r.j. fasenmyer annual lectureship metabolic control of ... 2... · r.j. fasenmyer annual...

RJ Fasenmyer Annual Lectureship Metabolic Control of Innate Immunity and Inflammation ndash The Rediscovered Frontier

Luke OrsquoNeill PhD Director Trinity Biomedical Sciences Institute School of Biochemistry and Immunology Trinity College Dublin Dublin Ireland

A RHEUMATOID JOINT IMAGED WITH 2-DEOXYGLUCOSE

- Where wersquove come from - What has 28 years of effort taught us about the mechanistic basis of inflammatory diseases

-Where we are now - what therapies have emerged

- Where we are going - a re-discovered frontier ndash the role of metabolism in innate immunity and inflammation

INNATE IMMUNITY

WHERE WErsquoVE COME FROM

PUBLISHED 1914

The diet ought to be as rich as can be digested ndash considerable amounts of meat good sound wine or stout and extracts of malt The best drug is syrup of the iodide of iron Sea sand should be heated in a tin and poured over the affected joint as hot as can be borne

TREATMENT FOR RHEUMATOID ARTHRITIS 1914 The Modern Family Doctor A Guide to Perfect Health Hygieia London 1914 p510

Another method which frequently does wonders consists in the application of large blisters over the spine these blisters being kept open for a week or ten days It is usual to give some opiate during the cure but the results are so gratifying in many cases as to warrant a trial even in seemingly hopeless cases

Without basic research there can be no applications After all electricity and the electric lightbulb were not invented by incremental improvements in the candle

THE NEED FOR BASIC RESEARCH

Without basic research there can be no applications After all anti-TNF medicines were not invented by incremental improvements in aspirin

INFLAMMATION

INFLAMMATORY DISEASES

PROSTAGLANDINS

1985

Cytokines IL-1 TNF IL6

Inflammatory gene expression

INFLAMMATION


1990

More and more Cytokines

Inflammatory gene expression (COX2 iNOS CAMs chemokines etc)

INFLAMMATION


NFkappaB p38 JAKs

Mid 90s

DCT cell interactions

T

DAMPs and PAMPs



INFLAMMATION


INNATE IMMUNE RECEPTORS

Late 90s ndash 2000s


NFkappaB p38 JAKs

T

DAMPs and PAMPs



INFLAMMATION



Today


NFkappaB p38 JAKs

Tregs Microbiome

WHERE WE ARE NOW THERAPIES

CYTOKINES AS KEY THERAPEUTIC TARGETS

Inaugural conference of the International Cytokine and Interferon Society (ICIS)

Sept 29th ndash Oct 3rd

ANTI-CYTOKINE THERAPIES 2013

Rheumatoid arthritis Anti-TNF Infliximab Adalimumab Etanercept Certolizumab Golimumab Anti-IL-1 Anakinra Anti-IL-6R Tocilizumab Psoriasis Anti-IL-1223 Ustekinumab Anti-TNF Crohnrsquos disease Anti-IL-6R Tocilizumab Anti-TNF Infliximab Adalimumab Phase III efficacy Anti-IL-1223 in Psoriasis Crohnrsquos disease and ulcerative colitis Anti-TNF in ulcerative colitis Anti-IL-17 in ulcerative colitis

WHAT ABOUT SIGNALS

- Tofacitinib (JAK inhibitor) approved in USA and Japan but not EU for rheumatoid arthritis in methotrexate non-responders

T

DAMPs and PAMPs



INFLAMMATION



Today


NFkappaB p38 JAKs

Tregs Microbiome

Endogenous ligands for TLR4 in RA

Hsp22 hsp60 hsp70 Biglycan Fibronectin HA fragments Tenascin-C Fibrinogen (Fibrinogen globe region) Citrullinated Fibrinogen (10X)

TLR4

TNF IL-6 etc

RA Synovial Tissue

gm

l)

Isotype (10

gm

l)

Humira (10

gm

l)

OPN-301

(10

gm

l)

OPN-301

(05

0

1000

2000

3000

4000

5000

6000

7000

Plt001

Plt001

Plt005

IL-6

pg

mlm

g o

f b

iop

sy

gm

l)

Isotype (10

gm

l)

Humira (10

gm

l)

OPN-301

(10

gm

l)

OPN-301

(05

0

10

20

30

40

50

60Plt001

Plt001

Pgt005

TN

F-

pg

ml

23 Doug Veale and colleagues St Vincentrsquos Hospital

Blocking TLR2 prevents cytokine production in

The discovery of inflammasomes has stimulated renewed interest in IL-1

as a target

INFLAMMASOMES

Park H et al (2012) Lighting the fires within the cell biology of autoinflammatory diseases Nat Rev Immunol 12 570-580

TYPE I DIABETES

AIRE Foxp3

Muckle Wells disease

-100 0 100 200

0

100

200

300

400

500

600

Days from infusion

SA

A m

gL

-100 0 100 2000

20

40

60

80

100

120

Days from infusion

CR

P m

gL

Immediately pre first treatment 24 hours post treatment

A Cure Canakinumab (Anti-IL1beta) induced a complete clinical and biochemical response

in all cases

Helen Lachman

Atherosclerosis Cholesterol

crystals

Nlrp3 in disease

The Nlrp3 inflammasome may be especially relevant to crystal arthropathies -Best data so far is in osteoarthritis ndash hydroxyapatite crystals

-Role in Gout less clear - inhibiting IL-1beta is efficacious ndash approved in the EU

Needles Rods

Cartilage integrity

Hydroxyapatite Nlrp3

IL-1

Joint inflammation

OA

NFkappaB JNK

2013

COX2 aggrecanases

-Where we are going - a re-discovered frontier ndash the role of metabolism in innate immunity and inflammation

TYPE 2 DIABETES AND IL-1

1990 2010 OBESITY

Type 2 diabetes

WHY CORN SYRUP

OBESITY AND TYPE 2 DIABETES

IRONY

IRONY

IL1 in T2D

IL1b

Beta cell proliferation Beta cell death

Hypoglycemia Insulin resistance

Appetite suppression

Inhibiting IL-1beta has shown benefits in the treatment of T2D 9 separate clinical trials

What induces IL-1beta in T2D

IAPP deposits in human pancreas

Production of IL-1b by IAPP requires Nlrp3

FAT TISSUE PANCREAS Lipids Insulin + IAPP (palmitate ceramide) Adipocytes and macrophages Macrophages in the pancreas Nlrp3 Caspase-1 activation Nlrp3 Caspase-1 activation IL-1beta IL-1beta Decreased fat oxidation obesity Beta cell death Insulin resistance Insulin resistance

A model for the pathogenesis of T2D Adipose tissue the pancreas Nlrp3 and T2D

IAPP (initial insulin resistence)

Nlrp3

IL-1

Further insulin resistance

Beta cell death

Type 2 diabetes

NFkappaB JNK

2011

GLUCOSE MUSCLE LIVER

INSULIN

+

NORMAL

WHY WOULD INFLAMMATION CAUSE INSULIN RESISTENCE


INSULIN

+

NORMAL

GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION


MACROPHAGE ACTIVATION (not resistant) HOST DEFENSE REPAIR

WHAT ROLE DOES GLUCOSE PLAY IN IL-1beta PRODUCTION

2DG decreases IL-1beta but not TNF expression at 24h in human

0

05

1

15

2

25

3

0 LPS

TN

Fa (

ng

ml)

LPS

LPS + 2DG

IL-1beta

TNFalpha

0

50

100

150

200

250

300

350

400

0 LPS

rela

tive I

L-1

beta

exp

ressio

n

LPS

LPS + 2DG

2DG blocks LPS-induced IL-1beta in vivo

Glucose 2-deoxyglucose

ONE OXYGEN MAKES ALL THE DIFFERENCE

BIOLOGY AT ITrsquoS SIMPLEST

Control 18h LPS treated 18h

LPS THE WARBURG EFFECT

CONTROL LPS LPS and 2-DG

4 8 16 240

5

10

15 unstimulated

LPS

Time (h)

Glu

co

se

util

isa

tio

nG

lyco

lysis

O

xp

ho

s

LPS increases flux through glycolyis and decreases mitochondrial respirations

0

2

4

6

unstimulated LPS

NA

DN

AD

H ra

tio p

mo

l10

6 c

ells

LPS decreases NAD production

Otto Warburg

The Warburg Effect Aerobic glycolysis

Otto Warburgrsquos grant application

lsquoThe lsquocancer metabolismrsquo of normal white blood cells is an artefact of mechanical and chemical damagersquo

Th17

Treg

HIF is required for RORgt

Nature (2013) 493 346-354

COMPARING SIGNATURE METABOLIC PROCESSES IN TUMORS AND INFLAMMATORY CELLS

Pro-inflammatory versus anti-inflammatory cells and metabolism

WARBURG EFFECT M1 macrophages activated dendritic cells activated fibroblasts Th17 cells OXIDATIVE PHOSPHORYLATION M2 macrophages (Sedoheptulose Kinase) Treg cells CD8+ memory cells

Glycolysis Oxidative Phosphorylation

First observation ndash Dingle JT and Page-Thomas DP (1956) Brit J of Exp Pathology 37 318-323

Enzymes in glycolysis are autoantigens in RA Glucose phosphate isomerase Enolase and aldolase Enolase is citrullinated

METBOLISM MEETS IMMUNOLOGY

Infection Injury Inflammation Immunity Restore Homeostasis


Infection Injury Nutrition Inflammation Metabolism Immunity Restore Maintain Homeostasis Homeostasis

EXTERNAL STRESSORS



EXTERNAL STRESSORS


Infection Injury Over-Nutrition Inflammation Altered Metabolism Immunity Metabolic diseases


Infection Injury Genetics Inflammation Altered Metabolism Immunity Inflammatory diseases

What are the precise molecular pathways that link the 2 systems

of immunity and metabolism

KEY QUESTION

METBOLISM MEETS IMMUNOLOGY TYPE 2 DIABETES

Infection Injury Over-Nutrition Nlrp3 Altered Metabolism IL-1beta Metabolic diseases

METABOLISM MEETS IMMUNOLOGY GOUT

Misawa T et al Nat Immunol 14 454-460 (2013)

Colchicine works in gout because if prevents tubulin polymerisation



Infection Injury Nutrition Inflammation Altered Metabolism Immunity -decreased NAD -decreased SIRT2 (deaceylase) - acetylated tubulin - Nlrp3 activation IL-1beta and inflammation

So in gout the decrease in NAD promotes Nlrp3 activation Another example inflammation inceases succinate to promote IL-1beta


0

05

1

15

2

25

3

0 LPS

TN

Fa (

ng

ml)

LPS

LPS + 2DG

IL-1beta

TNFalpha

0

50

100

150

200

250

300

350

400

0 LPSre

lati

ve I

L-1

beta

exp

ressio

n

LPS

LPS + 2DG

Two questions 1 What do the metabolic changes induced by LPS in macrophages mean for macrophage function 2 What is the mechanism by which they occur

WHAT ROLE DOES GLUCOSE PLAY IN ALL THIS CAN WE TREAT INFLAMMATION (INCLUDING TYPE 2 DIABETES) BY STARVING MACROPHAGES


-Anti-inflammatory agents might work in part by making a macrophage think itrsquos starvinghellippseudostarvation

Metabolomic Analysis

bull 2-DG inhibits glycolysis

bull LPS appears to promote glycolysis

bull What can we learn from the metabolome

in LPS activated macrophages

Profound metabolic changes in LPS-treated macrophages

THE GABA SHUNT

2-DG inhibits the LPS-induced increase in succinate

0

2times1006

4times1006

6times1006

su

ccin

ate

(p

ea

k a

rea

)

LPS

unstimulated

- 1 5 10

2DG (mM)

LPS induces succinate in part from glutamine being metabolised by thre

GABA shunt acetyl-CoA

citrate

a-ketoglutarate

succinate

fumarate

malate

oxaloacetateGlu [13C515N2]-Gln

12C

13C

14N

15NGABA

SinglepassofTCAcycle

What would inhibiting the GABA shunt with vigabatrin do to IL-1beta

production

IL-1beta

LPS LPS + sabril PBS sabril0

200

400

600

800

1000

IL-1b

eta (

pgm

l)IL-6


10

20

30

40IL-

6 (ng

ml)

TNFalpha


1

2

3

TNFa

(ngm

l)

Blocking the GABA shunt with Vigabatrin in vivo reduces LPS-induced IL-1b

Inhibition of the GABA shunt protects mice from LPS lethality

0 20 40 60 80 0

50

100

150 Co n t r o l

Vi g a + L PS

LP S

Ho u r s p o s t L P S

P e r c

e n

t s u r v

i v a l

At this stage

- LPS increases glycolysis and the GABA shunt in macrophages leading to an

increase in succinate

-What is the succinate doing

2DG inhibits LPS-induced IL-1beta promoter-driven luciferase activity

Increasing amounts of IL-1b luc +LPS +- 2DG

Could inhibition of HIF-1alpha explain why 2-DG blocks transcription of IL-1beta but not TNF

TNF promoter lacks sites for PU1 Spi1 and HIF-1alpha

LPS stabilises HIF1alpha in macrophages at 24h

WB HIF1α WB actin

LPS

2-DG

LPS - + - + - + - + HIF1 IL-1b B-actin

LPS-induced HIF1α protein is

inhibited by 2DG

Succinate KG and HIF1

HIF-1 HIF-1 hydroxylation and degradation

PHD

KG Succinate

Succinate will inhibit PHDs and stablise HIF1

HIF-1 HIF-1 stablised

PHD

KG Succinate

-

KG will antagonise the effect of succinate on PHD and destablise HIF1


PHD

KG Succinate

KG

Is Succinate involved in HIF-1 stabilisation in inflammation

-50

0

50

100

150

200

250

300

350

400

PGL3 IL-1 promoter IL-1 -HIF promoter

unstim

LPS

LPS plus 2DG

Mutating the HIF-1alpha site in the IL-1beta promoter abolishes LPS responsiveness

Effect of manipulation of HIF1a on IL-1 β expression

- LPS - LPS0

2000

4000

6000

8000

rela

tive I

L-1

b e

xp

ressio

n

+ succinate

0 LPS 0 LPS0

20000

40000

60000

rela

tive I

L-1

b e

xp

ressio

n

+ butylmalonate

EXOGENOUS SUCCINATE BOOSTS BLOCKING SUCCINATE METABOLISM BOOSTS



PHD

KG Succinate



PHD

KG Succinate

-



PHD

KG Succinate

KG

012

h

12h+αK

G24

h

24h+αK

G

00

05

10

Rela

tive E

nri

ch

men

t

0 LPS 0 LPS0

10000

20000

30000

rela

tiv

e IL

-1b

ex

pre

ss

ion

+ aKG

Alpha ketoglutarate ndash competes with succinate to degrade HIF1-alpha and inhibits IL-1beta induction

WBIL-1β

WBHIF-1α

- +LPSαKG αKG

WBβ-ac n

Summary

bull LPS promotes glycolysis and other metabolic processes (purine biosynthesis)

bull TCA cycle is limited and GABA shunt is activated ndash

succinate builds up bull Succinate is transported from the mitochondria and

stabilises HIF-1-alpha by inhibiting Prolyl Hydroxylase bull HIF-1-alpha promotes the transcription of IL-1beta and other proteins

Infectious Inflammatory signals

succinate

PHD

HIF-1 IL-1beta Inflammation

Comparison to Cytochrome C Succinate as a mitochondrial signal for inflammation


succinate

PHD


Multiple targets for succinate in innate immunity

Succinylation of target Proteins GPR91 synergy with TLR signaling

LPS increases protein succinylation in macrophages

2DG2DG

-LPS+LPS

WBsuccinyllysine

WBIL-1βWBβ-ac n

unstimulated LPS00

01

02

03

04

me

an

em

PA

I va

lue

LPS decreases expression of the desuccinylase Sirt5

Protein succinylation

bull We identified several metabolic enzymes that underwent succinylation

bull Notable example- bull malate dehydrogenase

bull GAPDH

bull Triose phosphate isomerase

bull PKM12

Succinylation is predicted to cause a major conformational change

Key finding

bull Inflammation promotes major metabolic changes ndash glycolysis succinate and HIF-1alpha that drives the inflammatory process forward




METABOLISM MEETS IMMUNOLOGY

Infection Injury Nutrition Inflammation Altered Metabolism Immunity -increased succinate -PHD inhibition - increased HIF1alpha - increased IL-1beta and other genes inflammation

THE IMMUNE SYSTEM AS A TURBO-CHARGED HYBRID CAR

When resting ndash battery (electricity) (Oxidative Phosphorylation) To activate ndash petrol (Glycolysis) Turbo charge Succinate from Glutamine

2-DG makes an activated macrophage think itrsquos starving

GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION

WHY WOULD A STARVING MACROPHAGE BE TURNED OFF

MACROPHAGE ACTIVATION HOST DEFENSE REPAIR

GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION



LOW GLUCOSE MACROPHAGE TURNED OFF GLUCOSE SPARED FOR VITAL ORGANS

LOW GLUCOSE

Starving Macrophage AMP kinase activation- less inflamed

ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE

The role of AMP kinase in promoting oxidative phosphorylation and limiting glycolysis in conserved in yeast

Low glucose

SNF2

YEAST MACROPHAGE

Low glucose 2DG

AMP kinase

Ox Phos Glycolysis Ethanol

Ox Phos Glycolysis Inflammation (HIF1alpha IL-1beta)

GLUCOSE

Anti-Diabetic drugs on a macrophage less inflamed

ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE

METFORMIN

(mM)

IL-1β qPCR

(mM)

Metformin inhibits transcription of IL-1beta but not TNF

Int Immunopharmacol 2013 Apr 116(1)85-92 doi 101016jintimp201303020 [Epub ahead of print] Metformin downregulates Th17 cells differentiation and attenuates murine autoimmune arthritis Kang KY Kim YK Yi H Kim J Jung HR Kim IJ Cho JH Park SH Kim HY Ju JH

GLUCOSE

Anti-inflammatory drugs on a macrophage less inflamed

ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE

METFORMIN SALICYLATES METHOTREXATE RESVERATROL

Do anti-inflammatory drugs cause Pseudo-starvation in a macrophage

turning it off

Succinate might be a very important signal- a driver

of innate immunity and inflammation

The take home messages

bull Inflammation promotes major metabolic changes ndash glycolysis succinate and HIF-1alpha

bull Pseudo-starvation might limit inflammation

via AMP kinase

Does inflammation lie at the heart of most diseases because it puts the body under metabolic stress This deprives tissues of nutrients ignoring the usual integrated cues that control metabolism leading to morbidity and shortening of life span

Infection

TLRs Nlrp3

NFkB

Cytokines (IL1 TNF IL6 IL12 IL17 IL23)

INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK

Genetic variation as the tipping point into disease

INNATE IMMUNITY AUTOINFLAMMATION AND AUTOIMMUNITY

Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK

HIF1

Metabolic shift NAD down succinate up


Metformin Salicylates


LUKE OrsquoNEILL

Rebecca Coll Beth Kelly

OrsquoNEILL LAB

Rebecca Coll Sinead Corr Annie Curtis Niamh Foley Anne McGettrick Claire McCoy Eva Palsson McDermott Gillian Tannahill Seth Masters (WEHI) Mustafa Alam Nick Bernard Moritz Haneklaus Beth Kelly Evanna Mills Sue Quinn Mirjam van den Bosch Paulina Wochal Kathy Banahan Cait Donoghue

Acknowledgment

Juraj Adamik Mike Murphy Phil Auron

University of Duquesne MRC Mitochondrial Biology Unit Cambridge

Univeristy College Dublin

Eoin Cummins Cormac Taylor

Beatson Institute for Cancer Research

Christian Frezza Eyal Gottlieb

Mass General HarvardMIT Broad Ramnik Xavier Clary Clish

Acknowledgment








IL4 Caloric restriction Nampt1 STAT6 NAD+ PGC-1b SIRT1

b-oxidation Mitochondrial biosynthesis

Metabolic Regulation of M2 macrophages and Th17 cells (a) M2 macrophages

CARKL

IL6 TORC1 STAT3 HIF1 Glycolysis

RORgt pVHL E3L Foxp3 degradation IL-17

(b) Th17 cells


TARGETING TLRs AGONISTS AND ANTAGONISTS

ONLY PUBLISHED ANTAGONIST IS ERITORAN (ANTI-TLR4) WHICH FAILED IN SEPSIS

Diethylfumarate (BG12) and MS

A natural metabolite in the TCA cycle Similar effect to AMP kinase activators ndash induces IL4 and IL10 (M2 macrophages) Approved by the FDA 27th March 2013 for relapsing remitting MS

210 binding site Human - UUUUCGCACAAU Mouse - UUUUCCCACCGU

Potential role of miR210 4th yr data

N=2

N=2

081211 bmdm SS

Ctl LPS (24 hr)0

1

2

3

4

Re

lative

miR

21

0 e

xp

ressio

n

181111 j774

Ctl LPS (24 hr)00

05

10

15

20

25

Re

lativ

e m

iR2

10

exp

ressio

n

310112 Bmdm ss 48h

Ctl LPS (48 hr)0

1

2

3

4

5

Re

lative

miR

21

0 e

xp

ressio

n

N=2

J774s

BMDM BMDM

miR210 SDHD succinate

LPS

HIF-1α IL-1β

Hypothesis

Confirmation that LPS-increases miR-210

080413 210

Ctl LPS Ctl LPS Ctl LPS00

05

10

15

20

0 MCSF2 FCS

2 MCSF2 FCS

10 MCSF10 FCS

Re

lativ

e m

iR-2

10

exp

ressio

n

27328140413 pooled 24h no SS n=8

Ctl LPS00

05

10

15

20

Re

lative

miR

-21

0 e

xp

ressio

n

N=8 N=3


LPS

HIF-1α IL-1β

Hypothesis

LPS decreases SDHD expression- through miR210

25030208160413 sdhd n=10

Ctl LPS00

05

10

15

Re

lative

sd

hd

exp

ressio

n

N=10


LPS

HIF-1α IL-1β

Hypothesis

Anti-miR210 may prevent LPS-induced decrease in SDHD and decreases IL-1β production


anti-miR210

HIF-1α IL-1β

n=1

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

- Where wersquove come from - What has 28 years of effort taught us about the mechanistic basis of inflammatory diseases

-Where we are now - what therapies have emerged

- Where we are going - a re-discovered frontier ndash the role of metabolism in innate immunity and inflammation

INNATE IMMUNITY

WHERE WErsquoVE COME FROM

PUBLISHED 1914







INFLAMMATION


PROSTAGLANDINS

1985



INFLAMMATION


1990



INFLAMMATION


NFkappaB p38 JAKs

Mid 90s


T

DAMPs and PAMPs



INFLAMMATION





NFkappaB p38 JAKs

T

DAMPs and PAMPs



INFLAMMATION



Today


NFkappaB p38 JAKs

Tregs Microbiome







WHAT ABOUT SIGNALS


T

DAMPs and PAMPs



INFLAMMATION



Today


NFkappaB p38 JAKs

Tregs Microbiome



TLR4

TNF IL-6 etc

RA Synovial Tissue

gm

l)

Isotype (10

gm

l)

Humira (10

gm

l)

OPN-301

(10

gm

l)

OPN-301

(05

0

1000

2000

3000

4000

5000

6000

7000

Plt001

Plt001

Plt005

IL-6

pg

mlm

g o

f b

iop

sy

gm

l)

Isotype (10

gm

l)

Humira (10

gm

l)

OPN-301

(10

gm

l)

OPN-301

(05

0

10

20

30

40

50

60Plt001

Plt001

Pgt005

TN

F-

pg

ml




as a target

INFLAMMASOMES


TYPE I DIABETES

AIRE Foxp3


-100 0 100 200

0

100

200

300

400

500

600

Days from infusion

SA

A m

gL

-100 0 100 2000

20

40

60

80

100

120

Days from infusion

CR

P m

gL



in all cases

Helen Lachman


crystals

Nlrp3 in disease



Needles Rods

Cartilage integrity


IL-1

Joint inflammation

OA

NFkappaB JNK

2013

COX2 aggrecanases



1990 2010 OBESITY

Type 2 diabetes

WHY CORN SYRUP


IRONY

IRONY

IL1 in T2D

IL1b











Nlrp3

IL-1


Beta cell death

Type 2 diabetes

NFkappaB JNK

2011


INSULIN

+

NORMAL



INSULIN

+

NORMAL

GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION





0

05

1

15

2

25

3

0 LPS

TN

Fa (

ng

ml)

LPS

LPS + 2DG

IL-1beta

TNFalpha

0

50

100

150

200

250

300

350

400

0 LPS

rela

tive I

L-1

beta

exp

ressio

n

LPS

LPS + 2DG








4 8 16 240

5

10

15 unstimulated

LPS

Time (h)

Glu

co

se

util

isa

tio

nG

lyco

lysis

O

xp

ho

s


0

2

4

6

unstimulated LPS

NA

DN

AD

H ra

tio p

mo

l10

6 c

ells


Otto Warburg




Th17

Treg


Nature (2013) 493 346-354











EXTERNAL STRESSORS



EXTERNAL STRESSORS







KEY QUESTION











0

05

1

15

2

25

3

0 LPS

TN

Fa (

ng

ml)

LPS

LPS + 2DG

IL-1beta

TNFalpha

0

50

100

150

200

250

300

350

400

0 LPSre

lati

ve I

L-1

beta

exp

ressio

n

LPS

LPS + 2DG











THE GABA SHUNT


0

2times1006

4times1006

6times1006

su

ccin

ate

(p

ea

k a

rea

)

LPS

unstimulated

- 1 5 10

2DG (mM)



citrate

a-ketoglutarate

succinate

fumarate

malate


12C

13C

14N

15NGABA



production

IL-1beta


200

400

600

800

1000

IL-1b

eta (

pgm

l)IL-6


10

20

30

40IL-

6 (ng

ml)

TNFalpha


1

2

3

TNFa

(ngm

l)



0 20 40 60 80 0

50

100

150 Co n t r o l

Vi g a + L PS

LP S


P e r c

e n

t s u r v

i v a l

At this stage









WB HIF1α WB actin

LPS

2-DG



inhibited by 2DG



PHD

KG Succinate



PHD

KG Succinate

-



PHD

KG Succinate

KG


-50

0

50

100

150

200

250

300

350

400


unstim

LPS

LPS plus 2DG



- LPS - LPS0

2000

4000

6000

8000

rela

tive I

L-1

b e

xp

ressio

n

+ succinate

0 LPS 0 LPS0

20000

40000

60000

rela

tive I

L-1

b e

xp

ressio

n

+ butylmalonate




PHD

KG Succinate



PHD

KG Succinate

-



PHD

KG Succinate

KG

012

h

12h+αK

G24

h

24h+αK

G

00

05

10

Rela

tive E

nri

ch

men

t

0 LPS 0 LPS0

10000

20000

30000

rela

tiv

e IL

-1b

ex

pre

ss

ion

+ aKG


WBIL-1β

WBHIF-1α

- +LPSαKG αKG

WBβ-ac n

Summary






succinate

PHD




succinate

PHD





2DG2DG

-LPS+LPS

WBsuccinyllysine

WBIL-1βWBβ-ac n

unstimulated LPS00

01

02

03

04

me

an

em

PA

I va

lue





bull GAPDH


bull PKM12


Key finding










GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION



GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION




LOW GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE


Low glucose

SNF2

YEAST MACROPHAGE

Low glucose 2DG

AMP kinase



GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE

METFORMIN

(mM)

IL-1β qPCR

(mM)



GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE



turning it off






via AMP kinase


Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK



Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK

HIF1





LUKE OrsquoNEILL


OrsquoNEILL LAB


Acknowledgment








Acknowledgment











CARKL



(b) Th17 cells








N=2

N=2

081211 bmdm SS

Ctl LPS (24 hr)0

1

2

3

4

Re

lative

miR

21

0 e

xp

ressio

n

181111 j774

Ctl LPS (24 hr)00

05

10

15

20

25

Re

lativ

e m

iR2

10

exp

ressio

n

310112 Bmdm ss 48h

Ctl LPS (48 hr)0

1

2

3

4

5

Re

lative

miR

21

0 e

xp

ressio

n

N=2

J774s

BMDM BMDM


LPS

HIF-1α IL-1β

Hypothesis


080413 210


05

10

15

20

0 MCSF2 FCS

2 MCSF2 FCS

10 MCSF10 FCS

Re

lativ

e m

iR-2

10

exp

ressio

n

27328140413 pooled 24h no SS n=8

Ctl LPS00

05

10

15

20

Re

lative

miR

-21

0 e

xp

ressio

n

N=8 N=3


LPS

HIF-1α IL-1β

Hypothesis


25030208160413 sdhd n=10

Ctl LPS00

05

10

15

Re

lative

sd

hd

exp

ressio

n

N=10


LPS

HIF-1α IL-1β

Hypothesis



anti-miR210

HIF-1α IL-1β

n=1

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

PUBLISHED 1914







INFLAMMATION


PROSTAGLANDINS

1985



INFLAMMATION


1990



INFLAMMATION


NFkappaB p38 JAKs

Mid 90s


T

DAMPs and PAMPs



INFLAMMATION





NFkappaB p38 JAKs

T

DAMPs and PAMPs



INFLAMMATION



Today


NFkappaB p38 JAKs

Tregs Microbiome







WHAT ABOUT SIGNALS


T

DAMPs and PAMPs



INFLAMMATION



Today


NFkappaB p38 JAKs

Tregs Microbiome



TLR4

TNF IL-6 etc

RA Synovial Tissue

gm

l)

Isotype (10

gm

l)

Humira (10

gm

l)

OPN-301

(10

gm

l)

OPN-301

(05

0

1000

2000

3000

4000

5000

6000

7000

Plt001

Plt001

Plt005

IL-6

pg

mlm

g o

f b

iop

sy

gm

l)

Isotype (10

gm

l)

Humira (10

gm

l)

OPN-301

(10

gm

l)

OPN-301

(05

0

10

20

30

40

50

60Plt001

Plt001

Pgt005

TN

F-

pg

ml




as a target

INFLAMMASOMES


TYPE I DIABETES

AIRE Foxp3


-100 0 100 200

0

100

200

300

400

500

600

Days from infusion

SA

A m

gL

-100 0 100 2000

20

40

60

80

100

120

Days from infusion

CR

P m

gL



in all cases

Helen Lachman


crystals

Nlrp3 in disease



Needles Rods

Cartilage integrity


IL-1

Joint inflammation

OA

NFkappaB JNK

2013

COX2 aggrecanases



1990 2010 OBESITY

Type 2 diabetes

WHY CORN SYRUP


IRONY

IRONY

IL1 in T2D

IL1b











Nlrp3

IL-1


Beta cell death

Type 2 diabetes

NFkappaB JNK

2011


INSULIN

+

NORMAL



INSULIN

+

NORMAL

GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION





0

05

1

15

2

25

3

0 LPS

TN

Fa (

ng

ml)

LPS

LPS + 2DG

IL-1beta

TNFalpha

0

50

100

150

200

250

300

350

400

0 LPS

rela

tive I

L-1

beta

exp

ressio

n

LPS

LPS + 2DG








4 8 16 240

5

10

15 unstimulated

LPS

Time (h)

Glu

co

se

util

isa

tio

nG

lyco

lysis

O

xp

ho

s


0

2

4

6

unstimulated LPS

NA

DN

AD

H ra

tio p

mo

l10

6 c

ells


Otto Warburg




Th17

Treg


Nature (2013) 493 346-354











EXTERNAL STRESSORS



EXTERNAL STRESSORS







KEY QUESTION











0

05

1

15

2

25

3

0 LPS

TN

Fa (

ng

ml)

LPS

LPS + 2DG

IL-1beta

TNFalpha

0

50

100

150

200

250

300

350

400

0 LPSre

lati

ve I

L-1

beta

exp

ressio

n

LPS

LPS + 2DG











THE GABA SHUNT


0

2times1006

4times1006

6times1006

su

ccin

ate

(p

ea

k a

rea

)

LPS

unstimulated

- 1 5 10

2DG (mM)



citrate

a-ketoglutarate

succinate

fumarate

malate


12C

13C

14N

15NGABA



production

IL-1beta


200

400

600

800

1000

IL-1b

eta (

pgm

l)IL-6


10

20

30

40IL-

6 (ng

ml)

TNFalpha


1

2

3

TNFa

(ngm

l)



0 20 40 60 80 0

50

100

150 Co n t r o l

Vi g a + L PS

LP S


P e r c

e n

t s u r v

i v a l

At this stage









WB HIF1α WB actin

LPS

2-DG



inhibited by 2DG



PHD

KG Succinate



PHD

KG Succinate

-



PHD

KG Succinate

KG


-50

0

50

100

150

200

250

300

350

400


unstim

LPS

LPS plus 2DG



- LPS - LPS0

2000

4000

6000

8000

rela

tive I

L-1

b e

xp

ressio

n

+ succinate

0 LPS 0 LPS0

20000

40000

60000

rela

tive I

L-1

b e

xp

ressio

n

+ butylmalonate




PHD

KG Succinate



PHD

KG Succinate

-



PHD

KG Succinate

KG

012

h

12h+αK

G24

h

24h+αK

G

00

05

10

Rela

tive E

nri

ch

men

t

0 LPS 0 LPS0

10000

20000

30000

rela

tiv

e IL

-1b

ex

pre

ss

ion

+ aKG


WBIL-1β

WBHIF-1α

- +LPSαKG αKG

WBβ-ac n

Summary






succinate

PHD




succinate

PHD





2DG2DG

-LPS+LPS

WBsuccinyllysine

WBIL-1βWBβ-ac n

unstimulated LPS00

01

02

03

04

me

an

em

PA

I va

lue





bull GAPDH


bull PKM12


Key finding










GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION



GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION




LOW GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE


Low glucose

SNF2

YEAST MACROPHAGE

Low glucose 2DG

AMP kinase



GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE

METFORMIN

(mM)

IL-1β qPCR

(mM)



GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE



turning it off






via AMP kinase


Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK



Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK

HIF1





LUKE OrsquoNEILL


OrsquoNEILL LAB


Acknowledgment








Acknowledgment











CARKL



(b) Th17 cells








N=2

N=2

081211 bmdm SS

Ctl LPS (24 hr)0

1

2

3

4

Re

lative

miR

21

0 e

xp

ressio

n

181111 j774

Ctl LPS (24 hr)00

05

10

15

20

25

Re

lativ

e m

iR2

10

exp

ressio

n

310112 Bmdm ss 48h

Ctl LPS (48 hr)0

1

2

3

4

5

Re

lative

miR

21

0 e

xp

ressio

n

N=2

J774s

BMDM BMDM


LPS

HIF-1α IL-1β

Hypothesis


080413 210


05

10

15

20

0 MCSF2 FCS

2 MCSF2 FCS

10 MCSF10 FCS

Re

lativ

e m

iR-2

10

exp

ressio

n

27328140413 pooled 24h no SS n=8

Ctl LPS00

05

10

15

20

Re

lative

miR

-21

0 e

xp

ressio

n

N=8 N=3


LPS

HIF-1α IL-1β

Hypothesis


25030208160413 sdhd n=10

Ctl LPS00

05

10

15

Re

lative

sd

hd

exp

ressio

n

N=10


LPS

HIF-1α IL-1β

Hypothesis



anti-miR210

HIF-1α IL-1β

n=1

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B





INFLAMMATION


PROSTAGLANDINS

1985



INFLAMMATION


1990



INFLAMMATION


NFkappaB p38 JAKs

Mid 90s


T

DAMPs and PAMPs



INFLAMMATION





NFkappaB p38 JAKs

T

DAMPs and PAMPs



INFLAMMATION



Today


NFkappaB p38 JAKs

Tregs Microbiome







WHAT ABOUT SIGNALS


T

DAMPs and PAMPs



INFLAMMATION



Today


NFkappaB p38 JAKs

Tregs Microbiome



TLR4

TNF IL-6 etc

RA Synovial Tissue

gm

l)

Isotype (10

gm

l)

Humira (10

gm

l)

OPN-301

(10

gm

l)

OPN-301

(05

0

1000

2000

3000

4000

5000

6000

7000

Plt001

Plt001

Plt005

IL-6

pg

mlm

g o

f b

iop

sy

gm

l)

Isotype (10

gm

l)

Humira (10

gm

l)

OPN-301

(10

gm

l)

OPN-301

(05

0

10

20

30

40

50

60Plt001

Plt001

Pgt005

TN

F-

pg

ml




as a target

INFLAMMASOMES


TYPE I DIABETES

AIRE Foxp3


-100 0 100 200

0

100

200

300

400

500

600

Days from infusion

SA

A m

gL

-100 0 100 2000

20

40

60

80

100

120

Days from infusion

CR

P m

gL



in all cases

Helen Lachman


crystals

Nlrp3 in disease



Needles Rods

Cartilage integrity


IL-1

Joint inflammation

OA

NFkappaB JNK

2013

COX2 aggrecanases



1990 2010 OBESITY

Type 2 diabetes

WHY CORN SYRUP


IRONY

IRONY

IL1 in T2D

IL1b











Nlrp3

IL-1


Beta cell death

Type 2 diabetes

NFkappaB JNK

2011


INSULIN

+

NORMAL



INSULIN

+

NORMAL

GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION





0

05

1

15

2

25

3

0 LPS

TN

Fa (

ng

ml)

LPS

LPS + 2DG

IL-1beta

TNFalpha

0

50

100

150

200

250

300

350

400

0 LPS

rela

tive I

L-1

beta

exp

ressio

n

LPS

LPS + 2DG








4 8 16 240

5

10

15 unstimulated

LPS

Time (h)

Glu

co

se

util

isa

tio

nG

lyco

lysis

O

xp

ho

s


0

2

4

6

unstimulated LPS

NA

DN

AD

H ra

tio p

mo

l10

6 c

ells


Otto Warburg




Th17

Treg


Nature (2013) 493 346-354











EXTERNAL STRESSORS



EXTERNAL STRESSORS







KEY QUESTION











0

05

1

15

2

25

3

0 LPS

TN

Fa (

ng

ml)

LPS

LPS + 2DG

IL-1beta

TNFalpha

0

50

100

150

200

250

300

350

400

0 LPSre

lati

ve I

L-1

beta

exp

ressio

n

LPS

LPS + 2DG











THE GABA SHUNT


0

2times1006

4times1006

6times1006

su

ccin

ate

(p

ea

k a

rea

)

LPS

unstimulated

- 1 5 10

2DG (mM)



citrate

a-ketoglutarate

succinate

fumarate

malate


12C

13C

14N

15NGABA



production

IL-1beta


200

400

600

800

1000

IL-1b

eta (

pgm

l)IL-6


10

20

30

40IL-

6 (ng

ml)

TNFalpha


1

2

3

TNFa

(ngm

l)



0 20 40 60 80 0

50

100

150 Co n t r o l

Vi g a + L PS

LP S


P e r c

e n

t s u r v

i v a l

At this stage









WB HIF1α WB actin

LPS

2-DG



inhibited by 2DG



PHD

KG Succinate



PHD

KG Succinate

-



PHD

KG Succinate

KG


-50

0

50

100

150

200

250

300

350

400


unstim

LPS

LPS plus 2DG



- LPS - LPS0

2000

4000

6000

8000

rela

tive I

L-1

b e

xp

ressio

n

+ succinate

0 LPS 0 LPS0

20000

40000

60000

rela

tive I

L-1

b e

xp

ressio

n

+ butylmalonate




PHD

KG Succinate



PHD

KG Succinate

-



PHD

KG Succinate

KG

012

h

12h+αK

G24

h

24h+αK

G

00

05

10

Rela

tive E

nri

ch

men

t

0 LPS 0 LPS0

10000

20000

30000

rela

tiv

e IL

-1b

ex

pre

ss

ion

+ aKG


WBIL-1β

WBHIF-1α

- +LPSαKG αKG

WBβ-ac n

Summary






succinate

PHD




succinate

PHD





2DG2DG

-LPS+LPS

WBsuccinyllysine

WBIL-1βWBβ-ac n

unstimulated LPS00

01

02

03

04

me

an

em

PA

I va

lue





bull GAPDH


bull PKM12


Key finding










GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION



GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION




LOW GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE


Low glucose

SNF2

YEAST MACROPHAGE

Low glucose 2DG

AMP kinase



GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE

METFORMIN

(mM)

IL-1β qPCR

(mM)



GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE



turning it off






via AMP kinase


Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK



Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK

HIF1





LUKE OrsquoNEILL


OrsquoNEILL LAB


Acknowledgment








Acknowledgment











CARKL



(b) Th17 cells








N=2

N=2

081211 bmdm SS

Ctl LPS (24 hr)0

1

2

3

4

Re

lative

miR

21

0 e

xp

ressio

n

181111 j774

Ctl LPS (24 hr)00

05

10

15

20

25

Re

lativ

e m

iR2

10

exp

ressio

n

310112 Bmdm ss 48h

Ctl LPS (48 hr)0

1

2

3

4

5

Re

lative

miR

21

0 e

xp

ressio

n

N=2

J774s

BMDM BMDM


LPS

HIF-1α IL-1β

Hypothesis


080413 210


05

10

15

20

0 MCSF2 FCS

2 MCSF2 FCS

10 MCSF10 FCS

Re

lativ

e m

iR-2

10

exp

ressio

n

27328140413 pooled 24h no SS n=8

Ctl LPS00

05

10

15

20

Re

lative

miR

-21

0 e

xp

ressio

n

N=8 N=3


LPS

HIF-1α IL-1β

Hypothesis


25030208160413 sdhd n=10

Ctl LPS00

05

10

15

Re

lative

sd

hd

exp

ressio

n

N=10


LPS

HIF-1α IL-1β

Hypothesis



anti-miR210

HIF-1α IL-1β

n=1

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B



INFLAMMATION


1990



INFLAMMATION


NFkappaB p38 JAKs

Mid 90s


T

DAMPs and PAMPs



INFLAMMATION





NFkappaB p38 JAKs

T

DAMPs and PAMPs



INFLAMMATION



Today


NFkappaB p38 JAKs

Tregs Microbiome







WHAT ABOUT SIGNALS


T

DAMPs and PAMPs



INFLAMMATION



Today


NFkappaB p38 JAKs

Tregs Microbiome



TLR4

TNF IL-6 etc

RA Synovial Tissue

gm

l)

Isotype (10

gm

l)

Humira (10

gm

l)

OPN-301

(10

gm

l)

OPN-301

(05

0

1000

2000

3000

4000

5000

6000

7000

Plt001

Plt001

Plt005

IL-6

pg

mlm

g o

f b

iop

sy

gm

l)

Isotype (10

gm

l)

Humira (10

gm

l)

OPN-301

(10

gm

l)

OPN-301

(05

0

10

20

30

40

50

60Plt001

Plt001

Pgt005

TN

F-

pg

ml




as a target

INFLAMMASOMES


TYPE I DIABETES

AIRE Foxp3


-100 0 100 200

0

100

200

300

400

500

600

Days from infusion

SA

A m

gL

-100 0 100 2000

20

40

60

80

100

120

Days from infusion

CR

P m

gL



in all cases

Helen Lachman


crystals

Nlrp3 in disease



Needles Rods

Cartilage integrity


IL-1

Joint inflammation

OA

NFkappaB JNK

2013

COX2 aggrecanases



1990 2010 OBESITY

Type 2 diabetes

WHY CORN SYRUP


IRONY

IRONY

IL1 in T2D

IL1b











Nlrp3

IL-1


Beta cell death

Type 2 diabetes

NFkappaB JNK

2011


INSULIN

+

NORMAL



INSULIN

+

NORMAL

GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION





0

05

1

15

2

25

3

0 LPS

TN

Fa (

ng

ml)

LPS

LPS + 2DG

IL-1beta

TNFalpha

0

50

100

150

200

250

300

350

400

0 LPS

rela

tive I

L-1

beta

exp

ressio

n

LPS

LPS + 2DG








4 8 16 240

5

10

15 unstimulated

LPS

Time (h)

Glu

co

se

util

isa

tio

nG

lyco

lysis

O

xp

ho

s


0

2

4

6

unstimulated LPS

NA

DN

AD

H ra

tio p

mo

l10

6 c

ells


Otto Warburg




Th17

Treg


Nature (2013) 493 346-354











EXTERNAL STRESSORS



EXTERNAL STRESSORS







KEY QUESTION











0

05

1

15

2

25

3

0 LPS

TN

Fa (

ng

ml)

LPS

LPS + 2DG

IL-1beta

TNFalpha

0

50

100

150

200

250

300

350

400

0 LPSre

lati

ve I

L-1

beta

exp

ressio

n

LPS

LPS + 2DG











THE GABA SHUNT


0

2times1006

4times1006

6times1006

su

ccin

ate

(p

ea

k a

rea

)

LPS

unstimulated

- 1 5 10

2DG (mM)



citrate

a-ketoglutarate

succinate

fumarate

malate


12C

13C

14N

15NGABA



production

IL-1beta


200

400

600

800

1000

IL-1b

eta (

pgm

l)IL-6


10

20

30

40IL-

6 (ng

ml)

TNFalpha


1

2

3

TNFa

(ngm

l)



0 20 40 60 80 0

50

100

150 Co n t r o l

Vi g a + L PS

LP S


P e r c

e n

t s u r v

i v a l

At this stage









WB HIF1α WB actin

LPS

2-DG



inhibited by 2DG



PHD

KG Succinate



PHD

KG Succinate

-



PHD

KG Succinate

KG


-50

0

50

100

150

200

250

300

350

400


unstim

LPS

LPS plus 2DG



- LPS - LPS0

2000

4000

6000

8000

rela

tive I

L-1

b e

xp

ressio

n

+ succinate

0 LPS 0 LPS0

20000

40000

60000

rela

tive I

L-1

b e

xp

ressio

n

+ butylmalonate




PHD

KG Succinate



PHD

KG Succinate

-



PHD

KG Succinate

KG

012

h

12h+αK

G24

h

24h+αK

G

00

05

10

Rela

tive E

nri

ch

men

t

0 LPS 0 LPS0

10000

20000

30000

rela

tiv

e IL

-1b

ex

pre

ss

ion

+ aKG


WBIL-1β

WBHIF-1α

- +LPSαKG αKG

WBβ-ac n

Summary






succinate

PHD




succinate

PHD





2DG2DG

-LPS+LPS

WBsuccinyllysine

WBIL-1βWBβ-ac n

unstimulated LPS00

01

02

03

04

me

an

em

PA

I va

lue





bull GAPDH


bull PKM12


Key finding










GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION



GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION




LOW GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE


Low glucose

SNF2

YEAST MACROPHAGE

Low glucose 2DG

AMP kinase



GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE

METFORMIN

(mM)

IL-1β qPCR

(mM)



GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE



turning it off






via AMP kinase


Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK



Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK

HIF1





LUKE OrsquoNEILL


OrsquoNEILL LAB


Acknowledgment








Acknowledgment











CARKL



(b) Th17 cells








N=2

N=2

081211 bmdm SS

Ctl LPS (24 hr)0

1

2

3

4

Re

lative

miR

21

0 e

xp

ressio

n

181111 j774

Ctl LPS (24 hr)00

05

10

15

20

25

Re

lativ

e m

iR2

10

exp

ressio

n

310112 Bmdm ss 48h

Ctl LPS (48 hr)0

1

2

3

4

5

Re

lative

miR

21

0 e

xp

ressio

n

N=2

J774s

BMDM BMDM


LPS

HIF-1α IL-1β

Hypothesis


080413 210


05

10

15

20

0 MCSF2 FCS

2 MCSF2 FCS

10 MCSF10 FCS

Re

lativ

e m

iR-2

10

exp

ressio

n

27328140413 pooled 24h no SS n=8

Ctl LPS00

05

10

15

20

Re

lative

miR

-21

0 e

xp

ressio

n

N=8 N=3


LPS

HIF-1α IL-1β

Hypothesis


25030208160413 sdhd n=10

Ctl LPS00

05

10

15

Re

lative

sd

hd

exp

ressio

n

N=10


LPS

HIF-1α IL-1β

Hypothesis



anti-miR210

HIF-1α IL-1β

n=1

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

T

DAMPs and PAMPs



INFLAMMATION





NFkappaB p38 JAKs

T

DAMPs and PAMPs



INFLAMMATION



Today


NFkappaB p38 JAKs

Tregs Microbiome







WHAT ABOUT SIGNALS


T

DAMPs and PAMPs



INFLAMMATION



Today


NFkappaB p38 JAKs

Tregs Microbiome



TLR4

TNF IL-6 etc

RA Synovial Tissue

gm

l)

Isotype (10

gm

l)

Humira (10

gm

l)

OPN-301

(10

gm

l)

OPN-301

(05

0

1000

2000

3000

4000

5000

6000

7000

Plt001

Plt001

Plt005

IL-6

pg

mlm

g o

f b

iop

sy

gm

l)

Isotype (10

gm

l)

Humira (10

gm

l)

OPN-301

(10

gm

l)

OPN-301

(05

0

10

20

30

40

50

60Plt001

Plt001

Pgt005

TN

F-

pg

ml




as a target

INFLAMMASOMES


TYPE I DIABETES

AIRE Foxp3


-100 0 100 200

0

100

200

300

400

500

600

Days from infusion

SA

A m

gL

-100 0 100 2000

20

40

60

80

100

120

Days from infusion

CR

P m

gL



in all cases

Helen Lachman


crystals

Nlrp3 in disease



Needles Rods

Cartilage integrity


IL-1

Joint inflammation

OA

NFkappaB JNK

2013

COX2 aggrecanases



1990 2010 OBESITY

Type 2 diabetes

WHY CORN SYRUP


IRONY

IRONY

IL1 in T2D

IL1b











Nlrp3

IL-1


Beta cell death

Type 2 diabetes

NFkappaB JNK

2011


INSULIN

+

NORMAL



INSULIN

+

NORMAL

GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION





0

05

1

15

2

25

3

0 LPS

TN

Fa (

ng

ml)

LPS

LPS + 2DG

IL-1beta

TNFalpha

0

50

100

150

200

250

300

350

400

0 LPS

rela

tive I

L-1

beta

exp

ressio

n

LPS

LPS + 2DG








4 8 16 240

5

10

15 unstimulated

LPS

Time (h)

Glu

co

se

util

isa

tio

nG

lyco

lysis

O

xp

ho

s


0

2

4

6

unstimulated LPS

NA

DN

AD

H ra

tio p

mo

l10

6 c

ells


Otto Warburg




Th17

Treg


Nature (2013) 493 346-354











EXTERNAL STRESSORS



EXTERNAL STRESSORS







KEY QUESTION











0

05

1

15

2

25

3

0 LPS

TN

Fa (

ng

ml)

LPS

LPS + 2DG

IL-1beta

TNFalpha

0

50

100

150

200

250

300

350

400

0 LPSre

lati

ve I

L-1

beta

exp

ressio

n

LPS

LPS + 2DG











THE GABA SHUNT


0

2times1006

4times1006

6times1006

su

ccin

ate

(p

ea

k a

rea

)

LPS

unstimulated

- 1 5 10

2DG (mM)



citrate

a-ketoglutarate

succinate

fumarate

malate


12C

13C

14N

15NGABA



production

IL-1beta


200

400

600

800

1000

IL-1b

eta (

pgm

l)IL-6


10

20

30

40IL-

6 (ng

ml)

TNFalpha


1

2

3

TNFa

(ngm

l)



0 20 40 60 80 0

50

100

150 Co n t r o l

Vi g a + L PS

LP S


P e r c

e n

t s u r v

i v a l

At this stage









WB HIF1α WB actin

LPS

2-DG



inhibited by 2DG



PHD

KG Succinate



PHD

KG Succinate

-



PHD

KG Succinate

KG


-50

0

50

100

150

200

250

300

350

400


unstim

LPS

LPS plus 2DG



- LPS - LPS0

2000

4000

6000

8000

rela

tive I

L-1

b e

xp

ressio

n

+ succinate

0 LPS 0 LPS0

20000

40000

60000

rela

tive I

L-1

b e

xp

ressio

n

+ butylmalonate




PHD

KG Succinate



PHD

KG Succinate

-



PHD

KG Succinate

KG

012

h

12h+αK

G24

h

24h+αK

G

00

05

10

Rela

tive E

nri

ch

men

t

0 LPS 0 LPS0

10000

20000

30000

rela

tiv

e IL

-1b

ex

pre

ss

ion

+ aKG


WBIL-1β

WBHIF-1α

- +LPSαKG αKG

WBβ-ac n

Summary






succinate

PHD




succinate

PHD





2DG2DG

-LPS+LPS

WBsuccinyllysine

WBIL-1βWBβ-ac n

unstimulated LPS00

01

02

03

04

me

an

em

PA

I va

lue





bull GAPDH


bull PKM12


Key finding










GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION



GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION




LOW GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE


Low glucose

SNF2

YEAST MACROPHAGE

Low glucose 2DG

AMP kinase



GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE

METFORMIN

(mM)

IL-1β qPCR

(mM)



GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE



turning it off






via AMP kinase


Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK



Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK

HIF1





LUKE OrsquoNEILL


OrsquoNEILL LAB


Acknowledgment








Acknowledgment











CARKL



(b) Th17 cells








N=2

N=2

081211 bmdm SS

Ctl LPS (24 hr)0

1

2

3

4

Re

lative

miR

21

0 e

xp

ressio

n

181111 j774

Ctl LPS (24 hr)00

05

10

15

20

25

Re

lativ

e m

iR2

10

exp

ressio

n

310112 Bmdm ss 48h

Ctl LPS (48 hr)0

1

2

3

4

5

Re

lative

miR

21

0 e

xp

ressio

n

N=2

J774s

BMDM BMDM


LPS

HIF-1α IL-1β

Hypothesis


080413 210


05

10

15

20

0 MCSF2 FCS

2 MCSF2 FCS

10 MCSF10 FCS

Re

lativ

e m

iR-2

10

exp

ressio

n

27328140413 pooled 24h no SS n=8

Ctl LPS00

05

10

15

20

Re

lative

miR

-21

0 e

xp

ressio

n

N=8 N=3


LPS

HIF-1α IL-1β

Hypothesis


25030208160413 sdhd n=10

Ctl LPS00

05

10

15

Re

lative

sd

hd

exp

ressio

n

N=10


LPS

HIF-1α IL-1β

Hypothesis



anti-miR210

HIF-1α IL-1β

n=1

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B







WHAT ABOUT SIGNALS


T

DAMPs and PAMPs



INFLAMMATION



Today


NFkappaB p38 JAKs

Tregs Microbiome



TLR4

TNF IL-6 etc

RA Synovial Tissue

gm

l)

Isotype (10

gm

l)

Humira (10

gm

l)

OPN-301

(10

gm

l)

OPN-301

(05

0

1000

2000

3000

4000

5000

6000

7000

Plt001

Plt001

Plt005

IL-6

pg

mlm

g o

f b

iop

sy

gm

l)

Isotype (10

gm

l)

Humira (10

gm

l)

OPN-301

(10

gm

l)

OPN-301

(05

0

10

20

30

40

50

60Plt001

Plt001

Pgt005

TN

F-

pg

ml




as a target

INFLAMMASOMES


TYPE I DIABETES

AIRE Foxp3


-100 0 100 200

0

100

200

300

400

500

600

Days from infusion

SA

A m

gL

-100 0 100 2000

20

40

60

80

100

120

Days from infusion

CR

P m

gL



in all cases

Helen Lachman


crystals

Nlrp3 in disease



Needles Rods

Cartilage integrity


IL-1

Joint inflammation

OA

NFkappaB JNK

2013

COX2 aggrecanases



1990 2010 OBESITY

Type 2 diabetes

WHY CORN SYRUP


IRONY

IRONY

IL1 in T2D

IL1b











Nlrp3

IL-1


Beta cell death

Type 2 diabetes

NFkappaB JNK

2011


INSULIN

+

NORMAL



INSULIN

+

NORMAL

GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION





0

05

1

15

2

25

3

0 LPS

TN

Fa (

ng

ml)

LPS

LPS + 2DG

IL-1beta

TNFalpha

0

50

100

150

200

250

300

350

400

0 LPS

rela

tive I

L-1

beta

exp

ressio

n

LPS

LPS + 2DG








4 8 16 240

5

10

15 unstimulated

LPS

Time (h)

Glu

co

se

util

isa

tio

nG

lyco

lysis

O

xp

ho

s


0

2

4

6

unstimulated LPS

NA

DN

AD

H ra

tio p

mo

l10

6 c

ells


Otto Warburg




Th17

Treg


Nature (2013) 493 346-354











EXTERNAL STRESSORS



EXTERNAL STRESSORS







KEY QUESTION











0

05

1

15

2

25

3

0 LPS

TN

Fa (

ng

ml)

LPS

LPS + 2DG

IL-1beta

TNFalpha

0

50

100

150

200

250

300

350

400

0 LPSre

lati

ve I

L-1

beta

exp

ressio

n

LPS

LPS + 2DG











THE GABA SHUNT


0

2times1006

4times1006

6times1006

su

ccin

ate

(p

ea

k a

rea

)

LPS

unstimulated

- 1 5 10

2DG (mM)



citrate

a-ketoglutarate

succinate

fumarate

malate


12C

13C

14N

15NGABA



production

IL-1beta


200

400

600

800

1000

IL-1b

eta (

pgm

l)IL-6


10

20

30

40IL-

6 (ng

ml)

TNFalpha


1

2

3

TNFa

(ngm

l)



0 20 40 60 80 0

50

100

150 Co n t r o l

Vi g a + L PS

LP S


P e r c

e n

t s u r v

i v a l

At this stage









WB HIF1α WB actin

LPS

2-DG



inhibited by 2DG



PHD

KG Succinate



PHD

KG Succinate

-



PHD

KG Succinate

KG


-50

0

50

100

150

200

250

300

350

400


unstim

LPS

LPS plus 2DG



- LPS - LPS0

2000

4000

6000

8000

rela

tive I

L-1

b e

xp

ressio

n

+ succinate

0 LPS 0 LPS0

20000

40000

60000

rela

tive I

L-1

b e

xp

ressio

n

+ butylmalonate




PHD

KG Succinate



PHD

KG Succinate

-



PHD

KG Succinate

KG

012

h

12h+αK

G24

h

24h+αK

G

00

05

10

Rela

tive E

nri

ch

men

t

0 LPS 0 LPS0

10000

20000

30000

rela

tiv

e IL

-1b

ex

pre

ss

ion

+ aKG


WBIL-1β

WBHIF-1α

- +LPSαKG αKG

WBβ-ac n

Summary






succinate

PHD




succinate

PHD





2DG2DG

-LPS+LPS

WBsuccinyllysine

WBIL-1βWBβ-ac n

unstimulated LPS00

01

02

03

04

me

an

em

PA

I va

lue





bull GAPDH


bull PKM12


Key finding










GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION



GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION




LOW GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE


Low glucose

SNF2

YEAST MACROPHAGE

Low glucose 2DG

AMP kinase



GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE

METFORMIN

(mM)

IL-1β qPCR

(mM)



GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE



turning it off






via AMP kinase


Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK



Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK

HIF1





LUKE OrsquoNEILL


OrsquoNEILL LAB


Acknowledgment








Acknowledgment











CARKL



(b) Th17 cells








N=2

N=2

081211 bmdm SS

Ctl LPS (24 hr)0

1

2

3

4

Re

lative

miR

21

0 e

xp

ressio

n

181111 j774

Ctl LPS (24 hr)00

05

10

15

20

25

Re

lativ

e m

iR2

10

exp

ressio

n

310112 Bmdm ss 48h

Ctl LPS (48 hr)0

1

2

3

4

5

Re

lative

miR

21

0 e

xp

ressio

n

N=2

J774s

BMDM BMDM


LPS

HIF-1α IL-1β

Hypothesis


080413 210


05

10

15

20

0 MCSF2 FCS

2 MCSF2 FCS

10 MCSF10 FCS

Re

lativ

e m

iR-2

10

exp

ressio

n

27328140413 pooled 24h no SS n=8

Ctl LPS00

05

10

15

20

Re

lative

miR

-21

0 e

xp

ressio

n

N=8 N=3


LPS

HIF-1α IL-1β

Hypothesis


25030208160413 sdhd n=10

Ctl LPS00

05

10

15

Re

lative

sd

hd

exp

ressio

n

N=10


LPS

HIF-1α IL-1β

Hypothesis



anti-miR210

HIF-1α IL-1β

n=1

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

T

DAMPs and PAMPs



INFLAMMATION



Today


NFkappaB p38 JAKs

Tregs Microbiome



TLR4

TNF IL-6 etc

RA Synovial Tissue

gm

l)

Isotype (10

gm

l)

Humira (10

gm

l)

OPN-301

(10

gm

l)

OPN-301

(05

0

1000

2000

3000

4000

5000

6000

7000

Plt001

Plt001

Plt005

IL-6

pg

mlm

g o

f b

iop

sy

gm

l)

Isotype (10

gm

l)

Humira (10

gm

l)

OPN-301

(10

gm

l)

OPN-301

(05

0

10

20

30

40

50

60Plt001

Plt001

Pgt005

TN

F-

pg

ml




as a target

INFLAMMASOMES


TYPE I DIABETES

AIRE Foxp3


-100 0 100 200

0

100

200

300

400

500

600

Days from infusion

SA

A m

gL

-100 0 100 2000

20

40

60

80

100

120

Days from infusion

CR

P m

gL



in all cases

Helen Lachman


crystals

Nlrp3 in disease



Needles Rods

Cartilage integrity


IL-1

Joint inflammation

OA

NFkappaB JNK

2013

COX2 aggrecanases



1990 2010 OBESITY

Type 2 diabetes

WHY CORN SYRUP


IRONY

IRONY

IL1 in T2D

IL1b











Nlrp3

IL-1


Beta cell death

Type 2 diabetes

NFkappaB JNK

2011


INSULIN

+

NORMAL



INSULIN

+

NORMAL

GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION





0

05

1

15

2

25

3

0 LPS

TN

Fa (

ng

ml)

LPS

LPS + 2DG

IL-1beta

TNFalpha

0

50

100

150

200

250

300

350

400

0 LPS

rela

tive I

L-1

beta

exp

ressio

n

LPS

LPS + 2DG








4 8 16 240

5

10

15 unstimulated

LPS

Time (h)

Glu

co

se

util

isa

tio

nG

lyco

lysis

O

xp

ho

s


0

2

4

6

unstimulated LPS

NA

DN

AD

H ra

tio p

mo

l10

6 c

ells


Otto Warburg




Th17

Treg


Nature (2013) 493 346-354











EXTERNAL STRESSORS



EXTERNAL STRESSORS







KEY QUESTION











0

05

1

15

2

25

3

0 LPS

TN

Fa (

ng

ml)

LPS

LPS + 2DG

IL-1beta

TNFalpha

0

50

100

150

200

250

300

350

400

0 LPSre

lati

ve I

L-1

beta

exp

ressio

n

LPS

LPS + 2DG











THE GABA SHUNT


0

2times1006

4times1006

6times1006

su

ccin

ate

(p

ea

k a

rea

)

LPS

unstimulated

- 1 5 10

2DG (mM)



citrate

a-ketoglutarate

succinate

fumarate

malate


12C

13C

14N

15NGABA



production

IL-1beta


200

400

600

800

1000

IL-1b

eta (

pgm

l)IL-6


10

20

30

40IL-

6 (ng

ml)

TNFalpha


1

2

3

TNFa

(ngm

l)



0 20 40 60 80 0

50

100

150 Co n t r o l

Vi g a + L PS

LP S


P e r c

e n

t s u r v

i v a l

At this stage









WB HIF1α WB actin

LPS

2-DG



inhibited by 2DG



PHD

KG Succinate



PHD

KG Succinate

-



PHD

KG Succinate

KG


-50

0

50

100

150

200

250

300

350

400


unstim

LPS

LPS plus 2DG



- LPS - LPS0

2000

4000

6000

8000

rela

tive I

L-1

b e

xp

ressio

n

+ succinate

0 LPS 0 LPS0

20000

40000

60000

rela

tive I

L-1

b e

xp

ressio

n

+ butylmalonate




PHD

KG Succinate



PHD

KG Succinate

-



PHD

KG Succinate

KG

012

h

12h+αK

G24

h

24h+αK

G

00

05

10

Rela

tive E

nri

ch

men

t

0 LPS 0 LPS0

10000

20000

30000

rela

tiv

e IL

-1b

ex

pre

ss

ion

+ aKG


WBIL-1β

WBHIF-1α

- +LPSαKG αKG

WBβ-ac n

Summary






succinate

PHD




succinate

PHD





2DG2DG

-LPS+LPS

WBsuccinyllysine

WBIL-1βWBβ-ac n

unstimulated LPS00

01

02

03

04

me

an

em

PA

I va

lue





bull GAPDH


bull PKM12


Key finding










GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION



GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION




LOW GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE


Low glucose

SNF2

YEAST MACROPHAGE

Low glucose 2DG

AMP kinase



GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE

METFORMIN

(mM)

IL-1β qPCR

(mM)



GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE



turning it off






via AMP kinase


Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK



Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK

HIF1





LUKE OrsquoNEILL


OrsquoNEILL LAB


Acknowledgment








Acknowledgment











CARKL



(b) Th17 cells








N=2

N=2

081211 bmdm SS

Ctl LPS (24 hr)0

1

2

3

4

Re

lative

miR

21

0 e

xp

ressio

n

181111 j774

Ctl LPS (24 hr)00

05

10

15

20

25

Re

lativ

e m

iR2

10

exp

ressio

n

310112 Bmdm ss 48h

Ctl LPS (48 hr)0

1

2

3

4

5

Re

lative

miR

21

0 e

xp

ressio

n

N=2

J774s

BMDM BMDM


LPS

HIF-1α IL-1β

Hypothesis


080413 210


05

10

15

20

0 MCSF2 FCS

2 MCSF2 FCS

10 MCSF10 FCS

Re

lativ

e m

iR-2

10

exp

ressio

n

27328140413 pooled 24h no SS n=8

Ctl LPS00

05

10

15

20

Re

lative

miR

-21

0 e

xp

ressio

n

N=8 N=3


LPS

HIF-1α IL-1β

Hypothesis


25030208160413 sdhd n=10

Ctl LPS00

05

10

15

Re

lative

sd

hd

exp

ressio

n

N=10


LPS

HIF-1α IL-1β

Hypothesis



anti-miR210

HIF-1α IL-1β

n=1

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

RA Synovial Tissue

gm

l)

Isotype (10

gm

l)

Humira (10

gm

l)

OPN-301

(10

gm

l)

OPN-301

(05

0

1000

2000

3000

4000

5000

6000

7000

Plt001

Plt001

Plt005

IL-6

pg

mlm

g o

f b

iop

sy

gm

l)

Isotype (10

gm

l)

Humira (10

gm

l)

OPN-301

(10

gm

l)

OPN-301

(05

0

10

20

30

40

50

60Plt001

Plt001

Pgt005

TN

F-

pg

ml




as a target

INFLAMMASOMES


TYPE I DIABETES

AIRE Foxp3


-100 0 100 200

0

100

200

300

400

500

600

Days from infusion

SA

A m

gL

-100 0 100 2000

20

40

60

80

100

120

Days from infusion

CR

P m

gL



in all cases

Helen Lachman


crystals

Nlrp3 in disease



Needles Rods

Cartilage integrity


IL-1

Joint inflammation

OA

NFkappaB JNK

2013

COX2 aggrecanases



1990 2010 OBESITY

Type 2 diabetes

WHY CORN SYRUP


IRONY

IRONY

IL1 in T2D

IL1b











Nlrp3

IL-1


Beta cell death

Type 2 diabetes

NFkappaB JNK

2011


INSULIN

+

NORMAL



INSULIN

+

NORMAL

GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION





0

05

1

15

2

25

3

0 LPS

TN

Fa (

ng

ml)

LPS

LPS + 2DG

IL-1beta

TNFalpha

0

50

100

150

200

250

300

350

400

0 LPS

rela

tive I

L-1

beta

exp

ressio

n

LPS

LPS + 2DG








4 8 16 240

5

10

15 unstimulated

LPS

Time (h)

Glu

co

se

util

isa

tio

nG

lyco

lysis

O

xp

ho

s


0

2

4

6

unstimulated LPS

NA

DN

AD

H ra

tio p

mo

l10

6 c

ells


Otto Warburg




Th17

Treg


Nature (2013) 493 346-354











EXTERNAL STRESSORS



EXTERNAL STRESSORS







KEY QUESTION











0

05

1

15

2

25

3

0 LPS

TN

Fa (

ng

ml)

LPS

LPS + 2DG

IL-1beta

TNFalpha

0

50

100

150

200

250

300

350

400

0 LPSre

lati

ve I

L-1

beta

exp

ressio

n

LPS

LPS + 2DG











THE GABA SHUNT


0

2times1006

4times1006

6times1006

su

ccin

ate

(p

ea

k a

rea

)

LPS

unstimulated

- 1 5 10

2DG (mM)



citrate

a-ketoglutarate

succinate

fumarate

malate


12C

13C

14N

15NGABA



production

IL-1beta


200

400

600

800

1000

IL-1b

eta (

pgm

l)IL-6


10

20

30

40IL-

6 (ng

ml)

TNFalpha


1

2

3

TNFa

(ngm

l)



0 20 40 60 80 0

50

100

150 Co n t r o l

Vi g a + L PS

LP S


P e r c

e n

t s u r v

i v a l

At this stage









WB HIF1α WB actin

LPS

2-DG



inhibited by 2DG



PHD

KG Succinate



PHD

KG Succinate

-



PHD

KG Succinate

KG


-50

0

50

100

150

200

250

300

350

400


unstim

LPS

LPS plus 2DG



- LPS - LPS0

2000

4000

6000

8000

rela

tive I

L-1

b e

xp

ressio

n

+ succinate

0 LPS 0 LPS0

20000

40000

60000

rela

tive I

L-1

b e

xp

ressio

n

+ butylmalonate




PHD

KG Succinate



PHD

KG Succinate

-



PHD

KG Succinate

KG

012

h

12h+αK

G24

h

24h+αK

G

00

05

10

Rela

tive E

nri

ch

men

t

0 LPS 0 LPS0

10000

20000

30000

rela

tiv

e IL

-1b

ex

pre

ss

ion

+ aKG


WBIL-1β

WBHIF-1α

- +LPSαKG αKG

WBβ-ac n

Summary






succinate

PHD




succinate

PHD





2DG2DG

-LPS+LPS

WBsuccinyllysine

WBIL-1βWBβ-ac n

unstimulated LPS00

01

02

03

04

me

an

em

PA

I va

lue





bull GAPDH


bull PKM12


Key finding










GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION



GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION




LOW GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE


Low glucose

SNF2

YEAST MACROPHAGE

Low glucose 2DG

AMP kinase



GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE

METFORMIN

(mM)

IL-1β qPCR

(mM)



GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE



turning it off






via AMP kinase


Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK



Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK

HIF1





LUKE OrsquoNEILL


OrsquoNEILL LAB


Acknowledgment








Acknowledgment











CARKL



(b) Th17 cells








N=2

N=2

081211 bmdm SS

Ctl LPS (24 hr)0

1

2

3

4

Re

lative

miR

21

0 e

xp

ressio

n

181111 j774

Ctl LPS (24 hr)00

05

10

15

20

25

Re

lativ

e m

iR2

10

exp

ressio

n

310112 Bmdm ss 48h

Ctl LPS (48 hr)0

1

2

3

4

5

Re

lative

miR

21

0 e

xp

ressio

n

N=2

J774s

BMDM BMDM


LPS

HIF-1α IL-1β

Hypothesis


080413 210


05

10

15

20

0 MCSF2 FCS

2 MCSF2 FCS

10 MCSF10 FCS

Re

lativ

e m

iR-2

10

exp

ressio

n

27328140413 pooled 24h no SS n=8

Ctl LPS00

05

10

15

20

Re

lative

miR

-21

0 e

xp

ressio

n

N=8 N=3


LPS

HIF-1α IL-1β

Hypothesis


25030208160413 sdhd n=10

Ctl LPS00

05

10

15

Re

lative

sd

hd

exp

ressio

n

N=10


LPS

HIF-1α IL-1β

Hypothesis



anti-miR210

HIF-1α IL-1β

n=1

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

INFLAMMASOMES


TYPE I DIABETES

AIRE Foxp3


-100 0 100 200

0

100

200

300

400

500

600

Days from infusion

SA

A m

gL

-100 0 100 2000

20

40

60

80

100

120

Days from infusion

CR

P m

gL



in all cases

Helen Lachman


crystals

Nlrp3 in disease



Needles Rods

Cartilage integrity


IL-1

Joint inflammation

OA

NFkappaB JNK

2013

COX2 aggrecanases



1990 2010 OBESITY

Type 2 diabetes

WHY CORN SYRUP


IRONY

IRONY

IL1 in T2D

IL1b











Nlrp3

IL-1


Beta cell death

Type 2 diabetes

NFkappaB JNK

2011


INSULIN

+

NORMAL



INSULIN

+

NORMAL

GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION





0

05

1

15

2

25

3

0 LPS

TN

Fa (

ng

ml)

LPS

LPS + 2DG

IL-1beta

TNFalpha

0

50

100

150

200

250

300

350

400

0 LPS

rela

tive I

L-1

beta

exp

ressio

n

LPS

LPS + 2DG








4 8 16 240

5

10

15 unstimulated

LPS

Time (h)

Glu

co

se

util

isa

tio

nG

lyco

lysis

O

xp

ho

s


0

2

4

6

unstimulated LPS

NA

DN

AD

H ra

tio p

mo

l10

6 c

ells


Otto Warburg




Th17

Treg


Nature (2013) 493 346-354











EXTERNAL STRESSORS



EXTERNAL STRESSORS







KEY QUESTION











0

05

1

15

2

25

3

0 LPS

TN

Fa (

ng

ml)

LPS

LPS + 2DG

IL-1beta

TNFalpha

0

50

100

150

200

250

300

350

400

0 LPSre

lati

ve I

L-1

beta

exp

ressio

n

LPS

LPS + 2DG











THE GABA SHUNT


0

2times1006

4times1006

6times1006

su

ccin

ate

(p

ea

k a

rea

)

LPS

unstimulated

- 1 5 10

2DG (mM)



citrate

a-ketoglutarate

succinate

fumarate

malate


12C

13C

14N

15NGABA



production

IL-1beta


200

400

600

800

1000

IL-1b

eta (

pgm

l)IL-6


10

20

30

40IL-

6 (ng

ml)

TNFalpha


1

2

3

TNFa

(ngm

l)



0 20 40 60 80 0

50

100

150 Co n t r o l

Vi g a + L PS

LP S


P e r c

e n

t s u r v

i v a l

At this stage









WB HIF1α WB actin

LPS

2-DG



inhibited by 2DG



PHD

KG Succinate



PHD

KG Succinate

-



PHD

KG Succinate

KG


-50

0

50

100

150

200

250

300

350

400


unstim

LPS

LPS plus 2DG



- LPS - LPS0

2000

4000

6000

8000

rela

tive I

L-1

b e

xp

ressio

n

+ succinate

0 LPS 0 LPS0

20000

40000

60000

rela

tive I

L-1

b e

xp

ressio

n

+ butylmalonate




PHD

KG Succinate



PHD

KG Succinate

-



PHD

KG Succinate

KG

012

h

12h+αK

G24

h

24h+αK

G

00

05

10

Rela

tive E

nri

ch

men

t

0 LPS 0 LPS0

10000

20000

30000

rela

tiv

e IL

-1b

ex

pre

ss

ion

+ aKG


WBIL-1β

WBHIF-1α

- +LPSαKG αKG

WBβ-ac n

Summary






succinate

PHD




succinate

PHD





2DG2DG

-LPS+LPS

WBsuccinyllysine

WBIL-1βWBβ-ac n

unstimulated LPS00

01

02

03

04

me

an

em

PA

I va

lue





bull GAPDH


bull PKM12


Key finding










GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION



GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION




LOW GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE


Low glucose

SNF2

YEAST MACROPHAGE

Low glucose 2DG

AMP kinase



GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE

METFORMIN

(mM)

IL-1β qPCR

(mM)



GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE



turning it off






via AMP kinase


Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK



Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK

HIF1





LUKE OrsquoNEILL


OrsquoNEILL LAB


Acknowledgment








Acknowledgment











CARKL



(b) Th17 cells








N=2

N=2

081211 bmdm SS

Ctl LPS (24 hr)0

1

2

3

4

Re

lative

miR

21

0 e

xp

ressio

n

181111 j774

Ctl LPS (24 hr)00

05

10

15

20

25

Re

lativ

e m

iR2

10

exp

ressio

n

310112 Bmdm ss 48h

Ctl LPS (48 hr)0

1

2

3

4

5

Re

lative

miR

21

0 e

xp

ressio

n

N=2

J774s

BMDM BMDM


LPS

HIF-1α IL-1β

Hypothesis


080413 210


05

10

15

20

0 MCSF2 FCS

2 MCSF2 FCS

10 MCSF10 FCS

Re

lativ

e m

iR-2

10

exp

ressio

n

27328140413 pooled 24h no SS n=8

Ctl LPS00

05

10

15

20

Re

lative

miR

-21

0 e

xp

ressio

n

N=8 N=3


LPS

HIF-1α IL-1β

Hypothesis


25030208160413 sdhd n=10

Ctl LPS00

05

10

15

Re

lative

sd

hd

exp

ressio

n

N=10


LPS

HIF-1α IL-1β

Hypothesis



anti-miR210

HIF-1α IL-1β

n=1

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B


-100 0 100 200

0

100

200

300

400

500

600

Days from infusion

SA

A m

gL

-100 0 100 2000

20

40

60

80

100

120

Days from infusion

CR

P m

gL



in all cases

Helen Lachman


crystals

Nlrp3 in disease



Needles Rods

Cartilage integrity


IL-1

Joint inflammation

OA

NFkappaB JNK

2013

COX2 aggrecanases



1990 2010 OBESITY

Type 2 diabetes

WHY CORN SYRUP


IRONY

IRONY

IL1 in T2D

IL1b











Nlrp3

IL-1


Beta cell death

Type 2 diabetes

NFkappaB JNK

2011


INSULIN

+

NORMAL



INSULIN

+

NORMAL

GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION





0

05

1

15

2

25

3

0 LPS

TN

Fa (

ng

ml)

LPS

LPS + 2DG

IL-1beta

TNFalpha

0

50

100

150

200

250

300

350

400

0 LPS

rela

tive I

L-1

beta

exp

ressio

n

LPS

LPS + 2DG








4 8 16 240

5

10

15 unstimulated

LPS

Time (h)

Glu

co

se

util

isa

tio

nG

lyco

lysis

O

xp

ho

s


0

2

4

6

unstimulated LPS

NA

DN

AD

H ra

tio p

mo

l10

6 c

ells


Otto Warburg




Th17

Treg


Nature (2013) 493 346-354











EXTERNAL STRESSORS



EXTERNAL STRESSORS







KEY QUESTION











0

05

1

15

2

25

3

0 LPS

TN

Fa (

ng

ml)

LPS

LPS + 2DG

IL-1beta

TNFalpha

0

50

100

150

200

250

300

350

400

0 LPSre

lati

ve I

L-1

beta

exp

ressio

n

LPS

LPS + 2DG











THE GABA SHUNT


0

2times1006

4times1006

6times1006

su

ccin

ate

(p

ea

k a

rea

)

LPS

unstimulated

- 1 5 10

2DG (mM)



citrate

a-ketoglutarate

succinate

fumarate

malate


12C

13C

14N

15NGABA



production

IL-1beta


200

400

600

800

1000

IL-1b

eta (

pgm

l)IL-6


10

20

30

40IL-

6 (ng

ml)

TNFalpha


1

2

3

TNFa

(ngm

l)



0 20 40 60 80 0

50

100

150 Co n t r o l

Vi g a + L PS

LP S


P e r c

e n

t s u r v

i v a l

At this stage









WB HIF1α WB actin

LPS

2-DG



inhibited by 2DG



PHD

KG Succinate



PHD

KG Succinate

-



PHD

KG Succinate

KG


-50

0

50

100

150

200

250

300

350

400


unstim

LPS

LPS plus 2DG



- LPS - LPS0

2000

4000

6000

8000

rela

tive I

L-1

b e

xp

ressio

n

+ succinate

0 LPS 0 LPS0

20000

40000

60000

rela

tive I

L-1

b e

xp

ressio

n

+ butylmalonate




PHD

KG Succinate



PHD

KG Succinate

-



PHD

KG Succinate

KG

012

h

12h+αK

G24

h

24h+αK

G

00

05

10

Rela

tive E

nri

ch

men

t

0 LPS 0 LPS0

10000

20000

30000

rela

tiv

e IL

-1b

ex

pre

ss

ion

+ aKG


WBIL-1β

WBHIF-1α

- +LPSαKG αKG

WBβ-ac n

Summary






succinate

PHD




succinate

PHD





2DG2DG

-LPS+LPS

WBsuccinyllysine

WBIL-1βWBβ-ac n

unstimulated LPS00

01

02

03

04

me

an

em

PA

I va

lue





bull GAPDH


bull PKM12


Key finding










GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION



GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION




LOW GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE


Low glucose

SNF2

YEAST MACROPHAGE

Low glucose 2DG

AMP kinase



GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE

METFORMIN

(mM)

IL-1β qPCR

(mM)



GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE



turning it off






via AMP kinase


Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK



Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK

HIF1





LUKE OrsquoNEILL


OrsquoNEILL LAB


Acknowledgment








Acknowledgment











CARKL



(b) Th17 cells








N=2

N=2

081211 bmdm SS

Ctl LPS (24 hr)0

1

2

3

4

Re

lative

miR

21

0 e

xp

ressio

n

181111 j774

Ctl LPS (24 hr)00

05

10

15

20

25

Re

lativ

e m

iR2

10

exp

ressio

n

310112 Bmdm ss 48h

Ctl LPS (48 hr)0

1

2

3

4

5

Re

lative

miR

21

0 e

xp

ressio

n

N=2

J774s

BMDM BMDM


LPS

HIF-1α IL-1β

Hypothesis


080413 210


05

10

15

20

0 MCSF2 FCS

2 MCSF2 FCS

10 MCSF10 FCS

Re

lativ

e m

iR-2

10

exp

ressio

n

27328140413 pooled 24h no SS n=8

Ctl LPS00

05

10

15

20

Re

lative

miR

-21

0 e

xp

ressio

n

N=8 N=3


LPS

HIF-1α IL-1β

Hypothesis


25030208160413 sdhd n=10

Ctl LPS00

05

10

15

Re

lative

sd

hd

exp

ressio

n

N=10


LPS

HIF-1α IL-1β

Hypothesis



anti-miR210

HIF-1α IL-1β

n=1

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B



Needles Rods

Cartilage integrity


IL-1

Joint inflammation

OA

NFkappaB JNK

2013

COX2 aggrecanases



1990 2010 OBESITY

Type 2 diabetes

WHY CORN SYRUP


IRONY

IRONY

IL1 in T2D

IL1b











Nlrp3

IL-1


Beta cell death

Type 2 diabetes

NFkappaB JNK

2011


INSULIN

+

NORMAL



INSULIN

+

NORMAL

GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION





0

05

1

15

2

25

3

0 LPS

TN

Fa (

ng

ml)

LPS

LPS + 2DG

IL-1beta

TNFalpha

0

50

100

150

200

250

300

350

400

0 LPS

rela

tive I

L-1

beta

exp

ressio

n

LPS

LPS + 2DG








4 8 16 240

5

10

15 unstimulated

LPS

Time (h)

Glu

co

se

util

isa

tio

nG

lyco

lysis

O

xp

ho

s


0

2

4

6

unstimulated LPS

NA

DN

AD

H ra

tio p

mo

l10

6 c

ells


Otto Warburg




Th17

Treg


Nature (2013) 493 346-354











EXTERNAL STRESSORS



EXTERNAL STRESSORS







KEY QUESTION











0

05

1

15

2

25

3

0 LPS

TN

Fa (

ng

ml)

LPS

LPS + 2DG

IL-1beta

TNFalpha

0

50

100

150

200

250

300

350

400

0 LPSre

lati

ve I

L-1

beta

exp

ressio

n

LPS

LPS + 2DG











THE GABA SHUNT


0

2times1006

4times1006

6times1006

su

ccin

ate

(p

ea

k a

rea

)

LPS

unstimulated

- 1 5 10

2DG (mM)



citrate

a-ketoglutarate

succinate

fumarate

malate


12C

13C

14N

15NGABA



production

IL-1beta


200

400

600

800

1000

IL-1b

eta (

pgm

l)IL-6


10

20

30

40IL-

6 (ng

ml)

TNFalpha


1

2

3

TNFa

(ngm

l)



0 20 40 60 80 0

50

100

150 Co n t r o l

Vi g a + L PS

LP S


P e r c

e n

t s u r v

i v a l

At this stage









WB HIF1α WB actin

LPS

2-DG



inhibited by 2DG



PHD

KG Succinate



PHD

KG Succinate

-



PHD

KG Succinate

KG


-50

0

50

100

150

200

250

300

350

400


unstim

LPS

LPS plus 2DG



- LPS - LPS0

2000

4000

6000

8000

rela

tive I

L-1

b e

xp

ressio

n

+ succinate

0 LPS 0 LPS0

20000

40000

60000

rela

tive I

L-1

b e

xp

ressio

n

+ butylmalonate




PHD

KG Succinate



PHD

KG Succinate

-



PHD

KG Succinate

KG

012

h

12h+αK

G24

h

24h+αK

G

00

05

10

Rela

tive E

nri

ch

men

t

0 LPS 0 LPS0

10000

20000

30000

rela

tiv

e IL

-1b

ex

pre

ss

ion

+ aKG


WBIL-1β

WBHIF-1α

- +LPSαKG αKG

WBβ-ac n

Summary






succinate

PHD




succinate

PHD





2DG2DG

-LPS+LPS

WBsuccinyllysine

WBIL-1βWBβ-ac n

unstimulated LPS00

01

02

03

04

me

an

em

PA

I va

lue





bull GAPDH


bull PKM12


Key finding










GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION



GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION




LOW GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE


Low glucose

SNF2

YEAST MACROPHAGE

Low glucose 2DG

AMP kinase



GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE

METFORMIN

(mM)

IL-1β qPCR

(mM)



GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE



turning it off






via AMP kinase


Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK



Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK

HIF1





LUKE OrsquoNEILL


OrsquoNEILL LAB


Acknowledgment








Acknowledgment











CARKL



(b) Th17 cells








N=2

N=2

081211 bmdm SS

Ctl LPS (24 hr)0

1

2

3

4

Re

lative

miR

21

0 e

xp

ressio

n

181111 j774

Ctl LPS (24 hr)00

05

10

15

20

25

Re

lativ

e m

iR2

10

exp

ressio

n

310112 Bmdm ss 48h

Ctl LPS (48 hr)0

1

2

3

4

5

Re

lative

miR

21

0 e

xp

ressio

n

N=2

J774s

BMDM BMDM


LPS

HIF-1α IL-1β

Hypothesis


080413 210


05

10

15

20

0 MCSF2 FCS

2 MCSF2 FCS

10 MCSF10 FCS

Re

lativ

e m

iR-2

10

exp

ressio

n

27328140413 pooled 24h no SS n=8

Ctl LPS00

05

10

15

20

Re

lative

miR

-21

0 e

xp

ressio

n

N=8 N=3


LPS

HIF-1α IL-1β

Hypothesis


25030208160413 sdhd n=10

Ctl LPS00

05

10

15

Re

lative

sd

hd

exp

ressio

n

N=10


LPS

HIF-1α IL-1β

Hypothesis



anti-miR210

HIF-1α IL-1β

n=1

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B



1990 2010 OBESITY

Type 2 diabetes

WHY CORN SYRUP


IRONY

IRONY

IL1 in T2D

IL1b











Nlrp3

IL-1


Beta cell death

Type 2 diabetes

NFkappaB JNK

2011


INSULIN

+

NORMAL



INSULIN

+

NORMAL

GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION





0

05

1

15

2

25

3

0 LPS

TN

Fa (

ng

ml)

LPS

LPS + 2DG

IL-1beta

TNFalpha

0

50

100

150

200

250

300

350

400

0 LPS

rela

tive I

L-1

beta

exp

ressio

n

LPS

LPS + 2DG








4 8 16 240

5

10

15 unstimulated

LPS

Time (h)

Glu

co

se

util

isa

tio

nG

lyco

lysis

O

xp

ho

s


0

2

4

6

unstimulated LPS

NA

DN

AD

H ra

tio p

mo

l10

6 c

ells


Otto Warburg




Th17

Treg


Nature (2013) 493 346-354











EXTERNAL STRESSORS



EXTERNAL STRESSORS







KEY QUESTION











0

05

1

15

2

25

3

0 LPS

TN

Fa (

ng

ml)

LPS

LPS + 2DG

IL-1beta

TNFalpha

0

50

100

150

200

250

300

350

400

0 LPSre

lati

ve I

L-1

beta

exp

ressio

n

LPS

LPS + 2DG











THE GABA SHUNT


0

2times1006

4times1006

6times1006

su

ccin

ate

(p

ea

k a

rea

)

LPS

unstimulated

- 1 5 10

2DG (mM)



citrate

a-ketoglutarate

succinate

fumarate

malate


12C

13C

14N

15NGABA



production

IL-1beta


200

400

600

800

1000

IL-1b

eta (

pgm

l)IL-6


10

20

30

40IL-

6 (ng

ml)

TNFalpha


1

2

3

TNFa

(ngm

l)



0 20 40 60 80 0

50

100

150 Co n t r o l

Vi g a + L PS

LP S


P e r c

e n

t s u r v

i v a l

At this stage









WB HIF1α WB actin

LPS

2-DG



inhibited by 2DG



PHD

KG Succinate



PHD

KG Succinate

-



PHD

KG Succinate

KG


-50

0

50

100

150

200

250

300

350

400


unstim

LPS

LPS plus 2DG



- LPS - LPS0

2000

4000

6000

8000

rela

tive I

L-1

b e

xp

ressio

n

+ succinate

0 LPS 0 LPS0

20000

40000

60000

rela

tive I

L-1

b e

xp

ressio

n

+ butylmalonate




PHD

KG Succinate



PHD

KG Succinate

-



PHD

KG Succinate

KG

012

h

12h+αK

G24

h

24h+αK

G

00

05

10

Rela

tive E

nri

ch

men

t

0 LPS 0 LPS0

10000

20000

30000

rela

tiv

e IL

-1b

ex

pre

ss

ion

+ aKG


WBIL-1β

WBHIF-1α

- +LPSαKG αKG

WBβ-ac n

Summary






succinate

PHD




succinate

PHD





2DG2DG

-LPS+LPS

WBsuccinyllysine

WBIL-1βWBβ-ac n

unstimulated LPS00

01

02

03

04

me

an

em

PA

I va

lue





bull GAPDH


bull PKM12


Key finding










GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION



GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION




LOW GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE


Low glucose

SNF2

YEAST MACROPHAGE

Low glucose 2DG

AMP kinase



GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE

METFORMIN

(mM)

IL-1β qPCR

(mM)



GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE



turning it off






via AMP kinase


Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK



Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK

HIF1





LUKE OrsquoNEILL


OrsquoNEILL LAB


Acknowledgment








Acknowledgment











CARKL



(b) Th17 cells








N=2

N=2

081211 bmdm SS

Ctl LPS (24 hr)0

1

2

3

4

Re

lative

miR

21

0 e

xp

ressio

n

181111 j774

Ctl LPS (24 hr)00

05

10

15

20

25

Re

lativ

e m

iR2

10

exp

ressio

n

310112 Bmdm ss 48h

Ctl LPS (48 hr)0

1

2

3

4

5

Re

lative

miR

21

0 e

xp

ressio

n

N=2

J774s

BMDM BMDM


LPS

HIF-1α IL-1β

Hypothesis


080413 210


05

10

15

20

0 MCSF2 FCS

2 MCSF2 FCS

10 MCSF10 FCS

Re

lativ

e m

iR-2

10

exp

ressio

n

27328140413 pooled 24h no SS n=8

Ctl LPS00

05

10

15

20

Re

lative

miR

-21

0 e

xp

ressio

n

N=8 N=3


LPS

HIF-1α IL-1β

Hypothesis


25030208160413 sdhd n=10

Ctl LPS00

05

10

15

Re

lative

sd

hd

exp

ressio

n

N=10


LPS

HIF-1α IL-1β

Hypothesis



anti-miR210

HIF-1α IL-1β

n=1

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

IRONY

IRONY

IL1 in T2D

IL1b











Nlrp3

IL-1


Beta cell death

Type 2 diabetes

NFkappaB JNK

2011


INSULIN

+

NORMAL



INSULIN

+

NORMAL

GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION





0

05

1

15

2

25

3

0 LPS

TN

Fa (

ng

ml)

LPS

LPS + 2DG

IL-1beta

TNFalpha

0

50

100

150

200

250

300

350

400

0 LPS

rela

tive I

L-1

beta

exp

ressio

n

LPS

LPS + 2DG








4 8 16 240

5

10

15 unstimulated

LPS

Time (h)

Glu

co

se

util

isa

tio

nG

lyco

lysis

O

xp

ho

s


0

2

4

6

unstimulated LPS

NA

DN

AD

H ra

tio p

mo

l10

6 c

ells


Otto Warburg




Th17

Treg


Nature (2013) 493 346-354











EXTERNAL STRESSORS



EXTERNAL STRESSORS







KEY QUESTION











0

05

1

15

2

25

3

0 LPS

TN

Fa (

ng

ml)

LPS

LPS + 2DG

IL-1beta

TNFalpha

0

50

100

150

200

250

300

350

400

0 LPSre

lati

ve I

L-1

beta

exp

ressio

n

LPS

LPS + 2DG











THE GABA SHUNT


0

2times1006

4times1006

6times1006

su

ccin

ate

(p

ea

k a

rea

)

LPS

unstimulated

- 1 5 10

2DG (mM)



citrate

a-ketoglutarate

succinate

fumarate

malate


12C

13C

14N

15NGABA



production

IL-1beta


200

400

600

800

1000

IL-1b

eta (

pgm

l)IL-6


10

20

30

40IL-

6 (ng

ml)

TNFalpha


1

2

3

TNFa

(ngm

l)



0 20 40 60 80 0

50

100

150 Co n t r o l

Vi g a + L PS

LP S


P e r c

e n

t s u r v

i v a l

At this stage









WB HIF1α WB actin

LPS

2-DG



inhibited by 2DG



PHD

KG Succinate



PHD

KG Succinate

-



PHD

KG Succinate

KG


-50

0

50

100

150

200

250

300

350

400


unstim

LPS

LPS plus 2DG



- LPS - LPS0

2000

4000

6000

8000

rela

tive I

L-1

b e

xp

ressio

n

+ succinate

0 LPS 0 LPS0

20000

40000

60000

rela

tive I

L-1

b e

xp

ressio

n

+ butylmalonate




PHD

KG Succinate



PHD

KG Succinate

-



PHD

KG Succinate

KG

012

h

12h+αK

G24

h

24h+αK

G

00

05

10

Rela

tive E

nri

ch

men

t

0 LPS 0 LPS0

10000

20000

30000

rela

tiv

e IL

-1b

ex

pre

ss

ion

+ aKG


WBIL-1β

WBHIF-1α

- +LPSαKG αKG

WBβ-ac n

Summary






succinate

PHD




succinate

PHD





2DG2DG

-LPS+LPS

WBsuccinyllysine

WBIL-1βWBβ-ac n

unstimulated LPS00

01

02

03

04

me

an

em

PA

I va

lue





bull GAPDH


bull PKM12


Key finding










GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION



GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION




LOW GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE


Low glucose

SNF2

YEAST MACROPHAGE

Low glucose 2DG

AMP kinase



GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE

METFORMIN

(mM)

IL-1β qPCR

(mM)



GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE



turning it off






via AMP kinase


Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK



Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK

HIF1





LUKE OrsquoNEILL


OrsquoNEILL LAB


Acknowledgment








Acknowledgment











CARKL



(b) Th17 cells








N=2

N=2

081211 bmdm SS

Ctl LPS (24 hr)0

1

2

3

4

Re

lative

miR

21

0 e

xp

ressio

n

181111 j774

Ctl LPS (24 hr)00

05

10

15

20

25

Re

lativ

e m

iR2

10

exp

ressio

n

310112 Bmdm ss 48h

Ctl LPS (48 hr)0

1

2

3

4

5

Re

lative

miR

21

0 e

xp

ressio

n

N=2

J774s

BMDM BMDM


LPS

HIF-1α IL-1β

Hypothesis


080413 210


05

10

15

20

0 MCSF2 FCS

2 MCSF2 FCS

10 MCSF10 FCS

Re

lativ

e m

iR-2

10

exp

ressio

n

27328140413 pooled 24h no SS n=8

Ctl LPS00

05

10

15

20

Re

lative

miR

-21

0 e

xp

ressio

n

N=8 N=3


LPS

HIF-1α IL-1β

Hypothesis


25030208160413 sdhd n=10

Ctl LPS00

05

10

15

Re

lative

sd

hd

exp

ressio

n

N=10


LPS

HIF-1α IL-1β

Hypothesis



anti-miR210

HIF-1α IL-1β

n=1

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

IL1 in T2D

IL1b











Nlrp3

IL-1


Beta cell death

Type 2 diabetes

NFkappaB JNK

2011


INSULIN

+

NORMAL



INSULIN

+

NORMAL

GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION





0

05

1

15

2

25

3

0 LPS

TN

Fa (

ng

ml)

LPS

LPS + 2DG

IL-1beta

TNFalpha

0

50

100

150

200

250

300

350

400

0 LPS

rela

tive I

L-1

beta

exp

ressio

n

LPS

LPS + 2DG








4 8 16 240

5

10

15 unstimulated

LPS

Time (h)

Glu

co

se

util

isa

tio

nG

lyco

lysis

O

xp

ho

s


0

2

4

6

unstimulated LPS

NA

DN

AD

H ra

tio p

mo

l10

6 c

ells


Otto Warburg




Th17

Treg


Nature (2013) 493 346-354











EXTERNAL STRESSORS



EXTERNAL STRESSORS







KEY QUESTION











0

05

1

15

2

25

3

0 LPS

TN

Fa (

ng

ml)

LPS

LPS + 2DG

IL-1beta

TNFalpha

0

50

100

150

200

250

300

350

400

0 LPSre

lati

ve I

L-1

beta

exp

ressio

n

LPS

LPS + 2DG











THE GABA SHUNT


0

2times1006

4times1006

6times1006

su

ccin

ate

(p

ea

k a

rea

)

LPS

unstimulated

- 1 5 10

2DG (mM)



citrate

a-ketoglutarate

succinate

fumarate

malate


12C

13C

14N

15NGABA



production

IL-1beta


200

400

600

800

1000

IL-1b

eta (

pgm

l)IL-6


10

20

30

40IL-

6 (ng

ml)

TNFalpha


1

2

3

TNFa

(ngm

l)



0 20 40 60 80 0

50

100

150 Co n t r o l

Vi g a + L PS

LP S


P e r c

e n

t s u r v

i v a l

At this stage









WB HIF1α WB actin

LPS

2-DG



inhibited by 2DG



PHD

KG Succinate



PHD

KG Succinate

-



PHD

KG Succinate

KG


-50

0

50

100

150

200

250

300

350

400


unstim

LPS

LPS plus 2DG



- LPS - LPS0

2000

4000

6000

8000

rela

tive I

L-1

b e

xp

ressio

n

+ succinate

0 LPS 0 LPS0

20000

40000

60000

rela

tive I

L-1

b e

xp

ressio

n

+ butylmalonate




PHD

KG Succinate



PHD

KG Succinate

-



PHD

KG Succinate

KG

012

h

12h+αK

G24

h

24h+αK

G

00

05

10

Rela

tive E

nri

ch

men

t

0 LPS 0 LPS0

10000

20000

30000

rela

tiv

e IL

-1b

ex

pre

ss

ion

+ aKG


WBIL-1β

WBHIF-1α

- +LPSαKG αKG

WBβ-ac n

Summary






succinate

PHD




succinate

PHD





2DG2DG

-LPS+LPS

WBsuccinyllysine

WBIL-1βWBβ-ac n

unstimulated LPS00

01

02

03

04

me

an

em

PA

I va

lue





bull GAPDH


bull PKM12


Key finding










GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION



GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION




LOW GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE


Low glucose

SNF2

YEAST MACROPHAGE

Low glucose 2DG

AMP kinase



GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE

METFORMIN

(mM)

IL-1β qPCR

(mM)



GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE



turning it off






via AMP kinase


Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK



Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK

HIF1





LUKE OrsquoNEILL


OrsquoNEILL LAB


Acknowledgment








Acknowledgment











CARKL



(b) Th17 cells








N=2

N=2

081211 bmdm SS

Ctl LPS (24 hr)0

1

2

3

4

Re

lative

miR

21

0 e

xp

ressio

n

181111 j774

Ctl LPS (24 hr)00

05

10

15

20

25

Re

lativ

e m

iR2

10

exp

ressio

n

310112 Bmdm ss 48h

Ctl LPS (48 hr)0

1

2

3

4

5

Re

lative

miR

21

0 e

xp

ressio

n

N=2

J774s

BMDM BMDM


LPS

HIF-1α IL-1β

Hypothesis


080413 210


05

10

15

20

0 MCSF2 FCS

2 MCSF2 FCS

10 MCSF10 FCS

Re

lativ

e m

iR-2

10

exp

ressio

n

27328140413 pooled 24h no SS n=8

Ctl LPS00

05

10

15

20

Re

lative

miR

-21

0 e

xp

ressio

n

N=8 N=3


LPS

HIF-1α IL-1β

Hypothesis


25030208160413 sdhd n=10

Ctl LPS00

05

10

15

Re

lative

sd

hd

exp

ressio

n

N=10


LPS

HIF-1α IL-1β

Hypothesis



anti-miR210

HIF-1α IL-1β

n=1

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B






Nlrp3

IL-1


Beta cell death

Type 2 diabetes

NFkappaB JNK

2011


INSULIN

+

NORMAL



INSULIN

+

NORMAL

GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION





0

05

1

15

2

25

3

0 LPS

TN

Fa (

ng

ml)

LPS

LPS + 2DG

IL-1beta

TNFalpha

0

50

100

150

200

250

300

350

400

0 LPS

rela

tive I

L-1

beta

exp

ressio

n

LPS

LPS + 2DG








4 8 16 240

5

10

15 unstimulated

LPS

Time (h)

Glu

co

se

util

isa

tio

nG

lyco

lysis

O

xp

ho

s


0

2

4

6

unstimulated LPS

NA

DN

AD

H ra

tio p

mo

l10

6 c

ells


Otto Warburg




Th17

Treg


Nature (2013) 493 346-354











EXTERNAL STRESSORS



EXTERNAL STRESSORS







KEY QUESTION











0

05

1

15

2

25

3

0 LPS

TN

Fa (

ng

ml)

LPS

LPS + 2DG

IL-1beta

TNFalpha

0

50

100

150

200

250

300

350

400

0 LPSre

lati

ve I

L-1

beta

exp

ressio

n

LPS

LPS + 2DG











THE GABA SHUNT


0

2times1006

4times1006

6times1006

su

ccin

ate

(p

ea

k a

rea

)

LPS

unstimulated

- 1 5 10

2DG (mM)



citrate

a-ketoglutarate

succinate

fumarate

malate


12C

13C

14N

15NGABA



production

IL-1beta


200

400

600

800

1000

IL-1b

eta (

pgm

l)IL-6


10

20

30

40IL-

6 (ng

ml)

TNFalpha


1

2

3

TNFa

(ngm

l)



0 20 40 60 80 0

50

100

150 Co n t r o l

Vi g a + L PS

LP S


P e r c

e n

t s u r v

i v a l

At this stage









WB HIF1α WB actin

LPS

2-DG



inhibited by 2DG



PHD

KG Succinate



PHD

KG Succinate

-



PHD

KG Succinate

KG


-50

0

50

100

150

200

250

300

350

400


unstim

LPS

LPS plus 2DG



- LPS - LPS0

2000

4000

6000

8000

rela

tive I

L-1

b e

xp

ressio

n

+ succinate

0 LPS 0 LPS0

20000

40000

60000

rela

tive I

L-1

b e

xp

ressio

n

+ butylmalonate




PHD

KG Succinate



PHD

KG Succinate

-



PHD

KG Succinate

KG

012

h

12h+αK

G24

h

24h+αK

G

00

05

10

Rela

tive E

nri

ch

men

t

0 LPS 0 LPS0

10000

20000

30000

rela

tiv

e IL

-1b

ex

pre

ss

ion

+ aKG


WBIL-1β

WBHIF-1α

- +LPSαKG αKG

WBβ-ac n

Summary






succinate

PHD




succinate

PHD





2DG2DG

-LPS+LPS

WBsuccinyllysine

WBIL-1βWBβ-ac n

unstimulated LPS00

01

02

03

04

me

an

em

PA

I va

lue





bull GAPDH


bull PKM12


Key finding










GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION



GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION




LOW GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE


Low glucose

SNF2

YEAST MACROPHAGE

Low glucose 2DG

AMP kinase



GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE

METFORMIN

(mM)

IL-1β qPCR

(mM)



GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE



turning it off






via AMP kinase


Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK



Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK

HIF1





LUKE OrsquoNEILL


OrsquoNEILL LAB


Acknowledgment








Acknowledgment











CARKL



(b) Th17 cells








N=2

N=2

081211 bmdm SS

Ctl LPS (24 hr)0

1

2

3

4

Re

lative

miR

21

0 e

xp

ressio

n

181111 j774

Ctl LPS (24 hr)00

05

10

15

20

25

Re

lativ

e m

iR2

10

exp

ressio

n

310112 Bmdm ss 48h

Ctl LPS (48 hr)0

1

2

3

4

5

Re

lative

miR

21

0 e

xp

ressio

n

N=2

J774s

BMDM BMDM


LPS

HIF-1α IL-1β

Hypothesis


080413 210


05

10

15

20

0 MCSF2 FCS

2 MCSF2 FCS

10 MCSF10 FCS

Re

lativ

e m

iR-2

10

exp

ressio

n

27328140413 pooled 24h no SS n=8

Ctl LPS00

05

10

15

20

Re

lative

miR

-21

0 e

xp

ressio

n

N=8 N=3


LPS

HIF-1α IL-1β

Hypothesis


25030208160413 sdhd n=10

Ctl LPS00

05

10

15

Re

lative

sd

hd

exp

ressio

n

N=10


LPS

HIF-1α IL-1β

Hypothesis



anti-miR210

HIF-1α IL-1β

n=1

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B


INSULIN

+

NORMAL



INSULIN

+

NORMAL

GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION





0

05

1

15

2

25

3

0 LPS

TN

Fa (

ng

ml)

LPS

LPS + 2DG

IL-1beta

TNFalpha

0

50

100

150

200

250

300

350

400

0 LPS

rela

tive I

L-1

beta

exp

ressio

n

LPS

LPS + 2DG








4 8 16 240

5

10

15 unstimulated

LPS

Time (h)

Glu

co

se

util

isa

tio

nG

lyco

lysis

O

xp

ho

s


0

2

4

6

unstimulated LPS

NA

DN

AD

H ra

tio p

mo

l10

6 c

ells


Otto Warburg




Th17

Treg


Nature (2013) 493 346-354











EXTERNAL STRESSORS



EXTERNAL STRESSORS







KEY QUESTION











0

05

1

15

2

25

3

0 LPS

TN

Fa (

ng

ml)

LPS

LPS + 2DG

IL-1beta

TNFalpha

0

50

100

150

200

250

300

350

400

0 LPSre

lati

ve I

L-1

beta

exp

ressio

n

LPS

LPS + 2DG











THE GABA SHUNT


0

2times1006

4times1006

6times1006

su

ccin

ate

(p

ea

k a

rea

)

LPS

unstimulated

- 1 5 10

2DG (mM)



citrate

a-ketoglutarate

succinate

fumarate

malate


12C

13C

14N

15NGABA



production

IL-1beta


200

400

600

800

1000

IL-1b

eta (

pgm

l)IL-6


10

20

30

40IL-

6 (ng

ml)

TNFalpha


1

2

3

TNFa

(ngm

l)



0 20 40 60 80 0

50

100

150 Co n t r o l

Vi g a + L PS

LP S


P e r c

e n

t s u r v

i v a l

At this stage









WB HIF1α WB actin

LPS

2-DG



inhibited by 2DG



PHD

KG Succinate



PHD

KG Succinate

-



PHD

KG Succinate

KG


-50

0

50

100

150

200

250

300

350

400


unstim

LPS

LPS plus 2DG



- LPS - LPS0

2000

4000

6000

8000

rela

tive I

L-1

b e

xp

ressio

n

+ succinate

0 LPS 0 LPS0

20000

40000

60000

rela

tive I

L-1

b e

xp

ressio

n

+ butylmalonate




PHD

KG Succinate



PHD

KG Succinate

-



PHD

KG Succinate

KG

012

h

12h+αK

G24

h

24h+αK

G

00

05

10

Rela

tive E

nri

ch

men

t

0 LPS 0 LPS0

10000

20000

30000

rela

tiv

e IL

-1b

ex

pre

ss

ion

+ aKG


WBIL-1β

WBHIF-1α

- +LPSαKG αKG

WBβ-ac n

Summary






succinate

PHD




succinate

PHD





2DG2DG

-LPS+LPS

WBsuccinyllysine

WBIL-1βWBβ-ac n

unstimulated LPS00

01

02

03

04

me

an

em

PA

I va

lue





bull GAPDH


bull PKM12


Key finding










GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION



GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION




LOW GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE


Low glucose

SNF2

YEAST MACROPHAGE

Low glucose 2DG

AMP kinase



GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE

METFORMIN

(mM)

IL-1β qPCR

(mM)



GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE



turning it off






via AMP kinase


Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK



Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK

HIF1





LUKE OrsquoNEILL


OrsquoNEILL LAB


Acknowledgment








Acknowledgment











CARKL



(b) Th17 cells








N=2

N=2

081211 bmdm SS

Ctl LPS (24 hr)0

1

2

3

4

Re

lative

miR

21

0 e

xp

ressio

n

181111 j774

Ctl LPS (24 hr)00

05

10

15

20

25

Re

lativ

e m

iR2

10

exp

ressio

n

310112 Bmdm ss 48h

Ctl LPS (48 hr)0

1

2

3

4

5

Re

lative

miR

21

0 e

xp

ressio

n

N=2

J774s

BMDM BMDM


LPS

HIF-1α IL-1β

Hypothesis


080413 210


05

10

15

20

0 MCSF2 FCS

2 MCSF2 FCS

10 MCSF10 FCS

Re

lativ

e m

iR-2

10

exp

ressio

n

27328140413 pooled 24h no SS n=8

Ctl LPS00

05

10

15

20

Re

lative

miR

-21

0 e

xp

ressio

n

N=8 N=3


LPS

HIF-1α IL-1β

Hypothesis


25030208160413 sdhd n=10

Ctl LPS00

05

10

15

Re

lative

sd

hd

exp

ressio

n

N=10


LPS

HIF-1α IL-1β

Hypothesis



anti-miR210

HIF-1α IL-1β

n=1

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B



0

05

1

15

2

25

3

0 LPS

TN

Fa (

ng

ml)

LPS

LPS + 2DG

IL-1beta

TNFalpha

0

50

100

150

200

250

300

350

400

0 LPS

rela

tive I

L-1

beta

exp

ressio

n

LPS

LPS + 2DG








4 8 16 240

5

10

15 unstimulated

LPS

Time (h)

Glu

co

se

util

isa

tio

nG

lyco

lysis

O

xp

ho

s


0

2

4

6

unstimulated LPS

NA

DN

AD

H ra

tio p

mo

l10

6 c

ells


Otto Warburg




Th17

Treg


Nature (2013) 493 346-354











EXTERNAL STRESSORS



EXTERNAL STRESSORS







KEY QUESTION











0

05

1

15

2

25

3

0 LPS

TN

Fa (

ng

ml)

LPS

LPS + 2DG

IL-1beta

TNFalpha

0

50

100

150

200

250

300

350

400

0 LPSre

lati

ve I

L-1

beta

exp

ressio

n

LPS

LPS + 2DG











THE GABA SHUNT


0

2times1006

4times1006

6times1006

su

ccin

ate

(p

ea

k a

rea

)

LPS

unstimulated

- 1 5 10

2DG (mM)



citrate

a-ketoglutarate

succinate

fumarate

malate


12C

13C

14N

15NGABA



production

IL-1beta


200

400

600

800

1000

IL-1b

eta (

pgm

l)IL-6


10

20

30

40IL-

6 (ng

ml)

TNFalpha


1

2

3

TNFa

(ngm

l)



0 20 40 60 80 0

50

100

150 Co n t r o l

Vi g a + L PS

LP S


P e r c

e n

t s u r v

i v a l

At this stage









WB HIF1α WB actin

LPS

2-DG



inhibited by 2DG



PHD

KG Succinate



PHD

KG Succinate

-



PHD

KG Succinate

KG


-50

0

50

100

150

200

250

300

350

400


unstim

LPS

LPS plus 2DG



- LPS - LPS0

2000

4000

6000

8000

rela

tive I

L-1

b e

xp

ressio

n

+ succinate

0 LPS 0 LPS0

20000

40000

60000

rela

tive I

L-1

b e

xp

ressio

n

+ butylmalonate




PHD

KG Succinate



PHD

KG Succinate

-



PHD

KG Succinate

KG

012

h

12h+αK

G24

h

24h+αK

G

00

05

10

Rela

tive E

nri

ch

men

t

0 LPS 0 LPS0

10000

20000

30000

rela

tiv

e IL

-1b

ex

pre

ss

ion

+ aKG


WBIL-1β

WBHIF-1α

- +LPSαKG αKG

WBβ-ac n

Summary






succinate

PHD




succinate

PHD





2DG2DG

-LPS+LPS

WBsuccinyllysine

WBIL-1βWBβ-ac n

unstimulated LPS00

01

02

03

04

me

an

em

PA

I va

lue





bull GAPDH


bull PKM12


Key finding










GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION



GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION




LOW GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE


Low glucose

SNF2

YEAST MACROPHAGE

Low glucose 2DG

AMP kinase



GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE

METFORMIN

(mM)

IL-1β qPCR

(mM)



GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE



turning it off






via AMP kinase


Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK



Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK

HIF1





LUKE OrsquoNEILL


OrsquoNEILL LAB


Acknowledgment








Acknowledgment











CARKL



(b) Th17 cells








N=2

N=2

081211 bmdm SS

Ctl LPS (24 hr)0

1

2

3

4

Re

lative

miR

21

0 e

xp

ressio

n

181111 j774

Ctl LPS (24 hr)00

05

10

15

20

25

Re

lativ

e m

iR2

10

exp

ressio

n

310112 Bmdm ss 48h

Ctl LPS (48 hr)0

1

2

3

4

5

Re

lative

miR

21

0 e

xp

ressio

n

N=2

J774s

BMDM BMDM


LPS

HIF-1α IL-1β

Hypothesis


080413 210


05

10

15

20

0 MCSF2 FCS

2 MCSF2 FCS

10 MCSF10 FCS

Re

lativ

e m

iR-2

10

exp

ressio

n

27328140413 pooled 24h no SS n=8

Ctl LPS00

05

10

15

20

Re

lative

miR

-21

0 e

xp

ressio

n

N=8 N=3


LPS

HIF-1α IL-1β

Hypothesis


25030208160413 sdhd n=10

Ctl LPS00

05

10

15

Re

lative

sd

hd

exp

ressio

n

N=10


LPS

HIF-1α IL-1β

Hypothesis



anti-miR210

HIF-1α IL-1β

n=1

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B








4 8 16 240

5

10

15 unstimulated

LPS

Time (h)

Glu

co

se

util

isa

tio

nG

lyco

lysis

O

xp

ho

s


0

2

4

6

unstimulated LPS

NA

DN

AD

H ra

tio p

mo

l10

6 c

ells


Otto Warburg




Th17

Treg


Nature (2013) 493 346-354











EXTERNAL STRESSORS



EXTERNAL STRESSORS







KEY QUESTION











0

05

1

15

2

25

3

0 LPS

TN

Fa (

ng

ml)

LPS

LPS + 2DG

IL-1beta

TNFalpha

0

50

100

150

200

250

300

350

400

0 LPSre

lati

ve I

L-1

beta

exp

ressio

n

LPS

LPS + 2DG











THE GABA SHUNT


0

2times1006

4times1006

6times1006

su

ccin

ate

(p

ea

k a

rea

)

LPS

unstimulated

- 1 5 10

2DG (mM)



citrate

a-ketoglutarate

succinate

fumarate

malate


12C

13C

14N

15NGABA



production

IL-1beta


200

400

600

800

1000

IL-1b

eta (

pgm

l)IL-6


10

20

30

40IL-

6 (ng

ml)

TNFalpha


1

2

3

TNFa

(ngm

l)



0 20 40 60 80 0

50

100

150 Co n t r o l

Vi g a + L PS

LP S


P e r c

e n

t s u r v

i v a l

At this stage









WB HIF1α WB actin

LPS

2-DG



inhibited by 2DG



PHD

KG Succinate



PHD

KG Succinate

-



PHD

KG Succinate

KG


-50

0

50

100

150

200

250

300

350

400


unstim

LPS

LPS plus 2DG



- LPS - LPS0

2000

4000

6000

8000

rela

tive I

L-1

b e

xp

ressio

n

+ succinate

0 LPS 0 LPS0

20000

40000

60000

rela

tive I

L-1

b e

xp

ressio

n

+ butylmalonate




PHD

KG Succinate



PHD

KG Succinate

-



PHD

KG Succinate

KG

012

h

12h+αK

G24

h

24h+αK

G

00

05

10

Rela

tive E

nri

ch

men

t

0 LPS 0 LPS0

10000

20000

30000

rela

tiv

e IL

-1b

ex

pre

ss

ion

+ aKG


WBIL-1β

WBHIF-1α

- +LPSαKG αKG

WBβ-ac n

Summary






succinate

PHD




succinate

PHD





2DG2DG

-LPS+LPS

WBsuccinyllysine

WBIL-1βWBβ-ac n

unstimulated LPS00

01

02

03

04

me

an

em

PA

I va

lue





bull GAPDH


bull PKM12


Key finding










GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION



GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION




LOW GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE


Low glucose

SNF2

YEAST MACROPHAGE

Low glucose 2DG

AMP kinase



GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE

METFORMIN

(mM)

IL-1β qPCR

(mM)



GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE



turning it off






via AMP kinase


Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK



Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK

HIF1





LUKE OrsquoNEILL


OrsquoNEILL LAB


Acknowledgment








Acknowledgment











CARKL



(b) Th17 cells








N=2

N=2

081211 bmdm SS

Ctl LPS (24 hr)0

1

2

3

4

Re

lative

miR

21

0 e

xp

ressio

n

181111 j774

Ctl LPS (24 hr)00

05

10

15

20

25

Re

lativ

e m

iR2

10

exp

ressio

n

310112 Bmdm ss 48h

Ctl LPS (48 hr)0

1

2

3

4

5

Re

lative

miR

21

0 e

xp

ressio

n

N=2

J774s

BMDM BMDM


LPS

HIF-1α IL-1β

Hypothesis


080413 210


05

10

15

20

0 MCSF2 FCS

2 MCSF2 FCS

10 MCSF10 FCS

Re

lativ

e m

iR-2

10

exp

ressio

n

27328140413 pooled 24h no SS n=8

Ctl LPS00

05

10

15

20

Re

lative

miR

-21

0 e

xp

ressio

n

N=8 N=3


LPS

HIF-1α IL-1β

Hypothesis


25030208160413 sdhd n=10

Ctl LPS00

05

10

15

Re

lative

sd

hd

exp

ressio

n

N=10


LPS

HIF-1α IL-1β

Hypothesis



anti-miR210

HIF-1α IL-1β

n=1

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

0

2

4

6

unstimulated LPS

NA

DN

AD

H ra

tio p

mo

l10

6 c

ells


Otto Warburg




Th17

Treg


Nature (2013) 493 346-354











EXTERNAL STRESSORS



EXTERNAL STRESSORS







KEY QUESTION











0

05

1

15

2

25

3

0 LPS

TN

Fa (

ng

ml)

LPS

LPS + 2DG

IL-1beta

TNFalpha

0

50

100

150

200

250

300

350

400

0 LPSre

lati

ve I

L-1

beta

exp

ressio

n

LPS

LPS + 2DG











THE GABA SHUNT


0

2times1006

4times1006

6times1006

su

ccin

ate

(p

ea

k a

rea

)

LPS

unstimulated

- 1 5 10

2DG (mM)



citrate

a-ketoglutarate

succinate

fumarate

malate


12C

13C

14N

15NGABA



production

IL-1beta


200

400

600

800

1000

IL-1b

eta (

pgm

l)IL-6


10

20

30

40IL-

6 (ng

ml)

TNFalpha


1

2

3

TNFa

(ngm

l)



0 20 40 60 80 0

50

100

150 Co n t r o l

Vi g a + L PS

LP S


P e r c

e n

t s u r v

i v a l

At this stage









WB HIF1α WB actin

LPS

2-DG



inhibited by 2DG



PHD

KG Succinate



PHD

KG Succinate

-



PHD

KG Succinate

KG


-50

0

50

100

150

200

250

300

350

400


unstim

LPS

LPS plus 2DG



- LPS - LPS0

2000

4000

6000

8000

rela

tive I

L-1

b e

xp

ressio

n

+ succinate

0 LPS 0 LPS0

20000

40000

60000

rela

tive I

L-1

b e

xp

ressio

n

+ butylmalonate




PHD

KG Succinate



PHD

KG Succinate

-



PHD

KG Succinate

KG

012

h

12h+αK

G24

h

24h+αK

G

00

05

10

Rela

tive E

nri

ch

men

t

0 LPS 0 LPS0

10000

20000

30000

rela

tiv

e IL

-1b

ex

pre

ss

ion

+ aKG


WBIL-1β

WBHIF-1α

- +LPSαKG αKG

WBβ-ac n

Summary






succinate

PHD




succinate

PHD





2DG2DG

-LPS+LPS

WBsuccinyllysine

WBIL-1βWBβ-ac n

unstimulated LPS00

01

02

03

04

me

an

em

PA

I va

lue





bull GAPDH


bull PKM12


Key finding










GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION



GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION




LOW GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE


Low glucose

SNF2

YEAST MACROPHAGE

Low glucose 2DG

AMP kinase



GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE

METFORMIN

(mM)

IL-1β qPCR

(mM)



GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE



turning it off






via AMP kinase


Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK



Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK

HIF1





LUKE OrsquoNEILL


OrsquoNEILL LAB


Acknowledgment








Acknowledgment











CARKL



(b) Th17 cells








N=2

N=2

081211 bmdm SS

Ctl LPS (24 hr)0

1

2

3

4

Re

lative

miR

21

0 e

xp

ressio

n

181111 j774

Ctl LPS (24 hr)00

05

10

15

20

25

Re

lativ

e m

iR2

10

exp

ressio

n

310112 Bmdm ss 48h

Ctl LPS (48 hr)0

1

2

3

4

5

Re

lative

miR

21

0 e

xp

ressio

n

N=2

J774s

BMDM BMDM


LPS

HIF-1α IL-1β

Hypothesis


080413 210


05

10

15

20

0 MCSF2 FCS

2 MCSF2 FCS

10 MCSF10 FCS

Re

lativ

e m

iR-2

10

exp

ressio

n

27328140413 pooled 24h no SS n=8

Ctl LPS00

05

10

15

20

Re

lative

miR

-21

0 e

xp

ressio

n

N=8 N=3


LPS

HIF-1α IL-1β

Hypothesis


25030208160413 sdhd n=10

Ctl LPS00

05

10

15

Re

lative

sd

hd

exp

ressio

n

N=10


LPS

HIF-1α IL-1β

Hypothesis



anti-miR210

HIF-1α IL-1β

n=1

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B




Th17

Treg


Nature (2013) 493 346-354











EXTERNAL STRESSORS



EXTERNAL STRESSORS







KEY QUESTION











0

05

1

15

2

25

3

0 LPS

TN

Fa (

ng

ml)

LPS

LPS + 2DG

IL-1beta

TNFalpha

0

50

100

150

200

250

300

350

400

0 LPSre

lati

ve I

L-1

beta

exp

ressio

n

LPS

LPS + 2DG











THE GABA SHUNT


0

2times1006

4times1006

6times1006

su

ccin

ate

(p

ea

k a

rea

)

LPS

unstimulated

- 1 5 10

2DG (mM)



citrate

a-ketoglutarate

succinate

fumarate

malate


12C

13C

14N

15NGABA



production

IL-1beta


200

400

600

800

1000

IL-1b

eta (

pgm

l)IL-6


10

20

30

40IL-

6 (ng

ml)

TNFalpha


1

2

3

TNFa

(ngm

l)



0 20 40 60 80 0

50

100

150 Co n t r o l

Vi g a + L PS

LP S


P e r c

e n

t s u r v

i v a l

At this stage









WB HIF1α WB actin

LPS

2-DG



inhibited by 2DG



PHD

KG Succinate



PHD

KG Succinate

-



PHD

KG Succinate

KG


-50

0

50

100

150

200

250

300

350

400


unstim

LPS

LPS plus 2DG



- LPS - LPS0

2000

4000

6000

8000

rela

tive I

L-1

b e

xp

ressio

n

+ succinate

0 LPS 0 LPS0

20000

40000

60000

rela

tive I

L-1

b e

xp

ressio

n

+ butylmalonate




PHD

KG Succinate



PHD

KG Succinate

-



PHD

KG Succinate

KG

012

h

12h+αK

G24

h

24h+αK

G

00

05

10

Rela

tive E

nri

ch

men

t

0 LPS 0 LPS0

10000

20000

30000

rela

tiv

e IL

-1b

ex

pre

ss

ion

+ aKG


WBIL-1β

WBHIF-1α

- +LPSαKG αKG

WBβ-ac n

Summary






succinate

PHD




succinate

PHD





2DG2DG

-LPS+LPS

WBsuccinyllysine

WBIL-1βWBβ-ac n

unstimulated LPS00

01

02

03

04

me

an

em

PA

I va

lue





bull GAPDH


bull PKM12


Key finding










GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION



GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION




LOW GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE


Low glucose

SNF2

YEAST MACROPHAGE

Low glucose 2DG

AMP kinase



GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE

METFORMIN

(mM)

IL-1β qPCR

(mM)



GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE



turning it off






via AMP kinase


Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK



Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK

HIF1





LUKE OrsquoNEILL


OrsquoNEILL LAB


Acknowledgment








Acknowledgment











CARKL



(b) Th17 cells








N=2

N=2

081211 bmdm SS

Ctl LPS (24 hr)0

1

2

3

4

Re

lative

miR

21

0 e

xp

ressio

n

181111 j774

Ctl LPS (24 hr)00

05

10

15

20

25

Re

lativ

e m

iR2

10

exp

ressio

n

310112 Bmdm ss 48h

Ctl LPS (48 hr)0

1

2

3

4

5

Re

lative

miR

21

0 e

xp

ressio

n

N=2

J774s

BMDM BMDM


LPS

HIF-1α IL-1β

Hypothesis


080413 210


05

10

15

20

0 MCSF2 FCS

2 MCSF2 FCS

10 MCSF10 FCS

Re

lativ

e m

iR-2

10

exp

ressio

n

27328140413 pooled 24h no SS n=8

Ctl LPS00

05

10

15

20

Re

lative

miR

-21

0 e

xp

ressio

n

N=8 N=3


LPS

HIF-1α IL-1β

Hypothesis


25030208160413 sdhd n=10

Ctl LPS00

05

10

15

Re

lative

sd

hd

exp

ressio

n

N=10


LPS

HIF-1α IL-1β

Hypothesis



anti-miR210

HIF-1α IL-1β

n=1

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

Nature (2013) 493 346-354











EXTERNAL STRESSORS



EXTERNAL STRESSORS







KEY QUESTION











0

05

1

15

2

25

3

0 LPS

TN

Fa (

ng

ml)

LPS

LPS + 2DG

IL-1beta

TNFalpha

0

50

100

150

200

250

300

350

400

0 LPSre

lati

ve I

L-1

beta

exp

ressio

n

LPS

LPS + 2DG











THE GABA SHUNT


0

2times1006

4times1006

6times1006

su

ccin

ate

(p

ea

k a

rea

)

LPS

unstimulated

- 1 5 10

2DG (mM)



citrate

a-ketoglutarate

succinate

fumarate

malate


12C

13C

14N

15NGABA



production

IL-1beta


200

400

600

800

1000

IL-1b

eta (

pgm

l)IL-6


10

20

30

40IL-

6 (ng

ml)

TNFalpha


1

2

3

TNFa

(ngm

l)



0 20 40 60 80 0

50

100

150 Co n t r o l

Vi g a + L PS

LP S


P e r c

e n

t s u r v

i v a l

At this stage









WB HIF1α WB actin

LPS

2-DG



inhibited by 2DG



PHD

KG Succinate



PHD

KG Succinate

-



PHD

KG Succinate

KG


-50

0

50

100

150

200

250

300

350

400


unstim

LPS

LPS plus 2DG



- LPS - LPS0

2000

4000

6000

8000

rela

tive I

L-1

b e

xp

ressio

n

+ succinate

0 LPS 0 LPS0

20000

40000

60000

rela

tive I

L-1

b e

xp

ressio

n

+ butylmalonate




PHD

KG Succinate



PHD

KG Succinate

-



PHD

KG Succinate

KG

012

h

12h+αK

G24

h

24h+αK

G

00

05

10

Rela

tive E

nri

ch

men

t

0 LPS 0 LPS0

10000

20000

30000

rela

tiv

e IL

-1b

ex

pre

ss

ion

+ aKG


WBIL-1β

WBHIF-1α

- +LPSαKG αKG

WBβ-ac n

Summary






succinate

PHD




succinate

PHD





2DG2DG

-LPS+LPS

WBsuccinyllysine

WBIL-1βWBβ-ac n

unstimulated LPS00

01

02

03

04

me

an

em

PA

I va

lue





bull GAPDH


bull PKM12


Key finding










GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION



GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION




LOW GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE


Low glucose

SNF2

YEAST MACROPHAGE

Low glucose 2DG

AMP kinase



GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE

METFORMIN

(mM)

IL-1β qPCR

(mM)



GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE



turning it off






via AMP kinase


Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK



Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK

HIF1





LUKE OrsquoNEILL


OrsquoNEILL LAB


Acknowledgment








Acknowledgment











CARKL



(b) Th17 cells








N=2

N=2

081211 bmdm SS

Ctl LPS (24 hr)0

1

2

3

4

Re

lative

miR

21

0 e

xp

ressio

n

181111 j774

Ctl LPS (24 hr)00

05

10

15

20

25

Re

lativ

e m

iR2

10

exp

ressio

n

310112 Bmdm ss 48h

Ctl LPS (48 hr)0

1

2

3

4

5

Re

lative

miR

21

0 e

xp

ressio

n

N=2

J774s

BMDM BMDM


LPS

HIF-1α IL-1β

Hypothesis


080413 210


05

10

15

20

0 MCSF2 FCS

2 MCSF2 FCS

10 MCSF10 FCS

Re

lativ

e m

iR-2

10

exp

ressio

n

27328140413 pooled 24h no SS n=8

Ctl LPS00

05

10

15

20

Re

lative

miR

-21

0 e

xp

ressio

n

N=8 N=3


LPS

HIF-1α IL-1β

Hypothesis


25030208160413 sdhd n=10

Ctl LPS00

05

10

15

Re

lative

sd

hd

exp

ressio

n

N=10


LPS

HIF-1α IL-1β

Hypothesis



anti-miR210

HIF-1α IL-1β

n=1

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B










EXTERNAL STRESSORS



EXTERNAL STRESSORS







KEY QUESTION











0

05

1

15

2

25

3

0 LPS

TN

Fa (

ng

ml)

LPS

LPS + 2DG

IL-1beta

TNFalpha

0

50

100

150

200

250

300

350

400

0 LPSre

lati

ve I

L-1

beta

exp

ressio

n

LPS

LPS + 2DG











THE GABA SHUNT


0

2times1006

4times1006

6times1006

su

ccin

ate

(p

ea

k a

rea

)

LPS

unstimulated

- 1 5 10

2DG (mM)



citrate

a-ketoglutarate

succinate

fumarate

malate


12C

13C

14N

15NGABA



production

IL-1beta


200

400

600

800

1000

IL-1b

eta (

pgm

l)IL-6


10

20

30

40IL-

6 (ng

ml)

TNFalpha


1

2

3

TNFa

(ngm

l)



0 20 40 60 80 0

50

100

150 Co n t r o l

Vi g a + L PS

LP S


P e r c

e n

t s u r v

i v a l

At this stage









WB HIF1α WB actin

LPS

2-DG



inhibited by 2DG



PHD

KG Succinate



PHD

KG Succinate

-



PHD

KG Succinate

KG


-50

0

50

100

150

200

250

300

350

400


unstim

LPS

LPS plus 2DG



- LPS - LPS0

2000

4000

6000

8000

rela

tive I

L-1

b e

xp

ressio

n

+ succinate

0 LPS 0 LPS0

20000

40000

60000

rela

tive I

L-1

b e

xp

ressio

n

+ butylmalonate




PHD

KG Succinate



PHD

KG Succinate

-



PHD

KG Succinate

KG

012

h

12h+αK

G24

h

24h+αK

G

00

05

10

Rela

tive E

nri

ch

men

t

0 LPS 0 LPS0

10000

20000

30000

rela

tiv

e IL

-1b

ex

pre

ss

ion

+ aKG


WBIL-1β

WBHIF-1α

- +LPSαKG αKG

WBβ-ac n

Summary






succinate

PHD




succinate

PHD





2DG2DG

-LPS+LPS

WBsuccinyllysine

WBIL-1βWBβ-ac n

unstimulated LPS00

01

02

03

04

me

an

em

PA

I va

lue





bull GAPDH


bull PKM12


Key finding










GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION



GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION




LOW GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE


Low glucose

SNF2

YEAST MACROPHAGE

Low glucose 2DG

AMP kinase



GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE

METFORMIN

(mM)

IL-1β qPCR

(mM)



GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE



turning it off






via AMP kinase


Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK



Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK

HIF1





LUKE OrsquoNEILL


OrsquoNEILL LAB


Acknowledgment








Acknowledgment











CARKL



(b) Th17 cells








N=2

N=2

081211 bmdm SS

Ctl LPS (24 hr)0

1

2

3

4

Re

lative

miR

21

0 e

xp

ressio

n

181111 j774

Ctl LPS (24 hr)00

05

10

15

20

25

Re

lativ

e m

iR2

10

exp

ressio

n

310112 Bmdm ss 48h

Ctl LPS (48 hr)0

1

2

3

4

5

Re

lative

miR

21

0 e

xp

ressio

n

N=2

J774s

BMDM BMDM


LPS

HIF-1α IL-1β

Hypothesis


080413 210


05

10

15

20

0 MCSF2 FCS

2 MCSF2 FCS

10 MCSF10 FCS

Re

lativ

e m

iR-2

10

exp

ressio

n

27328140413 pooled 24h no SS n=8

Ctl LPS00

05

10

15

20

Re

lative

miR

-21

0 e

xp

ressio

n

N=8 N=3


LPS

HIF-1α IL-1β

Hypothesis


25030208160413 sdhd n=10

Ctl LPS00

05

10

15

Re

lative

sd

hd

exp

ressio

n

N=10


LPS

HIF-1α IL-1β

Hypothesis



anti-miR210

HIF-1α IL-1β

n=1

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B



EXTERNAL STRESSORS







KEY QUESTION











0

05

1

15

2

25

3

0 LPS

TN

Fa (

ng

ml)

LPS

LPS + 2DG

IL-1beta

TNFalpha

0

50

100

150

200

250

300

350

400

0 LPSre

lati

ve I

L-1

beta

exp

ressio

n

LPS

LPS + 2DG











THE GABA SHUNT


0

2times1006

4times1006

6times1006

su

ccin

ate

(p

ea

k a

rea

)

LPS

unstimulated

- 1 5 10

2DG (mM)



citrate

a-ketoglutarate

succinate

fumarate

malate


12C

13C

14N

15NGABA



production

IL-1beta


200

400

600

800

1000

IL-1b

eta (

pgm

l)IL-6


10

20

30

40IL-

6 (ng

ml)

TNFalpha


1

2

3

TNFa

(ngm

l)



0 20 40 60 80 0

50

100

150 Co n t r o l

Vi g a + L PS

LP S


P e r c

e n

t s u r v

i v a l

At this stage









WB HIF1α WB actin

LPS

2-DG



inhibited by 2DG



PHD

KG Succinate



PHD

KG Succinate

-



PHD

KG Succinate

KG


-50

0

50

100

150

200

250

300

350

400


unstim

LPS

LPS plus 2DG



- LPS - LPS0

2000

4000

6000

8000

rela

tive I

L-1

b e

xp

ressio

n

+ succinate

0 LPS 0 LPS0

20000

40000

60000

rela

tive I

L-1

b e

xp

ressio

n

+ butylmalonate




PHD

KG Succinate



PHD

KG Succinate

-



PHD

KG Succinate

KG

012

h

12h+αK

G24

h

24h+αK

G

00

05

10

Rela

tive E

nri

ch

men

t

0 LPS 0 LPS0

10000

20000

30000

rela

tiv

e IL

-1b

ex

pre

ss

ion

+ aKG


WBIL-1β

WBHIF-1α

- +LPSαKG αKG

WBβ-ac n

Summary






succinate

PHD




succinate

PHD





2DG2DG

-LPS+LPS

WBsuccinyllysine

WBIL-1βWBβ-ac n

unstimulated LPS00

01

02

03

04

me

an

em

PA

I va

lue





bull GAPDH


bull PKM12


Key finding










GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION



GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION




LOW GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE


Low glucose

SNF2

YEAST MACROPHAGE

Low glucose 2DG

AMP kinase



GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE

METFORMIN

(mM)

IL-1β qPCR

(mM)



GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE



turning it off






via AMP kinase


Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK



Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK

HIF1





LUKE OrsquoNEILL


OrsquoNEILL LAB


Acknowledgment








Acknowledgment











CARKL



(b) Th17 cells








N=2

N=2

081211 bmdm SS

Ctl LPS (24 hr)0

1

2

3

4

Re

lative

miR

21

0 e

xp

ressio

n

181111 j774

Ctl LPS (24 hr)00

05

10

15

20

25

Re

lativ

e m

iR2

10

exp

ressio

n

310112 Bmdm ss 48h

Ctl LPS (48 hr)0

1

2

3

4

5

Re

lative

miR

21

0 e

xp

ressio

n

N=2

J774s

BMDM BMDM


LPS

HIF-1α IL-1β

Hypothesis


080413 210


05

10

15

20

0 MCSF2 FCS

2 MCSF2 FCS

10 MCSF10 FCS

Re

lativ

e m

iR-2

10

exp

ressio

n

27328140413 pooled 24h no SS n=8

Ctl LPS00

05

10

15

20

Re

lative

miR

-21

0 e

xp

ressio

n

N=8 N=3


LPS

HIF-1α IL-1β

Hypothesis


25030208160413 sdhd n=10

Ctl LPS00

05

10

15

Re

lative

sd

hd

exp

ressio

n

N=10


LPS

HIF-1α IL-1β

Hypothesis



anti-miR210

HIF-1α IL-1β

n=1

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B





KEY QUESTION











0

05

1

15

2

25

3

0 LPS

TN

Fa (

ng

ml)

LPS

LPS + 2DG

IL-1beta

TNFalpha

0

50

100

150

200

250

300

350

400

0 LPSre

lati

ve I

L-1

beta

exp

ressio

n

LPS

LPS + 2DG











THE GABA SHUNT


0

2times1006

4times1006

6times1006

su

ccin

ate

(p

ea

k a

rea

)

LPS

unstimulated

- 1 5 10

2DG (mM)



citrate

a-ketoglutarate

succinate

fumarate

malate


12C

13C

14N

15NGABA



production

IL-1beta


200

400

600

800

1000

IL-1b

eta (

pgm

l)IL-6


10

20

30

40IL-

6 (ng

ml)

TNFalpha


1

2

3

TNFa

(ngm

l)



0 20 40 60 80 0

50

100

150 Co n t r o l

Vi g a + L PS

LP S


P e r c

e n

t s u r v

i v a l

At this stage









WB HIF1α WB actin

LPS

2-DG



inhibited by 2DG



PHD

KG Succinate



PHD

KG Succinate

-



PHD

KG Succinate

KG


-50

0

50

100

150

200

250

300

350

400


unstim

LPS

LPS plus 2DG



- LPS - LPS0

2000

4000

6000

8000

rela

tive I

L-1

b e

xp

ressio

n

+ succinate

0 LPS 0 LPS0

20000

40000

60000

rela

tive I

L-1

b e

xp

ressio

n

+ butylmalonate




PHD

KG Succinate



PHD

KG Succinate

-



PHD

KG Succinate

KG

012

h

12h+αK

G24

h

24h+αK

G

00

05

10

Rela

tive E

nri

ch

men

t

0 LPS 0 LPS0

10000

20000

30000

rela

tiv

e IL

-1b

ex

pre

ss

ion

+ aKG


WBIL-1β

WBHIF-1α

- +LPSαKG αKG

WBβ-ac n

Summary






succinate

PHD




succinate

PHD





2DG2DG

-LPS+LPS

WBsuccinyllysine

WBIL-1βWBβ-ac n

unstimulated LPS00

01

02

03

04

me

an

em

PA

I va

lue





bull GAPDH


bull PKM12


Key finding










GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION



GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION




LOW GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE


Low glucose

SNF2

YEAST MACROPHAGE

Low glucose 2DG

AMP kinase



GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE

METFORMIN

(mM)

IL-1β qPCR

(mM)



GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE



turning it off






via AMP kinase


Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK



Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK

HIF1





LUKE OrsquoNEILL


OrsquoNEILL LAB


Acknowledgment








Acknowledgment











CARKL



(b) Th17 cells








N=2

N=2

081211 bmdm SS

Ctl LPS (24 hr)0

1

2

3

4

Re

lative

miR

21

0 e

xp

ressio

n

181111 j774

Ctl LPS (24 hr)00

05

10

15

20

25

Re

lativ

e m

iR2

10

exp

ressio

n

310112 Bmdm ss 48h

Ctl LPS (48 hr)0

1

2

3

4

5

Re

lative

miR

21

0 e

xp

ressio

n

N=2

J774s

BMDM BMDM


LPS

HIF-1α IL-1β

Hypothesis


080413 210


05

10

15

20

0 MCSF2 FCS

2 MCSF2 FCS

10 MCSF10 FCS

Re

lativ

e m

iR-2

10

exp

ressio

n

27328140413 pooled 24h no SS n=8

Ctl LPS00

05

10

15

20

Re

lative

miR

-21

0 e

xp

ressio

n

N=8 N=3


LPS

HIF-1α IL-1β

Hypothesis


25030208160413 sdhd n=10

Ctl LPS00

05

10

15

Re

lative

sd

hd

exp

ressio

n

N=10


LPS

HIF-1α IL-1β

Hypothesis



anti-miR210

HIF-1α IL-1β

n=1

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B











0

05

1

15

2

25

3

0 LPS

TN

Fa (

ng

ml)

LPS

LPS + 2DG

IL-1beta

TNFalpha

0

50

100

150

200

250

300

350

400

0 LPSre

lati

ve I

L-1

beta

exp

ressio

n

LPS

LPS + 2DG











THE GABA SHUNT


0

2times1006

4times1006

6times1006

su

ccin

ate

(p

ea

k a

rea

)

LPS

unstimulated

- 1 5 10

2DG (mM)



citrate

a-ketoglutarate

succinate

fumarate

malate


12C

13C

14N

15NGABA



production

IL-1beta


200

400

600

800

1000

IL-1b

eta (

pgm

l)IL-6


10

20

30

40IL-

6 (ng

ml)

TNFalpha


1

2

3

TNFa

(ngm

l)



0 20 40 60 80 0

50

100

150 Co n t r o l

Vi g a + L PS

LP S


P e r c

e n

t s u r v

i v a l

At this stage









WB HIF1α WB actin

LPS

2-DG



inhibited by 2DG



PHD

KG Succinate



PHD

KG Succinate

-



PHD

KG Succinate

KG


-50

0

50

100

150

200

250

300

350

400


unstim

LPS

LPS plus 2DG



- LPS - LPS0

2000

4000

6000

8000

rela

tive I

L-1

b e

xp

ressio

n

+ succinate

0 LPS 0 LPS0

20000

40000

60000

rela

tive I

L-1

b e

xp

ressio

n

+ butylmalonate




PHD

KG Succinate



PHD

KG Succinate

-



PHD

KG Succinate

KG

012

h

12h+αK

G24

h

24h+αK

G

00

05

10

Rela

tive E

nri

ch

men

t

0 LPS 0 LPS0

10000

20000

30000

rela

tiv

e IL

-1b

ex

pre

ss

ion

+ aKG


WBIL-1β

WBHIF-1α

- +LPSαKG αKG

WBβ-ac n

Summary






succinate

PHD




succinate

PHD





2DG2DG

-LPS+LPS

WBsuccinyllysine

WBIL-1βWBβ-ac n

unstimulated LPS00

01

02

03

04

me

an

em

PA

I va

lue





bull GAPDH


bull PKM12


Key finding










GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION



GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION




LOW GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE


Low glucose

SNF2

YEAST MACROPHAGE

Low glucose 2DG

AMP kinase



GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE

METFORMIN

(mM)

IL-1β qPCR

(mM)



GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE



turning it off






via AMP kinase


Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK



Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK

HIF1





LUKE OrsquoNEILL


OrsquoNEILL LAB


Acknowledgment








Acknowledgment











CARKL



(b) Th17 cells








N=2

N=2

081211 bmdm SS

Ctl LPS (24 hr)0

1

2

3

4

Re

lative

miR

21

0 e

xp

ressio

n

181111 j774

Ctl LPS (24 hr)00

05

10

15

20

25

Re

lativ

e m

iR2

10

exp

ressio

n

310112 Bmdm ss 48h

Ctl LPS (48 hr)0

1

2

3

4

5

Re

lative

miR

21

0 e

xp

ressio

n

N=2

J774s

BMDM BMDM


LPS

HIF-1α IL-1β

Hypothesis


080413 210


05

10

15

20

0 MCSF2 FCS

2 MCSF2 FCS

10 MCSF10 FCS

Re

lativ

e m

iR-2

10

exp

ressio

n

27328140413 pooled 24h no SS n=8

Ctl LPS00

05

10

15

20

Re

lative

miR

-21

0 e

xp

ressio

n

N=8 N=3


LPS

HIF-1α IL-1β

Hypothesis


25030208160413 sdhd n=10

Ctl LPS00

05

10

15

Re

lative

sd

hd

exp

ressio

n

N=10


LPS

HIF-1α IL-1β

Hypothesis



anti-miR210

HIF-1α IL-1β

n=1

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B










THE GABA SHUNT


0

2times1006

4times1006

6times1006

su

ccin

ate

(p

ea

k a

rea

)

LPS

unstimulated

- 1 5 10

2DG (mM)



citrate

a-ketoglutarate

succinate

fumarate

malate


12C

13C

14N

15NGABA



production

IL-1beta


200

400

600

800

1000

IL-1b

eta (

pgm

l)IL-6


10

20

30

40IL-

6 (ng

ml)

TNFalpha


1

2

3

TNFa

(ngm

l)



0 20 40 60 80 0

50

100

150 Co n t r o l

Vi g a + L PS

LP S


P e r c

e n

t s u r v

i v a l

At this stage









WB HIF1α WB actin

LPS

2-DG



inhibited by 2DG



PHD

KG Succinate



PHD

KG Succinate

-



PHD

KG Succinate

KG


-50

0

50

100

150

200

250

300

350

400


unstim

LPS

LPS plus 2DG



- LPS - LPS0

2000

4000

6000

8000

rela

tive I

L-1

b e

xp

ressio

n

+ succinate

0 LPS 0 LPS0

20000

40000

60000

rela

tive I

L-1

b e

xp

ressio

n

+ butylmalonate




PHD

KG Succinate



PHD

KG Succinate

-



PHD

KG Succinate

KG

012

h

12h+αK

G24

h

24h+αK

G

00

05

10

Rela

tive E

nri

ch

men

t

0 LPS 0 LPS0

10000

20000

30000

rela

tiv

e IL

-1b

ex

pre

ss

ion

+ aKG


WBIL-1β

WBHIF-1α

- +LPSαKG αKG

WBβ-ac n

Summary






succinate

PHD




succinate

PHD





2DG2DG

-LPS+LPS

WBsuccinyllysine

WBIL-1βWBβ-ac n

unstimulated LPS00

01

02

03

04

me

an

em

PA

I va

lue





bull GAPDH


bull PKM12


Key finding










GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION



GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION




LOW GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE


Low glucose

SNF2

YEAST MACROPHAGE

Low glucose 2DG

AMP kinase



GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE

METFORMIN

(mM)

IL-1β qPCR

(mM)



GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE



turning it off






via AMP kinase


Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK



Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK

HIF1





LUKE OrsquoNEILL


OrsquoNEILL LAB


Acknowledgment








Acknowledgment











CARKL



(b) Th17 cells








N=2

N=2

081211 bmdm SS

Ctl LPS (24 hr)0

1

2

3

4

Re

lative

miR

21

0 e

xp

ressio

n

181111 j774

Ctl LPS (24 hr)00

05

10

15

20

25

Re

lativ

e m

iR2

10

exp

ressio

n

310112 Bmdm ss 48h

Ctl LPS (48 hr)0

1

2

3

4

5

Re

lative

miR

21

0 e

xp

ressio

n

N=2

J774s

BMDM BMDM


LPS

HIF-1α IL-1β

Hypothesis


080413 210


05

10

15

20

0 MCSF2 FCS

2 MCSF2 FCS

10 MCSF10 FCS

Re

lativ

e m

iR-2

10

exp

ressio

n

27328140413 pooled 24h no SS n=8

Ctl LPS00

05

10

15

20

Re

lative

miR

-21

0 e

xp

ressio

n

N=8 N=3


LPS

HIF-1α IL-1β

Hypothesis


25030208160413 sdhd n=10

Ctl LPS00

05

10

15

Re

lative

sd

hd

exp

ressio

n

N=10


LPS

HIF-1α IL-1β

Hypothesis



anti-miR210

HIF-1α IL-1β

n=1

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B



citrate

a-ketoglutarate

succinate

fumarate

malate


12C

13C

14N

15NGABA



production

IL-1beta


200

400

600

800

1000

IL-1b

eta (

pgm

l)IL-6


10

20

30

40IL-

6 (ng

ml)

TNFalpha


1

2

3

TNFa

(ngm

l)



0 20 40 60 80 0

50

100

150 Co n t r o l

Vi g a + L PS

LP S


P e r c

e n

t s u r v

i v a l

At this stage









WB HIF1α WB actin

LPS

2-DG



inhibited by 2DG



PHD

KG Succinate



PHD

KG Succinate

-



PHD

KG Succinate

KG


-50

0

50

100

150

200

250

300

350

400


unstim

LPS

LPS plus 2DG



- LPS - LPS0

2000

4000

6000

8000

rela

tive I

L-1

b e

xp

ressio

n

+ succinate

0 LPS 0 LPS0

20000

40000

60000

rela

tive I

L-1

b e

xp

ressio

n

+ butylmalonate




PHD

KG Succinate



PHD

KG Succinate

-



PHD

KG Succinate

KG

012

h

12h+αK

G24

h

24h+αK

G

00

05

10

Rela

tive E

nri

ch

men

t

0 LPS 0 LPS0

10000

20000

30000

rela

tiv

e IL

-1b

ex

pre

ss

ion

+ aKG


WBIL-1β

WBHIF-1α

- +LPSαKG αKG

WBβ-ac n

Summary






succinate

PHD




succinate

PHD





2DG2DG

-LPS+LPS

WBsuccinyllysine

WBIL-1βWBβ-ac n

unstimulated LPS00

01

02

03

04

me

an

em

PA

I va

lue





bull GAPDH


bull PKM12


Key finding










GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION



GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION




LOW GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE


Low glucose

SNF2

YEAST MACROPHAGE

Low glucose 2DG

AMP kinase



GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE

METFORMIN

(mM)

IL-1β qPCR

(mM)



GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE



turning it off






via AMP kinase


Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK



Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK

HIF1





LUKE OrsquoNEILL


OrsquoNEILL LAB


Acknowledgment








Acknowledgment











CARKL



(b) Th17 cells








N=2

N=2

081211 bmdm SS

Ctl LPS (24 hr)0

1

2

3

4

Re

lative

miR

21

0 e

xp

ressio

n

181111 j774

Ctl LPS (24 hr)00

05

10

15

20

25

Re

lativ

e m

iR2

10

exp

ressio

n

310112 Bmdm ss 48h

Ctl LPS (48 hr)0

1

2

3

4

5

Re

lative

miR

21

0 e

xp

ressio

n

N=2

J774s

BMDM BMDM


LPS

HIF-1α IL-1β

Hypothesis


080413 210


05

10

15

20

0 MCSF2 FCS

2 MCSF2 FCS

10 MCSF10 FCS

Re

lativ

e m

iR-2

10

exp

ressio

n

27328140413 pooled 24h no SS n=8

Ctl LPS00

05

10

15

20

Re

lative

miR

-21

0 e

xp

ressio

n

N=8 N=3


LPS

HIF-1α IL-1β

Hypothesis


25030208160413 sdhd n=10

Ctl LPS00

05

10

15

Re

lative

sd

hd

exp

ressio

n

N=10


LPS

HIF-1α IL-1β

Hypothesis



anti-miR210

HIF-1α IL-1β

n=1

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

IL-1beta


200

400

600

800

1000

IL-1b

eta (

pgm

l)IL-6


10

20

30

40IL-

6 (ng

ml)

TNFalpha


1

2

3

TNFa

(ngm

l)



0 20 40 60 80 0

50

100

150 Co n t r o l

Vi g a + L PS

LP S


P e r c

e n

t s u r v

i v a l

At this stage









WB HIF1α WB actin

LPS

2-DG



inhibited by 2DG



PHD

KG Succinate



PHD

KG Succinate

-



PHD

KG Succinate

KG


-50

0

50

100

150

200

250

300

350

400


unstim

LPS

LPS plus 2DG



- LPS - LPS0

2000

4000

6000

8000

rela

tive I

L-1

b e

xp

ressio

n

+ succinate

0 LPS 0 LPS0

20000

40000

60000

rela

tive I

L-1

b e

xp

ressio

n

+ butylmalonate




PHD

KG Succinate



PHD

KG Succinate

-



PHD

KG Succinate

KG

012

h

12h+αK

G24

h

24h+αK

G

00

05

10

Rela

tive E

nri

ch

men

t

0 LPS 0 LPS0

10000

20000

30000

rela

tiv

e IL

-1b

ex

pre

ss

ion

+ aKG


WBIL-1β

WBHIF-1α

- +LPSαKG αKG

WBβ-ac n

Summary






succinate

PHD




succinate

PHD





2DG2DG

-LPS+LPS

WBsuccinyllysine

WBIL-1βWBβ-ac n

unstimulated LPS00

01

02

03

04

me

an

em

PA

I va

lue





bull GAPDH


bull PKM12


Key finding










GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION



GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION




LOW GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE


Low glucose

SNF2

YEAST MACROPHAGE

Low glucose 2DG

AMP kinase



GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE

METFORMIN

(mM)

IL-1β qPCR

(mM)



GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE



turning it off






via AMP kinase


Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK



Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK

HIF1





LUKE OrsquoNEILL


OrsquoNEILL LAB


Acknowledgment








Acknowledgment











CARKL



(b) Th17 cells








N=2

N=2

081211 bmdm SS

Ctl LPS (24 hr)0

1

2

3

4

Re

lative

miR

21

0 e

xp

ressio

n

181111 j774

Ctl LPS (24 hr)00

05

10

15

20

25

Re

lativ

e m

iR2

10

exp

ressio

n

310112 Bmdm ss 48h

Ctl LPS (48 hr)0

1

2

3

4

5

Re

lative

miR

21

0 e

xp

ressio

n

N=2

J774s

BMDM BMDM


LPS

HIF-1α IL-1β

Hypothesis


080413 210


05

10

15

20

0 MCSF2 FCS

2 MCSF2 FCS

10 MCSF10 FCS

Re

lativ

e m

iR-2

10

exp

ressio

n

27328140413 pooled 24h no SS n=8

Ctl LPS00

05

10

15

20

Re

lative

miR

-21

0 e

xp

ressio

n

N=8 N=3


LPS

HIF-1α IL-1β

Hypothesis


25030208160413 sdhd n=10

Ctl LPS00

05

10

15

Re

lative

sd

hd

exp

ressio

n

N=10


LPS

HIF-1α IL-1β

Hypothesis



anti-miR210

HIF-1α IL-1β

n=1

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

At this stage









WB HIF1α WB actin

LPS

2-DG



inhibited by 2DG



PHD

KG Succinate



PHD

KG Succinate

-



PHD

KG Succinate

KG


-50

0

50

100

150

200

250

300

350

400


unstim

LPS

LPS plus 2DG



- LPS - LPS0

2000

4000

6000

8000

rela

tive I

L-1

b e

xp

ressio

n

+ succinate

0 LPS 0 LPS0

20000

40000

60000

rela

tive I

L-1

b e

xp

ressio

n

+ butylmalonate




PHD

KG Succinate



PHD

KG Succinate

-



PHD

KG Succinate

KG

012

h

12h+αK

G24

h

24h+αK

G

00

05

10

Rela

tive E

nri

ch

men

t

0 LPS 0 LPS0

10000

20000

30000

rela

tiv

e IL

-1b

ex

pre

ss

ion

+ aKG


WBIL-1β

WBHIF-1α

- +LPSαKG αKG

WBβ-ac n

Summary






succinate

PHD




succinate

PHD





2DG2DG

-LPS+LPS

WBsuccinyllysine

WBIL-1βWBβ-ac n

unstimulated LPS00

01

02

03

04

me

an

em

PA

I va

lue





bull GAPDH


bull PKM12


Key finding










GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION



GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION




LOW GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE


Low glucose

SNF2

YEAST MACROPHAGE

Low glucose 2DG

AMP kinase



GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE

METFORMIN

(mM)

IL-1β qPCR

(mM)



GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE



turning it off






via AMP kinase


Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK



Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK

HIF1





LUKE OrsquoNEILL


OrsquoNEILL LAB


Acknowledgment








Acknowledgment











CARKL



(b) Th17 cells








N=2

N=2

081211 bmdm SS

Ctl LPS (24 hr)0

1

2

3

4

Re

lative

miR

21

0 e

xp

ressio

n

181111 j774

Ctl LPS (24 hr)00

05

10

15

20

25

Re

lativ

e m

iR2

10

exp

ressio

n

310112 Bmdm ss 48h

Ctl LPS (48 hr)0

1

2

3

4

5

Re

lative

miR

21

0 e

xp

ressio

n

N=2

J774s

BMDM BMDM


LPS

HIF-1α IL-1β

Hypothesis


080413 210


05

10

15

20

0 MCSF2 FCS

2 MCSF2 FCS

10 MCSF10 FCS

Re

lativ

e m

iR-2

10

exp

ressio

n

27328140413 pooled 24h no SS n=8

Ctl LPS00

05

10

15

20

Re

lative

miR

-21

0 e

xp

ressio

n

N=8 N=3


LPS

HIF-1α IL-1β

Hypothesis


25030208160413 sdhd n=10

Ctl LPS00

05

10

15

Re

lative

sd

hd

exp

ressio

n

N=10


LPS

HIF-1α IL-1β

Hypothesis



anti-miR210

HIF-1α IL-1β

n=1

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B




WB HIF1α WB actin

LPS

2-DG



inhibited by 2DG



PHD

KG Succinate



PHD

KG Succinate

-



PHD

KG Succinate

KG


-50

0

50

100

150

200

250

300

350

400


unstim

LPS

LPS plus 2DG



- LPS - LPS0

2000

4000

6000

8000

rela

tive I

L-1

b e

xp

ressio

n

+ succinate

0 LPS 0 LPS0

20000

40000

60000

rela

tive I

L-1

b e

xp

ressio

n

+ butylmalonate




PHD

KG Succinate



PHD

KG Succinate

-



PHD

KG Succinate

KG

012

h

12h+αK

G24

h

24h+αK

G

00

05

10

Rela

tive E

nri

ch

men

t

0 LPS 0 LPS0

10000

20000

30000

rela

tiv

e IL

-1b

ex

pre

ss

ion

+ aKG


WBIL-1β

WBHIF-1α

- +LPSαKG αKG

WBβ-ac n

Summary






succinate

PHD




succinate

PHD





2DG2DG

-LPS+LPS

WBsuccinyllysine

WBIL-1βWBβ-ac n

unstimulated LPS00

01

02

03

04

me

an

em

PA

I va

lue





bull GAPDH


bull PKM12


Key finding










GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION



GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION




LOW GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE


Low glucose

SNF2

YEAST MACROPHAGE

Low glucose 2DG

AMP kinase



GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE

METFORMIN

(mM)

IL-1β qPCR

(mM)



GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE



turning it off






via AMP kinase


Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK



Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK

HIF1





LUKE OrsquoNEILL


OrsquoNEILL LAB


Acknowledgment








Acknowledgment











CARKL



(b) Th17 cells








N=2

N=2

081211 bmdm SS

Ctl LPS (24 hr)0

1

2

3

4

Re

lative

miR

21

0 e

xp

ressio

n

181111 j774

Ctl LPS (24 hr)00

05

10

15

20

25

Re

lativ

e m

iR2

10

exp

ressio

n

310112 Bmdm ss 48h

Ctl LPS (48 hr)0

1

2

3

4

5

Re

lative

miR

21

0 e

xp

ressio

n

N=2

J774s

BMDM BMDM


LPS

HIF-1α IL-1β

Hypothesis


080413 210


05

10

15

20

0 MCSF2 FCS

2 MCSF2 FCS

10 MCSF10 FCS

Re

lativ

e m

iR-2

10

exp

ressio

n

27328140413 pooled 24h no SS n=8

Ctl LPS00

05

10

15

20

Re

lative

miR

-21

0 e

xp

ressio

n

N=8 N=3


LPS

HIF-1α IL-1β

Hypothesis


25030208160413 sdhd n=10

Ctl LPS00

05

10

15

Re

lative

sd

hd

exp

ressio

n

N=10


LPS

HIF-1α IL-1β

Hypothesis



anti-miR210

HIF-1α IL-1β

n=1

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

2-DG



inhibited by 2DG



PHD

KG Succinate



PHD

KG Succinate

-



PHD

KG Succinate

KG


-50

0

50

100

150

200

250

300

350

400


unstim

LPS

LPS plus 2DG



- LPS - LPS0

2000

4000

6000

8000

rela

tive I

L-1

b e

xp

ressio

n

+ succinate

0 LPS 0 LPS0

20000

40000

60000

rela

tive I

L-1

b e

xp

ressio

n

+ butylmalonate




PHD

KG Succinate



PHD

KG Succinate

-



PHD

KG Succinate

KG

012

h

12h+αK

G24

h

24h+αK

G

00

05

10

Rela

tive E

nri

ch

men

t

0 LPS 0 LPS0

10000

20000

30000

rela

tiv

e IL

-1b

ex

pre

ss

ion

+ aKG


WBIL-1β

WBHIF-1α

- +LPSαKG αKG

WBβ-ac n

Summary






succinate

PHD




succinate

PHD





2DG2DG

-LPS+LPS

WBsuccinyllysine

WBIL-1βWBβ-ac n

unstimulated LPS00

01

02

03

04

me

an

em

PA

I va

lue





bull GAPDH


bull PKM12


Key finding










GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION



GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION




LOW GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE


Low glucose

SNF2

YEAST MACROPHAGE

Low glucose 2DG

AMP kinase



GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE

METFORMIN

(mM)

IL-1β qPCR

(mM)



GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE



turning it off






via AMP kinase


Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK



Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK

HIF1





LUKE OrsquoNEILL


OrsquoNEILL LAB


Acknowledgment








Acknowledgment











CARKL



(b) Th17 cells








N=2

N=2

081211 bmdm SS

Ctl LPS (24 hr)0

1

2

3

4

Re

lative

miR

21

0 e

xp

ressio

n

181111 j774

Ctl LPS (24 hr)00

05

10

15

20

25

Re

lativ

e m

iR2

10

exp

ressio

n

310112 Bmdm ss 48h

Ctl LPS (48 hr)0

1

2

3

4

5

Re

lative

miR

21

0 e

xp

ressio

n

N=2

J774s

BMDM BMDM


LPS

HIF-1α IL-1β

Hypothesis


080413 210


05

10

15

20

0 MCSF2 FCS

2 MCSF2 FCS

10 MCSF10 FCS

Re

lativ

e m

iR-2

10

exp

ressio

n

27328140413 pooled 24h no SS n=8

Ctl LPS00

05

10

15

20

Re

lative

miR

-21

0 e

xp

ressio

n

N=8 N=3


LPS

HIF-1α IL-1β

Hypothesis


25030208160413 sdhd n=10

Ctl LPS00

05

10

15

Re

lative

sd

hd

exp

ressio

n

N=10


LPS

HIF-1α IL-1β

Hypothesis



anti-miR210

HIF-1α IL-1β

n=1

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B



PHD

KG Succinate

-



PHD

KG Succinate

KG


-50

0

50

100

150

200

250

300

350

400


unstim

LPS

LPS plus 2DG



- LPS - LPS0

2000

4000

6000

8000

rela

tive I

L-1

b e

xp

ressio

n

+ succinate

0 LPS 0 LPS0

20000

40000

60000

rela

tive I

L-1

b e

xp

ressio

n

+ butylmalonate




PHD

KG Succinate



PHD

KG Succinate

-



PHD

KG Succinate

KG

012

h

12h+αK

G24

h

24h+αK

G

00

05

10

Rela

tive E

nri

ch

men

t

0 LPS 0 LPS0

10000

20000

30000

rela

tiv

e IL

-1b

ex

pre

ss

ion

+ aKG


WBIL-1β

WBHIF-1α

- +LPSαKG αKG

WBβ-ac n

Summary






succinate

PHD




succinate

PHD





2DG2DG

-LPS+LPS

WBsuccinyllysine

WBIL-1βWBβ-ac n

unstimulated LPS00

01

02

03

04

me

an

em

PA

I va

lue





bull GAPDH


bull PKM12


Key finding










GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION



GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION




LOW GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE


Low glucose

SNF2

YEAST MACROPHAGE

Low glucose 2DG

AMP kinase



GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE

METFORMIN

(mM)

IL-1β qPCR

(mM)



GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE



turning it off






via AMP kinase


Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK



Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK

HIF1





LUKE OrsquoNEILL


OrsquoNEILL LAB


Acknowledgment








Acknowledgment











CARKL



(b) Th17 cells








N=2

N=2

081211 bmdm SS

Ctl LPS (24 hr)0

1

2

3

4

Re

lative

miR

21

0 e

xp

ressio

n

181111 j774

Ctl LPS (24 hr)00

05

10

15

20

25

Re

lativ

e m

iR2

10

exp

ressio

n

310112 Bmdm ss 48h

Ctl LPS (48 hr)0

1

2

3

4

5

Re

lative

miR

21

0 e

xp

ressio

n

N=2

J774s

BMDM BMDM


LPS

HIF-1α IL-1β

Hypothesis


080413 210


05

10

15

20

0 MCSF2 FCS

2 MCSF2 FCS

10 MCSF10 FCS

Re

lativ

e m

iR-2

10

exp

ressio

n

27328140413 pooled 24h no SS n=8

Ctl LPS00

05

10

15

20

Re

lative

miR

-21

0 e

xp

ressio

n

N=8 N=3


LPS

HIF-1α IL-1β

Hypothesis


25030208160413 sdhd n=10

Ctl LPS00

05

10

15

Re

lative

sd

hd

exp

ressio

n

N=10


LPS

HIF-1α IL-1β

Hypothesis



anti-miR210

HIF-1α IL-1β

n=1

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B


-50

0

50

100

150

200

250

300

350

400


unstim

LPS

LPS plus 2DG



- LPS - LPS0

2000

4000

6000

8000

rela

tive I

L-1

b e

xp

ressio

n

+ succinate

0 LPS 0 LPS0

20000

40000

60000

rela

tive I

L-1

b e

xp

ressio

n

+ butylmalonate




PHD

KG Succinate



PHD

KG Succinate

-



PHD

KG Succinate

KG

012

h

12h+αK

G24

h

24h+αK

G

00

05

10

Rela

tive E

nri

ch

men

t

0 LPS 0 LPS0

10000

20000

30000

rela

tiv

e IL

-1b

ex

pre

ss

ion

+ aKG


WBIL-1β

WBHIF-1α

- +LPSαKG αKG

WBβ-ac n

Summary






succinate

PHD




succinate

PHD





2DG2DG

-LPS+LPS

WBsuccinyllysine

WBIL-1βWBβ-ac n

unstimulated LPS00

01

02

03

04

me

an

em

PA

I va

lue





bull GAPDH


bull PKM12


Key finding










GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION



GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION




LOW GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE


Low glucose

SNF2

YEAST MACROPHAGE

Low glucose 2DG

AMP kinase



GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE

METFORMIN

(mM)

IL-1β qPCR

(mM)



GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE



turning it off






via AMP kinase


Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK



Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK

HIF1





LUKE OrsquoNEILL


OrsquoNEILL LAB


Acknowledgment








Acknowledgment











CARKL



(b) Th17 cells








N=2

N=2

081211 bmdm SS

Ctl LPS (24 hr)0

1

2

3

4

Re

lative

miR

21

0 e

xp

ressio

n

181111 j774

Ctl LPS (24 hr)00

05

10

15

20

25

Re

lativ

e m

iR2

10

exp

ressio

n

310112 Bmdm ss 48h

Ctl LPS (48 hr)0

1

2

3

4

5

Re

lative

miR

21

0 e

xp

ressio

n

N=2

J774s

BMDM BMDM


LPS

HIF-1α IL-1β

Hypothesis


080413 210


05

10

15

20

0 MCSF2 FCS

2 MCSF2 FCS

10 MCSF10 FCS

Re

lativ

e m

iR-2

10

exp

ressio

n

27328140413 pooled 24h no SS n=8

Ctl LPS00

05

10

15

20

Re

lative

miR

-21

0 e

xp

ressio

n

N=8 N=3


LPS

HIF-1α IL-1β

Hypothesis


25030208160413 sdhd n=10

Ctl LPS00

05

10

15

Re

lative

sd

hd

exp

ressio

n

N=10


LPS

HIF-1α IL-1β

Hypothesis



anti-miR210

HIF-1α IL-1β

n=1

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B


- LPS - LPS0

2000

4000

6000

8000

rela

tive I

L-1

b e

xp

ressio

n

+ succinate

0 LPS 0 LPS0

20000

40000

60000

rela

tive I

L-1

b e

xp

ressio

n

+ butylmalonate




PHD

KG Succinate



PHD

KG Succinate

-



PHD

KG Succinate

KG

012

h

12h+αK

G24

h

24h+αK

G

00

05

10

Rela

tive E

nri

ch

men

t

0 LPS 0 LPS0

10000

20000

30000

rela

tiv

e IL

-1b

ex

pre

ss

ion

+ aKG


WBIL-1β

WBHIF-1α

- +LPSαKG αKG

WBβ-ac n

Summary






succinate

PHD




succinate

PHD





2DG2DG

-LPS+LPS

WBsuccinyllysine

WBIL-1βWBβ-ac n

unstimulated LPS00

01

02

03

04

me

an

em

PA

I va

lue





bull GAPDH


bull PKM12


Key finding










GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION



GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION




LOW GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE


Low glucose

SNF2

YEAST MACROPHAGE

Low glucose 2DG

AMP kinase



GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE

METFORMIN

(mM)

IL-1β qPCR

(mM)



GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE



turning it off






via AMP kinase


Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK



Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK

HIF1





LUKE OrsquoNEILL


OrsquoNEILL LAB


Acknowledgment








Acknowledgment











CARKL



(b) Th17 cells








N=2

N=2

081211 bmdm SS

Ctl LPS (24 hr)0

1

2

3

4

Re

lative

miR

21

0 e

xp

ressio

n

181111 j774

Ctl LPS (24 hr)00

05

10

15

20

25

Re

lativ

e m

iR2

10

exp

ressio

n

310112 Bmdm ss 48h

Ctl LPS (48 hr)0

1

2

3

4

5

Re

lative

miR

21

0 e

xp

ressio

n

N=2

J774s

BMDM BMDM


LPS

HIF-1α IL-1β

Hypothesis


080413 210


05

10

15

20

0 MCSF2 FCS

2 MCSF2 FCS

10 MCSF10 FCS

Re

lativ

e m

iR-2

10

exp

ressio

n

27328140413 pooled 24h no SS n=8

Ctl LPS00

05

10

15

20

Re

lative

miR

-21

0 e

xp

ressio

n

N=8 N=3


LPS

HIF-1α IL-1β

Hypothesis


25030208160413 sdhd n=10

Ctl LPS00

05

10

15

Re

lative

sd

hd

exp

ressio

n

N=10


LPS

HIF-1α IL-1β

Hypothesis



anti-miR210

HIF-1α IL-1β

n=1

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B



PHD

KG Succinate

-



PHD

KG Succinate

KG

012

h

12h+αK

G24

h

24h+αK

G

00

05

10

Rela

tive E

nri

ch

men

t

0 LPS 0 LPS0

10000

20000

30000

rela

tiv

e IL

-1b

ex

pre

ss

ion

+ aKG


WBIL-1β

WBHIF-1α

- +LPSαKG αKG

WBβ-ac n

Summary






succinate

PHD




succinate

PHD





2DG2DG

-LPS+LPS

WBsuccinyllysine

WBIL-1βWBβ-ac n

unstimulated LPS00

01

02

03

04

me

an

em

PA

I va

lue





bull GAPDH


bull PKM12


Key finding










GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION



GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION




LOW GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE


Low glucose

SNF2

YEAST MACROPHAGE

Low glucose 2DG

AMP kinase



GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE

METFORMIN

(mM)

IL-1β qPCR

(mM)



GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE



turning it off






via AMP kinase


Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK



Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK

HIF1





LUKE OrsquoNEILL


OrsquoNEILL LAB


Acknowledgment








Acknowledgment











CARKL



(b) Th17 cells








N=2

N=2

081211 bmdm SS

Ctl LPS (24 hr)0

1

2

3

4

Re

lative

miR

21

0 e

xp

ressio

n

181111 j774

Ctl LPS (24 hr)00

05

10

15

20

25

Re

lativ

e m

iR2

10

exp

ressio

n

310112 Bmdm ss 48h

Ctl LPS (48 hr)0

1

2

3

4

5

Re

lative

miR

21

0 e

xp

ressio

n

N=2

J774s

BMDM BMDM


LPS

HIF-1α IL-1β

Hypothesis


080413 210


05

10

15

20

0 MCSF2 FCS

2 MCSF2 FCS

10 MCSF10 FCS

Re

lativ

e m

iR-2

10

exp

ressio

n

27328140413 pooled 24h no SS n=8

Ctl LPS00

05

10

15

20

Re

lative

miR

-21

0 e

xp

ressio

n

N=8 N=3


LPS

HIF-1α IL-1β

Hypothesis


25030208160413 sdhd n=10

Ctl LPS00

05

10

15

Re

lative

sd

hd

exp

ressio

n

N=10


LPS

HIF-1α IL-1β

Hypothesis



anti-miR210

HIF-1α IL-1β

n=1

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

012

h

12h+αK

G24

h

24h+αK

G

00

05

10

Rela

tive E

nri

ch

men

t

0 LPS 0 LPS0

10000

20000

30000

rela

tiv

e IL

-1b

ex

pre

ss

ion

+ aKG


WBIL-1β

WBHIF-1α

- +LPSαKG αKG

WBβ-ac n

Summary






succinate

PHD




succinate

PHD





2DG2DG

-LPS+LPS

WBsuccinyllysine

WBIL-1βWBβ-ac n

unstimulated LPS00

01

02

03

04

me

an

em

PA

I va

lue





bull GAPDH


bull PKM12


Key finding










GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION



GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION




LOW GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE


Low glucose

SNF2

YEAST MACROPHAGE

Low glucose 2DG

AMP kinase



GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE

METFORMIN

(mM)

IL-1β qPCR

(mM)



GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE



turning it off






via AMP kinase


Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK



Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK

HIF1





LUKE OrsquoNEILL


OrsquoNEILL LAB


Acknowledgment








Acknowledgment











CARKL



(b) Th17 cells








N=2

N=2

081211 bmdm SS

Ctl LPS (24 hr)0

1

2

3

4

Re

lative

miR

21

0 e

xp

ressio

n

181111 j774

Ctl LPS (24 hr)00

05

10

15

20

25

Re

lativ

e m

iR2

10

exp

ressio

n

310112 Bmdm ss 48h

Ctl LPS (48 hr)0

1

2

3

4

5

Re

lative

miR

21

0 e

xp

ressio

n

N=2

J774s

BMDM BMDM


LPS

HIF-1α IL-1β

Hypothesis


080413 210


05

10

15

20

0 MCSF2 FCS

2 MCSF2 FCS

10 MCSF10 FCS

Re

lativ

e m

iR-2

10

exp

ressio

n

27328140413 pooled 24h no SS n=8

Ctl LPS00

05

10

15

20

Re

lative

miR

-21

0 e

xp

ressio

n

N=8 N=3


LPS

HIF-1α IL-1β

Hypothesis


25030208160413 sdhd n=10

Ctl LPS00

05

10

15

Re

lative

sd

hd

exp

ressio

n

N=10


LPS

HIF-1α IL-1β

Hypothesis



anti-miR210

HIF-1α IL-1β

n=1

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B


succinate

PHD




succinate

PHD





2DG2DG

-LPS+LPS

WBsuccinyllysine

WBIL-1βWBβ-ac n

unstimulated LPS00

01

02

03

04

me

an

em

PA

I va

lue





bull GAPDH


bull PKM12


Key finding










GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION



GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION




LOW GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE


Low glucose

SNF2

YEAST MACROPHAGE

Low glucose 2DG

AMP kinase



GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE

METFORMIN

(mM)

IL-1β qPCR

(mM)



GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE



turning it off






via AMP kinase


Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK



Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK

HIF1





LUKE OrsquoNEILL


OrsquoNEILL LAB


Acknowledgment








Acknowledgment











CARKL



(b) Th17 cells








N=2

N=2

081211 bmdm SS

Ctl LPS (24 hr)0

1

2

3

4

Re

lative

miR

21

0 e

xp

ressio

n

181111 j774

Ctl LPS (24 hr)00

05

10

15

20

25

Re

lativ

e m

iR2

10

exp

ressio

n

310112 Bmdm ss 48h

Ctl LPS (48 hr)0

1

2

3

4

5

Re

lative

miR

21

0 e

xp

ressio

n

N=2

J774s

BMDM BMDM


LPS

HIF-1α IL-1β

Hypothesis


080413 210


05

10

15

20

0 MCSF2 FCS

2 MCSF2 FCS

10 MCSF10 FCS

Re

lativ

e m

iR-2

10

exp

ressio

n

27328140413 pooled 24h no SS n=8

Ctl LPS00

05

10

15

20

Re

lative

miR

-21

0 e

xp

ressio

n

N=8 N=3


LPS

HIF-1α IL-1β

Hypothesis


25030208160413 sdhd n=10

Ctl LPS00

05

10

15

Re

lative

sd

hd

exp

ressio

n

N=10


LPS

HIF-1α IL-1β

Hypothesis



anti-miR210

HIF-1α IL-1β

n=1

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B


2DG2DG

-LPS+LPS

WBsuccinyllysine

WBIL-1βWBβ-ac n

unstimulated LPS00

01

02

03

04

me

an

em

PA

I va

lue





bull GAPDH


bull PKM12


Key finding










GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION



GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION




LOW GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE


Low glucose

SNF2

YEAST MACROPHAGE

Low glucose 2DG

AMP kinase



GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE

METFORMIN

(mM)

IL-1β qPCR

(mM)



GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE



turning it off






via AMP kinase


Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK



Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK

HIF1





LUKE OrsquoNEILL


OrsquoNEILL LAB


Acknowledgment








Acknowledgment











CARKL



(b) Th17 cells








N=2

N=2

081211 bmdm SS

Ctl LPS (24 hr)0

1

2

3

4

Re

lative

miR

21

0 e

xp

ressio

n

181111 j774

Ctl LPS (24 hr)00

05

10

15

20

25

Re

lativ

e m

iR2

10

exp

ressio

n

310112 Bmdm ss 48h

Ctl LPS (48 hr)0

1

2

3

4

5

Re

lative

miR

21

0 e

xp

ressio

n

N=2

J774s

BMDM BMDM


LPS

HIF-1α IL-1β

Hypothesis


080413 210


05

10

15

20

0 MCSF2 FCS

2 MCSF2 FCS

10 MCSF10 FCS

Re

lativ

e m

iR-2

10

exp

ressio

n

27328140413 pooled 24h no SS n=8

Ctl LPS00

05

10

15

20

Re

lative

miR

-21

0 e

xp

ressio

n

N=8 N=3


LPS

HIF-1α IL-1β

Hypothesis


25030208160413 sdhd n=10

Ctl LPS00

05

10

15

Re

lative

sd

hd

exp

ressio

n

N=10


LPS

HIF-1α IL-1β

Hypothesis



anti-miR210

HIF-1α IL-1β

n=1

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B




bull GAPDH


bull PKM12


Key finding










GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION



GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION




LOW GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE


Low glucose

SNF2

YEAST MACROPHAGE

Low glucose 2DG

AMP kinase



GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE

METFORMIN

(mM)

IL-1β qPCR

(mM)



GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE



turning it off






via AMP kinase


Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK



Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK

HIF1





LUKE OrsquoNEILL


OrsquoNEILL LAB


Acknowledgment








Acknowledgment











CARKL



(b) Th17 cells








N=2

N=2

081211 bmdm SS

Ctl LPS (24 hr)0

1

2

3

4

Re

lative

miR

21

0 e

xp

ressio

n

181111 j774

Ctl LPS (24 hr)00

05

10

15

20

25

Re

lativ

e m

iR2

10

exp

ressio

n

310112 Bmdm ss 48h

Ctl LPS (48 hr)0

1

2

3

4

5

Re

lative

miR

21

0 e

xp

ressio

n

N=2

J774s

BMDM BMDM


LPS

HIF-1α IL-1β

Hypothesis


080413 210


05

10

15

20

0 MCSF2 FCS

2 MCSF2 FCS

10 MCSF10 FCS

Re

lativ

e m

iR-2

10

exp

ressio

n

27328140413 pooled 24h no SS n=8

Ctl LPS00

05

10

15

20

Re

lative

miR

-21

0 e

xp

ressio

n

N=8 N=3


LPS

HIF-1α IL-1β

Hypothesis


25030208160413 sdhd n=10

Ctl LPS00

05

10

15

Re

lative

sd

hd

exp

ressio

n

N=10


LPS

HIF-1α IL-1β

Hypothesis



anti-miR210

HIF-1α IL-1β

n=1

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

Key finding










GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION



GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION




LOW GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE


Low glucose

SNF2

YEAST MACROPHAGE

Low glucose 2DG

AMP kinase



GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE

METFORMIN

(mM)

IL-1β qPCR

(mM)



GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE



turning it off






via AMP kinase


Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK



Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK

HIF1





LUKE OrsquoNEILL


OrsquoNEILL LAB


Acknowledgment








Acknowledgment











CARKL



(b) Th17 cells








N=2

N=2

081211 bmdm SS

Ctl LPS (24 hr)0

1

2

3

4

Re

lative

miR

21

0 e

xp

ressio

n

181111 j774

Ctl LPS (24 hr)00

05

10

15

20

25

Re

lativ

e m

iR2

10

exp

ressio

n

310112 Bmdm ss 48h

Ctl LPS (48 hr)0

1

2

3

4

5

Re

lative

miR

21

0 e

xp

ressio

n

N=2

J774s

BMDM BMDM


LPS

HIF-1α IL-1β

Hypothesis


080413 210


05

10

15

20

0 MCSF2 FCS

2 MCSF2 FCS

10 MCSF10 FCS

Re

lativ

e m

iR-2

10

exp

ressio

n

27328140413 pooled 24h no SS n=8

Ctl LPS00

05

10

15

20

Re

lative

miR

-21

0 e

xp

ressio

n

N=8 N=3


LPS

HIF-1α IL-1β

Hypothesis


25030208160413 sdhd n=10

Ctl LPS00

05

10

15

Re

lative

sd

hd

exp

ressio

n

N=10


LPS

HIF-1α IL-1β

Hypothesis



anti-miR210

HIF-1α IL-1β

n=1

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B






GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION



GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION




LOW GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE


Low glucose

SNF2

YEAST MACROPHAGE

Low glucose 2DG

AMP kinase



GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE

METFORMIN

(mM)

IL-1β qPCR

(mM)



GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE



turning it off






via AMP kinase


Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK



Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK

HIF1





LUKE OrsquoNEILL


OrsquoNEILL LAB


Acknowledgment








Acknowledgment











CARKL



(b) Th17 cells








N=2

N=2

081211 bmdm SS

Ctl LPS (24 hr)0

1

2

3

4

Re

lative

miR

21

0 e

xp

ressio

n

181111 j774

Ctl LPS (24 hr)00

05

10

15

20

25

Re

lativ

e m

iR2

10

exp

ressio

n

310112 Bmdm ss 48h

Ctl LPS (48 hr)0

1

2

3

4

5

Re

lative

miR

21

0 e

xp

ressio

n

N=2

J774s

BMDM BMDM


LPS

HIF-1α IL-1β

Hypothesis


080413 210


05

10

15

20

0 MCSF2 FCS

2 MCSF2 FCS

10 MCSF10 FCS

Re

lativ

e m

iR-2

10

exp

ressio

n

27328140413 pooled 24h no SS n=8

Ctl LPS00

05

10

15

20

Re

lative

miR

-21

0 e

xp

ressio

n

N=8 N=3


LPS

HIF-1α IL-1β

Hypothesis


25030208160413 sdhd n=10

Ctl LPS00

05

10

15

Re

lative

sd

hd

exp

ressio

n

N=10


LPS

HIF-1α IL-1β

Hypothesis



anti-miR210

HIF-1α IL-1β

n=1

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

GLUCOSE BUILDS UP

MUSCLE LIVER

INSULIN

RESISTENCE

INFLAMMATION




LOW GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE


Low glucose

SNF2

YEAST MACROPHAGE

Low glucose 2DG

AMP kinase



GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE

METFORMIN

(mM)

IL-1β qPCR

(mM)



GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE



turning it off






via AMP kinase


Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK



Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK

HIF1





LUKE OrsquoNEILL


OrsquoNEILL LAB


Acknowledgment








Acknowledgment











CARKL



(b) Th17 cells








N=2

N=2

081211 bmdm SS

Ctl LPS (24 hr)0

1

2

3

4

Re

lative

miR

21

0 e

xp

ressio

n

181111 j774

Ctl LPS (24 hr)00

05

10

15

20

25

Re

lativ

e m

iR2

10

exp

ressio

n

310112 Bmdm ss 48h

Ctl LPS (48 hr)0

1

2

3

4

5

Re

lative

miR

21

0 e

xp

ressio

n

N=2

J774s

BMDM BMDM


LPS

HIF-1α IL-1β

Hypothesis


080413 210


05

10

15

20

0 MCSF2 FCS

2 MCSF2 FCS

10 MCSF10 FCS

Re

lativ

e m

iR-2

10

exp

ressio

n

27328140413 pooled 24h no SS n=8

Ctl LPS00

05

10

15

20

Re

lative

miR

-21

0 e

xp

ressio

n

N=8 N=3


LPS

HIF-1α IL-1β

Hypothesis


25030208160413 sdhd n=10

Ctl LPS00

05

10

15

Re

lative

sd

hd

exp

ressio

n

N=10


LPS

HIF-1α IL-1β

Hypothesis



anti-miR210

HIF-1α IL-1β

n=1

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B


Low glucose

SNF2

YEAST MACROPHAGE

Low glucose 2DG

AMP kinase



GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE

METFORMIN

(mM)

IL-1β qPCR

(mM)



GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE



turning it off






via AMP kinase


Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK



Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK

HIF1





LUKE OrsquoNEILL


OrsquoNEILL LAB


Acknowledgment








Acknowledgment











CARKL



(b) Th17 cells








N=2

N=2

081211 bmdm SS

Ctl LPS (24 hr)0

1

2

3

4

Re

lative

miR

21

0 e

xp

ressio

n

181111 j774

Ctl LPS (24 hr)00

05

10

15

20

25

Re

lativ

e m

iR2

10

exp

ressio

n

310112 Bmdm ss 48h

Ctl LPS (48 hr)0

1

2

3

4

5

Re

lative

miR

21

0 e

xp

ressio

n

N=2

J774s

BMDM BMDM


LPS

HIF-1α IL-1β

Hypothesis


080413 210


05

10

15

20

0 MCSF2 FCS

2 MCSF2 FCS

10 MCSF10 FCS

Re

lativ

e m

iR-2

10

exp

ressio

n

27328140413 pooled 24h no SS n=8

Ctl LPS00

05

10

15

20

Re

lative

miR

-21

0 e

xp

ressio

n

N=8 N=3


LPS

HIF-1α IL-1β

Hypothesis


25030208160413 sdhd n=10

Ctl LPS00

05

10

15

Re

lative

sd

hd

exp

ressio

n

N=10


LPS

HIF-1α IL-1β

Hypothesis



anti-miR210

HIF-1α IL-1β

n=1

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE

METFORMIN

(mM)

IL-1β qPCR

(mM)



GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE



turning it off






via AMP kinase


Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK



Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK

HIF1





LUKE OrsquoNEILL


OrsquoNEILL LAB


Acknowledgment








Acknowledgment











CARKL



(b) Th17 cells








N=2

N=2

081211 bmdm SS

Ctl LPS (24 hr)0

1

2

3

4

Re

lative

miR

21

0 e

xp

ressio

n

181111 j774

Ctl LPS (24 hr)00

05

10

15

20

25

Re

lativ

e m

iR2

10

exp

ressio

n

310112 Bmdm ss 48h

Ctl LPS (48 hr)0

1

2

3

4

5

Re

lative

miR

21

0 e

xp

ressio

n

N=2

J774s

BMDM BMDM


LPS

HIF-1α IL-1β

Hypothesis


080413 210


05

10

15

20

0 MCSF2 FCS

2 MCSF2 FCS

10 MCSF10 FCS

Re

lativ

e m

iR-2

10

exp

ressio

n

27328140413 pooled 24h no SS n=8

Ctl LPS00

05

10

15

20

Re

lative

miR

-21

0 e

xp

ressio

n

N=8 N=3


LPS

HIF-1α IL-1β

Hypothesis


25030208160413 sdhd n=10

Ctl LPS00

05

10

15

Re

lative

sd

hd

exp

ressio

n

N=10


LPS

HIF-1α IL-1β

Hypothesis



anti-miR210

HIF-1α IL-1β

n=1

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

(mM)

IL-1β qPCR

(mM)



GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE



turning it off






via AMP kinase


Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK



Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK

HIF1





LUKE OrsquoNEILL


OrsquoNEILL LAB


Acknowledgment








Acknowledgment











CARKL



(b) Th17 cells








N=2

N=2

081211 bmdm SS

Ctl LPS (24 hr)0

1

2

3

4

Re

lative

miR

21

0 e

xp

ressio

n

181111 j774

Ctl LPS (24 hr)00

05

10

15

20

25

Re

lativ

e m

iR2

10

exp

ressio

n

310112 Bmdm ss 48h

Ctl LPS (48 hr)0

1

2

3

4

5

Re

lative

miR

21

0 e

xp

ressio

n

N=2

J774s

BMDM BMDM


LPS

HIF-1α IL-1β

Hypothesis


080413 210


05

10

15

20

0 MCSF2 FCS

2 MCSF2 FCS

10 MCSF10 FCS

Re

lativ

e m

iR-2

10

exp

ressio

n

27328140413 pooled 24h no SS n=8

Ctl LPS00

05

10

15

20

Re

lative

miR

-21

0 e

xp

ressio

n

N=8 N=3


LPS

HIF-1α IL-1β

Hypothesis


25030208160413 sdhd n=10

Ctl LPS00

05

10

15

Re

lative

sd

hd

exp

ressio

n

N=10


LPS

HIF-1α IL-1β

Hypothesis



anti-miR210

HIF-1α IL-1β

n=1

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

GLUCOSE


ENERGY

TCA

ADP

ATP C02

H2O

mitochondria

STORED FATS

AMP KINASE



turning it off






via AMP kinase


Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK



Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK

HIF1





LUKE OrsquoNEILL


OrsquoNEILL LAB


Acknowledgment








Acknowledgment











CARKL



(b) Th17 cells








N=2

N=2

081211 bmdm SS

Ctl LPS (24 hr)0

1

2

3

4

Re

lative

miR

21

0 e

xp

ressio

n

181111 j774

Ctl LPS (24 hr)00

05

10

15

20

25

Re

lativ

e m

iR2

10

exp

ressio

n

310112 Bmdm ss 48h

Ctl LPS (48 hr)0

1

2

3

4

5

Re

lative

miR

21

0 e

xp

ressio

n

N=2

J774s

BMDM BMDM


LPS

HIF-1α IL-1β

Hypothesis


080413 210


05

10

15

20

0 MCSF2 FCS

2 MCSF2 FCS

10 MCSF10 FCS

Re

lativ

e m

iR-2

10

exp

ressio

n

27328140413 pooled 24h no SS n=8

Ctl LPS00

05

10

15

20

Re

lative

miR

-21

0 e

xp

ressio

n

N=8 N=3


LPS

HIF-1α IL-1β

Hypothesis


25030208160413 sdhd n=10

Ctl LPS00

05

10

15

Re

lative

sd

hd

exp

ressio

n

N=10


LPS

HIF-1α IL-1β

Hypothesis



anti-miR210

HIF-1α IL-1β

n=1

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B






via AMP kinase


Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK



Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK

HIF1





LUKE OrsquoNEILL


OrsquoNEILL LAB


Acknowledgment








Acknowledgment











CARKL



(b) Th17 cells








N=2

N=2

081211 bmdm SS

Ctl LPS (24 hr)0

1

2

3

4

Re

lative

miR

21

0 e

xp

ressio

n

181111 j774

Ctl LPS (24 hr)00

05

10

15

20

25

Re

lativ

e m

iR2

10

exp

ressio

n

310112 Bmdm ss 48h

Ctl LPS (48 hr)0

1

2

3

4

5

Re

lative

miR

21

0 e

xp

ressio

n

N=2

J774s

BMDM BMDM


LPS

HIF-1α IL-1β

Hypothesis


080413 210


05

10

15

20

0 MCSF2 FCS

2 MCSF2 FCS

10 MCSF10 FCS

Re

lativ

e m

iR-2

10

exp

ressio

n

27328140413 pooled 24h no SS n=8

Ctl LPS00

05

10

15

20

Re

lative

miR

-21

0 e

xp

ressio

n

N=8 N=3


LPS

HIF-1α IL-1β

Hypothesis


25030208160413 sdhd n=10

Ctl LPS00

05

10

15

Re

lative

sd

hd

exp

ressio

n

N=10


LPS

HIF-1α IL-1β

Hypothesis



anti-miR210

HIF-1α IL-1β

n=1

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B


Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK



Infection

TLRs Nlrp3

NFkB


INFLAMMATION

Tissue Injury

DAMPs

PAMPs Antigen

Autoantigen

Autoantibodies

T cell B cell

+

+

Abatacept Rituximab

Anti-cytokine

p38

Anti-JAK

HIF1





LUKE OrsquoNEILL


OrsquoNEILL LAB


Acknowledgment








Acknowledgment











CARKL



(b) Th17 cells








N=2

N=2

081211 bmdm SS

Ctl LPS (24 hr)0

1

2

3

4

Re

lative

miR

21

0 e

xp

ressio

n

181111 j774

Ctl LPS (24 hr)00

05

10

15

20

25

Re

lativ

e m

iR2

10

exp

ressio

n

310112 Bmdm ss 48h

Ctl LPS (48 hr)0

1

2

3

4

5

Re

lative

miR

21

0 e

xp

ressio

n

N=2

J774s

BMDM BMDM


LPS

HIF-1α IL-1β

Hypothesis


080413 210


05

10

15

20

0 MCSF2 FCS

2 MCSF2 FCS

10 MCSF10 FCS

Re

lativ

e m

iR-2

10

exp

ressio

n

27328140413 pooled 24h no SS n=8

Ctl LPS00

05

10

15

20

Re

lative

miR

-21

0 e

xp

ressio

n

N=8 N=3


LPS

HIF-1α IL-1β

Hypothesis


25030208160413 sdhd n=10

Ctl LPS00

05

10

15

Re

lative

sd

hd

exp

ressio

n

N=10


LPS

HIF-1α IL-1β

Hypothesis



anti-miR210

HIF-1α IL-1β

n=1

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

LUKE OrsquoNEILL


OrsquoNEILL LAB


Acknowledgment








Acknowledgment











CARKL



(b) Th17 cells








N=2

N=2

081211 bmdm SS

Ctl LPS (24 hr)0

1

2

3

4

Re

lative

miR

21

0 e

xp

ressio

n

181111 j774

Ctl LPS (24 hr)00

05

10

15

20

25

Re

lativ

e m

iR2

10

exp

ressio

n

310112 Bmdm ss 48h

Ctl LPS (48 hr)0

1

2

3

4

5

Re

lative

miR

21

0 e

xp

ressio

n

N=2

J774s

BMDM BMDM


LPS

HIF-1α IL-1β

Hypothesis


080413 210


05

10

15

20

0 MCSF2 FCS

2 MCSF2 FCS

10 MCSF10 FCS

Re

lativ

e m

iR-2

10

exp

ressio

n

27328140413 pooled 24h no SS n=8

Ctl LPS00

05

10

15

20

Re

lative

miR

-21

0 e

xp

ressio

n

N=8 N=3


LPS

HIF-1α IL-1β

Hypothesis


25030208160413 sdhd n=10

Ctl LPS00

05

10

15

Re

lative

sd

hd

exp

ressio

n

N=10


LPS

HIF-1α IL-1β

Hypothesis



anti-miR210

HIF-1α IL-1β

n=1

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

OrsquoNEILL LAB


Acknowledgment








Acknowledgment











CARKL



(b) Th17 cells








N=2

N=2

081211 bmdm SS

Ctl LPS (24 hr)0

1

2

3

4

Re

lative

miR

21

0 e

xp

ressio

n

181111 j774

Ctl LPS (24 hr)00

05

10

15

20

25

Re

lativ

e m

iR2

10

exp

ressio

n

310112 Bmdm ss 48h

Ctl LPS (48 hr)0

1

2

3

4

5

Re

lative

miR

21

0 e

xp

ressio

n

N=2

J774s

BMDM BMDM


LPS

HIF-1α IL-1β

Hypothesis


080413 210


05

10

15

20

0 MCSF2 FCS

2 MCSF2 FCS

10 MCSF10 FCS

Re

lativ

e m

iR-2

10

exp

ressio

n

27328140413 pooled 24h no SS n=8

Ctl LPS00

05

10

15

20

Re

lative

miR

-21

0 e

xp

ressio

n

N=8 N=3


LPS

HIF-1α IL-1β

Hypothesis


25030208160413 sdhd n=10

Ctl LPS00

05

10

15

Re

lative

sd

hd

exp

ressio

n

N=10


LPS

HIF-1α IL-1β

Hypothesis



anti-miR210

HIF-1α IL-1β

n=1

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

Acknowledgment











CARKL



(b) Th17 cells








N=2

N=2

081211 bmdm SS

Ctl LPS (24 hr)0

1

2

3

4

Re

lative

miR

21

0 e

xp

ressio

n

181111 j774

Ctl LPS (24 hr)00

05

10

15

20

25

Re

lativ

e m

iR2

10

exp

ressio

n

310112 Bmdm ss 48h

Ctl LPS (48 hr)0

1

2

3

4

5

Re

lative

miR

21

0 e

xp

ressio

n

N=2

J774s

BMDM BMDM


LPS

HIF-1α IL-1β

Hypothesis


080413 210


05

10

15

20

0 MCSF2 FCS

2 MCSF2 FCS

10 MCSF10 FCS

Re

lativ

e m

iR-2

10

exp

ressio

n

27328140413 pooled 24h no SS n=8

Ctl LPS00

05

10

15

20

Re

lative

miR

-21

0 e

xp

ressio

n

N=8 N=3


LPS

HIF-1α IL-1β

Hypothesis


25030208160413 sdhd n=10

Ctl LPS00

05

10

15

Re

lative

sd

hd

exp

ressio

n

N=10


LPS

HIF-1α IL-1β

Hypothesis



anti-miR210

HIF-1α IL-1β

n=1

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B




CARKL



(b) Th17 cells








N=2

N=2

081211 bmdm SS

Ctl LPS (24 hr)0

1

2

3

4

Re

lative

miR

21

0 e

xp

ressio

n

181111 j774

Ctl LPS (24 hr)00

05

10

15

20

25

Re

lativ

e m

iR2

10

exp

ressio

n

310112 Bmdm ss 48h

Ctl LPS (48 hr)0

1

2

3

4

5

Re

lative

miR

21

0 e

xp

ressio

n

N=2

J774s

BMDM BMDM


LPS

HIF-1α IL-1β

Hypothesis


080413 210


05

10

15

20

0 MCSF2 FCS

2 MCSF2 FCS

10 MCSF10 FCS

Re

lativ

e m

iR-2

10

exp

ressio

n

27328140413 pooled 24h no SS n=8

Ctl LPS00

05

10

15

20

Re

lative

miR

-21

0 e

xp

ressio

n

N=8 N=3


LPS

HIF-1α IL-1β

Hypothesis


25030208160413 sdhd n=10

Ctl LPS00

05

10

15

Re

lative

sd

hd

exp

ressio

n

N=10


LPS

HIF-1α IL-1β

Hypothesis



anti-miR210

HIF-1α IL-1β

n=1

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B








N=2

N=2

081211 bmdm SS

Ctl LPS (24 hr)0

1

2

3

4

Re

lative

miR

21

0 e

xp

ressio

n

181111 j774

Ctl LPS (24 hr)00

05

10

15

20

25

Re

lativ

e m

iR2

10

exp

ressio

n

310112 Bmdm ss 48h

Ctl LPS (48 hr)0

1

2

3

4

5

Re

lative

miR

21

0 e

xp

ressio

n

N=2

J774s

BMDM BMDM


LPS

HIF-1α IL-1β

Hypothesis


080413 210


05

10

15

20

0 MCSF2 FCS

2 MCSF2 FCS

10 MCSF10 FCS

Re

lativ

e m

iR-2

10

exp

ressio

n

27328140413 pooled 24h no SS n=8

Ctl LPS00

05

10

15

20

Re

lative

miR

-21

0 e

xp

ressio

n

N=8 N=3


LPS

HIF-1α IL-1β

Hypothesis


25030208160413 sdhd n=10

Ctl LPS00

05

10

15

Re

lative

sd

hd

exp

ressio

n

N=10


LPS

HIF-1α IL-1β

Hypothesis



anti-miR210

HIF-1α IL-1β

n=1

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B


25030208160413 sdhd n=10

Ctl LPS00

05

10

15

Re

lative

sd

hd

exp

ressio

n

N=10


LPS

HIF-1α IL-1β

Hypothesis



anti-miR210

HIF-1α IL-1β

n=1

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

00

05

10

15

20

25

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e S

DH

D e

xp

ressio

n

0

2000

4000

6000

8000

10000

anti-miR210 (10nM)

anti-miR210 (50nM)

NT NC

Unstimulated

LPS (1 ngml)

Re

lativ

e IL

-1b

exp

ressio

n

A

B

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