Genetic infectious susceptibility and TLR defects in human
Bacteria Mycobacteria Herpes simplex virus
Human Genetics of Infectious Diseases-INSERM U980, Necker Faculty, Paris Descartes University, Paris, France [email protected] Toll-like receptors (TLR)
• Receptor of innate immunity (11 in human)
• Membrane glycoproteines (90-115 kD)
• Leucin rich repeat (LRR)
• Recognize ligands derived from microorganisms: LPS – TLR4 or dsRNA-TLR3
• Initiation of inflammatory response
(Bell et al. TRENDS in Immunology 2003) Toll like receptors and IL-1Rs (TIRs) signalling pathway
(Picard et al. 2011 in press Clinical Microbiology Reviews) Inherited human IRAK-4 and MyD88 deficiencies
IL-1Rs TLRs NF-κB C IRAK4 -/- MyD88 -/-
ns TNFααα IL-1βββ ns TNFααα IL-1βββ ns TNFααα IL-1βββ TIR Domain
IRAK4 MyD88 p50/p50
p50/p65
NEMO IKK ααα βββ IkBααα MAPK p p IκκκBααα Iκκκ Bααα p50 p65 p50 p65 AP-1 MAPK C IRAK4 -/- MyD88 -/-
ns TNFααα IL-1βββ ns TNFααα IL-1βββ ns TNFααα IL-1βββ
Gene transcription P-p38 IL-1βββ. IL-6. IL-12. p50 p65 AP-1 TNF-ααα. IFN-γγγ...
p38 (Picard et al. Science 2003) (von Bernuth et al. Science 2008) Inherited human IRAK-4 and MyD88 deficiencies
3,000 4,000 C C 2,000 IRAK-4 -/- IRAK-4 -/- 2,000 IRAK-4-/- 1,000 TNF (pg/ml) TNF
TNF (pg/ml) TNF 0 0
o o K4 K4 N I I ium ium coli d S S PG LTA LPS d aureus E. me flagellinPMA+ me PMA+ zymosan S. PAM3CPAM2C M. tuberculosis 3 10 105 C+ = 6 MyD88-/- = 4 IRAK-4 -/- C 104 102 MyD88 -/- 103 2 10 10 IL-6 (pg/ml) IL-6
IL-6 (ratio) 10 1 1 ns IL-1β LPS P/Iono poly(I:C) IL-1β TNF-α PMA Blood cells of IRAK-4 and MyD88 deficient patients display an impaired responses to TLRs ligands (except TLR3) and IL-1β
(Picard et al. Science 2003. von Bernuth et al. Science 2008) IRAK-4 deficiency – patients
8 1 1 9 3 7 3 5 6 1 2 2 2 Picard et al. Science 2003 Medvedev et al. JEM 2003 Enders et al. J Ped 2004 Yang et al. Immunity 2005 Takada et al. J Ped 2006 2 Cardenes et al. J Ped 2006 McDonald et al. JACI 2006 Davidson et al. JI 2006 Ku et al. JEM 2007 Lavine et al. JACI 2007 Krause et al. CID 2009 Bouma et al. BJH 2009 Yoshikawa et al. J Ped 2010 52 patients from 33 kindreds in 14 countries Picard et al. Medicine 2010 Unpublished datas (Autosomal recessive disorder) MyD88 deficiency (AR) – patients
2 3 9 2 5 1
von Bernuth et al. 2008 Science 22 patients from 7 kindreds Conway et al. 2010 JACI Picard et al. 2010 Medicine in 6 countries Unpublished datas IRAK-4 deficiency – mutations R12C G298D M1V Y48X R183X Q293X E402X Y430X
NH2 1 460 COOH
I II III IV V VIVIIVIII IX X XI XII DD KD 5’ 1 1380 3’
1188+520A>G 1189-1G>T 1-1096_40+23del 523delA 631delG 1240insA 593delG 942-1481_1125+547del 118insA 620_621delAC 821delT 1126-1G>T 831+5G>T 897_900delCAAT Patients are homozygous or compound heterozygous for mutations in IRAK4 MyD88 deficiency – mutations E66X
E65del L106P R209C
NH2 1 296 COOH
DD I II III TIR IV V 5’ 1 888 3’
Patients are homozygous or compound heterozygous for mutations in MyD88 IRAK-4/MyD88 deficiencies (n=61)
Clinical manifestations by patient
Pneumonia MyD88 deficient patients
ENT infections IRAK-4 deficient patients
Skin abscess / orbital cellulitis IRAK-4/MyD88 deficient pts
Lymphadenitis
Deep inner organ/ abscess
Arthritis / osteomyelitis
Sepsis
Meningitis
0% 20% 40% 60% 80% 100% Patients (%)
Invasive bacterial infection: 148 reported episodes of InvBD (n=2.4 episodes/patient. range: 0 to 10) IRAK-4/MyD88 deficiencies: bacterial infections
Invasive infections Non invasive infections
IRAK-4 deficient pts IRAK-4 deficient pts S. pneumoniae
7.6% 8.1% 1.6% 16.1% S. aureus 18.1% 22.6% Other Gram-pos
5.7% 54.3% P. aeruginosa
8.1% Other Gram-neg 14.3% 43.5%
S. pneumoniae = 52% of MyD88 deficient pts MyD88 deficient pts invasive bacterial infections (Meningitis. sepsis. arthritis. 14.7% 13.3% 20% osteomyelitis. deep abscess)
13.3% 14.7% 44.1% S. aureus = 45.5% of non 5.9% invasive bacterial infections 53.3% (Cellulitis. omphalitis. sinusitis. ENT 20.6% infections. pneumonia)
S. pneumoniae isolated in 41 pts / 61 (IPD 67%) IRAK-4/MyD88 deficiencies: severe, narrow and transient phenotype
100 100 80 75 60 40 50 percent 20 25 0
0 0 1to yr 1 2to yr 2 3to yr 5 6to yr 6 7to yr 8 9to yr 0 10 20 30 40 50 3 4to yr 4 5to yr 7 8to yr 9 10 yr to asymptomatic patients (%) 20 to 25 25 yr 20 to 30 yr 25 to months 15 yr 10 to 20 yr 15 to 35 yr 30 to N patients 61 14 5 2 1 0 N pts 61 44 36 32 31 25 23 23 20 20 19 8 4 4 3 All infections InvBD NInvBD 100 Early clinical phenotype: 75 1st invasive infection occurred < 2 years
50 Severe clinical phenotype: 25 patients died of invasive bacterial infections 25 (16 caused by invasive bacterial S. pneumoniae infection) survival patients (%) 0 Transient clinical phenotype: 0 10 20 30 40 no severe invasive infections > 14 years years N patients 61 19 4 3 0 no deaths after the age of 8 years. Immunological explorations
• Blood lymphocyte and monocyte subsets: normal • T cell proliferations (mitogens and antigens): normal
• Complement pathways: normal
• lg: normal, except high levels IgG4 (35%) and IgE (65%) • Abs response to proteins: normal
• Abs response to polysaccharides: variable
• Inflammatory signs (clinical & biological): low IRAK-4/MyD88 deficiencies - summary
• Invasive infections by Gram-positive bacteria, but also Gram-negative
(S. pneumoniae, S. aureus and P. aeruginosa) < 14 years of age
• Recurrent episodes of cellulitis (S. aureus) and sinusitis persisting
• IMMUNODEFICIENCIES THAT IMPROVE WITH AGE
• Delayed or low inflammatory signs (fever, PMN, CRP…)
• Delayed separation of the umbilical cord (Takada. 2006 J.Ped)
• Deficient IL-1β and TLR signalling (except for TLR3)
• No other susceptibility to infection, in particular severe viral infection MyD88 and IRAK-4 deficiencies: phenocopies
Fibroblast cells (Damien Chaussabel, Baylor institute, Dallas, USA) Toll like receptors and IL-1Rs (TIRs) signalling pathway TLR-1, 2 , 5, 6, 10 IL-1Rs TLR-4
MD2
TLR-7, 8, 9 TRIF TLR-3
MyD88
TRAF3 UNC-93B UNC-93B IRAK-4 IKKεεε IRAK-1 TBK1
IκκκB MAPK NF-κκκB IRF3
NF-κκκB AP1 IRF3 IL-6,IL-1,TNFααα IL-6,IL-1,TNFααα IFNβββ Herpes Simplex virus Encephalitis (HSE): a devastating viral disease of unclear pathogenesis
HSV-1 reach the CNS via neurons and do not spread to epithelia and internal organs
Classical Primary Immunodeficiencies, do not usually predispose to HSE
HSE patients are normally resistant to most infectious agents, including neurotropic viruses
2-4 cases/million people/year
Incidence peaks 6 mos-6 yrs during primary infection by HSV-1
Up to 30% mortality in treated individuals HSE: a genetic epidemiological survey
50 100 10%10% consanguinity consanguinity 40 80
30 60 HSE 20 HSV+ 40
Number of HSE 10 20 Prevalence of HSV+
0 0 0 10 20 30 40 50 60 70 80 Age in yrs Autosomal Recessive UNC-93B deficiency: the first genetic etiology of isolated HSE
- UNC-93B is a 12-transmembrane domain protein in the ER. - UNC-93B delivers the nucleotide-sensing receptors TLR3, 7, 8 and 9 from the ER to endolysosomes TLR3 TLR7/8/9
Three individuals, from Portugal and France (2 gypsies)) from 2 consanguineous kindreds with homozygous mutations in UNC93B1 :
P1 HSE at 11, 14 and 42 months UNC-93B P2 HSE at 5 and 17 years P3 asymptomatic at 30 yrs (HSV1) ⇒⇒⇒ incomplete penetrance
NH2 1 597 COOH
I II III IV VVIVIIVIII IX X XI
IFNs 5’ 1 1794 3’ 781G>A 1034del4 (Casrouge et al. Science 2006) Autosomal Recessive UNC-93B deficiency: the first genetic etiology of isolated HSE
NH2 1 597 COOH
I II III IV VVIVIIVIII IX X XI TLR3 TLR7/8/9 5’ 1 1794 3’ 781G>A 1034del4
PBMCs Fibroblasts 104 104 C C UNC-93B-/- P1 P1 UNC-93B 103 UNC-93B-/- P2 103
(pg/ml) 2 2 10 (pg/ml) 10 λ λ λ λ α α α α IFN- IFN- 10 10
1 1 ) 5) 8 ) S C A S 13 -2 4 -C S C N I: D P 4) - M ) -8 8) G N I: y( /M L R M 7) 3 8 R 7/ p 9) y( ) IFNs ol -I L 3 R LR R C R l 3 IG (T TL (T L L o R P R ( (T (T P TL 3/ ( LR (T (Casrouge et al. Science 2006) Autosomal Dominant TLR3 deficiency: The second genetic etiology of HSE
P554S
NH2 1 904 COOH III III IV V
Leucin Rich Repeats TM TIR TLR3 5’ 1 2715 3’
UNC-93B TRIF
H539 N541 IRF-3 NF-κκκB Ins Ins IκBα P554
7 individuals bearing TLR3 AD mutations have been identified
from two unrelated kindreds, 2 had HSE (incomplete penetrance) IFNs P1 HSE at 5 years P2 HSE at 5 months
(Zhang Science 2007) Autosomal Dominant TLR3 deficiency:
C+ 103 60 C+ TRL3+/- P1 TLR3+/- TLR3+/- P2 UNC-93B -/- 102 40 (IU/ml) (IU/ml) β β β β
β 10 20 IFN- IFN- 1 0 1 I TLR3 S V V I V - I N S - polyI/C 0 1 5 25 0 1 5 25 V s s V a i S V e r C l a b (µg/ml) 12h 24h H s M P d a n E e i S 120 M UNC-93B C-Mock Fibroblasts (WT) TRIF 100 C-TLR3 WT 80 C-TLR3 P554S IRF-3 NF-κκκB
(pg/ml) 60 IκBα λ λ λ λ 40 IFN- 20 0 poly(I:C) 0 1 5 25 0 1 5 25 (µg/ml) 12h 24h IFNs Fibroblast cells from AD TLR3 deficient patients have impaired type 1 and 3 IFN production (Zhang Science 2007) Heterozygous TRAF3 mutation in a patient with HSE: The third genetic etiology of HSE
(Schneider et al. Nature Immunology, 2006) TRAF-3 has functions downstream from multiple TNF receptors and the receptors inducing IFN-ααα, IFN-βββ, and IFN-λλλ production, including TLR3. Heterozygous TRAF3 mutation in a patient with HSE
200 patients sequenced
P1 HSE at 4 years
TRAF3 c.705C>T TRAF3 protein R118W R118W
NH2 1 568 COOH
I II III IV V VI VII VIII IX X XI XII RING finger Zinc-finger Isoleucin TRAF domain zipper 5’ 1 1707 3’ (Rebeca Pérez de Diego et al. Immunity 2010) SV40 fibroblast studies: IFN type I and cytokines production
(Rebeca Pérez de Diego et al. Immunity 2010) SV40 fibroblast studies: Molecular phenotype
NF-κκκB pathway
IRF3 dimerization
Fibroblast cells from TRAF3 deficient pt: Reduced NF-kB nuclear translocation and IRF3 (Rebeca Pérez de Diego et al. Immunity 2010) dimerization in response to TLR3 agonist Complementation studies in SV40 fibroblast of P1
Stable transfectants
(Rebeca Pérez de Diego et al. Immunity 2010) TRAF3 in other pathways TLR4 and TLR7/8 Impaired IFN and cytokines production by MDDC and MDM
Monocyte derived dendritic cells (MDDC) Monocyte differentiated macrophages (MDM)
(Rebeca Pérez de Diego et al. Immunity 2010) TRAF3 in other pathways (alternative NF-kB pathway)
CD40 LTβR BAFFR Impaired IL-6 production after Diminished the production of Constitutive activation in B- CD40L activation by MDDC IL-8 by SV40-fibroblast EBV cells - IL10 production Negative dominant effect + ++ + _ Human AD TRAF3 deficiency
• Heterozygous R118W mutation in TRAF3 dominant-negative effect and results in impaired TLR3-dependent induction of IFN and predisposition to HSE
• TRAF3 patient displays a broad cellular phenotype: – with impaired cellular responses to IFN TLR3-independent pathways, – CD40, LTβββR and BAFFR, whose clinical consequences have so far remained silent. HSE-Specific Immunity
HSV-1 ISRE genes
dsRNA ssRNA CpG TLR7/8/9 IRF9 TLR3 MYD88 IRAK4 STAT1 STAT2 TRIF UNC93B ISGF3 TRAF3 complex α β NEMO IKK complex JAK1
IFNαR1
IRF3 IRF7 NFκB TYK2 IFNαR2
IFN-α,-β
Production of type I IFNs Response to type I IFNs Genetic infectious susceptibility and TLR defects in human RANK/VEGFR3 TCR/BCR TNFR-S TLR-1, 2 , 5, 6, 10 IL-1Rs TLR-4 / EDAR
MD2
TLR-7, 8, 9
MyD88 TRIF TLR-3
UNC-93B TRAF3 IRAK-4 UNC-93B IRAK-1 NEMO IKKεεε IKK ααα βββ TBK1 IκκκBααα p p IκκκBααα κκκ IκκκBααα NF- B p50 p50 p65 p65 MAPK IRF3
p50 AP1 p65 IL-6,IL-1,TNFααα IL-6,IL-1,TNFααα IRF3 IFNβββ Conclusions
• The TIRs signaling pathway (IRAK-4/MyD88-depend pathway) is crucial to control invasive pyogenic bacterial infection during childhood, by induction and propagation inflammation, and secondary initiation of adaptive immunity.
• The integrity of the TLR3-IFN pathway is crucial to control HSV-1 primary infection in the CNS.
• The exploration of « idiopathic infections » could contribute to diagnose new primary immunodeficiencies and to a better understanding of immunity against pathogens. Acknowledgements IRAK4 and MyD88 collaborators HGID-INSERM-U980 UK : Rainer Doffinger. D Kumararatne Anne Puel Helen Chapel. Graham Davies Horst von Bernuth. Peter Arkwright. Adrian Thrasher Pegah Ghandil. Claire Bethune Mayah Chrabieh. Cheng-Lung Ku. Canada : David Speert. Andrew C. Issekutz Laurent Abel Chaim Roifman Jean-Laurent Casanova Australia: Mimi Tang. Joanne Smart KSA: Sami Al-Hajjar Abdulaziz Al-Ghonaium Israel : Ben-Zion Garty Saleh Al-Mohsen Turkey: Yildiz Camcioglu USA: Noorbibi K. Day-Good Steven M. Holland. John Gallin. Spain : Carlos Rodriguez-Gallego Jens C. Krause. Clarence B Creech. Carlos Rodrigo, Maria Méndez Coleen K. Cunningham. Joseph Domachowske Francisco Almazán, Claudia Fortuny Ofer Levy. Douglas McDonald. Raif Geha Laia Alsina, Elena Colino, Juan Ignacio Aróstegui Germany: Stephan Ehl Portugal: Artur Bonito Vitor, Júlia Vasconcelos Margarida Guedes Switzerland: Janine Reichenbach Slovenia: Simona Eva Zitnik Hungary: László Maródi Japan: Hidetoshi Takada. France: Cyrille Hoarau Toshiro Hara Nicolas Sirvent, Dominique de Ricaud Hideto Yoshikawa Francois Dubos, Frédérique Delion Shigeaki Nonoyama Acknowledgements HSE TLR3
HGID Rockefeller Branch Shen-Ying Zhang Emmanuelle Jouanguy Institut Pasteur Yiqi Guo Lluis Quintana Melina Herman TRIF Jean-Laurent Casanova iFREC Osaka University Osamu Takeuchi
UCLA HGID Necker Branch Sang Hoon Rhee Vanessa Sancho Shimizu Lazaro Lorenzo-Diaz Rebeca Perez Cochin TRAF3 Annabelle Cardon Pierre Lebon, Soraya Boucherit Flore Rozenberg, Anne Puel S Plancoulaine KB Rebeca Pérez de Diego Marc Tardieu, Laurent Abel, Laboratory of Human Genetics of Infectious Diseases
Necker Branch - Paris Rockefeller Branch - New York
Dr Laurent Abel Pr Jean-Laurent Casanova Children and their families