伍安怡臺灣大學：免疫學研究所洪儷殷Hung, Li-YinLi-YinHung2007-11-292018-07-092007-11-292018-07-092005http://ntur.lib.ntu.edu.tw//handle/246246/63301組織胞漿菌是一種胞內病原性黴菌，其孢子被吸入生物體內後會在宿主體內轉變成酵母菌形式而進一步散播。巨噬細胞是組織胞漿菌的宿主細胞，在吞入酵母菌形式的病原體之後能讓此菌在胞內繁殖；另一方面，巨噬細胞也能引發免疫反應對抗此菌的感染。然而，在小鼠感染模式當中巨噬細胞用以和胞漿菌作用而媒介其吞噬的受體仍未被發現。本論文中，我們針對細胞表面能辨識病原菌上特定分子結構的受體做研究，希望了解這些受體是否參與在巨噬細胞吞入組織胞漿菌的過程中。利用免疫染色之系統觀察RAW264.7巨噬細胞株吞食組織胞漿菌的狀況，我們發現與黴菌細胞壁組成物質結構相似的mannan及laminarin可以抑制細胞吞噬作用。進一步我們確定了CR3以及TLR2參與在此吞噬作用當中。另外我們也發現當抑制這些受器與黴菌作用時，巨噬細胞產生TNF-α 的量也受到影響。此一結果有助於了解辨識特定分子構造之受器在對抗黴菌感染時所扮演的角色。Histoplasma capsulatum (Hc) is a dimorphic fungus which grows as a saprophyte in nature and is acquired by inhalation of airborne conidia and hyphal fragments. The fungus transforms to become yeast cells in the infected host. The macrophages, being the host cells, bind and ingest Hc yeasts, thereby providing the intracellular environment in which yeast cells thrive. Macrophages also act as effector cells in host defense against histoplasmosis. However, the receptors for Hc on macrophage have not been well defined, especially in mice model. In this study, we focus on pattern recognition receptors that are able to recognize cell wall components on fungus and see if they are involved in Hc internalization by macrophage. By using an immunofluorescence staining technique to monitor the uptake of Hc yeast cells by RAW264.7 cells, we found fungal cell wall structure analogous mannan and laminarin inhibited phagocytosis of macrophages. We further used specific blocking antibody to demonstrate the identity of these receptors and found both CR3 and TLR2 were involved in Hc uptake. In subsequent experiments, we also found blocking the interaction of Hc to its receptors also abolished TNF-α production by macrophages. The results of this study contribute to the understanding of fungus entry and the roles of pattern recognition receptors in anti-fungus immune response.Abstract…………………………………………………………i Abstract (Chinese)………………………………………………ii Chapter I. Introduction Part 1. Histoplasma capsulatum infection and host defense....2 Part 2. Pattern recognition receptors and immune response....4 Part 3. Rationale of studying receptors for Histoplasma capsulatum....6 Chapter II. Materials and Methods Part 1. Experimental Procedures………………………………9 1.1Cell line…………………………………………………… 9 1.2 Fungus…………………………………………………… 9 1.3 Fluorescein isothiocyanate (FITC) labeling of Histoplasma yeast……………………………………9 1.4 Phagocytosis assay…………………………………10 1.5 Carbohydrate and antibody inhibition experiments…10 1.6 Cell surface molecule staining and flow cytometric analysis…………………………………… 11 1.7 TNF-α ELISA assay……………………………… 11 Part 2. Experimental Materials…………………………12 Chapter III. Results Part 1. Comparing Raw 264.7 macrophage and primary mouse macrophage surface receptor expression........................................18 1.1 To identify macrophage surface receptor expression by specific antibodies……………………….........18 Part 2 Macrophage phagocytosis…………………………………18 2.1 Phagocytosis assay………………………………………… 18 2.2 Inhibition of RAW 264.7 cell phagocytosis by soluble saccharides………………………………………………….19 2.3 Inhibition of RAW 264.7 cell phagocytosis by anti-CR3 antibody………………………………………………………20 2.4 Combined treatment of laminarin and antibody to CR3 further inhibits phagocytosis……………………………21 2.5 Inhibition of RAW 264.7 cell phagocytosis by blocking TLR2………………………………………………………….21 Part 3. Cellular response after engulfing Histoplasma capsulatum………………………………………………………22 3.1 TNF-α production after internalization of the fungus....22 Chapter IV. Discussion Part 1. Receptor usage in Histoplasma internalization by macrophage………………………………………………………25 Part 2. Receptors and cellular responses in histoplasmosis ………28 Reference………………………………………………………32 Figures…………………………………………………………… Figure 1. Surface receptor expression on macrophages……42 Figure 2. Hc yeast cells were labeled by FITC and used to investigate phagocytosis of RAW cells…………………………44 Figure 3. Inhibiting macrophage phagocytosis of Histoplasma....46 Figure 4. Titrating the inhibitory effects of soluble saccharides on macrophage phagocytosis……………………………………48 Figure 5. Additive effects of mannan and laminarin in inhibiting macrophage phagocytosis.…………………………50 Figure 6. Anti-CR3 antibody inhibits macrophage phagocytosis...52 Figure 7. Additive effects of saccharides and anti-CR3 antibody in inhibiting macrophage phagocytosis……………54 Figure 8. The effect of anti-TLR2 antibody on macrophage phagocytosis.………………………………………………56 Figure 9. Additive effects in inhibiting phagocytosis by treatment of laminarin or TLR2 in addition to anti-CR3 antibody .....58 Figure 10. TNF-α production by RAW 264.7 cells after interaction with H. capsulatum.…………………………60en-US組織胞漿菌受器HistoplasmaReceptorsother