Regulation of Organized Intestinal Lymphoid Tissues by TRANCE/RANKL

描述（由申请方提供）：派尔集合淋巴结（PP）和分离淋巴滤泡（ILF）是肠道中的组织化粘膜淋巴组织，作为肠腔中抗原免疫应答的诱导部位。我们发现，RANKL（也称为TRANCE和TNFSF 11）是TNF超家族的一个成员，以前被证明在淋巴结发育、胸腺髓质上皮细胞分化、破骨细胞分化和乳腺泌乳中具有强制性作用，也存在于ILF和PP的基质细胞上。这些RANKL表达基质细胞集中在上皮下圆顶区域，与卵泡相关上皮紧密贴壁。先前将RANKL敲除小鼠的PP表征为小于野生型小鼠的PP，但未报告其他表型异常。我们发现RANKL基因敲除小鼠的PP在M细胞发育方面存在较大缺陷，M细胞是能够快速摄取颗粒抗原并将其递送至上皮内和上皮下发现的抗原呈递细胞（APC）的特化上皮细胞。平均而言，通过UEA-1凝集素染色检测到的RANKL敲除小鼠小肠中的M细胞比野生型对照小鼠少74倍。RANKL敲除小鼠中UEA-1+ M细胞的丢失与RANKL敲除PP将直径200 nm的荧光珠从肠腔转运至位于PP上皮下圆顶中的APC的能力几乎完全丧失相关。重组RANKL治疗7天可以挽救RANKL缺失小鼠中的M细胞发育。用中和抗RANKL抗体处理野生型小鼠重现了在RANKL缺失小鼠中观察到的M细胞损失。指导拟议实验的中心假设是RANKL通过其受体RANK作用于上皮细胞，是正常M细胞分化和功能所需的关键信号传导途径。虽然M细胞在30多年前首次在兔阑尾中被描述，并且已知在粘膜组织中颗粒抗原的摄取中具有核心作用，同时也被病原体用作入口，但这些细胞的组织发生的细节在很大程度上仍然是一个谜。该提案的第一个目的是确定基质细胞是否是参与FAE中M细胞分化诱导的RANKL的主要来源，以及RANKL诱导M细胞分化的作用是否仅限于圆顶相关隐窝中的干细胞。第二个目的是确定M细胞的功能活性是否可以通过用外源性RANKL和中和抗RANKL抗体操纵RANKL- RANK信号传导来改变。第三个目的是确定骨保护素（OPG）可溶性诱饵受体的局部表达和RANKL对肠DC和巨噬细胞的诱导作用如何影响基质细胞RANKL对表达RANK的肠细胞的作用。RANKL-RANK通路如何支持肠道免疫系统处理抗原的途径，促进肠腔中通常遇到的抗原耐受性的发展，这一机制的见解可能有助于开发口服疫苗接种策略和治疗人类炎症性肠病。 公共卫生相关性：本研究的目标是确定一种称为RANKL（RANK配体）和RANK（其受体）的蛋白质在小肠和大肠中含有有组织淋巴细胞的结构的建立和正常功能中的作用。这些淋巴样结构如派尔集合淋巴结通常有助于保护宿主免受病原体的侵害，并防止对肠道微环境中通常遇到的抗原的不适当的免疫应答。这些研究预计将有助于现有的知识如何在肠道免疫系统的特定失衡可能导致各种形式的人类炎症性肠病。

DESCRIPTION (provided by applicant): Peyer's patches (PP) and isolated lymphoid follicles (ILF) are organized mucosal lymphoid tissues in the intestine that serve as inductive sites for immune responses to antigens in the intestinal lumen. We discovered that RANKL (also known as TRANCE and TNFSF11), a member of the TNF superfamily previously shown to have obligatory roles in the development of lymph nodes, the differentiation of medullary thymic epithelial cells, the differentiation of osteoclasts, and mammary gland lactation, is also present on stromal cells in ILF and PP. These RANKL-expressing stromal cells are concentrated in the subepithelial dome area in close apposition to the follicle-associated epithelium. PP in RANKL null mice were previously characterized as smaller than those in wild type mice, but additional phenotypic abnormalities were not reported. We find that the PP of RANKL null mice have a large deficit in the development of M cells, specialized epithelial cells capable of rapid uptake and delivery of particulate antigens into antigen-presenting cells (APC) found within and beneath the epithelium. On average, the small intestine of RANKL null mice has 74-fold fewer M cells detected by staining with the UEA-1 lectin than wild type control mice. The loss of UEA-1+ M cells in RANKL null mice correlates with a nearly complete loss in the ability of RANKL null PP to translocate 200 nm diameter fluorescent beads from the intestinal lumen into APC located in the PP subepithelial dome. M cell development in RANKL null mice can be rescued by treatment with recombinant RANKL for 7 days. Treatment of wild type mice with neutralizing anti- RANKL antibodies reproduces the loss of M cells observed in RANKL null mice. The central hypothesis guiding the proposed experiments is that RANKL acting through its receptor RANK on epithelial cells is a critical signaling pathway required for normal M cell differentiation and function. While M cells were first described in the rabbit appendix over 30 years ago and are known to have a central role in uptake of particulate antigens in mucosal tissues while also being exploited as a portal of entry by pathogens, the details of the histogenesis of these cells have largely remained a mystery. The first aim of the proposal is to determine whether stromal cells are the major source of RANKL involved in induction of M cell differentiation in the FAE and whether the action of RANKL to induce M cell differentiation is restricted to stem cells in the dome-associated crypts. The second aim is to determine whether the functional activity of M cells can be altered by manipulation of RANKL- RANK signaling with exogenous RANKL and neutralizing anti-RANKL antibody. The third aim is to determine how local expression of the osteoprotegerin (OPG) soluble decoy receptor and RANKL-induced effects on intestinal DC and macrophages influence the effects of stromal cell RANKL on RANK-expressing enterocytes. Mechanistic insights into how the RANKL-RANK pathway supports pathways of antigen handling by the gut immune system that foster development of tolerance of antigens normally encountered in the gut lumen may be useful in developing oral vaccination strategies and in the treatment of human inflammatory bowel disease. PUBLIC HEALTH RELEVANCE: The goal of this research is to identify the role of a protein known as RANKL (RANK ligand) and RANK (its receptor) in the establishment and normal functioning of organized lymphocyte containing structures in the small and large intestine. These lymphoid structures such as Peyer's patches normally contribute to protecting the host from pathogens and preventing inappropriate immune responses to antigens normally encountered in the intestinal microenvironment. These studies are expected to contribute to existing knowledge of how specific imbalances in the intestinal immune system can lead to various forms of human inflammatory bowel disease.