a20 in inflammatory signaling and pathology · multiple sclerosis, ibd, atherosclerosis, diabetes,...

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  • A20 in inflammatory signalingand 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-1inflammasome

    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-KOβ

    -cate

    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