Immune Responses to Schistosome Egg Antigens

描述（由申请人提供）：该新提案集中于定义的Th 2 PAMP如何激活树突状细胞并使其成熟为DC 2，然后驱动适应性Th 2偏向的CD 4 + T细胞应答。当细胞表面受体连接病原体相关分子模式（PAMP）时，DC被激活。通常，PAMP激活APC以产生I型促炎介质，将DC驱动为DC 1型。DC 1将肽呈递给初始CD 4 + T细胞，诱导Th 1型应答（）。相比之下，人们对Th 2-PAMPs的分子性质或它们激活DC的机制知之甚少。乳糖-N-岩藻五糖III（LNFPIII）是第一个从蠕虫寄生虫中描述的Th 2 PAMP。该聚糖含有刘易斯X三糖。当作为载体上的缀合物（LNFP III-C）存在时，LNFP III在体内驱动Th 2应答并在体外将未成熟DC活化为功能性DC 2。该提议的总体目标是检查与Th 1 PAMP LPS相比，LNFPIII-C对DC和巨噬细胞的激活，以获得对Th 2 PAMP识别和激活这些细胞的生物学的理解。基于初步研究，我们假设LNFPIII-C需要Toll样受体4（TLR 4）和MD 2而不是TIR衔接蛋白MyD 88来驱动DC至DC 2。我们将通过从TLR 4和/或CD 14或MyD 88缺陷的小鼠中产生DC和巨噬细胞来测试这一点。我们还将使用用TLR 2、TLR 4、MD 2和CD 14单转染或双转染的HEK 293细胞，或使用作为TIR衔接蛋白TIRAP和MyD 88的显性阴性突变体的鼠巨噬细胞。我们假设MAP激酶ERK、NF-κ B和PGE 2是LNFP III-C激活DC所必需的，并且在LNFP III/刘易斯X激活的DC中存在差异表达的另外的信号分子和介质。我们将使用抑制剂和靶向微阵列分析来测试这些方面。我们相信LNFPIII-C/刘易斯X体外激活DC的模式代表了体内发生的情况，并将通过检查内源性脾DC的反应来测试这一点。LNFPIII-C活化巨噬细胞的能力是岩藻糖依赖性的，除了呈递LNFPIII的多个拷贝之外，载体分子没有明显的作用。然而，由于在糖脂和糖蛋白上发现了溶酶体刘易斯X，我们提出实验来测试糖脂尾的存在是否改变DC的LNFP III/刘易斯X活化。具体目标是：1）巨噬细胞的LNFPIII-C活化和DC 2的成熟是否需要TLR 4受体复合物和T1 R衔接蛋白？2)LNFPIII-C在巨噬细胞的活化和DC 2的成熟中需要或利用哪些信号分子和介质; 3）LNFPIII-C/刘易斯X是否会在体内驱动类似模式的DC活化。4)LNFP III/刘易斯X上脂质尾的存在是否改变了DC或巨噬细胞的识别或激活

DESCRIPTION (provided by applicant): This new proposal focuses on how a defined schistosome Th2 PAMP activates and matures dendritic cells to DC2s which then drive adaptive Th2 biased CD4+ T cell responses. DCs are activated when cell surface receptors ligate pathogen associated molecular patterns (PAMPs). In general, PAMPs activate APCs to produce Type-l, pro-inflammatory mediators, driving DCs to a DC1-type. DC1s present peptide to naive CD4+ T cells inducing a Th1-type response (). In contrast, little is known about the molecular nature of Th2-PAMPs or the mechanism by which they activate DCs. Lacto-N-fucopentaose III (LNFPIII) is the first Th2 PAMP described from helminth parasites. This glycan contains the Lewis X trisaccharide. When presented as a conjugate on a carrier (LNFPIII-C), LNFPIII drives Th2 responses in vivo and activates immature DCs to functional DC2s in vitro. The overall goal of this proposal is to examine activation of DCs and macrophages by LNFPIII-C compared to the Th1 PAMP LPS, to gain an understanding of the biology of recognition and activation of these cells by a schistosome Th2 PAMP. Based on preliminary studies we hypothesize that LNFPIII-C requires Toll Like Receptor 4 (TLR4) and MD2 but not the TIR adaptor protein MyD88 to to drive DCs to DC2s. We will test this by generating DCs and macrophages from mice deficient inTLR4 and/or CD14, or MyD88. We will also use HEK293 cells singly or doubly transfected with TLR2, TLR4, MD2 and CD14 or use murine macrophages that are dominant negative mutants for the TIR adaptor proteins TIRAP and MyD88. We hypothesize that the MAP kinase ERK, NF-kB and PGE2 are required for LNFPIII-C activation of DCs and that there are additional signaling molecules and mediators differentially expressed in LNFPIII/Lewis X activated DCs. We will test these aspects using inhibitors and targeted microarray analysis. We believe that the pattern of activation of DCs in vitro by LNFPIII-C/Lewis X is representative of what occurs in vivo and will test this by examining the responses of endogenous splenic DCs. The ability of LNFPIll-C to activate macrophages is fucose dependent, with no apparent role for carrier molecules other than to present multiple copies of LNFPIII. However, because schistosome Lewis X is found on glycolipids as well as glycoproteins we propose experiments to test whether the presence of a glycolipid tail alters LNFPIII/Lewis X activation of DCs. The specific aims are: 1) Does LNFPIII-C activation of macrophages and maturation of DC2s require the TLR4 receptor complex and the T1R adaptor proteins?; 2) What signaling molecules and mediators are required or utilized by LNFPIII-C in activation of macrophages and maturation of DC2s; 3) Will LNFPIII-C/Lewis X drive similar patterns of DC activation in vivo. 4) Does the presence of a lipid tail on LNFPIII/Lewis X alter DC or macrophage recognition or activation.