The structural network of Interleukin-10 and its implications in inflammation and cancer.

BACKGROUND: Inflammation has significant roles in all phases of tumor development, including initiation, progression and metastasis. Interleukin-10 (IL-10) is a well-known immuno-modulatory cytokine with an anti-inflammatory activity. Lack of IL-10 allows induction of pro-inflammatory cytokines and hinders anti-tumor immunity, thereby favoring tumor growth. The IL-10 network is among the most important paths linking cancer and inflammation. The simple node-and-edge network representation is useful, but limited, hampering the understanding of the mechanistic details of signaling pathways. Structural networks complete the missing parts, and provide details. The IL-10 structural network may shed light on the mechanisms through which disease-related mutations work and the pathogenesis of malignancies. RESULTS: Using PRISM (a PRotein Interactions by Structural Matching tool), we constructed the structural network of IL-10, which includes its first and second degree protein neighbor interactions. We predicted the structures of complexes involved in these interactions, thereby enriching the available structural data. In order to reveal the significance of the interactions, we exploited mutations identified in cancer patients, mapping them onto key proteins of this network. We analyzed the effect of these mutations on the interactions, and demonstrated a relation between these and inflammation and cancer. Our results suggest that mutations that disrupt the interactions of IL-10 with its receptors (IL-10RA and IL-10RB) and α2-macroglobulin (A2M) may enhance inflammation and modulate anti-tumor immunity. Likewise, mutations that weaken the A2M-APP (amyloid precursor protein) association may increase the proliferative effect of APP through preventing ß-amyloid degradation by the A2M receptor, and mutations that abolish the A2M-Kallikrein-13 (KLK13) interaction may lead to cell proliferation and metastasis through the destructive effect of KLK13 on the extracellular matrix. CONCLUSIONS: Prediction of protein-protein interactions through structural matching can enrich the available cellular pathways. In addition, the structural data of protein complexes suggest how oncogenic mutations influence the interactions and explain their potential impact on IL-10 signaling in cancer and inflammation.

Very early onset inflammatory bowel disease associated with aberrant trafficking of IL-10R1 and cure by T cell replete haploidentical bone marrow transplantation.

PURPOSE: Loss-of-function mutations in IL10 and IL10R cause very early onset inflammatory bowel disease (VEO-IBD). Here, we investigated the molecular pathomechanism of a novel intronic IL10RA mutation and describe a new therapeutic approach of T cell replete haploidentical hematopoietic stem cell transplantation (HSCT). METHODS: Clinical data were collected by chart review. Genotypes of IL10 and IL10R genes were determined by Sanger sequencing. Expression and function of mutated IL-10R1 were assessed by quantitative PCR, Western blot analysis, enzyme-linked immunosorbent assays, confocal microscopy, and flow cytometry. RESULTS: We identified a novel homozygous point mutation in intron 3 of the IL10RA (c.368-10C > G) in three related children with VEO-IBD. Bioinformatical analysis predicted an additional 3' splice site created by the mutation. Quantitative PCR analysis showed normal mRNA expression of mutated IL10RA. Sequencing of the patient's cDNA revealed an insertion of the last nine nucleotides of intron 3 as a result of aberrant splicing. Structure-based modeling suggested misfolding of mutated IL-10R1. Western blot analysis demonstrated a different N-linked glycosylation pattern of mutated protein. Immunofluorescence and FACS analysis revealed impaired expression of mutated IL-10R1 at the plasma membrane. In the absence of HLA-identical donors, T cell replete haploidentical HSCT was successfully performed in two patients. CONCLUSIONS: Our findings expand the spectrum of IL10R mutations in VEO-IBD and emphasize the need for genetic diagnosis of mutations in conserved non-coding sequences of candidate genes. Transplantation of haploidentical stem cells represents a curative therapy in IL-10R-deficient patients, but may be complicated by non-engraftment.

Exome sequencing identifies novel compound heterozygous mutations of IL-10 receptor 1 in neonatal-onset Crohn's disease.

Inflammatory bowel disease is well recognized for a strong genetic involvement in its pathogenesis. Homozygous mutations in interleukin-10 receptor 1 (IL-10R1) identified by linkage analysis were shown to be involved in this disorder. However, the underlying molecular mechanism and the causal nature of the mutations in the disease process remain to be clarified. In this study, using whole exome sequencing, we identified novel compound heterozygous missense mutations in the extracellular domain of IL-10R1 in a Crohn's disease patient from a non-consanguineous family. These mutations did not affect IL-10R1 expression, nor IL-10 binding. However, they abrogated IL-10R1 phosphorylation induced by IL-10, therefore leading to impaired STAT3 activation and suppression of inflammatory responses. After reconstitution with wild-type IL-10R1, the patient cells showed fully restored IL-10R function including IL-10-induced STAT3 activation and expression of suppressor of cytokine signaling 3. Thus, our results demonstrated that the mutations in IL-10R1 extracellular domain impair IL-10R1 activation rather than IL-10 binding, indicating these residues are important in IL-10 signal transduction through IL-10R1. The reconstitution data also confirmed the causality of the IL-10R1 mutations.

Inflammatory bowel disease and mutations affecting the interleukin-10 receptor.

BACKGROUND: The molecular cause of inflammatory bowel disease is largely unknown. METHODS: We performed genetic-linkage analysis and candidate-gene sequencing on samples from two unrelated consanguineous families with children who were affected by early-onset inflammatory bowel disease. We screened six additional patients with early-onset colitis for mutations in two candidate genes and carried out functional assays in patients' peripheral-blood mononuclear cells. We performed an allogeneic hematopoietic stem-cell transplantation in one patient. RESULTS: In four of nine patients with early-onset colitis, we identified three distinct homozygous mutations in genes IL10RA and IL10RB, encoding the IL10R1 and IL10R2 proteins, respectively, which form a heterotetramer to make up the interleukin-10 receptor. The mutations abrogate interleukin-10-induced signaling, as shown by deficient STAT3 (signal transducer and activator of transcription 3) phosphorylation on stimulation with interleukin-10. Consistent with this observation was the increased secretion of tumor necrosis factor alpha and other proinflammatory cytokines from peripheral-blood mononuclear cells from patients who were deficient in IL10R subunit proteins, suggesting that interleukin-10-dependent "negative feedback" regulation is disrupted in these cells. The allogeneic stem-cell transplantation performed in one patient was successful. CONCLUSIONS: Mutations in genes encoding the IL10R subunit proteins were found in patients with early-onset enterocolitis, involving hyperinflammatory immune responses in the intestine. Allogeneic stem-cell transplantation resulted in disease remission in one patient.

Structure-based decoupling of the pro- and anti-inflammatory functions of interleukin-10.

Interleukin-10 (IL-10) is an immunoregulatory cytokine with both anti-inflammatory and immunostimulatory properties and is frequently dysregulated in disease. We used a structure-based approach to deconvolute IL-10 pleiotropy by determining the structure of the IL-10 receptor (IL-10R) complex by cryo-electron microscopy at a resolution of 3.5 angstroms. The hexameric structure shows how IL-10 and IL-10Rα form a composite surface to engage the shared signaling receptor IL-10Rß, enabling the design of partial agonists. IL-10 variants with a range of IL-10Rß binding strengths uncovered substantial differences in response thresholds across immune cell populations, providing a means of manipulating IL-10 cell type selectivity. Some variants displayed myeloid-biased activity by suppressing macrophage activation without stimulating inflammatory CD8+ T cells, thereby uncoupling the major opposing functions of IL-10. These results provide a mechanistic blueprint for tuning the pleiotropic actions of IL-10.

Structure of IL-22 bound to its high-affinity IL-22R1 chain.

IL-22 is an IL-10 family cytokine that initiates innate immune responses against bacterial pathogens and contributes to immune disease. IL-22 biological activity is initiated by binding to a cell-surface complex composed of IL-22R1 and IL-10R2 receptor chains and further regulated by interactions with a soluble binding protein, IL-22BP, which shares sequence similarity with an extracellular region of IL-22R1 (sIL-22R1). IL-22R1 also pairs with the IL-20R2 chain to induce IL-20 and IL-24 signaling. To define the molecular basis of these diverse interactions, we have determined the structure of the IL-22/sIL-22R1 complex. The structure, combined with homology modeling and surface plasmon resonance studies, defines the molecular basis for the distinct affinities and specificities of IL-22 and IL-10 receptor chains that regulate cellular targeting and signal transduction to elicit effective immune responses.

The Structural Basis for Class II Cytokine Receptor Recognition by JAK1.

JAK1 is a member of the Janus kinase (JAK) family of non-receptor tyrosine kinases that are activated in response to cytokines and interferons. Here, we present two crystal structures of the human JAK1 FERM and SH2 domains bound to peptides derived from the class II cytokine receptors IFN-λ receptor 1 and IL-10 receptor 1 (IFNLR1 and IL10RA). These structures reveal an interaction site in the JAK1 FERM that accommodates the so-called "box1" membrane-proximal receptor peptide motif. Biophysical analysis of the JAK1-IFNLR1 interaction indicates that the receptor box1 is the primary driver of the JAK1 interaction, and identifies residues conserved among class II receptors as important for binding. In addition, we demonstrate that a second "box2" receptor motif further stabilizes the JAK1-IFNLR1 complex. Together, these data identify a conserved JAK binding site for receptor peptides and elucidate the mechanism by which class II cytokine receptors interact with JAK1.

Rational Structure-Based Rescaffolding Approach to De Novo Design of Interleukin 10 (IL-10) Receptor-1 Mimetics.

Tackling protein interfaces with small molecules capable of modulating protein-protein interactions remains a challenge in structure-based ligand design. Particularly arduous are cases in which the epitopes involved in molecular recognition have a non-structured and discontinuous nature. Here, the basic strategy of translating continuous binding epitopes into mimetic scaffolds cannot be applied, and other innovative approaches are therefore required. We present a structure-based rational approach involving the use of a regular expression syntax inspired in the well established PROSITE to define minimal descriptors of geometric and functional constraints signifying relevant functionalities for recognition in protein interfaces of non-continuous and unstructured nature. These descriptors feed a search engine that explores the currently available three-dimensional chemical space of the Protein Data Bank (PDB) in order to identify in a straightforward manner regular architectures containing the desired functionalities, which could be used as templates to guide the rational design of small natural-like scaffolds mimicking the targeted recognition site. The application of this rescaffolding strategy to the discovery of natural scaffolds incorporating a selection of functionalities of interleukin-10 receptor-1 (IL-10R1), which are relevant for its interaction with interleukin-10 (IL-10) has resulted in the de novo design of a new class of potent IL-10 peptidomimetic ligands.

Structural analysis of cytokines comprising the IL-10 family.

Interleukin-10 (IL-10) family of cytokines includes a number of its viral homologs and eight cellular cytokines (IL-19, IL-20, IL-22, IL-24, IL-26, IL-28A, IL-28B, and IL-29). The latter three proteins are also known as IFN-λ2, IFN-λ3, and IFN-λ1, and are recognized as type III (or λ) interferons. Most of the cellular homologs of IL-10 are monomeric in solution, whereas IL-10 and its viral homologs are intercalated dimers consisting of two helical bundle domains topologically similar to the monomeric members of the family. A classical four-helix bundle, a signature element of all helical cytokines, is always found as part of the domain of each member of the IL-10 family. The only crystal structures of these cytokine receptors that have been determined to date are for their extracellular domains (ECDs). Each ECD consists of two ß-sandwich domains connected in the middle by a linkage. Signal transduction occurs when a cytokine binds to its two appropriate receptor chains. IL-10 and its viral homologs use the same IL-10 receptor system, whereas the cellular homologs of IL-10 use their own receptors, which in some cases may overlap and be used in different pairwise combinations. The known structures of binary complexes allowed for marking of the receptor binding site, which always includes helix A, loop AB and helix F (IL-10 notations) on the side of a ligand, loops of the N-terminal and C-terminal domains directed toward the ligand, and the interdomain linkage of the ECD. An analysis of the published structures of both the binary and ternary complexes of all helical cytokines allowed for the generation of a model of the signaling complex of IL-10. The receptor binding site I of the high affinity receptor IL-10R1 is exactly the same as in the crystal structure of the binary IL-10/sIL-10R1 complex, whereas the receptor binding site II is located on the surface of the first and the third helices of the four-helix bundle. The receptor/receptor interface, or site III, is formed between the C-terminal domains of IL-10R1 and IL-10R2.