Hidenori Ohnishi

Structural basis for the multiple interactions of the MyD88 TIR domain in TLR4 signaling

Myeloid differentiating factor 88 (MyD88) and MyD88 adaptor-like (Mal) are adaptor molecules critically involved in the Toll-like receptor (TLR) 4 signaling pathway. While Mal has been proposed to serve as a membrane-sorting adaptor, MyD88 mediates signal transduction from activated TLR4 to downstream components. The Toll/Interleukin-1 receptor (TIR) domain of MyD88 is responsible for sorting and signaling via direct or indirect TIR−TIR interactions between Mal and TLR4. However, the molecular mechanisms involved in multiple interactions of the TIR domain remain unclear. The present study describes the solution structure of the MyD88 TIR domain. Reporter gene assays revealed that 3 discrete surface sites in the TIR domain of MyD88 are important for TLR4 signaling. Two of these sites were shown to mediate direct binding to the TIR domain of Mal. Interestingly, Mal-TIR, but not MyD88-TIR, directly binds to the cytosolic TIR domain of TLR4. These observations suggested that the heteromeric assembly of TIR domains of the receptor and adaptors constitutes the initial step of TLR4 intracellular signal transduction.

The structure and binding mode of interleukin-18.

Interleukin-18 (IL-18), a cytokine formerly known as interferon-γ- (IFN-γ-) inducing factor, has pleiotropic immunoregulatory functions, including augmentation of IFN-γ production, Fas-mediated cytotoxicity and developmental regulation of T-lymphocyte helper type I. We determined the solution structure of IL-18 as a first step toward understanding its receptor activation mechanism. It folds into a β-trefoil structure that resembles that of IL-1. Extensive mutagenesis revealed the presence of three sites that are important for receptor activation: two serve as binding sites for IL-18 receptor α (IL-18Rα), located at positions similar to those of IL-1 for IL-1 receptor type I (IL-1RI), whereas the third site may be involved in IL-18 receptor β (IL-18Rβ) binding. The structure and mutagenesis data provide a basis for understanding the IL-18-induced heterodimerization of receptor subunits, which is necessary for receptor activation.

Pediatric allergy and immunology in Japan

The Japanese Society of Pediatric Allergy and Clinical Immunology (JSPACI) was started in 1966 and currently has 3613 members as of August 1, 2012. The number of pediatricians specializing in allergies who have been certified by the Japanese Society of Allergology is 817. Among these, there are 125 training directors and training facilities for allergy and clinical immunology. The JSPACI first published an asthma guideline specific for children in 2000, and this has been revised every 3 yrs, contributing to better control of pediatric asthma. Food allergy management guidelines were first developed in 2005, which have helped to improve the care of food allergy patients. Among 514 pediatric training programs by the Japanese Society of Pediatrics, there are 312 facilities routinely performing oral food challenges. Among these, there were already 53 facilities performing oral immunotherapy at the end of 2011, treating 1400 cases of food allergy. The prevalence of pediatric allergic diseases has increased in Japan over the past 50 yrs. A number of International Study of Asthma and Allergies in Childhood surveys have been conducted in the past at specific times. The prevalence of wheezing among children aged 13–14 yrs in 2002 was 13.0%. Multi‐year surveys found a 1.5‐ to 2‐fold increase every 10 yrs until 2002. However, according to the latest data in 2012, asthma prevalence seems to have slightly decreased in Japan. Food allergy mainly associated with infantile atopic eczema among infants younger than 1 yr of age is the most common form as with other developed countries. The estimated food allergy prevalence based on data from several surveys is 5–10% among infants (0–6 yrs) and 1–2% among schoolchildren (6–15 yrs). A variety of patients suffering from primary deficiency syndrome have been actively analyzed. Previously, antibody defects and well‐defined syndromes with immunodeficiency were analyzed, but recent research is focusing on not only acquired immune disorders but also on innate immune disorders. In contrast to the widespread use of oral immunotherapy, one immediate issue is to develop and reassess subcutaneous and sublingual immunotherapies for mite and Japanese cedar pollen antigens that have been disused in Japan since the 1990s.

A rapid screening method to detect autosomal-dominant ectodermal dysplasia with immune deficiency syndrome.

7. Spergel JM, Paller AS. Atopic dermatitis and the atopic march. J Allergy Clin Immunol 2003;112(suppl):S118-27. 8. Tran DQ, Andersson J, Wang R, Ramsey H, Unutmaz D, Shevach EM. GARP (LRRC32) is essential for the surface expression of latent TGF-beta on platelets and activated FOXP31 regulatory T cells. Proc Natl Acad Sci U S A 2009;106: 13445-50. 9. Marenholz I, Bauerfeind A, Esparza-Gordillo J, Kerscher T, Granell R, Nickel R, et al. The eczema risk variant on chromosome 11q13 (rs7927894) in the population-based ALSPAC cohort: a novel susceptibility factor for asthma and hay fever. Hum Mol Genet 2011;20:2443-9.

Phosphatase and tensin homolog (PTEN) mutation can cause activated phosphatidylinositol 3-kinase δ syndrome–like immunodeficiency

BACKGROUND Activated phosphatidylinositol 3-kinase δ syndrome (APDS) is a recently discovered primary immunodeficiency disease (PID). Excess phosphatidylinositol 3-kinase (PI3K) activity linked to mutations in 2 PI3K genes, PIK3CD and PIK3R1, causes APDS through hyperphosphorylation of AKT, mammalian target of rapamycin (mTOR), and S6. OBJECTIVE This study aimed to identify novel genes responsible for APDS. METHODS Whole-exome sequencing was performed in Japanese patients with PIDs. Immunophenotype was assessed through flow cytometry. Hyperphosphorylation of AKT, mTOR, and S6 in lymphocytes was examined through immunoblotting, flow cytometry, and multiplex assays. RESULTS We identified heterozygous mutations of phosphatase and tensin homolog (PTEN) in patients with PIDs. Immunoblotting and quantitative PCR analyses indicated that PTEN expression was decreased in these patients. Patients with PTEN mutations and those with PIK3CD mutations, including a novel E525A mutation, were further analyzed. The clinical symptoms and immunologic defects of patients with PTEN mutations, including lymphocytic AKT, mTOR, and S6 hyperphosphorylation, resemble those of patients with APDS. Because PTEN is known to suppress the PI3K pathway, it is likely that defective PTEN results in activation of the PI3K pathway. CONCLUSION PTEN loss-of-function mutations can cause APDS-like immunodeficiency because of aberrant PI3K pathway activation in lymphocytes.

The structural basis for receptor recognition of human interleukin-18

Interleukin (IL)-18 is a proinflammatory cytokine that belongs to the IL-1 family and plays an important role in inflammation. The uncontrolled release of this cytokine is associated with severe chronic inflammatory disease. IL-18 forms a signalling complex with the IL-18 receptor α (Rα) and β (Rβ) chains at the plasma membrane, which induces multiple inflammatory cytokines. Here, we present a crystal structure of human IL-18 bound to the two receptor extracellular domains. Generally, the receptors’ recognition mode for IL-18 is similar to IL-1β; however, certain notable differences were observed. The architecture of the IL-18 receptor second domain (D2) is unique among the other IL-1R family members, which presumably distinguishes them from the IL-1 receptors that exhibit a more promiscuous ligand recognition mode. The structures and associated biochemical and cellular data should aid in developing novel drugs to neutralize IL-18 activity.

Autosomal Dominant Anhidrotic Ectodermal Dysplasia with Immunodeficiency Caused by a Novel NFKBIA Mutation, p.Ser36Tyr, Presents with Mild Ectodermal Dysplasia and Non-Infectious Systemic Inflammation

PurposeAnhidrotic ectodermal dysplasia with immunodeficiency (EDA-ID) is characterized by hypohidrosis, dental abnormalities, sparse hair, and immunodeficiency. Autosomal dominant (AD)-EDA-ID, caused by a heterozygous mutation within NFKBIA, is very rare and its clinical features remain largely unknown. This study describes a patient with AD-EDA-ID harboring a novel NFKBIA mutation who presented with mild EDA and non-infectious systemic inflammation.MethodsThe clinical presentation of an AD-EDA-ID patient was described and immunological, genetic, and biochemical analyses were performed, with a focus on nuclear factor kappa B (NF-κB) activation.ResultsThe patient presented with symptoms of mild EDA-ID, namely sparse hair and hypohidrosis, although a skin biopsy confirmed the presence of sweat glands. There were no dental abnormalities. The patient also suffered from non-infectious inflammation, which responded to systemic corticosteroid therapy; however, the patient remained ill. Immunological analyses revealed reduced Toll-like receptor/IL-1 (TLR/IL-1) and tumor necrosis factor (TNF) receptor family responses to various stimuli. Genetic analysis identified a de novo heterozygous missense mutation, p.Ser36Tyr, in NFKBIA, resulting in defective NFKBIA degradation and impaired NF-κB activation. The patient was diagnosed with AD-EDA-ID and underwent hematopoietic stem cell transplantation. Engraftment was successful, with few signs of acute graft versus host disease. However, the patient suffered hemolytic anemia and thrombocytopenia, and died from a brain hemorrhage due to intractable thrombocytopenia.ConclusionAD-EDA-ID patients can present with mild ectodermal dysplasia and non-infectious inflammation, rather than with recurrent infections. Also, hematopoietic stem cell transplantation for AD-EDA-ID is still a clinical challenge.

Expression, purification and structural analysis of human IL-18 binding protein: a potent therapeutic molecule for allergy.

BACKGROUND While interleukin-18 (IL-18) plays an important role in the innate and adaptive immune responses, it can also cause severe allergic inflammatory reactions. Thus it is a molecule currently being targeted for therapy. The natural intrinsic inhibitor of IL-18 receptor activation, IL-18 binding protein (IL-18BP), shows a great potential for the treatment of allergy. METHODS Expression and purification of recombinant human IL-18BP (rhIL-18BP) were performed using the baculovirus system to develop a therapeutic molecule for the treatment of IL-18-related diseases and to investigate the structural basis of its inhibitory mechanism. RESULTS Purified rhIL-18BP potently inhibited the production of interferon-gamma by peripheral blood mononuclear cells in the presence of lipopolysaccharide and by human myelomonocytic KG-1 cells in the presence of IL-18 (IC50 = 0.4 nM). Surface plasmon resonance showed a high affinity (Kd = 0.46 nM) for rhIL-18BP in binding hIL-18. Structural analysis indicated that the stoichiometry between IL-18 and IL-18BP is 1 : 1 in solution and the model structure of the complex suggests that the key residues on IL-18 (L5, K53, S55) and the estimated key residues on IL-18BP (F93,Y97, F104) could have interactions. The structural mechanism of IL-18BP inhibition might be a competition for Site 2 on rIL-18 so that IL-18BP can prevent IL-18 receptor alpha from binding to Site 2 and inhibit IL-18 receptor activation. CONCLUSIONS IL-18BP has unique features with respect to its structure, binding mode and inhibitory mechanism. It is a molecule that has a great potential for the therapy of allergy.

TRAM Is Involved in IL-18 Signaling and Functions as a Sorting Adaptor for MyD88

MyD88, a Toll/interleukin-1 receptor homology (TIR) domain-containing adaptor protein, mediates signals from the Toll-like receptors (TLR) or IL-1/IL-18 receptors to downstream kinases. In MyD88-dependent TLR4 signaling, the function of MyD88 is enhanced by another TIR domain-containing adaptor, Mal/TIRAP, which brings MyD88 to the plasma membrane and promotes its interaction with the cytosolic region of TLR4. Hence, Mal is recognized as the “sorting adaptor” for MyD88. In this study, a direct interaction between MyD88-TIR and another membrane-sorting adaptor, TRAM/TICAM-2, was demonstrated in vitro. Cell-based assays including RNA interference experiments and TRAM deficient mice revealed that the interplay between MyD88 and TRAM in cells is important in mediating IL-18 signal transduction. Live cell imaging further demonstrated the co-localized accumulation of MyD88 and TRAM in the membrane regions in HEK293 cells. These findings suggest that TRAM serves as the sorting adaptor for MyD88 in IL-18 signaling, which then facilitates the signal transduction. The binding sites for TRAM are located in the TIR domain of MyD88 and actually overlap with the binding sites for Mal. MyD88, the multifunctional signaling adaptor that works together with most of the TLR members and with the IL-1/IL-18 receptors, can interact with two distinct sorting adaptors, TRAM and Mal, in a conserved manner in a distinct context.

Functional assessment of the mutational effects of human IRAK4 and MyD88 genes.

Human interleukin-1 receptor-associated kinase 4 (IRAK4) deficiency and myeloid differentiating factor 88 (MyD88) deficiency syndromes are two primary immune-deficiency disorders with innate immune defects. Although new genetic variations of IRAK4 and MyD88 have recently been deposited in the single nucleotide polymorphism (SNP) database, the clinical significance of these variants has not yet been established. Therefore, it is important to establish methods for assessing the association of each gene variation with human diseases. Because cell-based assays, western blotting and an NF-κB reporter gene assay, showed no difference in protein expression and NF-κB activity between R12C and wild-type IRAK4, we examined protein-protein interactions of purified recombinant IRAK4 and MyD88 proteins by analytical gel filtration and NMR titration. We found that the variant of IRAK4, R12C, as well as R20W, located in the death domain of IRAK4 and regarded as a SNP, caused a loss of interaction with MyD88. Our studies suggest that not only the loss of protein expression but also the defect of Myddosome formation could cause IRAK4 and MyD88 deficiency syndromes. Moreover a combination of in vitro functional assays is effective for confirming the pathogenicity of mutants found in IRAK4 and MyD88-deficiency patients.