Transcript Pyogenic Bacterial Infections in Humans with MyD88 Deficiency

Ines Hahn
April 6th 2009
Science 1 August 2008:
Vol. 321. no. 5889, pp. 691 - 696
Pyogenic Bacterial Infections in Humans
with MyD88 Deficiency
Horst von Bernuth et al.
Capucine Picard, Zhongbo Jin, Rungnapa Pankla, Hui Xiao, Cheng-Lung Ku, Maya
Chrabieh, Imen Ben Mustapha, Pegah Ghandil, Yildiz Camcioglu, Júlia Vasconcelos,
Nicolas Sirvent, Margarida Guedes, Artur Bonito Vitor, María José Herrero-Mata, Juan
Ignacio Aróstegui, Carlos Rodrigo, Laia Alsina, Estibaliz Ruiz-Ortiz, Manel Juan, Claudia
Fortuny, Jordi Yagüe, Jordi Antón, Mariona Pascal, Huey-Hsuan Chang, Lucile Janniere,
Yoann Rose, Ben-Zion Garty, Helen Chapel, Andrew Issekutz, László Maródi, Carlos
Rodriguez-Gallego, Jacques Banchereau, Laurent Abel, Xiaoxia Li, Damien Chaussabel,
Anne Puel, Jean-Laurent Casanova
Department of Pulmonary Medicine, Laboratory for Experimental Lung Research
Hannover Medical School
What is this study about?
•
9 clinical cases of children with invasive pyogenic bacterial diseases:
– patients suffered from life-threatening, often recurring pyogenic bacterial
infections e.g. invasive pneumococcal disease
– these patients were otherwise healthy, with normal resistance to other microbes!
•
Patients show no IRAK4-deficiency even though previous reports had associated
invasive pneumococcal disease in children with a lack of IL-1 receptor-associated
kinase 4 (IRAK-4)
Same disease, different causes!
Hannover Medical School
Result: Genetic analysis
•
→ 3 different mutations of the MYD88 gene:
in-frame deletion:
E52del
patients: P1, P6, P8 and P9
heterozygous missense mutation:
L93P, R196C
patient: P2
homozygous missense mutation:
R196C
patients: P3 and P4
Why have these mutations such dramatic,
life-threatening consequences?
Hannover Medical School
Introduction
→ Signaling through TLRs 1/2/6, 5, 7, 8 and 9 and signal transduction through
IL-1R are exclusively mediated by MyD88 !!!
MyD88-indepenent signaling:
•
TLR4 utilizes both MyD88 and
TRIF (TIR domain-containing
adaptor inducing interferon)
•
TLR3 exclusively utilizes TRIF for
signaling
•
Signal transduction through
TNF receptor is MyD88independent !
West, AP et al, 2006, Annu Rev Cell Dev Biol 22:409-37
Introduction
•
MyD88 has a C-terminal Toll/IL-1 receptor (TIR) domain and a N-terminal
death domain (DD)
•
MyD88 associates with the TIR domain of the TLR via its TIR domain
•
Via its DD, MyD88 binds DD of other molecules (e.g. IRAK)
TIR: Toll/IL-1 receptor
DD: death domain
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Introduction
•
Upon stimulation, MyD88 associates with TIR
•
Receptor-associated MyD88 recruits IRAK-4 and
IRAK-1 which then associate with TRAF6
•
TRAF6 activates TAK1 which subsequently
activates IKK, JNK and p38
•
this leads to NF-κB and AP-1 activation
•
the MyD88/IRAK/TRAF6 complex also mediates
the activation of IRF5 and IRF7 leading to IFN-α
production
•
TRIF-dependent signaling additionally activates
IFN-β production via IRF3
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Result: Genetic analysis
•
→ 3 different mutations of the MYD88 gene:
•
Mutations are non-conservative and affect residues that are conserved
across species
Residues 195-197 are crucial for Toll/TIR-1 receptor (TIR/TIR) interactions
•
E52del → P1, P6, P8 and P9
R196C → P3 and P4
R196C and L93P → P2
TIR: Toll/IL-1 receptor
DD: death domain
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Results
Northern and Western blotting
•
•
MYD88 mRNA in fibroblasts was of normal molecular weight and abundance
(Patients P1 to P4 representing the 3 combinations of alleles)
MyD88 protein levels were reduced (P1 and P2) and normal in P3 and P4
I3A: MyD88-deficient HEK cell line
Hannover Medical School
Functionality of MyD88 protein
→ Loss of function!
•
No phosphorylation and subsequent degradation of IRAK-1 in response to
IL-1β in patients with MyD88-defect or IRAK-4-deficiency
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Functionality of MyD88 protein
→ Loss of function!
•
•
No IL-1β-incuced downstream activation of JNK and p38
No DNA-binding activity of NF-κB in response to IL-1β
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Cytokine production
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No production of IL-6, IL-8, IFN-β and IFN-λ in response to IL-1β in
patients with MyD88-defect or IRAK-4-deficiency
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Transfection
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Transfection of fibroblasts with expression vectors encoding MyD88 or IRAK-4
•
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MYD88-gene transfected cells from P1 and P2 regained IL-1β responsiveness
the MyD88-deficient HEK cell line I3A regained IL-1β responsiveness only when
transfected with WT MYD88 allele, confirming that all MYD88 mutant alleles are
loss-of-function
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Immunoprecipitation and Western Blotting
•
•
R196C mutation in the TIR domain prevents interaction with IL-1R
E52del and L93P mutations in the death domain prevent interaction with
IRAK-4
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All nine patients have complete MyD88-deficiency resulting from the
inheritance of two loss-of-function alleles!
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Stimulation with various TLR-agonists
→ Secretion of cytokines by whole-blood cells
•
•
Whole blood from MyD88-deficient patients showed no cytokine response to
six of the eight TLR agonists!
Activation with PolyI:C and LPS induced expression of several cytokines to
levels similar to those in healthy controls
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IL-1R pathway of MyD88- and IRAK-4-deficient patients
•
Analysis of genome wide transcriptional profiles of fibroblasts from healthy
controls and patients stimulated with IL-1β, TNF- and poly(I:C)
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Healthy controls:
– 275 / 1451 transcripts regulated by IL-1β,
TNF- and poly(I:C) in 2 hours / 8 hours
•
IRAK-4 and MyD88-deficient patients:
– unresponsive to IL-1β
– Similar response to Poly(I:C) and TNF as
compared to healthy controls
→ same phenotype
→ complete and specific lack of IL-1β responsiveness as a characteristic
of IRAK-4 and MyD88-deficiency!
Hannover Medical School
Summary
•
9 children with mutations in MYD88 gene suffered from life-threatening,
often recurring pyogenic bacterial infections
– 3 children died between the age of 1 and 11 months
•
MYD88 mRNA in fibroblasts was of normal molecular weight and
abundance but patients have functional MyD88-deficiency with low or
normal protein levels
•
All nine patients have complete MyD88-deficiency resulting from two lossof-function alleles!
•
IRAK-4 and MyD88-deficient patients: → indistinguishable cellular
phenotype
•
Complete and specific lack of IL-1β responsiveness as a characteristic of
IRAK-4 and MyD88-deficiency
Hannover Medical School
Conclusion
•
MyD88- and IRAK-4-dependent TLRs and IL-1Rs play a narrow nonredundant role in protective immunity
•
TIR-signaling seems to be less important for survival later in life
maybe due to the compensatory effect of adaptive immunity (Be
critical with this statement!)
•
Is it surprising that MyD88-deficient mice suffer so much more than
MyD88-deficient human?
– Actually, it`s not! Because human get long-term treatment (antibiotics etc)!
Also, it`s impossible to test susceptibility of humans to different pathogen
as it is done with mice!
ZIB
Ines Hahn
Department of Pulmonary Medicine
Laboratory for Experimental Lung Research
Hannover Medical School