2013-08-012024-05-14https://scholars.lib.ntu.edu.tw/handle/123456789/656141摘要：Histoplasma為雙型態真菌，其酵母型菌體寄宿於巨噬細胞中，吞噬細胞藉由模式識別受體與病原交互作用並引起抗菌反應，有效抑制病原生長，因此研究巨噬細胞與Histoplasma交互作用，對了解宿主抵抗真菌的機制相當重要。我們先前發表的研究指出，CR3參與在巨噬細胞吞噬Histoplasma的反應中，並與Dectin-1合作促使細胞產生激素，而細胞的吞噬反應及產生細胞激素皆須仰賴Syk之活化，暗示CR3與Dectin-1同時透過Syk及其下游訊號分子，合作調控Histoplasma所引起的細胞激素生成。在病原刺激下，CR3與Dectin-1的合作關係至今尚無文獻探討，因此值得深入研究CR3與Dectin-1所傳遞的訊號途徑間交互作用。本研究計畫目標1. 研究巨噬細胞中參與在Histoplasma引起的細胞激素反應之訊號分子。利用kinase抑制劑篩選出參與在反應中之訊號分子。2. 剖析巨噬細胞上的CR3與Dectin-1下游的訊號傳遞並研究兩者下游傳遞途徑的交互作用。利用從CR3-/-、Dectin-1-/-、以及CR3-/-Dectin-1-/-巨噬細胞進行實驗，釐清CR3及Dectin-1兩者傳遞之訊號途徑的交會點。將Histoplasma與專一性配體（iC3b和curdlan）刺激所活化訊號相較，可進一步確定兩受體所傳遞的訊號途徑。3. 研究Syk及其下游訊號分子在CR3與Dectin-1交互作用中的貢獻。利用Syk-/-的巨噬細胞、專一性抑制劑及shRNA抑制特定激酶活化，可釐清參與在CR3與Dectin-1下游共同訊號分子的活化順序。<br> Abstract: Interaction between innate immune cells with pathogens through pattern recognition receptors (PRRs) induces antimicrobial responses including phagocytosis, cytokine secretion, productions of nitric oxygen (NO) and reactive oxygen species (ROS). Histoplasma capsulatium is a dimorphic fungal pathogen. The yeast cells of the fungus reside primarily in macrophages. Therefore, investigating the interaction between macrophage and Histoplasma is critical to the understanding of host response to the fungal pathogen.We have previously reported that CR3 is responsible for macrophage phagocytosis of Histoplasma yeast and that CR3 and Dectin-1 together mediate cytokine response. Furthermore, phagocytosis of and cytokine response to Histoplasma are both Syk kinase-dependent. These results show that CR3 and Dectin-1 collaboratively regulate cytokine response to Histoplasma and suggest that Syk and its downstream signaling molecules are the key regulators in the crosstalk between CR3 and Dectin-1. To our knowledge, collaboration between CR3 and Dectin-1 in cellular response to a pathogen has never been reported. In this proposal, we will decipher the signal pathways downstream of CR3 and Dectin-1 in macrophage response to Histoplasma and study the crosstalk of the signal pathways downstream of these two receptors.The study in this proposal will be carried out in 3 aims. Aim #1, to investigate the signaling molecules that are involved in macrophage cytokine response to Histoplasma. Using kinase inhibitors, the candidate molecules that are involved in macrophage cytokine response to Histoplasma will be identified. Aim #2, to delineate the signaling pathways downstream of CR3 and Dectin-1 on macrophage and study their crosstalk. Macrophages from CR3-/-, Dectin-1-/- and CR3-/-Dectin-1-/- mice will be employed. The results will reveal at what point the CR3 and Dectin-1 signaling pathways converge. By comparing the signaling in single and double knockout macrophages stimulated by Histoplasma and to stimulation by iC3b and curdlan, we will also define the signaling pathway downstream of each receptor. Aim #3, to decipher the contribution of Syk and its downstream signaling molecules in the interplay between CR3 and Dectin-1. By using Syk-deficient macrophages, specific inhibitors and shRNA against specific downstream kinases, the order of sequence of the signaling molecules involved in the interplay between CR3 and Dectin-1 will be elucidated.Investigating the collaborative relationship between CR3 and Dectin-1 in cytokine response to Histoplasma will reveal the complex interplay between PPRs in innate cells. Many Dectin-1-recognizing fungal pathogens threaten the lives of immunocompromised patients. Our study will help the understanding of whether exogenously applying agonist of PPR can facilitate fungal clearance through increase of Dectin-1-mediated proinflammatory responses. This should help the development of a new strategy for fungal therapeutics.