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.
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