8/5/2012

1

Mendelian Susceptibility to Fungal Infections

Bodo GrimbacherBodo GrimbacherCentre of Chronic Immune Deficiency (CCI)

University Hospital Freiburg

bodo.grimbacher@uniklinik-freiburg.de

Immunity to Immunity to C. albicansC. albicans

Nat Rev Microbiology 2008

C. albicansC. albicans recognitionrecognition

Pattern recognition receptors for C. albicans:- Toll-like receptors: TLR2, TLR6, TLR4

- C-type lectin receptors: Dectin-1, Dectin-2, Mannose receptor, DC-SIGN…

Role of Th17 cell cytokines in Candida controlRole of Th17 cell cytokines in Candida control

Skin & mucosal immunity to Skin & mucosal immunity to C. albicansC. albicans

PMN recruitment and activationAntimicrobial peptide secretion

Pathogen clearance

Biblia Latina. Northern Italy 1273

8/5/2012

2

AD-HIES and STAT3

• In 2007 we described mutations in the Signal Transducer and Activator of Transcription 3 (STAT3) to cause AD-HIES

• STAT3 was also called an “acute phase response factor”

• To date more than 250 patients carrying mutations in STAT3 have been published

• The mutations found cluster mainly in the DNA-binding and the SH2 domain of STAT3

• All mutations found are heterozygous

AD-HIES and STAT3

R382L

R382W

R382Q

R384L

R384S

T389I H437Y

V463del

S465AV637L

V637M

V638G

K642E

Q644P E690_P699del

P639A

205 of 256 HIES patients have heterozygous mutations in STAT3

N terminaldomain

Coiled-coildomain

DNA-binding domain SH2 domainTransactivation

domain Linkerdomain

H332Y

R335W

K340N/T341del

V343L

R382L

T412S

R423Q

V432M

H437P Q469H

N472D

D371_G380del

S611N

T620A

F621V

T622I

V637L

N647D

E652K

Y657C

I665N K709E

V713L

S636FQ644del

F384L

R382L

R382W

R382Q

T389I N466S

N466T

Q469HT622I

S636Y

V637M

P639S

T663S

S668F

K591E

R423Q

N466KV637A

Y657C

D371_G380del

H58Y

C328_P330dup

G342D

V463delT714A

T708S

c.2144+1G>Ap.?

F710C

JAK-STAT Signaling

JAK2Tyk2JAK2Tyk2

P

PP

P

JAK2Tyk2P

P

P

P

TAT PP

TAT

STA

T

ST

STA

P

STA

T

P

STA

T

P

STA

T

P

STA

T

Gen

STA

T

P

STA

T

STA

T

STA

T

25% 25%50%

xxxx

Naïve

Stat1Stat4T-bet

Stat6GATA3

Th1IFN-

Th2IL-4IL-5IL-13

IFN-

IL-4

IL-12IL-4

Protection against intracellular pathogens(eg. viruses, bacteria)

Protection against extracellular pathogens(eg. parasites, bacteria)

STAT3 and Th17 cells

T cell

Stat3RORt

FoxP3

Th17IL-17A/FIL-22

Treg

TGF-

+ IL6

IL-21TGF-

IL-23

Protection against extracellular pathogens(eg. fungi, bacteria)

E. Deenick & S. Tangye, Immunol. and Cell Biol, 2007

STAT3STAT3+/+/-- patients display low proportionspatients display low proportionsof circulating ILof circulating IL--1717--producing T cellsproducing T cells

Role of Th17 cell cytokines in Candida controlRole of Th17 cell cytokines in Candida control

Skin & mucosal immunity to Skin & mucosal immunity to C. albicansC. albicans

PMN recruitment and activationAntimicrobial peptide secretion

Pathogen clearance

8/5/2012

3

B Clinical Manifestations

A Family 1

P1

Patient 1 Patient 2

P3P2

Patient 1 Patient 3

Figure1. Pedigreeof Family1withAutosomal Dominant ChronicMucocu-taneousCandidiasis(CMC) andClinical SignsinAffectedFamilyMembers.

Panel A shows the pedigree of a Dutch family in which three members of two generations have clinical symptoms characteristic of CMC (black sym-bols). Squares indicate male family members, and circles female family members. The affected family members have severe dermatophytosis and candidiasis of the feet and severe oropharyngeal chronic candidiasis (Panel B). Patients 1 and 2 also have other autoimmune disorders, although the manifestations of CMC in Patient 3 are limited to the feet. Van de Veerdonk et al, NEJM, July 2011

Van de Veerdonk et al, NEJM, July 2011

Kindred A Kindred B Kindred C Kindred D Kindred E

Kindred I Kindred J

Kindred F

Kindred K Kindred M

R274Q/WT

R274Q/WT

K286I/WT

K286I/WT

R274Q/WT

R274Q/WT

M202V/WT

M202V/WT

C174R/WT

C174R/WT

C174R/WT

C174R/WT

C174R/WT C174R/WT

WT/WT

R274W/WT

R274W/WT

R274W/WTWT/WT

Kindred H

1 2

1 2

1 2 3 4 5

1 1

1 2

1 2

2 3

1 2

1 2

1 2

1 2

1 2 3

1 2

1 2 3 4

1 2 3 4 5

1 2 1 2 1 2

1 2

1 2

1 2 3

Kindred L

1 2 1 2

Kindred G

1 2

WT/WT

WT/WTWT/WT

1 2

WT/WT

1 2

WT/WT

WT/WT

WT/WT

I

II

III

IV

I

B.

Kindred O Kindred P Kindred Q

D165H/WT

M202I/WT A267V/WT A267V/WT R274W/WT

T288A/WT

T288A/WT

T288A/WT WT/WT

Y170N/WT

WT/WT

WT/WT WT/WTR274Q/WT

WT/WT

R274Q/WT

WT/WT

Kindred R

M202V/WT

Kindred S

M202I/WT

M202I/WT

Kindred N

WT/WT

WT/WT

1 2 3 1 2 31 21 2 3 4 5

WT/WT R274Q/WT

R274Q/WT WT/WT

1 2 1 2

1 21 1 1 1 2 3

1 2 1 2 1 2 1 2 1 21 2

D165G/WT

1 2

Kindred T

Q271P/WT

1 2

WT/WT

WT/WT

WT/WT

WT/WT

WT/WT WT/WT

II

I

II

WT/WT WT/WT

1 2 3

R274W/WT

WT/WT

1 2

Liu et al., J.Exp.Med. 2011

Mock WT

IFN

-

R274Q L706S

IFN

-

IFN

-

IFN

-

IL-2

7

IL-2

7

IL-2

7

IL-2

7

IFN

-

IFN

-

IFN

-

IFN

-

GA

S (

fold

indu

ctio

n)

A. Mock WT R274Q L706S

IFN

-

IL-2

7

IFN

-

NS

IFN

-

IL-2

7

IFN

-

NS

IFN

-

IL-2

7

IFN

-

NS

IFN

-

IL-2

7

IFN

-

NS

p-STAT3

p-STAT1

-tubulin

STAT1

STAT3

90 KDa

90 KDa

90 KDa

90 KDa

55 KDa

B.

A (

fold

indu

ctio

n)

C.

NA

(fo

ld in

duct

ion)

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E; 'F- G'

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R274Q is a gain-of-function mutation R274Q acts in a dominant fashion

Mock WT R274Q L706S

Time (h)

0 2 8 0 2 8 0 2 8 0 2 8

CX

CL

9 m

RN

A

Mock WT R274Q L706S

Time (h)

0 2 8 0 2 8 0 2 8 0 2 8 CX

CL

10 m

RN

Staurosporine IFN-

-

- + + + +

- 15 30 60 -

WT R274Q F77A Mock

p-STAT1

STAT1

-tubulin

-

- + + + +

- 15 30 60 -

- + + + +

- 15 30 60

+

90 KDa

90 KDa

55 KDa

E. IFN-

p-STAT1

STAT1

-tubulin

-

-

+

-

+

+

-

-

+

-

+

+

-

-

+

-

+

+

-

-

+

-

+

+

+

+

Mock WT R274Q D165G F77A

Pervanadate

90 KDa

90 KDa

55 KDa

F.

Liu et al., J.Exp.Med. 2011

45

34

45

34

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8/5/2012

4

Liu et al., J.Exp.Med. 2011

Published mutations in STAT1

Liu et al., J.Exp.Med. 2011

Brown - recessive, associated with complete STAT1 deficiency and intracellular bacterial and viral disease

Blue - recessive and associated with partial STAT1 deficiencyand intracellular bacterial and/or viral disease

Green - dominant and associated with partial STAT1 deficiencyand Mendelian susceptibility to mycobacterial disease

Red - gain-of-function (phosphorylation) mutations, the phenotype is CMC

STAT1 = Th17 cells

STAT3

STAT3 = Th1 cells

STAT1

STAT1 mutations detected in Freiburg:8/12 unrelated families and 3/10 sporadic patients

I

II

III

I

I

II

III

I I

II

III

IV

I

II

III

I

II

I

II

III

The fungal host defence

8/5/2012

5

CME-Summary Please identify the one mistake:Susceptibility to fungal infections may be caused by:

- homozygous mutations in the IL17-receptor

- autoantibodies against IL17 and/or IL22

Casanova, Science 2011

Meager, JExpMed 2010Casanova, JExpMed 2010

gain of function- dominant negative mutations in STAT1

- dominant negative mutations in STAT3

- homozygous mutations in CARD9

- mutations in Dectin-1 confer risk

- dominant-negative mutations in IL17

Netea NEJM, 2011Casanova, JExpMed 2011

Minegishi, Nature 2007Grimbacher, NEJM 2007

Grimbacher, NEJM 2010

Netea, NEJM 2010

Casanova, Science 2011

gain-of-function-------------------------------------------------------------------------

Acknowledgements

Freiburg, CCIMark DepnerJan Raabe

Royal Free Hospital UCLSaad PatanErik Glocker

L b t i M lé l i

Centre d'Infectiologie Necker PasteurHôpital Necker Enfants malades Fanny Lanternier Luyan LiuCapucine PiccardAnne Puel

Laboratoire MoléculaireUniversité Montpellier Gerard Lefranc

Faculté de Médecine Tlemcen AlgérieOmar Boudghene -Stambouli

Rockefeller UniversitySophie Cypowij Jean-Laurent Casanova

Funding:• EU Marie-Curie Grant MEXT-CT-2006-042316

All patients

and their families

A new gene causing early-onset CVID

identified by Gabriela Lopez-Herrera

< 2 years< 13 years

IgG lowIgA lowIgM low

ITPArthirtis

Age of 

onset

Immuno

globulin

s levels

unit

0 1 2 3 4 5

Age of onset

Immunoglobulin's levels

Clinical phenotype in 5 patients with autosomal-recessive CVID

# Patients

ArthirtisIFA

MyasteniaHypotiroydism

Reccurent pneumoniaBrohchiectasis

AsthmaUrticaria

Alelrgic dermatitis

WartsMolluscum contagiosum

Histoplasma

DiarrheaGrowth retardationCerebral granuloma

Autoim

mu

y

Respir

atory 

tract

Atopic 

sympto

ms

Intracell

ular 

infection

sOther

s

Autoimmunity

Respiratory tract

Atopicsymptoms

Intracellular Infections

Others

Patient # Patient 1 Patient 2 Patient 3 Patient 4 Patient 5 Mutation I2657S I2657S R1683X E59X I2 del

Lymphocyte counts: Age at measurement 5y 14y 29y 19y 19y

CD3 (cells/µl) 900 (900-4.500) 2.073

(800-3.500) 2.350 (700-2.100) ↑ 5.716

(700-2.100) ↑ 355(700-2.100) ↓

CD4 (cells/µl) 685 (500-2.400) 767

(400-2.100) 1.290 (300-1.400) 1.061

(300-1.400) 262(300-1.400)

↓

CD4 CD45RA N.D 179 (230-770)a

201 (27-833)b

72 (27-833)b N.D

CD4 CD45RO 553 887 588

Laboratory values in patients with homozygous mutations in LRBA

CD4 CD45RO N.D 553(240-700)a

887 (167-670)b ↑ 588

(167-670)b N.D

CD8 (cells/µl) 380 (300-1.600) 1.200

(200-1.200) 1032 (200-900) ↑ 4.707

(200-900) ↑ 165 (200-900)

↓

CD8 CD45RA N.D 1.003 (240-710)a ↑ 941

(19-508)b ↑ 1.991

(19-508)b ↑ N.D

CD8 CD45RO N.D 473 (10-142)b

↑ 627 (15-275)b

↑ 374 (15-275)b ↑ N.D

Age at measurement 5y 10y 16y 16y 19y

NK cells (cells/µl) 260 (100-1.000) 190

(70-1.200) 35 (70-1.200) ↓ N.D. N.D.

CD19 (cells/µl) 280 (200-2.100) 300

(200-600) 219 (200-600) 69

(200-600) ↓ 121(100-500)

Switched memory B cells (CD19+ CD27+ IgM , % of total B-cells)

0 (3.9-16.2) ↓ 1

(3.85-16.5) ↓ 0.8 (4-22.8) ↓ 0.89

(4-22.8) ↓ N.D.

Family A

I.1 I.2

Linkage analysis in one AR-CVID family

P1 P2

II.1 II.2 II.3 II.4 II.5 II.6

Chromosome 4q, 5 and 12LOD=2.08

8/5/2012

6

Family A

P1 P2

I.1 I.2

II.1 II.2 II.3 II.4 II.5 II.6

Missense mutation in Lipopolysaccharide responsive beige-like anchor protein (LRBA)

Missense MutationATT AGT I2657S

Highly conserved residue, mutation not found in 128 healthy individuals of arabic heritage

ROYAL FREE HOSPITAL & UNIVERSITY COLLEGE LONDON

Lipopolysaccharide responsive beige-like anchor protein (LRBA)

Family A

P1 P2

I.1 I.2

II.1 II.2 II.3 II.4 II.5 II.6

Family B

P3

I.1 I.2

II.1 II.2 II.3

P4

I.1 I.2

II.1 II.2

Family C

I.1 I.2

II.1 II.2

P5

Family D

Missense MutationATT AGT I2657S

Nonsense MutationCGA TGAR1683X

Nonsense MutationGAA TAAE59X

111114bp Deletionincl. E1 + E2

T T T T T TC A

200 alleles from Caucasian healthy donors

16 consanguineous families analyzed

No protein expression in LRBA-deficient patients

ROYAL FREE HOSPITAL & UNIVERSITY COLLEGE LONDON

LYST is a beige-like protein mutated in Chediak-Higashi syndrome (CHS)

gDNA: 752,397 bp (58 exons); mRNA: 9,899 bp; Protein: 2863 aa (319KDa)

The LRBA gene

Armadillo/beta-catenin-like repeats Hatzfeld M. The armadillo family of structural proteins. 1999

T b i

WDL

WD40Xu, C. Structure and function of WD40 domain proteins. 2002

WD40Xu, C. Structure and function of WD40 domain proteins. 2002

N- -CWD40BEACH

DHC-N1Based on sequence homology

PKA RII binding sitesDe Lozanne, A. The role of BEACH proteins in Dictyostelium. 2003

Transmembrane region

Beige and Chediak-HigashiWu WI. Structure-function analysis of the BEACH protein LvsA.2004Ward DM, Chediak-Higashi syndrome: a clinical and molecular view of a rare lysosomal storage disorder. 2002

WBW DomainARMDHC-N1 TM

Y site

SH3 site

SH2 site

mLRBA has diverse isoforms and orthologous

LBAα

Human LRBA shares 85% homology with murine LRBA

Wang JW et al, 2001

8/5/2012

7

Wang JW et al, 2001

pre-B Macrophages

Murine LRBA

LRBA

GAPDH

PBMCs from healthy individuals

Human LRBA

LRBA-deficiency shows reduced T and B cell proliferation

p=0.0004*** p=0.0039** p=0.0058**

Healthy LRBA-deficiency

Unstimulated

LRBA deficient cells PMA+Ionomycin

Healthy control PMA+Ionomycin

Unstimulated PHA αCD3 +αCD28 PWM

R1683X I2657S

CFSE

CD3+ gate

Deficient T cell activation

Unstimulated

R1683X I2657S

CD3+ cells

LRBA deficient cells PMA+Ionomycin

Healthy control PMA+Ionomycin

CD69

ICOS

LRBA-deficiency leads to an increased susceptibility to apoptosis in B and T cells

HeLa cellsp=0.0172*

p=0.0057**

% o

f An

ne

xin

V p

osi

tive

ce

lls

50

40

30

20

10

0

EBV cell lines

Wang JW et al, 2004.

Control EBVR1683X (P3)I2657S (P2)I2657S (P1)

p=0.0038**p=0.0198*

p=0.0027**

D0 D5 D7 D9% o

f An

ne

xin

V p

osi

tive

ce

lls

Day of cell blast culture (CD3+ cells)

60

40

20

0

PBMCsPHA+IL2

BAD phosphorylation is reduced in starved EBV cells and is restored after LRBA reconstitution

BADBAD

Phospho-S112

Cytoplasm, cell survival

BADBAD

S112

cAMP-Protein Kinase

Mitochondria, apoptosis

Starved EBV cellsPMA (-) (+) (+)

Mock

R1683X

Mock LRBA

pS112 BAD

BAD

GAPDH

PMA:(-) (-) (+) (+) (+) (+)

pS112-BAD

BAD

8/5/2012

8

Possible role of BEACH proteins in autophagy

GFP-mLRBA-BEACH-WD Lysosomes Merge

BEACH proteins have been suggested to participate inAutophagy (Kaplan et al, 2008)

Lysosomal destabilization contributes to apoptosis ingerminal centre formation (van Nierop et al, 2006)

Autophagy: Is a controlled cellular degradation pathway important for recycling of organelles and proteins.It is important in adaptation to starvation, in cell survival, immunity, development and cancer.

Wang JW et al, 2001;

LysosomeIsolationmembrane

LC3

Lysosomestained by“LysoID”

Autophagosome

Autolysosome

StarvationThis is the colocalisation of LysoID and LC3 (BDS)

Simon, K., et al, under revisionImageStream

Reduced autophagy and organelle accumulation in LRBA-deficiency

+

Healthy R1683X

CD19+ cells

HD E59X

CD19+ cells

% C

D19

+LC

3+Ly

soID

+B

DS

hi

Control Control+I Starved+I

Data from Katja Simon

E59XE59X

G = Golgi apparatus, M = Mitochondria, * = autophagosomes,(arrow)= centrioles

Data from Lennart Hammarstroem

We keep screening for LRBA deficiency Summary

LRBA is a new genetic defect associated with earlyonset CVID

LRBA deficiency is characterized by:

Lo B and T cell acti ationLow B and T cell activation

Increased susceptibility to apoptosis

Reduced BAD phosphorylation

Reduced proliferation

Reduced autophagy

Acknowledgements

University College Londonabriela Lopez-Herreralaudia M Trujillo-Vargaseer Herholz

NIH-Beteshda, USAlejandro Schäfferichael Edward Gertz

Karolinska institute, Sweden

•Oxford UniversityKatja SimonKanchan Phadwal

•Rappaport School of Medicine, IsraelAmos EtzioniAdi MoryIzhak Srugo

•Tehran University of Medical Sciences

iang-Pan Hammarströmennart Hammarströmhonghai Liu

reiburg University, Germanyietmar Pfeiferermann Eibellrich Salzer

University of Brescia, Italylessandro Plebaniiacomo Tampellaassilios Lougaris

Ashgar AghmohammadiNima Rezai

•University of Liége, BelgiumMichel MoutschenVinciane DidebergPierre Philippet

EURO-PADnet

Bodo Grimbacher: bodo.grimbacher@uniklinik-freiburg.de