Genetic Factors Governing

Susceptibilities to Severe Infections

GSK-Chair of Infectious Diseases

Pr Jean-Paul MIRA

Polymorphisms of Toll Like Receptors

and Variability of Innate Immunity

Institute of Cellular PathologyFebruary 16, 2005

Pr. Jean-Paul MIRAMedical Medical ICU & ICU & DeptDept. of . of Cell BiologyCell Biology

Cochin Cochin UniversityUniversity HospitalHospital & Cochin & Cochin InstituteInstitute, Paris, F , Paris, F

Septic Shock EpidemiologySeptic Shock Epidemiology

Annane D. Am J Respir Crit Care Med 2003;168:165–172

Variability of Severe Sepsis Phenotype

MIEUX COMPRENDREMIEUX COMPRENDRE

POUR MIEUX GUERIRPOUR MIEUX GUERIR

«« Today we are learning the language in which God created life. Today we are learning the language in which God created life. It will revolutionize the diagnosis, prevention and treatment ofIt will revolutionize the diagnosis, prevention and treatment of most, most, if not all human diseases.if not all human diseases. »» William J. Clinton, June 26, 2000

« If it were not for the great variability among individuals medicine might as well be a science and not an art »Sir William Osler, 1892

19531953 20012001--20032003

AS HUMANS, WE ALL SHARE AS HUMANS, WE ALL SHARE THE SAME BASIC GENESTHE SAME BASIC GENES

BUT…BUT…

Very small differences in genotype make beautiful differences in phenotype…

Small differences in genotype make big differences to phenotype

Evidences Evidences for a for a genetic genetic component to component to sepsissepsis

Animal Studies

- Susceptibility/resistance to certain infection in mice

Mice Susceptibility Mice Susceptibility to Infection to Infection with with Group A Group A StreptococciStreptococci

103 cfu Strepto Subcutaneous

d2Goldman O. J Infect Dis 2003;187:854-61.

Evidences Evidences for a for a genetic genetic component to component to sepsissepsis

Animal Studies

- Susceptibility/resistance to certain infection in mice

Human Studies- Clinical Evidences

- Ethnic Differences

- Twin Studies

- Adoptee Studies

GeneticGenetic andand environmentalenvironmental influences influences on on premature deathpremature death in in adult adopteesadult adoptees

Cause of Death(Parent Dead before the age of 50)

Relative risk for the adopteeto die from the same cause

All causesBiologicAdoptive

1.710.71

BiologicAdoptive

Infection5.80.73

VascularBiologicAdoptive

4.53.1

Sørensen TI, et al. NEJM 1988; 318:727-32.

STUDY NATURE AND OBJECTIVESSTUDY NATURE AND OBJECTIVESSTUDY METHODS

Population based associations

Genetic association with clinical outcomes

Case control cohort comparisons

Familial studies

In vivo physiologic responses

In vitro responsiveness

In vitro transfection

Knockout models

Large, observational(characterizing genetic risk and clinical usefulness)

Small, highly manipulated(enhancing understanding of the biology of disease mechanisms)

Genetic Polymorphisms and Severe Sepsis

Meningococcemia; Severe sepsisMeningococcemia; Severe sepsis

PAI-1FactorV Leiden

Meningococcemia;Septic Shock; MalariaSevere SepsisSevere Sepsis, MeningococcemiaSevere sepsis

TNF locusIL-18IL-10IL-6

Meningococcemia; PneumococcemiaFCγRII Receptor

Meningococcemia, PneumococcemiaSevere sepsis

Mannose Binding Lectin

Susceptibility and/or OutcomeGene

Gram negative/positive Septic Shock

Legionnaire’s Disease

Septic Shock

Toll-Like Receptor 4/2

Toll-Like Receptor 5

CD14

IL-1 locusIL-4Caspase12

Severe SepsisViral PneumoniaSeptic shock

Pathogen Detection

HumanHuman TollToll--like Receptorslike Receptors

•• 10 10 Receptors Receptors (TLR1 (TLR1 –– TLR10)TLR10)

•• Specific pathogen detection Specific pathogen detection

•• Mediate the innate Mediate the innate immune immune responseresponse

•• Induce the inflammatory reaction Induce the inflammatory reaction (NF(NF--κκBB))

•• InduceInduce thethe adaptative immune adaptative immune responseresponse

•• ResolutionResolution of of the the inflammation (inflammation (apoptosisapoptosis))1 2 3 4 5 6 7 8 9

XX 3 394 4 66 110

chromosome

Polymorphisms of the CD14 receptor

CD14 receptor and TLRs

% P

atie

nts

605040302010

ControlSeptic Shock

-159CC -159CT -159TT

Genotypes

P<0.05

CD14/159T CD14/159T and Susceptibilityand Susceptibility to to Septic ShockSeptic Shock

Le Van TD et al. J Immunol 2001;167:5838

CD14/159T increases sCD14 and mCD14 levels

Gibot S et al. Crit Care Med 2002; 30:969-73

CD14/159T CD14/159T and Mortalityand Mortality to to Septic ShockSeptic Shock

Characteristics C/CN=19

C/TN=43

T/TN=28

p

Age (mean±SD) 53±15 59±13 59±17 .18

SAPSII (mean±SD) 53±18 56±21 60±19 .21

OSF (mean±SD) 3±1.2 2.8±0.9 3.1±1 .42

Gibot S et al. Crit Care Med 2002; 30:969-73

CD14/159T CD14/159T and Mortalityand Mortality to to Septic ShockSeptic Shock

Characteristics C/CN=19

C/TN=43

T/TN=28

p

Age (mean±SD) 53±15 59±13 59±17 .18

SAPSII (mean±SD) 53±18 56±21 60±19 .21

OSF (mean±SD) 3±1.2 2.8±0.9 3.1±1 .42

Mortality (%) 26.3 58.1 71.4 <.0001

CD14/159TT RR= 5.1; 95%CI [3.2-7,9]

Gibot S et al. Crit Care Med 2002; 30:969-73

Immunomodulatory genes

NF-κB Signalisation

Adaptative Response

Cellular Immunity

LipoproteinsGram+ BacteriaFungi

LPSGram- Bacteria

TLR4TLR3 TLR5 TLRx

CpG DNA?

Monocyte orDendritic cell

Apoptosis SepticShock

Tissue Injury

Bacteria Lysis

Double-stranded RNA

TLR2+/- TLR1/6

Flagellin

TLR9

Impaired Impaired LPS LPS responsiveness responsiveness in TLR4in TLR4--//-- macrophagesmacrophages

Hoshino Hoshino et al., J. Immunol. 1999; 162:3749et al., J. Immunol. 1999; 162:3749

NFNF--kB ActivitykB Activity in in TransfectedTransfected THPTHP--1 1 cellscells

Wild type Thr399Ile Asp299Gly

NFκ

B a

ctiv

ity (L

.U.x

103 /m

g)

100

80

60

40

20

(-) LPS

(+) LPS

Arbour NC. Nature Genetics 2000;25:187

TLR4 mutation TLR4 mutation andand LPS LPS responsivenessresponsiveness

40

20

IL-1α

(pg/

ml)

WT/WT WT/Asp299Gly&

Thr399Ile

Arbour NC. Nature Genetics 2000;25:187

TLR4 Mutant ExpressionTLR4 Mutant Expression

Human Airway Epithelia

TLR4 WT TLR4 WT/299 Gly

Arbour NC. Nature Genetics 2000;25:187

TLR4 TLR4 Polymorphisms and Septic ShockPolymorphisms and Septic Shock

ControlControl

% T

LR4

% T

LR4

mut

ated

m

utat

ed p

atie

nts

patie

nts 30

25

20

15

10

5

Gram Gram negativenegativeSeptic shockSeptic shock

Lorenz , Arch. Intern. Med. 2002 162:1028

TLR4 Variants TLR4 Variants and Predispositionand Predisposition to Gram to Gram Negative SepsisNegative Sepsis

Agnese DM, JID 2002; 186:1522

WithoutWithout TLR4TLR4MutationMutation

% G

ram

%

Gra

m n

egat

ive

nega

tive

infe

ctio

nin

fect

ion WithWith TLR4 TLR4 MutationMutation

100

80

60

40

20

P = 0.004

Immunomodulatory genes

NF-κB Signalisation

Adaptative Response

Cellular Immunity

LipoproteinsGram+ BacteriaFungi

LipopolysaccharideGram- Bacteria

TLR4TLR3 TLR5 TLRx

CpG DNA?

Monocyte orDendritic cell

Apoptosis SepticShock

Tissue Injury

Bacteria Lysis

Double-stranded RNA

TLR2+/- TLR1/6

Flagellin

TLR9

TLR2TLR2--Knockout Mice Knockout Mice

TLR2 and Streptococcus pneumoniae meningitis

WT

TLR2 -/-

Echchannaoui H et al. JID 2002;186:798

TLR2TLR2--deficient mice and Gram positive infectionsdeficient mice and Gram positive infections

IV challenge of lived Staphylococcus aureus

Takeuchi et al., J. Immunol. 1999; 165:5392

TLR2TLR2--deficient mice and Candida Infectionsdeficient mice and Candida Infections

Candida albicans IP 50 x 106 Candida albicans IV 106

Villamon E., Microbes Infect 2004; 6:1-7

TLR2 Polymorphisms in HumansTLR2 Polymorphisms in Humans

Tlr2 location: Chromosome 4q32

http://www.ncbi.nlm.nih.gov/entrez/query.fcqihttp://innateimmunity.net

88 SNPs

• 5’UTR: 26 SNPs

• 3’UTR: 17 SNPs

• Intron: 29 SNPs

• Exon: 16 SNPs

TLR Structur

e

Non-synonymous

Position rs number

411(T/I) 5743699

556 (I/T) 5743702

579 (R/H) 5743703

631 (P/H) 5743704677 (R/W) 5743706

715 (Y/N) 5743707715 (Y/Amb)753 (R/Q) 5743708

Synonymous

Position rs number35 5743697

199 3804099213 5743698

450 3804100

541 5743700542 5743701

707 IIPGA-TLR2-31410

781 5743709

Location of R753 in the TIR domain

R753Q Polymorphism in the TLR2 gene R753Q Polymorphism in the TLR2 gene 743743 765765

Human TLR2Human TLR2 K A I P Q R P C K L K A I P Q R P C K L RR K I M N T K T Y L E W P K I M N T K T Y L E W P Mouse TLR2Mouse TLR2 K A I P Q R P C K L K A I P Q R P C K L RR K I M N T K T Y L E W P K I M N T K T Y L E W P

No stimulationNo stimulation

LipoproteinsLipoproteins

NFNF --

kB a

ctiv

atio

n (x

fold

incr

ease

)kB

act

ivat

ion

(x fo

ld in

crea

se)

66

55

44

33

22

11

TLR2 wt TLR2 R753QTLR2 wt TLR2 R753Q

QQ

Lorenz, Infect. Immun. 2OOOLorenz, Infect. Immun. 2OOO

Dominant Dominant Negative EffectNegative Effect of TLR2 R753Qof TLR2 R753Q

0

2000

4000

6000

pCMV6-TLR2 pCMV6-TLR2 R753Q

HKSABasal

0

5000

10000

15000

250 50 100 250

HKSABasal

pCMV6 pCMV6-TLR2 R753Q

A.

B.

ng ng ng ng

RR753753Q Q andand NFNF--κκB B translocationtranslocation

- + - +TLR2 R753Q

p50/p65 -

RLU

(arb

itrar

y un

its)

RLU

(arb

itrar

y un

its)

IRAK

HKSA

h Toll2h Toll2

HKSA

IRAK

P

TRAF6TAK

NIK

IKKα IKKβ

p50p50 p65p65

IIκκBB

NucleusTranslocation

p50p50 p65p65

MyD88

p50 p65

IRAK

HKSA

h Toll2h Toll2

HKSA

Rac1p85

p110

IRAK

P

TRAF6TAK

NIK

IKKα IKKβ

p50p50 p65p65

IIκκBBp50 p65

Nucleus

Basal Complex

CBPP

CytokinesCytokinesActive Active AktAkt

P

P

TranslocationTranslocation

TransactivationTransactivation

p50p50 p65p65

MyD88

IRAK

HKSA

TLR2 753QTLR2 753Q

HKSA

Rac1p85

p110

IRAK

P

TRAF6TAK

NIK

IKKα IKKβ

p50p50 p65p65

IIκκBBp50 p65

Nucleus

Basal Complex

CBPP

CytokinesCytokinesActive Active AktAkt

P

P

TranslocationTranslocation

TransactivationTransactivation

p50p50 p65p65

MyD88

- + - +TLR2 R753Q

p50/p65 -

TLR2-R753Q

TLR2

Immunoblot p85

0 3 5 10 20 30HKSA

TLR2R753Q and p65 phosphorylation

2020151510100020201515101000

TLR2TLR2--R753QR753QTLR2TLR2

PhosphoPhospho--P65 P65 --

P65 P65 --

ImagingImaging HKSAHKSA--inducedinduced PIPI--3K activation3K activation

TLR2TLR2 R753Q

QuickTime™ et un décompresseurCinepak sont requis pour visualiser

cette image.

QuickTime™ et un décompresseurCinepak sont requis pour visualiser

cette image.

TLR2 TLR2 stablystably--transfectedtransfected 293 293 cellscellsGFPGFP--AKTAKT--PHPHHKSAHKSA

TLR2 TLR2 Polymorphisms Polymorphisms in in HumansHumans

TLR2R753Q and Cytokine Production

Monocytes from 8 TLR2wt Pts Monocytes from 10 TLR2R753Q Pts

Manuscript in preparation

TNF-α (pg/mL)

0

2000

4000

6000

8000

Basal HKSA0

2000

4000

6000

8000

Basal HKSA

IL-10 (pg/mL)

p50/p65 -

HKSA - + - +P1 P2

TLR2TLR2R753QR753Q in MICU in MICU 1103 Caucasian ICU Pts (322 SS) 28 Pts TLR2R753Q (2,5%)

• Age 47,2 ± 18 • SAPS 2 40,5 ± 19• Survivors 23/28• Infections 16/28 16 Septic Shock (5.1% of SS group)• Origin of Infections

•Pneumonia 10•Meningitis 4•Septicemia 5•Others 4

• Microorganisms•S. pneumoniae 8•S. aureus 5•Candida sp. 2•Aspergillus sp. 2•Others 6

11.1% of Gram positive SS group

Location of R753 and R677 in the TIR domain

R753Q & R677W inhibit NFR753Q & R677W inhibit NF--kB activation kB activation

00

100100

200200

300300

400400

500500

pCMV6pCMV6--TLR2TLR2 pCMV6pCMV6--TLR2TLR2 R753QR753Q pCMV6pCMV6--TLR2TLR2 R677WR677W

HKSAHKSABasalBasal

RU

L (U

. arb

itrai

res)

RU

L (U

. arb

itrai

res)

Clinical Importance of TLR2Clinical Importance of TLR2R677W

Association of TLR2 R677W with:

- susceptibility to tuberculosis in Tunisian patientsBen Ali M; Clin Diagn Lab Immunol. 2004;11:625Ben Ali M; Clin Diagn Lab Immunol. 2004;11:625--66

- susceptibility to Lepromatous LeprosyKang TJ, FEMS Immunol Med Microbiol. 2001;31:53-8.

No association of TLR R677W with:

- chronic mucocutaneous candidiasis van der Graaf CA, Neth J med 2003:61:365-9

- lymphatic filariasisHise AG, Genes Immun 2003; 4:524-7

PolymorphismPolymorphismR677W in the in the TLR2TLR2 gene gene

Bochud PY. J Immunol 2003;170:3451Bochud PY. J Immunol 2003;170:3451

IRAK

HKSA

TLR2 R677WTLR2 R677W

HKSA

Rac1p85

p110

IRAK

P

TRAF6TAK

NIK

IKKα IKKβ

p50p50 p65p65

IIκκBBp50 p65

Nucleus

Basal Complex

CBPP

CytokinesCytokinesActive AktActive Akt

P

P

TranslocationTranslocation

TransactivationTransactivation

p50p50 p65p65

MyD88

HKSAR677WTLR2

- + +-

p50/p65-

Rac-PBD test

0 3 5 10 20 30 min

TLR2

R677W

HKSA

Polymorphisms Polymorphisms of TLR of TLR Signalling ProteinsSignalling Proteins

Cook DN. Nat Immunol 2004;5:975

Conclusions and Perspectives

• TLR functional genomics might explain some inter-individual variabilities of innate immune response

• Functional genomics validates signal transduction in Humans

• Importance of TLR1 and TLR6 polymorphisms on TLR2 signalling

Surface coSurface co--localisation of TLR1 and TLR2localisation of TLR1 and TLR2

Surface antibody-patching of TLR2

Sandor F; J Cell Biol 2003;162:1099-1100

Complementarities of TLR2 and TLR1 for signallingComplementarities of TLR2 and TLR1 for signalling

TLR1: 77 SNPs TLR6: 53 SNPs

Sandor F; J Cell Biol 2003;162:1099-1100

Conclusions and Perspectives

• TLR functional genomics might explain inter-individual variabilityof innate immune response

• Functional genomics validates signal transduction in Humans

• Importance of TLR1 and TLR6 polymorphisms on TLR2 signalling

• TLR2 polymorphism detection for severe sepsis prevention

Immunization of TLR2Immunization of TLR2--deficient micedeficient mice

IV infection with 106 virulent C. albicans

Villamon E; Microbes Infect 2004;6:542-548

Clinique

Diagnostic Pronostic Therapeutic

MARKERSMARKERS

Génomique Proteomic

Biology

Acknowledgments

Cochin Institute - Paris CHICHE JeanCHICHE Jean--Daniel Daniel

DHAINAUT JeanDHAINAUT Jean--François François

TEXEREAU JoelleTEXEREAU Joelle

GRIMALDI DavidGRIMALDI David

ROGET KarineROGET Karine

ROUSSEAU ChristopheROUSSEAU Christophe

COMBA BéatriceCOMBA Béatrice

CHRABIEH MayaCHRABIEH Maya

CollaborationsCollaborations

Cochin Institute - ParisBISMUTH GeorgesBISMUTH Georges

CONJAUD HélèneCONJAUD Hélène

Duke University, Durham, USASCHWARTZ DavidSCHWARTZ David

LORENZ LORENZ EvaEva

University University of Colorado, Denver USAof Colorado, Denver USAABRAHAM ABRAHAM EdwardEdward