Identification of Target Molecules for Immune Proteins and its Biological Function
Date Issued
2015
Date
2015
Author(s)
Lin, Chih-Ru
Abstract
The immune system mounts robust immune response against invading microorganisms while retaining the capacity to keep natural autoimmunity under control. This thesis contains two parts to study the immune modulation through different manners including a novel herbal derived immunostimulator “maltoheptaose” and the identification of CD69 receptor-ligand mediated immune regulation. Wheatgrass is one of the most widely used health foods, but its functional components and mechanisms remain unexplored. The wheatgrass-derived oligosaccharides (WG-PS3) were previously isolated and found to induce CD69 and TH1 cytokine expression in human peripheral blood mononuclear cells (hPBMC). In particular, WG-PS3 directly activated the purified monocytes by inducing the expression of CD69, CD80, CD86, IL-12, and TNF-α but affected NK and T cells only in the presence of monocytes. After further purification and structural analysis, maltoheptaose was identified from WG-PS3 as an immunomodulator. Herein, maltoheptaose was demonstrated to activate monocytes via Toll-like receptor 2 (TLR-2) signaling, which was discovered by pretreatment of blocking antibodies against Toll-like receptors (TLRs) and by use of alkyl diazirine-based photo-affinity probes and click chemistry. The TLR-2 downstream signaling NF-κB activation was also observed in maltoheptaose-treated THP-1 cells, as determined by NF-κB-driven luciferase activity. This study is the first to reveal the immunostimulatory component of wheatgrass with well defined molecular structures and mechanisms. The second part describes the identification of endogenous ligands for the CD69 receptor and its biological function. CD69 is a leukocyte activation receptor that functions as the regulatory factor in the maintenance of immune homeostasis and is positively selected on the surface of activated regulatory T cell (Treg) lineage. However, whether and how CD69 is involved in the generation of Treg cells has yet to be elucidated. Here we show that CD69 per se is not sufficient to support the switch of the CD4+ naive T cells into Tregs in the absence of antigen-presenting cells, suggesting that additional ligand-receptor interaction is likely required for the stimulation. Using immunoprecipitation and mass spectrometry, we identified the S100A8/S100A9 complex as the natural ligand of CD69 receptor in hPBMC. The in vitro binding assay and competition assay further confirmed a specific interaction existing between CD69 and S100A8/S100A9 complex. Mechanistically, as peptide N-glycosidase treatment of CD69 abolished its interaction with S100A8/S100A9, the ligand-receptor binding of CD69 most likely requires N-linked glycans of CD69. To be more precise, we also determined the glycosylation site and the N-glycan compositions of CD69 through the nanoflow liquid chromatography coupled with tandem mass spectrometry. Functionally speaking, we showed that interaction between CD69 and S100A8/S100A9 is required for the up-regulation of SOCS3 expression leading to inhibited STAT3 signaling during conversion into Treg cells. In addition, CD69-S100A8/S100A9 association increases the secretion of functional cytokines of Treg cells TGF-β leading to reduced IL-4 production in hPBMC. Overall, our results not only define a novel ligand-receptor binging between CD69 and S100A8/S100A9 but also provide their functional and mechanistic interplays involved in Treg cell differentiation.
Subjects
Wheatgrass
Immunomodulation
Human peripheral blood mononuclear cells
Ligand
Regulatory T cell
SDGs
Type
thesis
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