Pathways regulating plasmacytoid dendritic cells in Peyers Patches

描述（由申请人提供）：浆细胞样树突状细胞（pDCs）是骨髓和淋巴器官中主要的I型干扰素（IFN）产生细胞，被认为在抗病毒免疫中起关键作用。通过toll样受体（TLRs）激活后，pDCs分泌IFN并成熟到抗原呈递状态，在这种状态下，它们能够引发适应性免疫反应，包括CD4+ T细胞极化。pDC的发育途径是通过一个共同的DC祖细胞（CDP），它可以产生pDC和传统的DC。PDC的产生依赖于受体酪氨酸激酶Flt3和转录因子STAT3和E2-2的信号传导。在肠道Peyer's Patches （PP）中发现了一个pDC亚群，在TLR触发后不能产生I型ifn。这个亚群与口服耐受有关；然而，PP - pDCs在引发适应性免疫反应中的发育起源和作用在很大程度上是未知的。我们发现PP pDC的积累需要通过I型ifn和STAT1进行细胞自主信号传导，这表明与BM或脾pDC相比，PP pDC具有不同的发育或调节途径。此外，我们发现PP中pDCs与il -17分泌CD4+ T （Th17）细胞相关。我们假设PP pDCs与定位于BM和脾脏的pDCs来自相同的发育途径（即通过响应Flt3-STAT3信号的CDPs），然而，肠道微环境中的调节可能具有不同的功能属性，包括诱导Th17细胞生成的能力。我们将通过研究调控PPs内pDCs积累的途径来检验这一假设（目的1）。我们将评估纯化的BM祖细胞（如CDPs）是否在致命辐照小鼠或使用缺乏PP pDCs的Ifnar-/-动物的新型移植模型中重建PP pDCs。PP pDC生成所需的分子途径将通过移植实验来检验，这些移植实验来自稳态或对诱导BM和脾脏pDC生成的细胞因子（即Flt3L， IFN-1）的BM祖细胞，这些祖细胞来自Stat1-/-、stat3缺陷、Ifnar-/-或Tcf4-/- （e2 -2缺陷）小鼠。在Aim 2中，我们将使用纯化的PP、BM和脾pDCs以及原生CD4+ T细胞进行体外CD4+ T细胞极化实验，确定PP pDCs在诱导CD4+ T细胞反应方面是否在功能上与BM或脾pDCs不同。此外，在Aim 1中，PPs内的CD4+ T细胞群将与PP pDC重构相关，以进一步研究PP pDC与Th细胞反应之间的关系，并通过测量候选细胞因子的产生、激活和抑制分子的表达以及转录谱，将PP pDC的激活谱与BM和脾脏pDC进行比较。该项目的成功完成将提供PP - pDC发育途径的机制视图，因此可能有助于开发与胃肠道炎症相关疾病的免疫应答重定向治疗应用。

DESCRIPTION (provided by applicant): Plasmacytoid dendritic cells (pDCs) are principal type I interferon(IFN)-producing cells in bone marrow and lymphoid organs that are thought to play critical roles in anti-viral immunity. Upon activation through Toll-like receptors (TLRs), pDCs secrete IFN and mature to an antigen-presenting state in which they are able to elicit adaptive immune responses including CD4+ T cell polarization. The pDC developmental pathway proceeds through a common DC progenitor (CDP), which generates both pDCs and conventional DCs. PDC production depends on signaling by the receptor tyrosine kinase Flt3 and the transcription factors STAT3 and E2-2. A pDC subset was identified within Peyer's Patches (PP) in the gut that is unable to produce type I IFNs upon TLR triggering. This subset is implicated in oral tolerance; however the developmental origin and role for PP pDCs in eliciting adaptive immune responses are largely unknown. We found that PP pDC accrual required cell autonomous signaling via type I IFNs and STAT1, suggesting a distinct developmental or conditioning pathway compared to BM or splenic pDCs. Furthermore, we found that PP pDCs correlate with IL-17-secreting CD4+ T (Th17) cells in PPs. We hypothesize that PP pDCs arise from the same developmental pathway as pDCs localized in BM and spleen (i.e., via CDPs in response to Flt3-STAT3 signaling), however conditioning within the gut microenvironment may render distinct functional attributes including the ability to induce Th17 cell generation. We will test this hypothesis by investigating the pathways that regulate the accrual of pDCs within PPs (Aim 1). We will assess whether purified BM progenitors such as CDPs reconstitute PP pDCs in lethally irradiated mice or in a novel transplant model using Ifnar-/- animals that lack PP pDCs. Molecular pathways required for PP pDC generation will be examined by transplant experiments with BM progenitors from Stat1-/-, STAT3-deficient, Ifnar-/- or Tcf4-/- (E2-2-deficient) mice in steady state or in response to cytokines that elicit BM and spleen pDC generation (i.e., Flt3L, IFN-1). In Aim 2, we will determine whether PP pDCs are functionally distinct from BM or splenic pDCs in terms of eliciting CD4+ T cell responses, using in vitro CD4+ T cell polarization assays with purified PP, BM and splenic pDCs and naove CD4+ T cells. In addition, CD4+ T cell populations within PPs will be correlated with PP pDC reconstitution in Aim 1 to further examine the relationship between PP pDCs and Th cell responses, and the activation profiles of PP pDCs will be compared to BM and spleen pDCs by measuring candidate cytokine production, activating and inhibitory molecule expression and transcriptional profiles. Successful completion of this project will provide a mechanistic view of the PP pDC developmental pathway, and thus may contribute to development of therapeutic applications that enable redirection of the immune response in diseases associated with gastrointestinal inflammation.