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Pyogenic Bacterial Infections in Humans with MyD88 Deficiency Horst von Bernuth et al. Capucine Picard, Zhongbo Jin, Rungnapa Pankla, Hui Xiao, Cheng-Lung.

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Presentation on theme: "Pyogenic Bacterial Infections in Humans with MyD88 Deficiency Horst von Bernuth et al. Capucine Picard, Zhongbo Jin, Rungnapa Pankla, Hui Xiao, Cheng-Lung."— Presentation transcript:

1 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 no. 5889, pp Department of Pulmonary Medicine, Laboratory for Experimental Lung Research Hannover Medical School Ines Hahn April 6 th 2009

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) Hannover Medical School Same disease, different causes!

3 Result: Genetic analysis Hannover Medical School → 3 different mutations of the MYD88 gene: in-frame deletion:E52delpatients: P1, P6, P8 and P9 heterozygous missense mutation:L93P, R196C patient: P2 homozygous missense mutation:R196Cpatients: P3 and P4 Why have these mutations such dramatic, life-threatening consequences?

4 West, AP et al, 2006, Annu Rev Cell Dev Biol 22: 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 !!!

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

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) Hannover Medical School TIR: Toll/IL-1 receptor DD: death domain

7 Hannover Medical School 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 -κ B and AP-1 activation the MyD88/IRAK/TRAF6 complex also mediates the activation of IRF5 and IRF7 leading to IFN- α production

8 Result: Genetic analysis Hannover Medical School → 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

9 Results Northern and Western blotting Hannover Medical School 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

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- κ B 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 Hannover Medical School 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

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

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!

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 Hannover Medical School 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

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


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