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Pyogenic Bacterial Infections in Humans with MyD88 Deficiency

Ines Hahn April 6 th 2009. Pyogenic Bacterial Infections in Humans with MyD88 Deficiency. Science 1 August 2008: Vol. 321. no. 5889, pp. 691 - 696. Horst von Bernuth et al.

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Pyogenic Bacterial Infections in Humans with MyD88 Deficiency

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  1. Ines Hahn April 6th 2009 Pyogenic Bacterial Infections in Humans with MyD88 Deficiency Science 1 August 2008:Vol. 321. no. 5889, pp. 691 - 696 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: 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 • 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

  6. 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

  7. 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 Hannover Medical School

  8. ResultsNorthern 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

  9. 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

  10. 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

  11. 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

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

  13. 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

  14. 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

  15. 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

  16. 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

  17. 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

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