探討細胞受體以及訊息傳遞在組織胞漿菌感染下引發炎性體活化

To identify receptor(s) and signals that are involved in inflammasome activation by Histoplasma capsulatum

張子軒(Tzu-Hsuan Chang)

指導教授 ： 伍安怡

國立臺灣大學/醫學院/免疫學研究所/碩士(2013年)

https://doi.org/10.6342/NTU.2013.01448

摘要

先天免疫系統(innate immunity system)是對抗外來病原菌感染時的第一道防線。在我的研究中探討樹突細胞 (BMDCs) 以及巨噬細胞 (BMDMs ＆ thio-pMac) 在感染組織包漿菌時引發炎性體的活化機制。
組織胞漿菌是一種雙型態的真菌，自然狀況下以菌絲的型態生長於土壤中，一旦吸入動物體內，會寄生在巨噬細胞 (macrophages) 吞噬小體內進行繁衍。除此之外，研究發現酵母型組織胞漿菌可以被樹突細胞 (dendritic cells) 所吞噬並且呈現抗原給Ｔ細胞。因此，在先天免疫系統中，巨噬細胞和樹突細胞扮演著抵禦組織包漿菌感染的重要角色。
炎性體 (inflammasome) 是一多蛋白組合而成的胞內感應平台。有別於胞外的細胞受體，炎性體做為細胞質內感應外來物 (PAMPs) 的胞內受體 (PRRs)。在我的研究裡，探討組織胞漿菌感染樹突細胞以及巨噬細胞後是否會引起的炎性體的活化。在細胞吞噬組織胞漿菌之後，會傳遞訊號生成pro-IL-1β，以及活化細胞內的 caspase-1，進而導致活化的caspase-1 去切pro-IL-1β使之成為成熟型的IL-1β而分泌出細胞外。在使用NLRP3 的基因剔除鼠的樹突細胞感染組織胞漿菌的實驗結果顯示少了NLRP3的細胞，在感染組織胞漿菌後便無法產生成熟型的IL-1β同時也沒有caspase-1的活化，顯示NLRP3 在胞內會辨識組織胞漿菌並且引發炎性體的組成與活化。而用抗體抑制住樹突細胞外的細胞受體，再感染組織胞漿菌後，可以發現Dectin-2 扮演著辨識組織胞漿菌並且傳遞訊息活化炎性體的角色。從我的實驗結果顯示，Dectin-2會誘導MAPK活化傳遞下游訊息去引起訊息一：Il-1β mRNA 的轉錄導致pro-IL-1β的生成，以及訊息二：caspase-1的活化。 因此在組織胞漿菌感染引起的炎性體活化中，Dectin-2會辨識組織胞漿菌並且傳遞訊息一以及訊息二去活化炎性體。

關鍵字

組織胞漿菌 ； 炎性體

並列摘要

The innate immune system is the first line to defend the invasion of microbes. In this study, I investigated inflammation activation in bone marrow-derived dendritic cells (BMDCs), macrophages (BMDMs) and thioglycollate-elicited peritoneal macrophages (thio-pMacs) after infection by Histoplasma capsulatum.
Histoplasma capsulatum is an opportunistic dimorphic fungal pathogen. It grows in the soil in nature as mycelium form and transforms into yeast after the microconidia enter into the lungs through inhalation. The yeast form of Histoplasma is an intracellular pathogen of the macrophage. Dendritic cells take up Histoplasma and present fungal antigens to both CD4 and CD8 T cells. Therefore, the roles of macrophages and dendritic cells and their interactions with the fungus are important to defense against Histoplasma infection.
Inflammasome is an intracellular protein sensor platform that composes of multiple protein complexes. Unlike membrane bound Toll-like receptors (TLRs), Nod-like receptors (NLRs) serve as cytosolic pattern recognition receptors to recognize PAMPs in the host. Here, I investigated the mechanism of inflammasome activation in the BMDCs, BMDMs and thio-pMacs after stimulation with Histoplasma. Both BMDCs and thio-pMacs stimulated with Histoplasma triggered caspase-1 activation and production of interleukin-1β (IL-1β). I used NLRP3 knockout mice to investigate whether Histoplasma engages NLRP3 in BMDCs. Cells with NLRP3-deficiency failed to produce IL-1β after Histoplasma infection. These results demonstrated that NLRP3 is the major PRR in the cytosol to recognize Histoplasma and mediates inflammasome activation. Using blocking antibodies I demonstrated that Dectin-2 played an important role in the induction of IL-β production and Caspase-1 activation after Histoplasam infection in BMDCs. Dectin-2 induced downstream MAPK activation to trigger both the first signal (the transcription of Il-1β) and the second signal (activation of caspase-1) in BMDCs.

並列關鍵字

Histoplasma ； inflammasome

參考文獻

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