自噬作用 (autophagy) 是一種藉由自噬小體 (autophagosome) 將細胞內異常堆積的蛋白質或是細胞內老化或受損的胞器 (如粒線體) 進行包裹作用。自噬小體與溶酶體融合形成自噬溶酶體 (autophagolysosome) 並利用溶酶體內的酵素將包裹物清除以維持細胞存活及代謝平衡。近幾年的報導指出自噬作用參與在細胞對抗結核菌的感染過程中，被認為是一種細胞抵禦外來感染的機制。除此之外，丙型干擾素 (IFN-γ) 誘導的Irgs (IFN-inducible immunity-related GTPases) 蛋白能引發自噬作用的發生，然而其詳細的分子機制並不清楚。本研究中，我們證明自噬作用能促進丙型干擾素的訊號傳遞係以透過調控Jak2-STAT1進而影響細胞的發炎反應。在Atg5基因剔除 (Atg5-/-) 的小鼠胚胎纖維母細胞中，丙型干擾素刺激無法誘發自噬作用的產生；同時，丙型干擾素下游基因－干擾素調節因子1 (IRF-1) 的表現量也顯著的較為低下。利用第三型 PI3K (class III phosphatidylinositol 3-kinase) 的抑制劑3-MA (3-methyladenine) 抑制自噬作用的產生，也可以發現不管在小鼠胚胎纖維母細胞或是RAW264.7巨噬細胞皆可以抑制丙型干擾素誘發的自噬現象以及細胞發炎反應。在另一個Atg7基因剔除 (Atg7-/-) 的小鼠胚胎纖維母細胞也可以觀察到與Atg5-/-小鼠胚胎纖維母細胞有著同樣的效果，諸如無法形成自噬作用、較低的細胞發炎反應抑或是Jak2-STAT1活化受到抑制。在丙型干擾素訊息傳遞過程，負向調控因子包括SOCS1 (suppressor of cytokine signaling-1)、SOCS3和SHP2 (dual-phosphatase Src homology-2 domain-containing phosphatase) 參與其中的調控。透過shRNA (lentiviral-based short hairpin RNA) 抑制了SHP2的表現可增加丙型干擾素誘導的STAT1磷酸化表現量，顯示在Atg5-/-的細胞中SHP2扮演抑制丙型干擾素訊息傳遞的角色。此外，在缺乏自噬作用的細胞中，ROS (reactive oxygen species) 的表現和粒線體的含量都較高，並能活化SHP2進而抑制丙型干擾素的訊息傳遞及細胞發炎反應。綜合以上實驗結果，自噬作用能透過調控Jak2-STAT1的活性促進丙型干擾素的訊號傳遞，進而影響細胞的發炎反應。

Autophagy is a mechanism for degrading aggregated proteins and damaged organelles such as mitochondria, which has important roles in development, immune defense, programmed cell death, and neurodegeneration. Recent report showed that autophagy is a defense mechanism, which participated in host cell resistance to Mycobacterium. One way that autophagy participates in regulation of immunity is it enhances interferon (IFN)-γ-mediated antimicrobial efficacy; however, the effects of autophagy on IFN-γ signaling and bioactivaties remain unclear. In this study, we investigate that autophagy facilitates IFN-γ signal activation by regulating Janus kinase (Jak) 2 and signal transducer and activator of transcription (STAT) 1 signal and influencing cellular inflammation. Autophagy protein (Atg) 5 deficient (Atg5-/-) mouse embryonic fibroblasts (MEFs) are resistant to IFN-γ-induced light chain 3 (LC3) conversion as well as autophagosome formation. At the same time, IFN-γ-induced IFN regulatory factor 1 expression was significantly lower. We used 3-MA (3-methyladenine) which is a PI3K (class III phosphatidylinositol 3-kinase) inhibitor, inhibiting autophagy formation. Either MEFs or RAW264.7, which is macrophage like cell, inhibited IFN-γ-induced autophagy and cellular inflammation. Both of Atg5-/- and Atg7-/- MEFs are resistant to IFN-γ-activated Jak2-STAT1, which suggests autophagy has a potent role in IFN-γsignaling. There are negative regulator such as SOCS1 (suppressor of cytokine signaling-1), SOCS3, and SHP2 (dual-phosphatase Src homology-2 domain-containing phosphatase) in IFN-γ signal pathway. Inhibited SHP2 expression increase IFN-γ-induced STAT1 phosphorylation by shRNA (lentiviral-based short hairpin RNA), SHP2 play the inhibition role of IFN-γ signaling in Atg5-/- MEFs. In addition, increased reactive oxygen species (ROS) result from the absence of autophagy positively regulates SHP2, which inactivates STAT1. Image analysis shows that IFN-γ promotes co-localization of autophagosomes and ROS-producing mitochondria, which lead to the clearance of aged mitochondria. Atg5-/- MEFs fail to generate autophagy result in abundant ROS produced mitochondria exist. This study reveals a link between autophagy and IFN-γ signaling and bioactivities, and autophagy plays a pivotal role of Jak2-STAT1 through inhibition of ROS and SHP2 and influencing on cellular inflammation.

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