1. Immunology and Inflammation
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Glutathione de novo synthesis but not recycling process coordinates with glutamine catabolism to control redox homeostasis and directs murine T cell differentiation

  1. Gaojian Lian
  2. JN Rashida Gnanaprakasam
  3. Tingting Wang
  4. Ruohan Wu
  5. Xuyong Chen
  6. Lingling Liu
  7. Yuqing Shen
  8. Mao Yang
  9. Jun Yang
  10. Ying Chen
  11. Vasilis Vasiliou
  12. Teresa A Cassel
  13. Douglas R Green
  14. Yusen Liu
  15. Teresa WM Fan
  16. Ruoning Wang  Is a corresponding author
  1. The Research Institute at Nationwide Children's Hospital, Ohio State University, United States
  2. University of South China, China
  3. St. Jude Children’s Research Hospital, United States
  4. Yale University, United States
  5. University of Kentucky, United States
  6. The Research Institute at Nationwide Children's Hospital, Ohio State University, Ohio, United States
Research Article
Cite this article as: eLife 2018;7:e36158 doi: 10.7554/eLife.36158
6 figures, 1 table and 2 additional files

Figures

Figure 1 with 1 supplement
De novo synthesis but not recycling of GSSG is required for producing GSH and fine-tuning ROS upon TCR stimulation.

(A) Diagram of GSH biosynthesis, with metabolic pathways highlighted in red and enzymes highlighted in blue. (B) Naive CD4+T cells from WT and Gclm KO (left), or WT (CD4-Cre-, Gclcfl/fl) and Gclc KO (CD4-Cre+, Gclcfl/fl, (middle), or WT and Gsr KO (right) were activated by plate-bound anti-CD3 plus anti-CD28 for 24 hr, followed by the measurement of GSH levels. (C) Naive CD4+T cells from WT and Gclm KO (left), or WT (CD4-Cre-, Gclcfl/fl) and Gclc KO (CD4-Cre+, Gclcfl/fl, (middle), or WT and Gsr KO (right) were activated by plate-bound anti-CD3 plus anti-CD28 for 24 hr, followed by the measurement of ROS levels. Data in Figure 1B–C are representative of two independent experiments. Data represent the mean ± S.D.

https://doi.org/10.7554/eLife.36158.002
Figure 1—source data 1

Source data for B and C.

https://doi.org/10.7554/eLife.36158.005
Figure 1—figure supplement 1
TCR stimulation drives GSH and ROS production in T cells.

(A–B) Naive CD4 +T cells from C57BL/6 mice were either cultured in the presence of IL-7 (naive) or activated by plate-bound anti-CD3 and anti-CD28 for 24 hr, followed by measuring intracellular GSH (A) and ROS (B) by FACS. (C) RNAs were isolated from naïve or activated T cells for indicated times, and used for real-time qPCR analyses of indicated genes. Expression levels in naive cells were set to 1. (D) The protein levels of Gclm (left) or Gclc (middle) in total T cells from mice with indicated genotypes were determined by western blot. RNAs were isolated from WT or Gsr KO T cells and used for real-time qPCR analyses of Gsr gene. Expression levels in WT sample were set to 1. Data in Figure A-C are representative of two independent experiments. Data are represented the mean ± S.D.

https://doi.org/10.7554/eLife.36158.003
Figure 1—figure supplement 1—source data 1

Source data for A, B, C and D.

https://doi.org/10.7554/eLife.36158.004
Figure 2 with 1 supplement
Severe depletion of GSH by blocking de novo synthesis suppresses T cell activation and proliferation.

(A–C) Naive CD4 +T cells from WT and Gclm KO (A), or WT (CD4-Cre-, Gclcfl/fl) and Gclc KO (CD4-Cre+, Gclcfl/fl) (B), or WT and Gsr KO (C) mice were activated by plate-bound anti-CD3 plus anti-CD28 for 24 hr, followed by cell surface expression of CD25 (upper panel) and CD69 (lower panel). (D–F) Cell proliferation of active CD4 +T cells (72 hr) with indicated genotypes was determined by CFSE dilution. (G) Naive CD4 +T cells isolated from mice with indicated genotypes were activated by plate-bound anti-CD3 and anti-CD28 for 24 hr. Cell viability was determined by FACS. Figure 2A–G are representative of three independent experiments.

https://doi.org/10.7554/eLife.36158.006
Figure 2—figure supplement 1
Severe depletion of GSH by blocking de novo synthesis suppresses T cell activation and proliferation.

(A–C) Distribution of CD4+ and CD8+ cells in the spleen, lymph nodes or thymus from mice with indicated genotypes were determined by FACS. Figure 2A -D are representative of two independent experiments (D) Bar graph represents the number of T cells calculated from total splenocytes, lymph node cells and thymocytes.

https://doi.org/10.7554/eLife.36158.007
Figure 2—figure supplement 1—source data 1

Source data for A, B, C and D.

https://doi.org/10.7554/eLife.36158.008
Figure 3 with 1 supplement
Ablation of de novo synthesis but not recycling of GSSG reciprocally alters TH17 and iTreg cell differentiation.

(A–F) Naive CD4+ T cells from WT and Gclm KO, or WT (Cre-,) and CreER+ (Gclc-KO- in the presence of 100 nM of 4-hydroxytamoxifen (4OHT)), or WT and Gsr KO mice were stained with 4 µm CFSE and differentiated under TH17 or iTreg -inducing conditions for 5 days, followed by intracellular staining of IL-17 and Foxp3. (G–H) mice with indicated genotypes were immunized with MOG to induce EAE and pathological progressions were scored daily. Data in Figure 4A–H are representative of two-three independent experiments.

https://doi.org/10.7554/eLife.36158.009
Figure 3—source data 1

Source data for G and H.

https://doi.org/10.7554/eLife.36158.012
Figure 3—figure supplement 1
Ablation of de novo synthesis but not recycling of GSSG reciprocally alters TH17 and iTreg cell differentiation.

(A) The protein levels of Gclc in cells collected two days following 4OHT treatment were determined by western blot. (B) Naïve CD4+ T cells from WT (CD4-Cre-, Gclcfl/fl) and Gclc KO (CD4-Cre+, Gclcfl/fl) mice were differentiated under TH17-inducing conditions with or without 5 mM N-acetyl cysteine (NAC) for 5 days, followed by intracellular staining of IL-17. (C) Naive CD4+ T cells were activated for 24 hr with or without 5 mM NAC, followed by measuring ROS and (D) GSH by FACS. (E) WT, Gclm-/- (KO) and CD4-Cre, Gclcfl/fl (KO) mice were immunized with MOG/CFA. After 20 days, cervical spinal cord sections were analyzed by H and E (bars, 200 μm) and anti-Mac2 and anti-CD3 immunohistochemistry (bars, 50 μm).Data are representative of two independent trials. Data in Figure A-D are representative of two-three independent experiments.

https://doi.org/10.7554/eLife.36158.010
Figure 3—figure supplement 1—source data 1

Source data for C and D.

https://doi.org/10.7554/eLife.36158.011
Figure 4 with 1 supplement
TH17 cells preferentially maintain higher level of GSH than iTreg cells.

(A–B) Naive CD4+ T cells from C57BL/6 mice were differentiated under iTreg or TH17–inducing conditions and cells were collected at indicated times, followed by measuring intracellular GSH (A) and ROS (B) by FACS. (C) Naive CD4+ T cells from C57BL/6 mice were differentiated under TH17 or iTreg–inducing conditions for 5 days. The intracellular levels of GSH and GSSG were determined by mass spectrometry. (D) Naive CD4+T cells from WT and Gclm KO (top) or WT (CD4-Cre-, Gclcfl/fl) and Gclc KO (CD4-Cre+, Gclcfl/fl, (bottom) were differentiated under TH17-inducing conditions for 5 days, followed by the measurement of GSH levels. Data in Figure 4A–D are representative of three independent experiments. Data represent the mean ± S.D.

https://doi.org/10.7554/eLife.36158.013
Figure 4—source data 1

Source data for A, B, C and D.

https://doi.org/10.7554/eLife.36158.016
Figure 4—figure supplement 1
De novo synthesis but not recycling of GSSG is required for providing GSH and suppressing ROS during TH17 cell differentiation.

(A) RNAs were isolated from cells differentiated under TH17 or iTreg-inducing conditions for indicated times, and used for real-time qPCR analyses of indicated genes. Expression levels in day 0 were set to 1 (B–D) Naive CD4+ T cells from WT and Gclm KO, or WT (CD4-Cre-, Gclcfl/fl) and Gclc KO (CD4-Cre+, Gclcfl/fl), or WT and Gsr KO mice were differentiated under TH17-inducing conditions for 5 days, followed by measuring ROS by FACS. Data in Figure B-D are representative of two independent experiments. Data represent the mean ±S.D.

https://doi.org/10.7554/eLife.36158.014
Figure 4—figure supplement 1—source data 1

Source data for A, B and C.

https://doi.org/10.7554/eLife.36158.015
Figure 5 with 1 supplement
DMF suppresses TH17 differentiation by augmenting ROS generation.

(A) Naive CD4+ T cells from C57BL/6 mice were differentiated under TH17 or iTreg-inducing conditions with or without H2O2 (1 µM) for 5 days, followed by intracellular staining of IL-17 and Foxp3. (B) Cell proliferation of active CD4+ T cells (72 hr) with or without H2O2 (1 µM) was determined as CFSE dilution. (C–D) Naive CD4+ T cells from C57BL/6 mice were differentiated under TH17-inducing conditions with indicated dose of DMF for 5 days, followed by intracellular staining of IL-17 (C) and ROS (D). (E) Naive CD4+ T cells from C57BL/6 mice were differentiated under TH17-inducing conditions with indicated treatment for 5 days, followed by intracellular staining of IL-17. Data in Figure 5 are representative of two-three independent experiments. Data represent the mean ±S.D.

https://doi.org/10.7554/eLife.36158.017
Figure 5—figure supplement 1
DMF suppresses TH17 differentiation through augmenting ROS generation.

(A) Cell proliferation of active CD4 +T cells (72 hr) with indicated treatments was determined as CFSE dilution (B). Naive CD4 +T cells from C57BL/6 mice were differentiated with 5 and 20 µM of DMF under iTreg cell -inducing conditions for 5 days, followed by intracellular staining of Foxp3.

https://doi.org/10.7554/eLife.36158.018
Figure 6 with 2 supplements
Glutamate that fuels GSH de novo synthesis is partially derived from glutamine in T cells.

(A) Diagram of metabolic steps linked to the GSH production, with metabolic pathways highlighted in red. (B) Naive CD4+ T cells from C57BL/6 mice were differentiated under TH17 and iTreg–inducing conditions for 5 days, followed by culturing in media containing 13C515N2-glutamine. The intracellular levels of Glutamate and GSH including 13C-, 13C,15N-, and 12C-unlabeled forms were determined by IC-UHRFTMS. (C) Naive CD4+ T cells from C57BL/6 mice were differentiated under TH17 or iTreg cell–inducing conditions for 5 days, were used for measuring the generation of 14CO2 from [U-14C]-glutamine (glutaminolysis), from [2-14C]-pyrvuate (TCA). (D–E) Naive CD4+ T cells from C57BL/6 mice were differentiated in completed, glutamine-free (Q free) or glucose-free (G free) medium under TH17 or iTreg cell-inducing conditions for 5 days, followed by intracellular staining of IL-17 and Foxp3. (F) Naive CD4+ T cells from C57BL/6 mice were activated in complete medium for 24 hr and cells were washed with PBS and switch to conditional medium in presence or absence of glutamine, 3 mM αKG or 100 μM hypoxanthine and 16 μM thymidine (HT) for 5 days followed by intracellular staining of IL-17 and (G) and ROS. Data represent the mean ±S.D.

https://doi.org/10.7554/eLife.36158.020
Figure 6—source data 1

Source data for B, C and G.

https://doi.org/10.7554/eLife.36158.024
Figure 6—figure supplement 1
Glutamine catabolism is required for driving TH17 and iTreg cell differentiation.

(A) Naive CD4 +T cells from C57BL/6 mice were differentiated with TH17 cell -inducing conditions for 5 days in complete medium and cells were washed and cultured in Q-free medium as indicated time followed by the measurement of GSH. (B–C) RNAs were isolated from cells differentiated under TH17 or iTreg-inducing conditions for 3–5 days, and used for real-time qPCR analyses of indicated metabolic genes. Expression levels in TH17 group were set to 1. (D) Naive CD4+ T cells from C57BL/6 mice were differentiated in presence of 30 µM DON, 25 µM BPTES and 25 µM CB-839 under TH17 cell -inducing conditions for 5 days, followed by intracellular staining of IL-17 and Foxp3 (E) ROS production by measuring the DCF-fluorescence intensity and the data was represented by histogram (upper panel) and relative intensity by bar graph (lower panel). (F) Naive CD4+ T cells from C57BL/6 mice were differentiated in presence and absence of glutamine and 5 mM NAC, 100 μM hypoxanthine and 16 μM thymidine (HT) and in combination of HT and NAC under TH17 cell-inducing conditions for 5 days, followed by intracellular staining of IL-17 (G). Bar graph shows the relative value of mean fluorescence intensity of ROS measurement. Data represent the mean ±S.D of two-three independent experiments.

https://doi.org/10.7554/eLife.36158.021
Figure 6—figure supplement 1—source data 1

Source data for A, B, C, E and G.

https://doi.org/10.7554/eLife.36158.022
Figure 6—figure supplement 2
Glutamine catabolism coordinates with GSH metabolism in modulating ROS homeostasis and T cell differentiation.

Modulation of GSH biosynthesis and ROS production in directing T cell differentiation.

https://doi.org/10.7554/eLife.36158.023

Tables

Key resources table
Reagent type
(species) or
resource
DesignationSource or referenceIdentifiersAdditional
information
Strain, strain
background
(Mus musculus)
C57BL/6 (B6) miceTaconic
Strain, strain
background
(Mus musculus)
C3H/HeNEnvigo
Genetic
reagent
(Mus musculus)
CD4-Cre
Gclcflox/flox
PMID:23226398
Genetic
reagent
(Mus musculus)
Gclm-KOPMID:12384496
Genetic
reagent
(Mus musculus)
ROSA26CreERT2RRID:
IMSR_JAX:008463
The Jackson
Laboratory
Genetic
reagent
(Mus musculus)
GSR-KOPMID: 10218442
AntibodyMouse anti-
CD3 mAb
Cat. #:BE0001-1,
RRID:AB_1107634
BioXcell
AntibodyMouse anti-
CD28 mAb
Cat. #BE0015-1
RRID:AB_1107624
BioXcell
AntibodyMouse anti
-IL2 mAb
Cat.#BE0043
RRID::AB_1107702
BioXcell
AntibodyMouse anti-
IL4 mAb
Cat. #BE0045
RRID:AB_1107707
BioXcell
AntibodyMouse anti-
IFNγ mAb
Cat. #BE0055
RRID:AB_1107694
BioXcell
AntibodyAnti mouse
CD4-FITC
Cat. #11–0042
RRID:AB_464897
eBioscience(1:200)
AntibodyAnti mouse
CD4-APC
Cat. #17-0041-81
RRID:AB_469319
eBioscience(1:200)
AntibodyAnti mouse
CD8-APC-Cy7
Cat. #100714
RRID:AB_312753
Biolegend(1:200)
AntibodyAnti mouse
Foxp3-APC
Cat. #
RRID:AB_469456
eBioscience(1:200)
AntibodyAnti mouse
IL-17A-PECy7
Cat. #25-7177-82
RRID:AB_10732356
eBioscience(1:200)
AntibodyAnti GCLC antibody
(rabbit monoclonal)
Cat. #ab190685
RRID:AB_10975474
AbcamWB (1:1000)
AntibodyAnti GCLM antibody
(rabbit monoclonal)
Cat. #ab124827
RRID:AB_10975474
AbcamWB (1:1000)
Antibodyanti-mouse
CD25 -PE
Cat. #101904
RRID:AB_312847
Biolegend(1:200)
Antibodyanti-mouse
CD69-PECy7
Cat. #552879
RRID:AB_394508
BD Bioscience(1:200)
AntibodyAnti mouse
monoclonal CD3
Cat. #sc-101442
RRID:AB_1120355
Santa CruzIHC (1:50)
AntibodyAnti mouse
monoclonal
galectin-3
Cat. #sc-32790,
RRID:AB_627657
Santa CruzIHC (1:50)
Peptide,
recombinant protein
MOG35-55 peptidesynthesized
and HPLC-purified
St. Jude
Hartwell Center
for Biotechnology
Peptide,
recombinant protein
Recombinant
mouse IL-6
216–16Peprotech
Peptide,
recombinant protein
Recombinant
human TGFb
100–21 cPeprotech
Peptide,
recombinant protein
Recombinant
human or mouse IL-2
200–02Peprotech
Commercial
assay or kit
Foxp3/Transcription
Factor Staining
Buffer Set
00-5523-00e-Bioscience
Commercial
assay or kit
Naive CD4 + T cell
isolation kit,mouse
5160725186Miltenyi Biotec
Commercial
assay or kit
CD45R(B220)
microbeads, mouse
5150309030Miltenyi Biotec
Commercial
assay or kit
ABC kitPK-7200Vector
laboratories
Commercial
assay or kit
MojoSort Mouse
naive CD4 T Cell
Isolation Kit
480031Biolegend
Chemical
compound, drug
Diethly FumerateSigma AldrichD95654
Chemical
compound, drug
N-Acetyl-L-cysteineSigma-AldrichA7250
Chemical
compound, drug
TamofixenSigma-AldrichT5648
Chemical
compound, drug
4-hydroxytamoxifenSigmaH7904
Chemical
compound, drug
Dimethy a-keto
glutarate/aKG
Sigma-Aldrich34963–1
Chemical
compound, drug
HypoxathineSigma-AldrichH9377
Chemical
compound, drug
ThymidineSigmaT9250
Chemical
compound, drug
H2O2Sigma-Aldrich7722-84-1
Chemical
compound, drug
carboxyfluorescein
diacetate
succinimidyl
ester(CFSE)
InvitrogenC1157
Chemical
compound, drug
DM-H2DCFDAInvitrogenC6827
Chemical
compound, drug
DABVector
Laboratories
SK-4100
Chemical
compound, drug
MonobromobimaneInvitrogenM1378
Chemical
compound, drug
7-amino-
actinomycin D(7AAD)
Biolegend420404
Chemical
compound, drug
Pertussis toxin181List Biological
Laboratories
Chemical
compound, drug
Mycobacterium
tuberculosum
231141Difco
Chemical
compound, drug
Incomplete
Freund’s Adjuvant
263910Difco
Chemical
compound, drug
[U-14C]-glutamineMC 1124Moravek
Chemical
compound, drug
[2–14C]-pyruvateARC 0222American
Radiolabeled
Chemicals
Chemical
compound, drug
Cell Stimulation
Cocktail (plus
protein transport
inhibitors) (500X)
00-4975-93eBioscience
Chemical
compound, drug
Iscove's Modified
Dulbecco's Media
- Glucose free
conditional medium
ME17058P1Thermo
Fisher Scientific
Chemical
compound, drug
Iscove's Modified
Dulbecco's Media
- without L-glutamine
12–726 fLonza
Chemical
compound, drug
RPMI 1640
Medium, No Glucose
11-879-020Gibco
Chemical
compound, drug
Hyclone RPMI
1640 Medium,
no glutamine
sh30096.10Thermo
Fisher Scientific
Chemical
compound, drug
U-13C6-glutamineCNLM-1275–0.1Cambridge
Isotope Lab
Chemical
compound, drug
6-Diazo-5-oxo-L
-norleucine
D2141-5MGSigma-aldrich
Chemical
compound, drug
Bis-2-
(5-phenylacetamido
-1,3,4-thiadiazol-2-yl)
ethyl sulfide (BPTES)
SML0601Sigma-aldrich
Chemical
compound, drug
CB-83922038Cayman
Software,
algorithm
Graphpad PrismRRID:SCR_002798
Software,
algorithm
FlowJoRRID:SCR_008520

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files.

Additional files

Supplementary file 1

List of primer sequences used for RT-PCR analysis.

https://doi.org/10.7554/eLife.36158.025
Transparent reporting form
https://doi.org/10.7554/eLife.36158.026

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