Antigen Processing And Presentation In The Intestine

抗原在肠道中的加工和呈现

• 批准号: 8555836
• 负责人: BRIAN KELSALL
• 金额: $ 145.68万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8555836
• 关键词: Accounting; Address; Adjuvant; Adoptive Transfer; Affect; Agonist; Antigen Presentation; Antigen-Presenting Cells; Antigens; Apoptotic; Area; Autoimmune Diseases; Automobile Driving; B cell differentiation; Biological Preservation; Blood; CD4 Positive T Lymphocytes; CX3CL1 gene; Cell physiology; Cells; Colitis; Colon; Complex; Data; Defect; Dendritic Cells; Development; Dose; Drug Formulations; Enzyme-Linked Immunosorbent Assay; Epithelial Cells; Equilibrium; Flow Cytometry; Gene Expression; Gene Expression Profile; Genetic; Goals; Granulocyte-Macrophage Colony-Stimulating Factor; Greater sac of peritoneum; Growth Factor; HIV; Homeostasis; Human; ITGAM gene; ITGAX gene; Immune; Immune response; Immunity; Immunofluorescence Microscopy; Immunoglobulin A; Immunosuppression; In Situ; In Situ Hybridization; Infection; Infection prevention; Inflammation; Inflammatory; Inflammatory Bowel Diseases; Inflammatory disease of the intestine; Interferon Type II; Interferons; Interleukin-10; Interleukin-12; Interleukin-15; Intestines; Invaded; Knock-in Mouse; Laboratories; Lamina Propria; Macaca; Mediating; Methods; Microarray Analysis; Modeling; Molecular; Mucosal Immune Responses; Mus; Mutant Strains Mice; N Domain; Natural Immunity; Natural Killer Cells; Opportunistic Infections; Oral; Organ Transplantation; Organism; Outcome; Patients; Phenotype; Physiological; Play; Population; Predisposition; Process; Production; Proteins; Recruitment Activity; Regulation; Regulatory T-Lymphocyte; Reoviridae Infections; Reovirus Type 1; Reverse Transcriptase Polymerase Chain Reaction; Role; SIV; STAT5A gene; Structure of aggregated lymphoid follicle of small intestine; Surface; T cell differentiation; T cell response; T-Lymphocyte; Time; Tissues; Toxoplasmosis; Transplant Recipients; Treatment Efficacy; Vaccines; Viral Antigens; Virus; Virus Diseases; acquired immunity; antigen processing; base; cell type; commensal microbes; cytokine; dimer; improved; in vitro Assay; in vivo; interleukin-23; macrophage; migration; monocyte; mouse model; neutrophil; novel; novel strategies; oral tolerance; oral vaccine; pathogen; peripheral blood; prevent; repaired; research study; response; trafficking

This project focuses on how antigens are processed in the intestine of mice and presented by different populations of dendritic cells (DC) and macrophages influence immune responses in the intestine. While it is clear that the outcome of oral antigen exposure can be either positive, i.e., the development of mucosal IgA responses, and in some cases the induction of systemic immunity as well, or negative, i.e., the induction of oral tolerance, the details of why one or the other outcome occurs is complex and poorly understood. Furthermore, the normal intestinal immune response to symbiotic/commensal bacteria, which allows for one to tolerate these organisms without the onset of inflammation, is essential for immune homeostasis in the intestine, as a defect in this homeostasis results in inflammatory bowel disease. Furthermore, while it is known that the antigen formulation, the presence of adjuvants, and the antigen dose, as well as genetic factors, can affect mucosal immune responses, how these act together to influence immunity has never been established. Therefore, this project focuses on how immune responses are regulated in the intestine with a focus on the roles of dendritic cells and macrophages in this regulation, and on factors that control inflammatory functions of these cells. In prior studies we defined different antigen-presenting cell populations in the Peyer's patch (PP) and lamina propria and have detailed the surface phenotype, function, and migration of DCs in the PP using in situ immunofluorescence microscopy and in situ hybridization, flow cytometry of purified cells, and in vitro assays of cytokine production (ELISA and quantitative RT-PCR) and T cell differentiation. PP DCs have the unique capacity to induce the differentiation of T cells that produce high levels of IL-10, a cytokine important for the IgA B cell differentiation. These studies thus were some of the first to directly demonstrate that DCs from different tissues may be unique in their ability to induce tissue specific immunity. We also demonstrated that DCs in the subepithelial dome region of the PP process viral antigen from virally infected apoptotic epithelial cells following reovirus infection. Furthermore, we determined that clearance of lethal experimental infection with a model mucosal virus infection, type 1 reovirus, is dependent on type-1 interferon production in the PP, that type-1 interferon production by dendritic cells within the PP is a primary determinant of whether this mucosal pathogen survives and is disseminated to other tissues. In other studies, we localized and studied the function of plasmacytoid DCs (pDCs) from the Peyer's patch. Furthermore, we have performed studies in humans the ability of granulocyte-macrophage colony stimulating factor (GM-CSF), a growth factor that is commonly used to treat organ transplant patients to improve their production of neutrophil and monocyte after immunosuppression, to treat patients with inflammatory bowel disease. Treatment efficacy was correlated with changes in peripheral blood T cell production of IL-10, and sustained changes in the numbers of plasmacytoid DCs, cells known to drive non-pathogenic Th2 responses, suggesting that treatments that enhance the number of plasmacytoid DCs in the blood may have therapeutic efficacy in Th1-mediated autoimmune disorders. In FY2012, we have focused our efforts on two areas. First, we have defined sub-populations of macrophages and DCs in the mouse colon and are exploring their role in maintaining immune homeostasis in steady-state conditions and during inflammation in murine models of inflammatory bowel disease. We demonstrated four populations of cells based on surface markers that correlate with either a macrophage or DC phenotype, and have begun to understand their function in vivo. More specifically, we found that F4/80hi CX3CR1hi (CD11b+CD103-) cells account for 80% of mouse colonic lamina propria (cLP) MHC-IIhi cells. Both CD11c+ and CD11c- cells within this population were identified as MPs based on multiple criteria, including a MP transcriptome revealed by microarray analysis. These MPs constitutively released high levels of IL-10 at least partially in response to the microbiota via an MyD88-independent mechanism. In contrast, cells expressing low to intermediate levels of F4/80 and CX3CR1 were identified as DCs, based on phenotypic and functional analysis and comprise three separate CD11chi cell populations: CD103+CX3CR1-CD11b- DCs, CD103+CX3CR1-CD11b+ DCs and CD103-CX3CR1intCD11b+ DCs. In non-inflammatory conditions, Ly6Chi monocytes differentiated primarily into CD11c+, but not CD11c- MPs. In contrast, during colitis, Ly6Chi monocytes massively invaded the colon and differentiated into pro-inflammatory CD103-CX3CR1intCD11b+ DCs, which produced high levels of IL-12, IL-23, iNOS and TNFalpha. These findings demonstrated the dual capacity of Ly6Chi blood monocytes to differentiate into either regulatory MP or inflammatory DCs in the colon, and that the balance of these immunologically antagonistic cell types is dictated by micro-environmental conditions. These studies are important in that they delineate for the first time the precise definitions of macrophages and dendritic cells in the colon based on the use of a comprehensive array of surface markers, gene expression analysis, and development from defined circulating precursors. They also demonstrate the primary role played by peripheral blood monocytes in contributing to intestinal inflammation, and thus indicate that blocking the trafficking of this cell type to the inflamed colon could provide a novel strategy for treatment of inflammatory bowel disease. Second, we have addressed the role of type-1 interferons in the regulation of intestinal immunity and found an essential role for type-1 interferons in preventing abnormal inflammation in mouse models of inflammatory bowel disease. Therefore, type-1 interferons have essential roles both in prevention of infection by some, but not all, intestinal viruses, as well as in controlling abnormal intestinal inflammation. We are currently delineating mechanisms by which type-1 interferons control intestinal immune homeostasis. In collaborative studies we helped demonstrate and essential role for NK-cell- derived IFN-gamma in both inducing the loss of resident monocyte/macrophage populations and driving the differentiation of newly recruited monocytes into inflammatory monocyte-derived dendritic cells in the peritoneal cavity during Toxoplasma gondii infection in mice. In addition, we conducted experiments to show the importance of tetramerization of STAT5 in driving functional regulatory T cell responses. In these experiments, regulatory T cells from Stat5a-Stat5b double knock-in (DKI) N-domain mutant mice in which STAT5 proteins form dimers but not tetramers were not able to control colitis in an adoptive transfer model of inflammatory bowel disease. These studies were important as they provided the first data that IFN-gamma, in particular produced by NK-cells was essential for orchestrating both the cellular dynamics and cytokine production capacities of antigen-presenting cells during infection by T. gondii, an important human pathogen, and demonstrated an essential molecular role for a STAT tetramerization in regulating immunity. Finally, in ongoing collaborative efforts with Dr. Berzofskys laboratory, we helped perform studies to demonstrate that IL-15 and TLR agonists included in a vaccine formulation for SIV infection, the macaque model of HIV in humans, resulted in the preservation of CD4+ T cells in the intestine. SIV and HIV normally target intestinal CD4+ T cells in the intestine and their loss results in susceptibility to opportunistic infections, loss of intestinal barrier function, and SIV/HIV progression.

该项目重点研究抗原在小鼠肠道中的加工以及不同群体的树突状细胞 (DC) 和巨噬细胞的呈递如何影响肠道的免疫反应。虽然很明显，口服抗原暴露的结果可以是阳性的，即产生粘膜 IgA 反应，并且在某些情况下还诱导全身免疫，也可以是阴性的，即诱导口服耐受，但为什么发生一种或另一种结果的细节是复杂的且知之甚少。 此外，对共生/共生细菌的正常肠道免疫反应使得人们能够耐受这些微生物而不发生炎症，这对于肠道内的免疫稳态至关重要，因为这种稳态的缺陷会导致炎症性肠病。 此外，虽然已知抗原配方、佐剂的存在、抗原剂量以及遗传因素可以影响粘膜免疫反应，但这些因素如何共同作用以影响免疫尚未确定。 因此，该项目重点研究肠道中免疫反应的调节方式，重点关注树突状细胞和巨噬细胞在这种调节中的作用，以及控制这些细胞炎症功能的因素。 在之前的研究中，我们定义了派尔氏淋巴结 (PP) 和固有层中不同的抗原呈递细胞群，并使用原位免疫荧光显微镜和原位杂交、纯化细胞的流式细胞术以及细胞因子产生（ELISA 和定量 RT-PCR）和 T 细胞的体外测定，详细介绍了 PP 中 DC 的表面表型、功能和迁移。 差异化。 PP DC 具有诱导 T 细胞分化的独特能力，可产生高水平的 IL-10（一种对 IgA B 细胞分化很重要的细胞因子）。因此，这些研究首次直接证明来自不同组织的树突状细胞在诱导组织特异性免疫方面可能是独特的。 我们还证明，呼肠孤病毒感染后，PP 上皮下圆顶区域的 DC 可以处理来自病毒感染的凋亡上皮细胞的病毒抗原。此外，我们确定，模型粘膜病毒感染（1型呼肠孤病毒）的致命实验感染的清除取决于PP中1型干扰素的产生，PP内树突状细胞产生的1型干扰素是该粘膜病原体是否存活并传播到其他组织的主要决定因素。在其他研究中，我们定位并研究了派尔氏淋巴结中的浆细胞样 DC (pDC) 的功能。 此外，我们还对人类进行了粒细胞巨噬细胞集落刺激因子（GM-CSF）治疗炎症性肠病患者的研究，该因子是一种常用于治疗器官移植患者的生长因子，可在免疫抑制后提高其中性粒细胞和单核细胞的产生。治疗效果与外周血 T 细胞产生 IL-10 的变化以及浆细胞样 DC（已知驱动非致病性 Th2 反应的细胞）数量的持续变化相关，这表明增加血液中浆细胞样 DC 数量的治疗可能对 Th1 介导的自身免疫性疾病具有治疗效果。 2012财年，我们重点抓好两个方面的工作。 首先，我们定义了小鼠结肠中的巨噬细胞和 DC 亚群，并正在探索它们在炎症性肠病小鼠模型的稳态条件下和炎症期间维持免疫稳态中的作用。 我们基于与巨噬细胞或 DC 表型相关的表面标记展示了四个细胞群，并已开始了解它们的体内功能。 更具体地说，我们发现 F4/80hi CX3CR1hi (CD11b+CD103-) 细胞占小鼠结肠固有层 (cLP) MHC-IIhi 细胞的 80%。根据多种标准，包括通过微阵列分析揭示的 MP 转录组，该群体中的 CD11c+ 和 CD11c- 细胞均被鉴定为 MP。这些 MP 持续释放高水平的 IL-10，至少部分是通过 MyD88 独立机制对微生物群做出反应。相反，基于表型和功能分析，表达低至中等水平的 F4/80 和 CX3CR1 的细胞被鉴定为 DC，并包含三个独立的 CD11chi 细胞群：CD103+CX3CR1-CD11b- DC、CD103+CX3CR1-CD11b+ DC 和 CD103-CX3CR1intCD11b+ DC。在非炎症条件下，Ly6Chi 单核细胞主要分化为 CD11c+，而不是 CD11c- MP。相反，在结肠炎期间，Ly6Chi单核细胞大量侵入结肠并分化为促炎性CD103-CX3CR1intCD11b+ DC，产生高水平的IL-12、IL-23、iNOS和TNFα。这些发现证明了 Ly6Chi 血单核细胞在结肠中分化为调节性 MP 或炎症 DC 的双重能力，并且这些免疫拮抗细胞类型的平衡由微环境条件决定。 这些研究很重要，因为它们基于使用一系列全面的表面标记、基因表达分析和确定的循环前体细胞的发育，首次描述了结肠中巨噬细胞和树突状细胞的精确定义。 他们还证明了外周血单核细胞在导致肠道炎症中发挥的主要作用，因此表明阻止这种细胞类型向发炎结肠的运输可以为治疗炎症性肠病提供一种新策略。 其次，我们研究了 1 型干扰素在肠道免疫调节中的作用，并发现 1 型干扰素在预防炎症性肠病小鼠模型中的异常炎症中具有重要作用。 因此，1 型干扰素在预防某些（但不是全部）肠道病毒感染以及控制异常肠道炎症方面具有重要作用。 我们目前正在描绘 1 型干扰素控制肠道免疫稳态的机制。 在合作研究中，我们帮助证明了 NK 细胞衍生的 IFN-γ 在小鼠弓形虫感染期间诱导驻留单核细胞/巨噬细胞群的损失以及驱动新招募的单核细胞分化为腹腔内炎性单核细胞衍生树突状细胞的重要作用。 此外，我们还进行了实验来证明 STAT5 四聚化在驱动功能性调节性 T 细胞反应中的重要性。 在这些实验中，来自 Stat5a-Stat5b 双敲入 (DKI) N 结构域突变小鼠的调节性 T 细胞（其中 STAT5 蛋白形成二聚体而不是四聚体）无法在炎症性肠病过继转移模型中控制结肠炎。这些研究非常重要，因为它们提供了第一批数据，表明IFN-γ，特别是由NK细胞产生的IFN-γ，对于在弓形虫（一种重要的人类病原体）感染期间协调细胞动力学和抗原呈递细胞的细胞因子产生能力至关重要，并证明了STAT四聚化在调节免疫中的重要分子作用。 最后，在与 Berzofsky 博士实验室持续合作的过程中，我们帮助进行了研究，证明 SIV 感染（人类 HIV 猕猴模型）疫苗配方中包含的 IL-15 和 TLR 激动剂可导致肠道中 CD4+ T 细胞的保存。 SIV 和 HIV 通常以肠道中的肠道 CD4+ T 细胞为目标，它们的丢失会导致机会性感染的易感性、肠道屏障功能的丧失以及 SIV/HIV 的进展。