A20 in inflammatory signaling

and pathology

Geert van Loo

VIB Inflammation Research Center

Gent University

FAPESP/EU-LIFE Scientific Workshop

Sao Paulo - Brasil

June 8, 2016

IBD Optineurin

NF-kB

InflammationTLR

TNFNecroptosis FADD

caspase

ER stress

Autophagy

Apoptosis

A20IL-1

inflammasome

Rheumatoid arthritis

psoriasis

Tissue homeostasis

Multiple sclerosis

MALT

RIP1

inflammasome

SLE

Autophagy

ER stress

A20NIKOtulin

NF-kB signalling pathways

ProinflammatoryUV Oxidants VirusesBacteria

Cytokines(TNF, IL-1)

NF-kB

INFLAMMATION(Rheumatoid arthritis, asthma,

multiple sclerosis, IBD, atherosclerosis,

diabetes, septic shock)

Cytokines Chemokines Enzymes Adhesion

proteinsAnti-apoptotic

proteinsIL-6,

GM-CSF,IL-8 iNOS ICAM-1 IAP-1/2

TNF, IL-1,

IL-2

RANTES COX VCAM-1 IEX-1L

A20MIP-1 E-selectinMCP-2

Cell cycle

C-myc

Cyclin D1

REGULATION !

stimulus

nucleus

NEMO

receptor

IKK-complex

a bP P

p65p50

IkBa

p65p50

IkBa

p65p50

IkBaP P

p65p50

P PUb

UbUb

Ub

Regulation of NF-kB signalling by A20

OTUA20

NF-kB responsive genes

stimulus

nucleus

NEMO

receptor

IKK-complex

a bP P

p65p50

IkBa

p65p50

IkBa

p65p50

IkBaP P

p65p50

P PUb

UbUb

Ub

Regulation of NF-kB/apoptosis signalling by A20

OTUA20

NF-kB responsive genes

8 8

apoptosis

FA

DD

FA

DD

The Zn-finger protein A20

LIVER

KID

NEY

CO

LON

JOIN

TSK

IN

Lee et al., Science, 2000

The Zn-finger protein A20

A20 and autoimmune disease

XE3

A20FL/FL gene

E4

LysM CRE

E5

A20 deletion in myeloid cells

WT A20myel-KOA20myel-KO WT

0

10

20

30

40

LysMA20 WT

Weig

ht (g

)

A20myel-KO WT

Matmati, Jacques et al., Nat. Genet., 2011

A20myel-KO mice develop spontaneous arthritis

In vitro

TNF secretion by resident pMΦ

WT A20myel-KO0

10

20

30

40

50

60 *

TN

F (

pg

/ml)

TNF secretion by thioglycollate-induced pMΦ

WT A20myel-KO0

50

100

150

200

250 *IL

-6 (

pg

/ml)

WT A20myel-KO0

500

1000

1500

2000

*

TN

F (

pg

/ml)

WT A20myel-KO

/ 15’ 30’ 45’ 60’ 120’ / 15’ 30’ 45’ 60’ 120’

IkBa

actin

Matmati, Jacques et al., Nat. Genet., 2011

A20myel-KO mice spontaneously produce inflammatory cytokines

A20 controls inflammatory cytokine production in myeloid cells

Arthritis in myeloid A20 knockout mice is MyD88 and IL-6 dependent

Matmati, Jacques et al., Nat. Genet., 2011

Arthritis in myeloid A20 knockout mice is IL1 dependent

Vande Walle et al., Nature, 2014

Collaboration with Mohamed Lamkanfi

A20myel-KO BMDMs are hypersensitive to activation of

the Nlrp3 inflammasome

Vande Walle et al., Nature, 2014

Step 1

(priming)

Step 2

A20 inhibits Nlrp3 inflammasome priming

A20 inhibits Nlrp3 inflammasome priming

Arthritis in A20myel-KO mice is Nlrp3 inflammasome dependent

Vande Walle et al., Nature, 2014

Spontaneous NLRP3 inflammasome activity in macrophages from

patients with TNFAIP3 mutations

Myeloid A20 deficiency promotes osteoclastogenesis

Splenocyte cultures 6 days treated with M-CSF+RANKL

WT A20myel-KO

0

10

20

30

40 *

Nu

mb

er

TR

AP

+ c

ell

s

WT A20myel-KO

0

10

20

30

40

50 *

Perc

en

t P

it f

orm

ati

on

Myeloid /osteoclast A20 deficiency

OTU

A20

TNFR1

TNF

p65p50

IkBaP

P

NEMO IKK-complex

a b

P P

p65p50

RIP1

Inflammatory responses

RANK

RANKL

p65p50

IkBaP

P

NEMO IKK-complex

a b

P P

p65p50

TRAF6

osteoclastogenesis

Osteoclast A20 deficiency induces severe osteoporosis

A20 FL/FL Ctsk/Cre +/+ A20 FL/FL Ctsk/Cre Tg/+

Micro-CT

Martens et al., unpublished

A20 inhibits RANK-induced NF-kB signaling

Transient transfection HEK-293T

ctrl RANK A20 RANK + A200

1000

2000

rela

tive l

ucif

era

se a

cti

vit

y (

*E3) *

Martens et al., unpublished

XE3

A20FL/FL gene

E4

Villin CRE

E5

A20 deletion in the intestinal epithelium

A20 in IECs is dispensible for normal intestinal development

A20IEC-KO mice are highly sensitive to DSS colitis and TNF toxicity

Vereecke et al., J. Exp. Med., 2010

0 1 2 3 4 5 6 7 8 9 10 11 12 13

0

1

2

3

4

WT

A20 IEC-KO

1.5% DSS H2O

time (days)

Clin

ica

l s

co

re

* * *

DSS colitis TNF lethality

A20IEC-KO mice are highly sensitive to DSS colitis and TNF toxicity

TUNEL

WT A20IEC-KO

A20 deficient IECs are highly sensitive to inflammatory cytokines

Vereecke et al., Nat. Commun., 2014

A20 protects IECs from TNF-induced apoptosis

A20 deletion in IECs and myeloid cells

XE3

A20FL/FL gene

E4

Villin CRE

E5

LysM CRE

A20IEC/myel-KO mice spontaneously develop intestinal inflammation

Vereecke et al., Nat. Commun., 2014

A20IEC/myel-KO mice have reduced Paneth and goblet cell numbers

Cryp

t-1 in

situ

WT A20IEC/MYEL-KO

Epithelial hyperproliferation in A20IEC/myel-KO mice

Severe colitis in aged A20IEC/myel-KO mice

Aged A20IEC/myel-KO mice develop colorectal tumors

WT A20IEC/Myel-KO

A20IEC/myel-KO

β-c

ate

nin

Sox-9

Olfm

4

A20IEC/myel-KO mice

X

A20FL/FL

CNS CRE

A20 in neuroinflammation

Hisahara et al. , 2003

oligodendrocyte

target cells vs. effector cells

neuron

target cells vs. effector cells

astrocytemicroglia

A20 in CNS inflammation and MS pathology

Mc Guire et al., unpublished

CNS (nestin-cre) neurons (Thy1.2-cre)

astrocytes (GFAP-cre) oligodendrocytes (MOGi-cre)

Microglia A20 deficiency strongly sensitizes to EAE

Voet, Mc Guire et al., unpublished

Microglia A20 deficiency strongly sensitizes to EAE

A20FL A20Cx3Cr1-KO

A20 controls Nlrp3 inflammasome activation in microglia

A20 controls Nlrp3 inflammasome activation in microglia

Voet, Mc Guire et al., unpublished

Acknowledgements

CollaboratorsMohamed Lamkanfi, Lieselotte Vande Walle, VIB and UGent, Belgium

Dirk Elewaut, Peggy Jacques, VIB and UGhent, Belgium

Rudi Beyaert, VIB and Ugent, Belgium

Hans Clevers, Johan Van Es, Hubrecht Institute, Utrecht, The Netherlands

Marco Prinz, Peter Wieghofer, Freiburg, Germany

Conor Mc Guire (ex)

Mourad Matmati (ex)

Jonathan Maelfait (ex)

Lars

Vereecke

Esther

Hoste

Leen

Catrysse

Karolina

Slowicke

Sofie

Voet

Arne

Martens

Mozes

Sze

Hanna

Vikkula