發炎的機制以及其在中樞代謝調控中的作用研究

Abstract

本研究包括兩個部分。第一部分是發現Ifitm基因剔除鼠中產生的自發性發炎反應表型。Ifitm基因是一組會受干擾素所誘導表現的基因，干擾素刺激後所轉譯出的蛋白質在抗病毒功能中有著重要作用。然而，這些蛋白質的生化性質和生物功能上還不清楚。在先前的研究中，剔除5個Ifitm基因的IfitmDel小鼠被用來探討Ifitm基因的生物功能，我們在IfitmDel小鼠中觀察到隨著年齡的增加會產生低度發炎，在周邊血中單核細胞活化標誌物如CD86和MHCII的水平升高。IfitmDel小鼠在文獻中被指出有肥胖和食慾過盛的表現，我們發現這些小鼠的下視丘中Nos2表現顯著增加，表示著下視丘有發炎的情況。為了測試是否是機能不全的骨髓細胞或是過多的發炎訊號引起IfitmDel小鼠低度發炎，我們取野生型小鼠及IfitmDel小鼠的骨髓，並將其誘導成巨噬細胞來，比較其對發炎刺激的反應。在脂多醣體(LPS)和干擾素-γ(Interferon-γ)的刺激下，IfitmDel小鼠骨髓誘導巨噬細胞被活化的程度顯著較高。我們的結果顯示IFITM蛋白在巨噬細胞中具有調節發炎反應的功能，Ifitm基因的缺失導致在刺激下有較強烈的發炎反應。本研究的第二部分是研究促發炎細胞因子如何改變POMC神經元的代謝信號。位於下視丘弓狀核的POMC神經元在飲食調控和能量平衡中扮演重要角色，透過接受周邊代謝信號，如瘦蛋白和胰島素，來調控食慾下降並增加能量消耗，最近的研究中表明發炎可能會干擾POMC神經元中的代謝調節路徑。在我們的研究中，檢測了促發炎因子的增加如何影響mHypoA-POMC / GFP細胞株的代謝信號傳導。此外，考慮到造成發炎的物質並不局限於單一種促發炎細胞因子，所以將以LPS處理過的誘導巨噬細胞培養液應用為條件培養液加入POMC神經元中，此條件培養液中含有促炎細胞因子混合物。我們首先檢測POMC神經元對混合促炎細胞因子的反應，調控POMC神經元厭食反應的基因，如：POMC和Socs3，在發炎信號的刺激下升高。之後檢測在有條件培養液的環境下，POMC神經元中瘦素或胰島素誘導的信號傳導途徑的活化是否會受影響，瘦素或胰島素信號傳導在促炎細胞因子存在下會被破壞。另外，POMC神經元中的關鍵代謝調節蛋白：單磷酸腺苷活化蛋白質激酶(AMPK)的活化會被促炎細胞因子抑制。綜合上述觀察結果，我們認為POMC神經元的代謝調控會在發炎的環境下遭到破壞。

This study consists of two major parts. The first part is to characterize the inflammation developed in Ifitm genes knockout mutants. Ifitm genes are a group of interferon inducible genes that are transcriptional activated upon interferon stimulation and play important roles in interferon meditated antiviral functions. However, the biochemical properties and other biological functions of these proteins are not fully understood. Previously IfitmDel mutant mice that lack 5 Ifitm genes were generated to investigate the biological functions of Ifitm genes. We recently observed age dependent low-grade inflammation phenotype in IfitmDel mice. Mutants at 4-month of age started to exhibit elevated levels of monocyte activation markers such as CD86 and MHCII in peripheral blood. Obesity and hyperphagia have previously been reported in IfitmDel mutants. The expression of hypothalamic nos2 is upregulated in the mutants, indicating that hypothalamic inflammation is also developed. To test whether inflammation is caused by malfunctioned mutant myeloid cells or by excessive inflammatory signals, we further generate bone marrow derived macrophages from both IfitmDel mutants and wild type controls. Upon LPS and interferon-γ stimulation, mutant macrophages are drastically activated and the activation status is significantly higher than that of wild type macrophages. Our data suggest the anti-inflammatory roles of IFITM proteins. Ablating Ifitm genes augment the inflammatory response upon stimulation. The second part of this study is to investigate how proinflammatory cytokines altered the metabolic signaling in POMC neurons. POMC neurons in arcuate nucleus play pivotal roles in feeding controls and energy homeostasis. This group of neurons respond to peripheral metabolic signals, such as leptin and insulin, and mediate the anorexigenic response and increase energy expenditure in the hypothalamus. Recently accumulating data suggest that inflammation might interrupt metabolic regulations by affecting this group of neurons. In this study, we examined how increase of proinflammatory molecules influence metabolic signaling in the cell line mHypoA-POMC/GFP, which is generated by immortalizing the primary hypothalamic POMC neurons cultures. Also, considering that the effect of inflammation might not be limited to single kind of proinflammatory cytokines, we applied conditioned medium of LPS-primed macrophages, which contained a mixture of proinflammatory cytokines, to the POMC neuron culture. We first examined the response of the POMC neurons to the mixed proinflammatory cytokines. The genes mediating anorexigenic response of POMC neurons, such as POMC and Socs3, are elevated upon the stimulation of the inflammatory signals. Additionally, the activation of signaling pathways of the POMC neurons induced by leptin or insulin were examined with the conditioned medium. Both leptin or insulin signaling are disrupted at the presence of proinflammatory cytokines. Also, the activation of AMP-activated protein kinase (AMPK), a key metabolic regulator in POMC neurons, is also inhibited by proinflammatory cytokines. Combining all the observations, we conclude that metabolic signaling of POMC neurons are disrupted with inflammation.