1. Pyogenic Bacterial Infections in Humans with MyD88 Deficiency
Ines Hahn
April 6th 2009
Science 1 August 2008: Vol no. 5889, pp
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

2. 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

3. 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

4. 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 MyD88-independent !
West, AP et al, 2006, Annu Rev Cell Dev Biol 22:409-37

5. Introduction
→ Signal transduction through IL-1R is exclusively mediated by MyD88
TIR: Toll/IL-1 receptor DD: death domain

6. 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
Hannover Medical School

7. 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
Hannover Medical School

8. Result: Genetic analysis
→ 3 different mutations of the MYD88 gene:
Mutations are non-conservative and affect residues that are conserved across species
Residues 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
Hannover Medical School

9. 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

10. 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
Hannover Medical School

11. 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
Hannover Medical School

12. 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
Hannover Medical School

13. Transfection
Transfection of fibroblasts with expression vectors encoding MyD88 or IRAK-4
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
Hannover Medical School

14. 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
All nine patients have complete MyD88-deficiency resulting from the inheritance of two loss-of-function alleles!
Hannover Medical School

15. 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
Hannover Medical School

16. 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!
Hannover Medical School

17. 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 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
Hannover Medical School

18. 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!
Ines Hahn
Department of Pulmonary Medicine
Laboratory for Experimental Lung Research
Hannover Medical School
ZIB