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

Science 1 August 2008:Vol. 321. no. 5889, pp. 691 - 696

Department of Pulmonary Medicine, Laboratory for Experimental Lung Research

Hannover Medical School

Ines Hahn

April 6th 2009

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)

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Same disease, different causes!

Result: Genetic analysis

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• → 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?

West, AP et al, 2006, Annu Rev Cell Dev Biol 22:409-37

Introduction

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 MyD88-independent !

→ Signaling through TLRs 1/2/6, 5, 7, 8 and 9 and signal transduction through IL-1R are exclusively mediated by MyD88 !!!

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)

Hannover Medical SchoolTIR: Toll/IL-1 receptor DD: death domain

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• TRIF-dependent signaling additionally activates

IFN-β production via IRF3

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

Result: Genetic analysis

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• → 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 P9R196C → P3 and P4R196C and L93P → P2

TIR: Toll/IL-1 receptor DD: death domain

ResultsNorthern and Western blotting

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• 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

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

• 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

• 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

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!

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)

• 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!

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

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• All nine patients have complete MyD88-deficiency resulting from two loss-of-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

Conclusion

• MyD88- and IRAK-4-dependent TLRs and IL-1Rs play a narrow non-redundant 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