探討LC3相關之吞噬作用在巨噬細胞與組織胞漿菌交互作用之下的角色

Abstract

LC3 相關之吞噬作用 (LC3-associated phagocytosis；LAP) 是一種參與在吞噬體成熟 (phagosome maturation) 的非典型的自噬作用(autophagy)。病原菌被細胞表面的模式識別受體 (pattern-recognition receptor) 辨認後，活化下游訊號使脂化的LC3 （LC3-II） 聚集並鑲嵌於吞噬體的細胞膜上，協助吞噬體和溶酶體 (lysosome) 之間的融合。過去的研究中指出，LAP 在抵禦白色念珠菌、黃麴黴菌以及新型隱球菌這些真菌中扮演著重要的角色。然而，LAP 在組織胞漿菌的感染中的角色尚未被釐清。 組織胞漿菌 （Histoplasma capsulatum）是一種雙型態的致病性真菌，在免疫低下的個體中會造成嚴重的肺部感染。巨噬細胞 (macrophage) 在組織胞漿菌的感染中非常重要且扮演多重的角色，它是組織胞漿菌的宿主細胞、感染後產生細胞激素之細胞、做為提供抗原的細胞，在被活化之後也能作為作用細胞清除胞內的組織胞漿菌。因此，了解 LAP 在巨噬細胞與組織胞漿菌交互作用中所扮演的角色是相當重要的。 本篇研究的目的在探討巨噬細胞受到組織胞漿菌感染後，所引起之 LAP 形成的作用機制。我們發現 LC3-II 在組織胞漿菌刺激後的 30 分鐘內即會形成，並且在 60 分鐘時被招募至吞噬體的細胞膜上。透過電子顯微鏡的觀察，我們發現包裹在被吞噬的組織胞漿菌外圍的吞噬體膜呈現單層膜結構，進一步證實組織胞漿菌可以引發巨噬細胞形成 LAP。藉由封阻型抗體以及 Dectin-1、CR3 缺失的巨噬細胞，我們發現 Dectin-1 參與了組織胞漿菌引發的 LAP，且排除 CR3、Dectin-2、TLR2 參與 LAP 形成的可能性。利用化學抑制劑抑制 Syk 激酶的活化會呈現劑量依存性的 LC3-II 形成下降。此外，專一性的抑制 NOX2 產生活性氧化物質（reactive oxygen species； ROS）會使組織胞漿菌引起的 LC3-II 形成下降，但抑制粒線體產生 ROS 以及一氧化氮合酶 (nitric oxide synthases) 則對於 LC3-II 形成沒有影響。已知黃麴黴菌需要藉由 Rubicon 來引發 LAP。但在本篇研究中，我們發現利用 siRNA 使 Rubicon 表現下降並不影響 LC3-II 的形成、招募，以及吞噬體和溶酶體間的融合。 這些結果顯示在巨噬細胞與組織胞漿菌交互作用時，需要藉由 Dectin-1/Syk/NOX2 訊息傳遞所釋放之 ROS 來引發 LAP。除此之外，我們的實驗結果也指出組織胞漿菌所引發的 LAP 不需要透過 Rubicon。

LC3-associated phagocytosis (LAP) is a non-canonical autophagy pathway involved in phagosome maturation. Pathogens interacting with pattern recognition receptor (PRR) triggers LC3-II recruitment to phagosomal membrane and facilitates its fusion with lysosome. Previous studies show that LAP plays a significant role in defense against fungal pathogens including Candida albicans, Aspergillus fumigatus, and Cryptococcus neoformans. However, the role of LAP in Histoplasma capsulatum infection is not clear. H. capsulatum is a dimorphic fungal pathogen that causes severe pulmonary infections in immunocompromised individuals. Macrophage being the host cell, cytokine-producing cell, antigen-donor cell and effector cell, is important for H. capsulatum infection. Therefore, it is critical to understand the role of LAP in the interaction between macrophage and H. capsulatum. This study aims to investigate the mechanism of LAP formation in macrophages upon encountering H. capsulatum. We found that LC3-II was formed within 30 min and was recruited to phagosomes by1 h after interaction with H. capsulatum. Electron microscopic imaging showed that H. capsulatum-containing phagosomes was a single-membrane but not a double-membrane structure, confirming that H. capsulatum can trigger LAP formation in macrophages. Employing receptor blocking antibodies and Dectin-1-/- and CR3-/- macrophages, we found that Dectin-1, but not CR3, Dectin-2 or TLR2, was involved in H. capsulatum-induced LAP. Chemical inhibition of Syk signaling reduced LC3-II formation in a dose dependent manner. We also showed that specific inhibition of ROS production by NOX2 but not mitochondria or nitric oxide synthases reduced H. capsulatum-induced LC3-II formation. While Rubicon is known to be required for Aspergillus-induced LAP, we found that silencing Rubicon did not affect LC3-II formation, its recruitment or fusion of H. capsulatum-containing phagosome with lysosome. These data together suggest that Dectin-1/Syk/NOX2-mediated release of ROS is required for LAP formation in macrophages upon encountering H. captulatum. Moreover, we demonstrated that LC3-associated phagocytosis of H. captulatum is independent of Rubicon.