伍安怡臺灣大學：免疫學研究所彭榮桂Peng, Jung-KueiJung-KueiPeng2007-11-292018-07-092007-11-292018-07-092004http://ntur.lib.ntu.edu.tw//handle/246246/63298在小鼠的全身性組織胞漿菌炎感染模式中會誘發強烈之第一型細胞激素產生，我們利用此模式來探討在組織胞漿菌感染之免疫反應中，若在第一型T細胞priming階段存在第二型拮抗型細胞激素介白素(IL-4，IL-10)下會否改變第一型T細胞之極性。小鼠在感染組織胞漿菌之前先注射二劑山羊抗鼠IgD免疫球蛋白血清(GDominant type-1 cytokine production is induced in a murine model of systemic histoplasmosis. We used this model to investigate whether the presence of antagonistic cytokines during T cell priming changes the polarity of T cells in response to Histoplasma infection. Before infection with Histoplasma capsulatum, mice were injected twice with goat anti-mouse IgD antiserum (GaMd), which induced expression of dominant type-2 cytokines. At days 7 and 14 after infection, the GaMd-treated mice had suppressed IFN-g response and a significantly greater fungal burden in their spleens and lungs. The number of IFN-g-producing cells as well as the level of IFN-g produced per cell was greatly reduced. Not only CD4+ T cells but also CD8+ T cells were affected. Reduction of IFN-g-producing T cells is not a result in T cell expansion, T cell death, or intrinsic defect in TCR signaling. There was no difference in the numbers of CD4 and CD8 T cells in infected mice with or without GaMd treatment. The number of Histoplasma-induced IFN-g-producing cells was partially restored in GaMd-treated IL-4-/- and IL-10-/- mice and completely restored in IL-4-/-IL-10-/- mice. Thus, the combined effect of IL-4 and IL-10 suppressed the generation of IFN-g-producing cells. A longitudinal study demonstrated that as IL-4 and IL-10 decreased, the number of Histoplasma-induced IFN-g-producing cells rapidly increased, and fungal clearance improved, demonstrating that the presence of IL-4 and IL-10 did not permanently change the polarity of T cells.Abstract (Chinese)……………………………………………………………………... 2 Abstract…………………………………………………………………………………. 3 Chapter I. Introduction………………………………………………………………. 8 1.1 Background………………………………………………………………………… 9 1.1.1 Type-1 and type-2 T cells differentiation………………………………….. 9 1.1.1.1 T cells subsets…………………………………………………………. 9 1.1.1.2 Th1 cells differentiation……………………………………………….. 9 1.1.1.3 Th2 cells differentiation……………………………………………….. 11 1.1.2 Goat anti-mouse IgD antiserum (GaMd) induces immunoglobulin production and cytokines expression in mice……………………………….. 14 1.1.3 Histoplasma capsulatum infection and host defense……………………. 15 1.1.4 Regulation of type-1 and type-2 cytokines in experimental model…….. 16 1.2 Objectives.…………………………………………………………….…………… 18 Chapter II. Materials and Methods………………………………………………… 19 2.1 Experimental Methods…………………………………………………………….. 20 2.1.1 Mice…………………………………………………………………………… 20 2.1.2 Experimental design………………………………………………………… 20 2.1.3 Fungus infection………………………………………………………..…… 21 2.1.4 Preparation of mouse splenic leukocytes………………………………… 21 2.1.5 Fungus counts………………………………………………………………. 21 2.1.6 Genotyping of IL-4-/-IL-10-/- mice from tail DNA………………………..… 22 2.1.7 Total RNA isolation and reverse transcription (RT)…………………….… 23 2.1.8 Competitive RT-PCR……………………………………………………...… 24 2.1.9 7-amino-actinomycin D staining………………………………………….… 25 2.1.10 Cell surface marker and intracytoplasmic cytokines staining…………. 25 2.1.11 IgE ELISA assay……………………………………………………….…… 26 2.1.12 IFN-g ELISA assay…………………………………………………….…… 27 2.1.13 IL-2 ELISA assay……………………………………………………...…… 27 2.1.14 Statistical analysis…………………………………………………….…… 28 2.2 Experimental Materials………………………………………………………….… 29 2.2.1 Hank’s balanced salt solution (HBSS)………………………………….…. 29 2.2.2 Complete RPMI-1640 medium…………………………………………..… 29 2.2.3 RBC lysis buffer……………………………………………………………… 30 2.2.4 Enriched BHI agar slant………………………………………………..…… 30 2.2.5 Sabourand’s agar plate………………………………………………...…… 30 2.2.6 10 × PBS buffer……………………………………………………………… 30 2.2.7 Intracellular cytokines staining buffer……………………………………… 31 2.2.8 Intracellular cytokines staining fixation buffer…………………………..… 31 2.2.9 Intracellular cytokines staining permeabilization buffer……………..…… 31 2.2.10 ELISA coating buffer…………………………………………………..…… 32 2.2.11 ELISA blocking buffer……………………………………………………… 32 2.2.12 ELISA washing buffer……………………………………………………… 32 2.2.13 IgE ELISA substrate buffer…………………………………………...…… 32 2.2.14 ELISA stop solution………………………………………………...……… 32 2.2.15 DEPC H2O…………………………………………………………..……… 33 2.2.16 5 × TBE buffer…………………………………………….…………...…… 33 2.2.17 Reagents………………………………………………………………….… 33 2.2.18 Antibodies…………………………………………………………………... 34 2.2.19 Sequences of primers………………………………………………….….. 35 2.2.20 Instruments……………………………………………………………….… 36 Chapter III. Results…………………………………………………………………… 38 3.1 Establishment of type-1 Histoplasma infectious response and type-2 GaMd-induced cytokines response in vivo……………………………………. 39 3.1.1 Histoplasma infection induces a dominant type-1 response…………… 39 3.1.2 GaMd treatment induces a dominant type-2 cytokine response………. 39 3.2 The effect of GaMd-induced type-2 cytokines response on the regulation of Histoplasma infectious type-1 response…………………………….………… 40 3.2.1 GaMd treatment profoundly suppresses Histoplasma-induced IFN-g response……………………………………………………………………… 40 3.2.2 GaMd treatment retards fungal clearance……………………………..…. 41 3.2.3 IL-4 is partially responsible for GaMd–induced suppression……………. 42 3.2.4 IL-4 and IL-10 together mediate GaMd–induced suppression of IFN-669577 bytesapplication/pdfen-US組織胞漿菌炎第一型第二型免疫反應干擾素HistoplasmosisType-1 and type-2 immune responseinterferonotherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/63298/1/ntu-93-R87449004-1.pdf