Functional characterization of IL-40, a novel cytokine

新型细胞因子 IL-40 的功能表征

• 批准号: 8872593
• 负责人: Albert Zlotnik
• 金额: $ 23.18万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-02-01 至 2017-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8872593
• 关键词: Activated B-Lymphocyte; Affect; Amino Acids; B cell differentiation; B-Cell Lymphomas; B-Lymphocyte Subsets; B-Lymphocytes; Biological; Biological Assay; Biological Process; Biology; Bone Marrow; CD4 Positive T Lymphocytes; Cell Line; Cells; Cellular biology; Characteristics; Data; Databases; Defect; Exhibits; Family; Feces; Fetal Liver; GPR2 gene; Gene Expression; Genes; Genomics; Growth; Helper-Inducer T-Lymphocyte; Human; Human body; IL4 gene; IgA Deficiency; Immune; Immune system; Immunoglobulin A; Immunoglobulin Class Switching; Immunoglobulin Switch Recombination; Immunoglobulins; Immunohistochemistry; In Vitro; Interleukin-4; Interleukins; Knockout Mice; Lactation; Lead; Link; Lipopolysaccharides; Lymphoid Cell; Lymphoma; MHC Class II Genes; Mammary gland; Measures; Membrane; Mitogens; Molecular; Monoclonal Antibodies; Mus; Names; Organ; Peptide Signal Sequences; Peptides; Physiology; Population; Production; Proteins; RNA Interference; Recombinant Interleukins; Recombinant Proteins; Research; Role; Source; Stimulus; Structure of aggregated lymphoid follicle of small intestine; T-Lymphocyte; TNFRSF5 gene; TNFSF5 gene; Testing; Tissues; Transforming Growth Factor beta; Wild Type Mouse; anti-IgM; cytokine; human genome sequencing; in vivo; mammalian genome; microbiome; novel; protein expression; public health relevance; receptor; response; screening

 DESCRIPTION (provided by applicant): We have identified a novel secreted protein encoded by an uncharacterized gene (C17Orf99) that is specifically expressed in the fetal liver, bone marrow and activated B cells. The gene sequence predicts a ~27KDa secreted protein with a signal peptide and exhibits no homology to any known cytokine family. However, it is strongly induced upon activation in several B cell lines or in normal B cells activated with stimuli such as CD40L, IL-4 or Lipopolysaccharide (LPS). These characteristics (small secreted protein, expression restricted to activated lymphoid cells) suggest that this gene encodes a novel cytokine which we have named Interleukin-40. In this proposal, we aim to functionally characterize IL-40. We have cloned and expressed mouse and human IL-40, and will use these recombinant proteins to explore its biological functions. We have produced monoclonal antibodies against IL-40 peptides which we are currently screening with recombinant protein. We have also obtained an IL-40-/- mouse that exhibits abnormalities in the Peyer's patches and low levels of IgA in the mammary gland and feces. In Specific Aim 1, we will undertake the biological characterization of IL-40 in vitro by identifying the cellular sources of IL-40. We hypothesize that IL-40 is produced by certain B cell subsets. We will next determine the cells that produce IL-40 in the fetal liver and bone marrow. These results will help us understand its biological function in these organs. We hypothesize that IL-40 production is linked to the production of IL-4. We will therefore explore whether other strong IL-4 producing cells (like iNKT and Th2 CD4+ cells) also produce IL-40. IL4 is a B cell costimulatory factor that induces proliferation in combination with various B cell mitogens, and these are the same conditions that induce IL-40 production by B cells. We therefore hypothesize that IL-40 may be involved in some of the known biological activities of IL-4. We will test this by performing IL-4 driven proliferation and differentiation assays using either IL-40-/- or wild type (WT) mouse B cells. In Specific Aim 2, we will explore the physiology of IL-40 using an IL-40-/- mouse, which are viable and fertile. We will explore the Peyer's patches of these mice to explore the cellular changes that have taken place due to the lack of IL-40, and we will quantify the number of IgA-producing cells in the Peyer's patches of IL-40-/- mice. We will measure the expression of other genes that have been shown to influence IgA expression like CCL28 and its receptor CCR10, as well as TGFβ. IgA levels are linked to the gut microbiome, so we will analyze the gut microbiome of the IL-40-/- mouse for abnormalities. Finally, we will search for molecular defects that may affect the production of Immunoglobulin production including class switch recombination in the B cells of the IL-40-/- mouse. Through these specific aims we will open a new field of research, namely, the study of the biology of IL-40.

 描述（由申请人提供）：我们已经鉴定出一种由未表征的基因（C17Orf99）编码的新型分泌蛋白，该蛋白在胎儿肝脏、骨髓和活化的 B 细胞中特异性表达。该基因序列预测具有信号肽的约 27KDa 分泌蛋白，并且与任何已知的细胞因子家族没有同源性。然而，在几种 B 细胞系或用刺激激活的正常 B 细胞中，它会被强烈诱导，例如 CD40L、IL-4 或脂多糖 (LPS)。这些特征（小分泌蛋白，表达仅限于活化的淋巴细胞）表明该基因编码一种新的细胞因子，我们将其命名为 Interleukin-40。在本提案中，我们的目标是对 IL-40 进行功能表征。我们已经克隆并表达了小鼠和人IL-40，并将利用这些重组蛋白来探索其生物学功能。我们已经生产了针对 IL-40 肽的单克隆抗体，目前正在用重组蛋白对其进行筛选。我们还获得了一只 IL-40-/- 小鼠，其派尔氏淋巴结异常，乳腺和粪便中 IgA 水平低。在具体目标 1 中，我们将通过鉴定 IL-40 的细胞来源来进行 IL-40 的体外生物学表征。我们假设 IL-40 由某些 B 细胞亚群产生。接下来我们将确定胎儿肝脏和骨髓中产生 IL-40 的细胞。这些结果将帮助我们了解其在这些器官中的生物学功能。我们假设 IL-40 的产生与 IL-4 的产生相关。因此，我们将探讨其他强 IL-4 产生细胞（如 iNKT 和 Th2 CD4+ 细胞）是否也产生 IL-40。 IL4 是一种 B 细胞共刺激因子，与各种 B 细胞有丝分裂原结合诱导增殖，这些条件与诱导 B 细胞产生 IL-40 的条件相同。因此，我们假设 IL-40 可能参与 IL-4 的一些已知生物活性。我们将通过使用 IL-40-/- 或野生型 (WT) 小鼠 B 细胞进行 IL-4 驱动的增殖和分化测定来测试这一点。在具体目标 2 中，我们将使用具有活力和生育能力的 IL-40-/- 小鼠探索 IL-40 的生理学。我们将探索这些小鼠的派尔氏集结区，以探索由于缺乏IL-40而发生的细胞变化，并且我们将量化IL-40-/-小鼠的派尔氏集结区中产生IgA的细胞的数量。我们将测量已被证明影响 IgA 表达的其他基因的表达，例如 CCL28 及其受体 CCR10 以及 TGFβ。 IgA 水平与肠道微生物组相关，因此我们将分析 IL-40-/- 小鼠的肠道微生物组是否存在异常。最后，我们将寻找可能影响的分子缺陷 免疫球蛋白的生产，包括 IL-40-/- 小鼠 B 细胞中的类别转换重组。通过这些具体目标，我们将开辟一个新的研究领域，即IL-40的生物学研究。