5'-AACTGCAGCTCCTTCTGGATTC-3' |
| Sequence-based reagent | mcl-1 | IDT DNA | PowerSYBR Green PCR Master Mix | Forward: 5'-AGCTTCATCGAACCATTAGCAGAA-3'; reverse: 5'-CCTTCTAGGTCCTGTACGTGGA-3' |
| Sequence-based reagent | mcmv ie1 | IDT DNA | TAQman assay | Forward: 5′-CCCTCTCCTAACTCTCCCTTT-3′;reverse: 5′-TGGTGCTCTTTTCCCGTG 3′;probe: 5′-TCTCTTGCCCCGTCCTGAAAACC-3′ |
| Sequence-based reagent | pdk1 | IDT DNA | PowerSYBR Green PCR Master Mix | Forward: 5'-GATTCAGGTTCACGTCACGCT-3'; reverse: 5'-GACGGATTCTGTCGACAGAG-3' |
| Sequence-based reagent | pkm2 | IDT DNA | PowerSYBR Green PCR Master Mix | Forward: 5'-CAGGAGTGCTCACCAAGTGG-3'; reverse: 5'-CATCAAGGTACAGGCACTACAC-3' |
| Commercial assay or kit | XF Cell Mito Stress Test Kit | Agilent | Cat# 103015-100 | |
| Commercial assay or kit | CYTO-ID Autophagy detection kit | Enzo Life Sciences, Inc. | Cat# ENZ-51031-0050 | |
| Commercial assay or kit | Cell Event Caspase 3/7 Green Flow Cytometry Assay Kit | Thermo Fisher Scientific | Cat# C10423 | |
| Chemical compound, drug | Recombinant mouse IL-18 | MBL | Cat# B002-5 | |
| Chemical compound, drug | Recombinant murine IL-15 | Peptrotech | Cat# 210-15 | |
| Chemical compound, drug | Interferon-beta | PBL Assay Science | Cat# 12401-1, RRID:AB_605469 | |
| Chemical compound, drug | Percoll | Sigma | Cat# P1644 | |
| Software, algorithm | FlowJo 10 | Treestar | https://www.flowjo.com/; RRID:SCR_008520 | |
| Software, algorithm | Prism 6 | GraphPad | https://www.graphpad.com/; RRID:SCR_002798 |
