Metabolomics Analysis Reveals Differential T-Cell Serine...

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Metabolomics Analysis Reveals Differential T-Cell Serine Metabolism as a Target in Autoimmunity Gabriela Andrejeva Jeffrey Rathmell lab Vanderbilt University Medical Center gabriela.andrejeva@ vanderbilt.edu 2018 UK Metabolism Symposium Monday, 30 th July 2018

Transcript of Metabolomics Analysis Reveals Differential T-Cell Serine...

Page 1: Metabolomics Analysis Reveals Differential T-Cell Serine ...bioinformatics.cesb.uky.edu/pub/RCSIRM/Workshop...CD4 T Cells and Metabolism 5 Adapted from Swain et al., Nat Rev Immunol,

Metabolomics Analysis Reveals Differential T-Cell Serine Metabolism as a Target in Autoimmunity Gabriela Andrejeva Jeffrey Rathmell lab Vanderbilt University Medical Center [email protected] 2018 UK Metabolism Symposium Monday, 30th July 2018

Page 2: Metabolomics Analysis Reveals Differential T-Cell Serine ...bioinformatics.cesb.uky.edu/pub/RCSIRM/Workshop...CD4 T Cells and Metabolism 5 Adapted from Swain et al., Nat Rev Immunol,

Outline

Background and hypothesis

Global metabolomics and proteomics studies of CD4 T cells

Serine pathway in CD4 T cell subsets

Serine pathway and MTHFD2 in EAE and multiple sclerosis

Summary and future directions

2

AGXT2L-Cystine

DLD

L-Arginine

CoA

ADC

5,10-Methylenetetrahydrofolate"

CDO1

GOT2

4-Aminobutanoate

MPST

GOT1

2-OxoglutarateMercaptopyruvate

Pyruvate

L-Cysteate

Cyanide

3-Sulfino-L-alanine

GPT

Taurine

GAD1

beta-Alanine

GAD2

GPT2

CSAD

Hypotaurine

SDS

5-FormiminotetrahydrofolateThiocysteine

2-Oxobutanoate

2-Aminoacrylate

L-Glutamate

L-Cysteine

L-Alanine

Oxaloacetate

AGXTCTH

L-Cystathionine

4-Guanidinobutanoate

Succinyl-CoA

GAMT

Guanidinoacetate

Creatine

GSS

DMGDH

S-Adenosyl-L-homocysteine

Betaine aldehyde

CO2

S-Aminomethyldihydrolipoylprotein

L-Pipecolate

PDPR

CHDH

Choline

Sarcosine

GLDC

SARDH

ALAS1

Glutathione

ALAS2

PIPOX

GNMT

GCAT

L-2-Amino-3-oxobutanoic acid

5-Aminolevulinate

MTRBHMT2

"N,N-Dimethylglycine"

BHMT

Selenohomocysteine

AHCYL1

L-Homocysteine

BetaineCob(II)alamin

AHCY

PPCS

ANPEPHydroxypyruvate

CP

Acetyl-CoA

GATM

Se-Adenosylselenomethionine

L-Methionine

MAT1AMAT2B

S-Adenosyl-L-methionine

MAT2A

CBS

PPIGGlycine

IL4I1Cystathionine

ALDH1L2

ALDH1L1

Tetrahydrofolyl-[Glu](n)

Poly-L-glutamate

10-Formyltetrahydrofolylpolyglutamate 10-Formyltetrahydrofolyl L-glutamate

GGH

ATIC

MTHFD2

DHFR

MTHFD1

10-Formyltetrahydrofolate

Tetrahydrofolate

MTHFD1L

"5,10-Methylenetetrahydrofolate"

FPGS

"5,10-Methenyltetrahydrofolate"

Formate

O-Phospho-L-serine

5-MethyltetrahydrofolateMTHFR

MTHFS

FTCD

MTFMT

N-Formimino-L-glutamatePSPH

GART

PHGDH

3-Phosphonooxypyruvate

2-Oxo-3-hydroxy-4-phosphobutanoate

3-Phospho-D-glycerate

O-Phospho-4-hydroxy-L-threonine PSAT1

NAGS

SHMT1

NH3 L-Serine

AMT5-Formyltetrahydrofolate

SHMT2

dUMP dTMP

Folate

DihydropteroateDihydrofolate

TYMS

Glucose

Serine

Methylene-THF

Serine

ExtracelularSerine

Methylene-THF

FormateFormate

MTHFD2

NADPH NADPH/NADHPURINES

GSH

METHIONINE

GSH

MITOCHONDRION

CYTOSOL

Treg

Th17

Th1

Page 3: Metabolomics Analysis Reveals Differential T-Cell Serine ...bioinformatics.cesb.uky.edu/pub/RCSIRM/Workshop...CD4 T Cells and Metabolism 5 Adapted from Swain et al., Nat Rev Immunol,

Outline

Background and hypothesis

Global metabolomics and proteomics studies of CD4 T cells

Serine pathway in CD4 T cell subsets

Serine pathway and MTHFD2 in EAE and multiple sclerosis

Summary and future directions

3

Page 4: Metabolomics Analysis Reveals Differential T-Cell Serine ...bioinformatics.cesb.uky.edu/pub/RCSIRM/Workshop...CD4 T Cells and Metabolism 5 Adapted from Swain et al., Nat Rev Immunol,

CD4 T Cells and Metabolism 4

Adapted from Swain et al., Nat Rev Immunol, 2012

Th2 GATA3

Treg FoxP3

Th17 Ror𝛾t

Th1 T-bet

TGFβ IL-6

IL-23 TGFβ IL-2

Antigen Recognition & Co-Stimulation

IL-12 IFN𝛾 IL-4

IL-10 IL-17A, IL-17F, IL-22

IFN𝛾, TNF IL-4, IL-5, IL-13

POLARIZING MILEU

EFFECTOR MEDIATORS

EFFECTOR FUNCTIONS

Regulation, suppression of inflammatory responses

Inflammation Allergic and helminth

responses

Macrophage activation,

inflammation

Naïve

TCR

Page 5: Metabolomics Analysis Reveals Differential T-Cell Serine ...bioinformatics.cesb.uky.edu/pub/RCSIRM/Workshop...CD4 T Cells and Metabolism 5 Adapted from Swain et al., Nat Rev Immunol,

CD4 T Cells and Metabolism 5

Adapted from Swain et al., Nat Rev Immunol, 2012

Th2 GATA3

Treg FoxP3

Th17 Ror𝛾t

Th1 T-bet

TGFβ IL-6

IL-23 TGFβ IL-2

Antigen Recognition & Co-Stimulation

IL-12 IFN𝛾 IL-4

IL-10 IL-17A, IL-17F, IL-22

IFN𝛾, TNF IL-4, IL-5, IL-13

POLARIZING MILEU

EFFECTOR MEDIATORS

EFFECTOR FUNCTIONS

Regulation, suppression of inflammatory responses

Inflammation Allergic and helminth

responses

Macrophage activation,

inflammation

Naïve

TCR

Sugiura & Rathmell., J Immunol, 2018

Page 6: Metabolomics Analysis Reveals Differential T-Cell Serine ...bioinformatics.cesb.uky.edu/pub/RCSIRM/Workshop...CD4 T Cells and Metabolism 5 Adapted from Swain et al., Nat Rev Immunol,

CD4 T Cells and the Immunological Balance 6

Th2 Treg

Tolerance Normal Immunity

Th17

Th1 Th2

Treg

Immunosuppression Cancer

Th17

Th1

Th2

Treg

Inflammation Autoimmunity

Th17

Th1

Page 7: Metabolomics Analysis Reveals Differential T-Cell Serine ...bioinformatics.cesb.uky.edu/pub/RCSIRM/Workshop...CD4 T Cells and Metabolism 5 Adapted from Swain et al., Nat Rev Immunol,

Hypothesis

Cytokine signaling during CD4 T cell development induces differential

metabolic rewiring to fulfill biosynthetic, signaling and epigenetic

roles.

These are selectively essential for the development and function of

different CD4 T cell subsets.

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Page 8: Metabolomics Analysis Reveals Differential T-Cell Serine ...bioinformatics.cesb.uky.edu/pub/RCSIRM/Workshop...CD4 T Cells and Metabolism 5 Adapted from Swain et al., Nat Rev Immunol,

Outline

Background and hypothesis

Global metabolomics and proteomics studies of CD4 T cells

Serine pathway in CD4 T cell subsets

Serine pathway and MTHFD2 in EAE and multiple sclerosis

Summary and future directions

8

Page 9: Metabolomics Analysis Reveals Differential T-Cell Serine ...bioinformatics.cesb.uky.edu/pub/RCSIRM/Workshop...CD4 T Cells and Metabolism 5 Adapted from Swain et al., Nat Rev Immunol,

Experimental System for Metabolomics and Proteomics

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Cytokines added Outcome TGFβ Treg TGFβ + IL-2 Treg proliferative IL-12 Th1 IL-12 + IL-2 Th1 TGFβ + IL-6 Th17 TGFβ + IL-6 + IL-23 More pathogenic Th17 TGFβ + IL-6 + IL-23 + IL-1β

Pathogenic Th17

Nothing Th0

Cytokines added Outcome IL-7 Naïve T cell

Isolated naïve

mouse CD4+ T

cell

anti-CD3 anti-CD28

CD28 CD3

TCR

SIGNAL 1 SIGNAL 2

anti-CD3 and anti-CD28 coated plate (5ug/ml each)

Isolated naïve

mouse CD4+ T

cell

Immune Characterization •  Transcription Factor Expression •  Cytokine Secretion

Metabolic Characterization •  Intracellular Metabolite MS •  Medium Analysis by NMR •  Proteomics

24h, 48h, 72h

Page 10: Metabolomics Analysis Reveals Differential T-Cell Serine ...bioinformatics.cesb.uky.edu/pub/RCSIRM/Workshop...CD4 T Cells and Metabolism 5 Adapted from Swain et al., Nat Rev Immunol,

Outline

Background and hypothesis

Global metabolomics and proteomics studies of CD4 T cells

Serine pathway in CD4 T cell subsets

Serine pathway and MTHFD2 in EAE and multiple sclerosis

Summary and future directions

10

Page 11: Metabolomics Analysis Reveals Differential T-Cell Serine ...bioinformatics.cesb.uky.edu/pub/RCSIRM/Workshop...CD4 T Cells and Metabolism 5 Adapted from Swain et al., Nat Rev Immunol,

Serine/Glycine/One-Carbon Metabolism 11

Glucose

Serine

Methylene-THF

Serine

Extracellular Serine

Methylene-THF

Formate Formate

NADPH NADPH/ NADH PURINES

GSH

METHIONINE

GSH

MITOCHONDRION

CYTOSOL

Glycine

Glycine

Page 12: Metabolomics Analysis Reveals Differential T-Cell Serine ...bioinformatics.cesb.uky.edu/pub/RCSIRM/Workshop...CD4 T Cells and Metabolism 5 Adapted from Swain et al., Nat Rev Immunol,

Serine/Glycine/One-Carbon Metabolism 12

Glucose

Serine

Methylene-THF

Serine

Extracellular Serine

Methylene-THF

Formate Formate

MTHFD2

NADPH NADPH/ NADH PURINES

GSH

METHIONINE

GSH

MITOCHONDRION

CYTOSOL

Glycine

Glycine

Page 13: Metabolomics Analysis Reveals Differential T-Cell Serine ...bioinformatics.cesb.uky.edu/pub/RCSIRM/Workshop...CD4 T Cells and Metabolism 5 Adapted from Swain et al., Nat Rev Immunol,

Serine/Glycine/One-Carbon Metabolism 13

Glucose

Serine

Methylene-THF

Serine

Extracellular Serine

Methylene-THF

Formate Formate

MTHFD2

NADPH NADPH/ NADH PURINES

GSH

METHIONINE

GSH

MITOCHONDRION

CYTOSOL

Th17 ? Glycine

Glycine

TReg

Th1

Page 14: Metabolomics Analysis Reveals Differential T-Cell Serine ...bioinformatics.cesb.uky.edu/pub/RCSIRM/Workshop...CD4 T Cells and Metabolism 5 Adapted from Swain et al., Nat Rev Immunol,

Possible Mechanisms of MTHFD2 Inhibitor Action

Nucleotide Synthesis Reactive Oxygen Species

Page 15: Metabolomics Analysis Reveals Differential T-Cell Serine ...bioinformatics.cesb.uky.edu/pub/RCSIRM/Workshop...CD4 T Cells and Metabolism 5 Adapted from Swain et al., Nat Rev Immunol,

The Model of MTHFD2 Inhibitor Action 15

Glucose

Serine

Methylene-THF

Serine

Extracellular Serine

Methylene-THF

Formate Formate

MTHFD2

NADPH NADPH/ NADH PURINES

GSH

METHIONINE

GSH

MITOCHONDRION

CYTOSOL

Th17

Glycine

Glycine

TReg

Th1

AICAR

AMPK

mTORC1

Page 16: Metabolomics Analysis Reveals Differential T-Cell Serine ...bioinformatics.cesb.uky.edu/pub/RCSIRM/Workshop...CD4 T Cells and Metabolism 5 Adapted from Swain et al., Nat Rev Immunol,

Outline

Background and hypothesis

Global metabolomics and proteomics studies of CD4 T cells

Serine pathway in CD4 T cell subsets

Serine pathway and MTHFD2 in EAE and multiple sclerosis

Summary and future directions

16

Page 17: Metabolomics Analysis Reveals Differential T-Cell Serine ...bioinformatics.cesb.uky.edu/pub/RCSIRM/Workshop...CD4 T Cells and Metabolism 5 Adapted from Swain et al., Nat Rev Immunol,

Outline

Background and hypothesis

Global metabolomics and proteomics studies of CD4 T cells

Serine pathway in CD4 T cell subsets

Serine pathway and MTHFD2 in EAE and multiple sclerosis

Summary and future directions

17

Page 18: Metabolomics Analysis Reveals Differential T-Cell Serine ...bioinformatics.cesb.uky.edu/pub/RCSIRM/Workshop...CD4 T Cells and Metabolism 5 Adapted from Swain et al., Nat Rev Immunol,

Summary and Future Directions

Global metabolomics analysis can reveal pathways that are important in CD4 T cell activation and subset function

Naïve CD4 T cell differentiation with lineage-specific cytokines results in significant alterations of serine metabolism

Targeting MTHFD2 might be a viable strategy to shift the balance between Th1/Th17/Treg in human inflammatory disease In vivo: Does targeting MTHFD2 suppress autoimmunity and promote Treg stability?

In humans: Is MTHFD2 expression increased in human multiple sclerosis patient brain lesions?

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AGXT2L-Cystine

DLD

L-Arginine

CoA

ADC

5,10-Methylenetetrahydrofolate"

CDO1

GOT2

4-Aminobutanoate

MPST

GOT1

2-OxoglutarateMercaptopyruvate

Pyruvate

L-Cysteate

Cyanide

3-Sulfino-L-alanine

GPT

Taurine

GAD1

beta-Alanine

GAD2

GPT2

CSAD

Hypotaurine

SDS

5-FormiminotetrahydrofolateThiocysteine

2-Oxobutanoate

2-Aminoacrylate

L-Glutamate

L-Cysteine

L-Alanine

Oxaloacetate

AGXTCTH

L-Cystathionine

4-Guanidinobutanoate

Succinyl-CoA

GAMT

Guanidinoacetate

Creatine

GSS

DMGDH

S-Adenosyl-L-homocysteine

Betaine aldehyde

CO2

S-Aminomethyldihydrolipoylprotein

L-Pipecolate

PDPR

CHDH

Choline

Sarcosine

GLDC

SARDH

ALAS1

Glutathione

ALAS2

PIPOX

GNMT

GCAT

L-2-Amino-3-oxobutanoic acid

5-Aminolevulinate

MTRBHMT2

"N,N-Dimethylglycine"

BHMT

Selenohomocysteine

AHCYL1

L-Homocysteine

BetaineCob(II)alamin

AHCY

PPCS

ANPEPHydroxypyruvate

CP

Acetyl-CoA

GATM

Se-Adenosylselenomethionine

L-Methionine

MAT1AMAT2B

S-Adenosyl-L-methionine

MAT2A

CBS

PPIGGlycine

IL4I1Cystathionine

ALDH1L2

ALDH1L1

Tetrahydrofolyl-[Glu](n)

Poly-L-glutamate

10-Formyltetrahydrofolylpolyglutamate 10-Formyltetrahydrofolyl L-glutamate

GGH

ATIC

MTHFD2

DHFR

MTHFD1

10-Formyltetrahydrofolate

Tetrahydrofolate

MTHFD1L

"5,10-Methylenetetrahydrofolate"

FPGS

"5,10-Methenyltetrahydrofolate"

Formate

O-Phospho-L-serine

5-MethyltetrahydrofolateMTHFR

MTHFS

FTCD

MTFMT

N-Formimino-L-glutamatePSPH

GART

PHGDH

3-Phosphonooxypyruvate

2-Oxo-3-hydroxy-4-phosphobutanoate

3-Phospho-D-glycerate

O-Phospho-4-hydroxy-L-threonine PSAT1

NAGS

SHMT1

NH3 L-Serine

AMT5-Formyltetrahydrofolate

SHMT2

dUMP dTMP

Folate

DihydropteroateDihydrofolate

TYMS

Glucose

Serine

Methylene-THF

Serine

ExtracelularSerine

Methylene-THF

FormateFormate

MTHFD2

NADPH NADPH/NADHPURINES

GSH

METHIONINE

GSH

MITOCHONDRION

CYTOSOL

Treg

Th17

Th1

Page 19: Metabolomics Analysis Reveals Differential T-Cell Serine ...bioinformatics.cesb.uky.edu/pub/RCSIRM/Workshop...CD4 T Cells and Metabolism 5 Adapted from Swain et al., Nat Rev Immunol,

Acknowledgements 19

Rathmells’ labs •  Jeffrey Rathmell •  Kimryn Rathmell •  Kathryn Beckermann •  Anna Chytil •  Diana Contreras •  Aguirre De cubas •  Stephanie Dudzinski •  Marc Johnson •  Aaron Lim •  Matthew Madden •  Damian Maseda •  Frank Mason •  Stephen Norris •  Bradley Reinfeld •  Ayaka Sugiura •  Esteban Terzo •  Jamie Weyandt •  Melissa Wolf •  Kirsten Young

Young lab •  Jamey Young •  Irina Trenary •  Ali McAtee

Vanderbilt NMR cores •  Donald Stec •  Markus Voehler

Sriram lab •  Subramaniam Sriram

Aune lab •  Thomas Aune

McLean lab •  Stacy Sherrod •  Alexandra Rutledge •  Simona Codreanu

Vanderbilt MS core •  Kevin Schey •  Wade Calcutt •  Emilio Rivera •  Kristie Lindsey Rose

Norris lab •  Jeremy Norris •  Danielle Guttierez


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serine Cbz

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Government Relations Col Swain

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J Immunol 2009 GriffSith 5208 20

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J Immunol 2000 Kasprowicz 680 90

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Mol Immunol 2007 Cultivo Celular

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