Antigen Processing And Presentation In The Intestine

抗原在肠道中的加工和呈现

• 批准号: 8745368
• 负责人: BRIAN KELSALL
• 金额: $ 123.56万
• 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/8745368
• 关键词: 5 year old; Address; Adjuvant; Adoptive Transfer; Affect; Antibody Formation; Antigen Presentation; Antigen-Presenting Cells; Antigens; Apoptosis; Apoptotic; B cell differentiation; C57BL/10 Mouse; C57BL/6 Mouse; CD4 Positive T Lymphocytes; Cell Count; Cell physiology; Cells; Child; Chronic; Colitis; Colon; Complex; Defect; Dendritic Cells; Development; Disease; Dose; Drug Formulations; Enzyme-Linked Immunosorbent Assay; Epithelial Cells; Equilibrium; Eye diseases; Flow Cytometry; Gene Expression Profiling; Generations; Genes; Genetic; Genetic Polymorphism; Goals; Granulocyte-Macrophage Colony-Stimulating Factor; Homeostasis; ITGAX gene; Immune; Immune response; Immunity; Immunofluorescence Microscopy; Immunoglobulin A; In Situ; In Situ Hybridization; Infection; Infection prevention; Inflammation; Inflammatory; Inflammatory Bowel Diseases; Inflammatory disease of the intestine; Interferon Type II; Interferons; Interleukin-1; Interleukin-10; Interleukin-12; Intestines; Invaded; Laboratories; Lamina Propria; Link; Lymphoid Tissue; Methods; Modeling; Molecular; Mucosal Immune Responses; Mus; Natural Immunity; Oral; Organism; Outcome; Pathogenesis; Patients; Phenotype; Physiological; Play; Population; Predisposition; Process; Production; Proliferating; Regulation; Regulatory T-Lymphocyte; Reoviridae Infections; Reovirus Type 1; Reverse Transcriptase Polymerase Chain Reaction; Role; Rotavirus; Rotavirus Infections; Severities; Signal Pathway; Signal Transduction; Site; Structure of aggregated lymphoid follicle of small intestine; Surface; T cell differentiation; T-Lymphocyte; Time; Tissues; Viral; Viral Antigens; Virus; Virus Diseases; acquired immunity; antigen processing; base; cell type; commensal microbes; cytokine; human disease; in vitro Assay; in vivo; inhibitor/antagonist; interleukin-12 subunit p40; interleukin-23; killings; macrophage; migration; monocyte; mouse model; novel; oral infection; oral tolerance; oral vaccine; pathogen; prevent; repaired; response; skin disorder

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. Furthermore, 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 (MP) or DC phenotype, and have begun to understand their function in vivo. Cells defined to be MPs constitutively released high levels of IL-10 at least partially in response to the microbiota via an MyD88-independent mechanism. In contrast, cells identified as DCs comprise three separate cell populations. 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 DC/macropahges. 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 delineated 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. In 2013, we have extended our studies of 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. We demonstrated that type 1 interferon signaling on non- T cells in the adoptive transfer model of colitis helps prevent inflammation via a mechanism that involves the induction of IL-10 production and the suppression of IFNbeta function, at least partially by enhancing the production IL-1RA, a natural inhibitor of IL-1 activity. 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. This opens the possibility of looking further into the functional effects of type-1 interferon signaling pathways in the pathogenesis of IBD and in re-addressing therapy with type-1 interferons in selected patients. In separate studies, we furthered our understanding of intestinal virus infection by understanding the role of PPs in the generation of IgA to intestinal rotavirus infection. Rotavirus is an important human disease that kills an estimated 450,000 children under the age of 5 years. We demonstrated that PPs are a primary initial site for rotavirus infection in mice that PP dendritic cells capture viral antigens very similar to what we had shown with the related type-1 reovirus, and that lymphoid tissues, including PPs and MLNs are absolutely required for a primary IgA responses to rotavirus that then results in viral clearance. Importantly, systemic antibody responses were effective in clearing systemically disseminated virus after oral infection, but were insufficient for the clearance of intestinal virus. This was the first time that the importance of IgA generated in local lymphoid tissues has been shown to directly result in viral clearance, e.g., that the lack of intestinal IgA responses resulted in chronic rotavirus infection. We also addressed a longstanding issue in the laboratory, the enhanced susceptibility of RAG-deficient mice on the C57BL/10 background to inflammatory disease following adoptive transfer of nave CD4+ T cells. In prior studies, we have shown that RAG-deficient C57BL/10 mice have a more rapid progression and increased severity of colitis, as well as develop inflammatory skin and eye disease in particular environmental conditions. We found that adoptively transferred T cells proliferate at an increased rate, and have a defect in the de novo induction of CD4+Foxp3+ regulatory T cells in the MLNs of RAG-deficient C57BL/10 compared to in the MLNs of RAG-deficient C57BL/6 mice. We also demonstrated that this skewed differentiation was not likely due to differences in bacterial flora, but was likely dependent on enhanced IL-23 and IL-12 production by DCs from C57BL/10 mice following adoptive transfer. These studies linked prior studies showing a genetic propensity of C57BL/10 mice to produce higher levels of IL-12 and IL-23 (due to a polymorphism in the IL-12p40 gene) to those showing an enhanced susceptibility to colitis induction in this mouse model; and described one mechanism by which this propensity enhances disease. In collaborative studies with Warren Leonards laboratory, we demonstrated the functional consequences of controlling dendritic cell apoptosis by IL-21 and GM-CSF, by showing enhanced inflammation of IL-21-deficient RAG-/- mice to T cell transfer colitis; that was associated with increased dendritic cell numbers, as well as increased IFN-gamma production T cells. These studies showed that IL-21 can enhance and GM-CSF can reduce apoptosis of conventional dendritic cells that has consequences for the development of intestinal inflammation.

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，但详细信息为什么会出现一种或另一种结果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、流式细胞术详细介绍了 PP 中 DC 的表面表型、功能和迁移。纯化的细胞，以及细胞因子产生的体外测定（ELISA 和定量 RT-PCR）和 T 细胞分化。 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 型干扰素是这种粘膜病原体是否存活并传播到其他组织。 此外，我们已经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 (MP) 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。 被定义为 MP 的细胞至少部分通过 MyD88 独立机制响应微生物群而持续释放高水平的 IL-10。相反，被鉴定为 DC 的细胞包含三个独立的细胞群。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DC/巨噬细胞。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。 2013年，我们对1型干扰素在肠道免疫调节中的作用进行了扩展研究，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方面具有重要作用。 我们证明，在结肠炎的过继转移模型中，非 T 细胞上的 1 型干扰素信号传导有助于通过一种涉及诱导 IL-10 产生和抑制 IFNβ 功能的机制来预防炎症，至少部分是通过增强 IL-10 的产生来预防炎症。 1RA，IL-1 活性的天然抑制剂。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。 这为进一步研究 1 型干扰素信号通路在 IBD 发病机制中的功能作用以及重新解决选定患者的 1 型干扰素治疗提供了可能性。 在单独的研究中，我们通过了解 PP 在肠道轮状病毒感染 IgA 生成中的作用，进一步加深了我们对肠道病毒感染的了解。 轮状病毒是一种重要的人类疾病，估计导致 450,000 名 5 岁以下儿童死亡。 我们证明，PP 是小鼠轮状病毒感染的主要起始位点，PP 树突状细胞捕获的病毒抗原与我们在相关 1 型呼肠孤病毒中所显示的非常相似，并且包括 PP 和 MLN 在内的淋巴组织对于轮状病毒感染绝对是必需的。对轮状病毒的初级 IgA 反应，然后导致病毒清除。 重要的是，全身抗体反应可有效清除口腔感染后全身传播的病毒，但不足以清除肠道病毒。这是第一次证明局部淋巴组织中产生的 IgA 的重要性直接导致病毒清除，例如肠道 IgA 反应的缺乏导致慢性轮状病毒感染。 我们还解决了实验室中长期存在的问题，即 C57BL/10 背景的 RAG 缺陷小鼠在过继转移初始 CD4+ T 细胞后对炎症性疾病的易感性增强。 在之前的研究中，我们已经表明，RAG 缺陷的 C57BL/10 小鼠的结肠炎进展更快，严重程度更高，并且在特定环境条件下会出现炎症性皮肤和眼部疾病。 我们发现，与 RAG 缺陷的 C57BL/ 的 MLN 相比，过继转移的 T 细胞增殖速度加快，并且在 RAG 缺陷的 C57BL/10 的 MLN 中从头诱导 CD4+Foxp3+ 调节性 T 细胞存在缺陷。 6只老鼠。我们还证明，这种倾斜的分化不太可能是由于细菌菌群的差异，而是可能依赖于过继转移后来自 C57BL/10 小鼠的 DC 产生的 IL-23 和 IL-12 的增强。 这些研究将先前的研究与显示 C57BL/10 小鼠产生较高水平 IL-12 和 IL-23（由于 IL-12p40 基因多态性）的遗传倾向与该小鼠对结肠炎诱导的易感性增强的研究联系起来模型;并描述了这种倾向加剧疾病的一种机制。 在与 Warren Leonards 实验室的合作研究中，我们通过显示 IL-21 缺陷的 RAG-/- 小鼠对 T 细胞转移性结肠炎的炎症增强，证明了 IL-21 和 GM-CSF 控制树突状细胞凋亡的功能后果；这与树突状细胞数量的增加以及产生 IFN-γ 的 T 细胞的增加有关。这些研究表明，IL-21 可以增强传统树突状细胞的凋亡，而 GM-CSF 可以减少其凋亡，从而导致肠道炎症的发生。