Pathways regulating plasmacytoid dendritic cells in Peyers Patches

描述(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(干扰素)产生细胞，被认为在抗病毒免疫中发挥关键作用。PDCs通过Toll样受体(TLRs)激活后，分泌干扰素，并成熟到抗原呈递状态，在此状态下能够诱导包括CD4+T细胞极化在内的适应性免疫反应。PDC的发育途径通过一个共同的DC前体(CDP)进行，它既产生PDCs又产生传统的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。在肠道的Peyer‘s Patches(PP)中发现了一个PDC子集，该子集在TLR触发时不能产生I型IFN。这个亚群与口服耐受有关；然而，PP pDC的发育起源和在诱导适应性免疫反应中的作用在很大程度上尚不清楚。我们发现，PP PDC的累加需要通过I型IFN和STAT1的细胞自主信号，这表明与BM或脾PDCs相比，有一条不同的发育或条件通路。此外，我们还发现PP pDC与PPS中分泌IL-17的CD4+T(Th17)细胞相关。我们假设PP PDCs与PDCs起源于相同的发育途径，定位于骨髓和脾(即通过CDPs响应Flt3-STAT3信号)，然而肠道微环境中的条件作用可能产生不同的功能属性，包括诱导Th17细胞生成的能力。我们将通过调查在PPS(目标1)内调节pDC累积的途径来检验这一假说。我们将评估纯化的骨髓祖细胞，如CDPs，是否在致死性照射的小鼠中重建PP PDCs，或者在使用缺乏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STAT1/-、STAT3缺失、Ifnar-/-或TCF4-/-(E2-2缺失)小鼠的BM前体细胞在稳定状态或对诱导BM和脾PDC生成的细胞因子(即Flt3L、干扰素-1)的反应来检测。在目标2中，我们将利用纯化的PP、BM、脾PDCs和NAOVE CD4+T细胞进行体外CD4+T细胞极化试验，以确定PP PDCs在诱导CD4+T细胞反应方面是否与骨髓或脾PDCs的功能不同。此外，在目标1中，PPS内的CD4+T细胞群将与PP PDC重建相关，以进一步研究PP pDC与Th细胞反应之间的关系，并将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来比较PP pDC与骨髓和脾pDC的激活情况。该项目的成功完成将提供PP PDC发育途径的机械观点，从而可能有助于开发治疗应用，使与胃肠道炎症相关的疾病的免疫反应能够重新定向。

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.