SP110異構蛋白在巨噬細胞中的功能之研究

Abstract

肺結核主要是由結核分枝桿菌所造成的慢性傳染性疾病，可以透過患者的飛沫進行傳染。根據統計，全世界約有三分之一的人口曾經感染過結核分枝桿菌，然而，在這些感染者中卻只有約十分之一的人會發展為肺結核。目前研究認為宿主的免疫力以及基因多型性(polymorphism)可能是導致宿主是否能抵抗病原菌的重要因素。由先前在小鼠的研究發現一段調控先天免疫的基因座sst1 (Supersusceptibility to tuberculosis 1)，並且在此區域找到一個基因Ipr1 (Intracellular pathogen resistance 1)，它可能參與小鼠感染結核分枝桿菌時的先天免疫反應。人類的核蛋白SP110為小鼠Ipr1蛋白的同源蛋白有高達41%的相似度，在轉錄過程中經過選擇性剪接(Alternative splicing)可以產生三種不同的異構體SP110a、SP110b及SP110c；然而，三者在免疫反應扮演的角色與功能目前尚不明確。 我們成功建立由Doxycycline誘導分別表現SP110a、SP110b及SP110c的THP-1細胞株，透過phorbol 12-myristate 13-acetate (PMA)誘導THP-1細胞株分化成巨噬細胞，並能分別穩定表現SP110 isoforms。利用流式細胞儀分析，我們發現表現SP110b時會降低參與先天免疫反應重要的表面受體CD14，而表現SP110a及SP110c對於表面受體CD14沒有顯著影響。這個結果暗示SP110b可能在先天免疫反應中扮演重要的角色。接著我們進一步分析CD14/TLR4訊息傳遞路徑下游促發炎細胞激素mRNA表現量，結果發現SP110 isoforms在THP-1細胞株分化前能促進促發炎細胞激素mRNA表現量，但是在分化後的THP-1細胞株中卻發現SP110b會抑制促發炎細胞激素mRNA表現量。SP110 isoforms 不只在分化前後分別扮演不同功能，而且對於促發炎細胞激素mRNA表現量分別有著不同的調控。此結果暗示，SP110 isoforms在先天免疫反應中，可能有維持促發炎細胞激素恆定的功能。另外，在Interferon-γ作用下，我們分別表現SP110a、SP110b及SP110c並用流式細胞儀分析巨噬細胞的細胞凋亡程度，結果發現SP110b能有效抑制Interferon-γ誘導的發炎環境下巨噬細胞的細胞凋亡。這些結果顯示，SP110b在先天免疫反應中，可能扮演調控者的角色，避免過度免疫反應造成組織的損傷，而SP110a和SP110c相較SP110b在先天免疫反應中可能扮演次要的角色。SP110 isoforms是如何調控促發炎細胞激素目前尚不清楚是否有其他分子參與其中，未來可藉由分析細胞中與SP110 isoforms結合的分子，來釐清由SP110 isoforms所調控的促發炎細胞激素。

關鍵字：核蛋白； 巨噬細胞； 肺結核

Tuberculosis (TB) is an infectious disease caused by the bacillus Mycobacterium tuberculosis (Mtb) and is the second leading cause of death in infectious disease worldwide. It’s estimated that one-third of the world’s population is currently infected with Mtb, and only less than 10% of people will develop TB disease during their lifetime. In the previous study, a gene Ipr1 (Intracellular pathogen resistance 1) was identified within sst1 (Supersusceptibility to tuberculosis 1) locus on mouse chromosome 1, which participates in innate immunity in mouse models of Mtb infection. SP110 nuclear body protein (SP110) is the human homologous of Ipr1 protein and the protein has three major isoforms: SP110a, SP110b and SP110c. The alternative splicing gives rise to different isoforms of SP110; however, the distinct roles and functions among these isoforms are still unclear. In this study, we have successfully generated several THP-1 clones which express SP110a, SP110b and SP110c upon Doxycycline induction. We treated THP-1 clones with phorbol 12-myristate 13-acetate (PMA) to differentiate into macrophages, which stably expressed SP110a, SP110b and SP110c. By flow cytometric analysis of macrophages surface markers, we found that the expression of CD14, a marker of M1 macrophage, as well as a co-receptor of the Toll-like receptor4, was decreased when expressing SP110b, but not in PMA-induced macrophage when expressing SP110a or SP110c. By analyzing NF-κB mediated pro-inflammatory cytokines mRNA expression, we found that SP110 isoforms functioned diversely in THP-1 monocytes and PMA-induced macrophages. During M. tb infection, IFN-γ activation renders the macrophages' ability of killing intracellular mycobacteria by overcoming the phagosome maturation block and inducing apoptosis in macrophages. After IFN-γ stimulation, Anneix-V apoptosis assay showed that IFN-γ stimulated apoptosis of PMA-induced macrophages, while the expression of SP110b can prevent apoptosis. Based on our findings, we suggested that SP110 isoforms play important roles as immune regulators in regulation of innate immune homeostasis. SP110b also played a role in IFN-γ-induced apoptosis by reducing apoptosis in PMA-induced macrophages. SP110b might demonstrate different cellular functions from SP110a/SP110c did, of which could reduce over-active immune responses after PMA or IFN-γ stimulation. The mechanisms of how SP110 isoforms regulate pro-inflammtory cytokines mRNA and how they prevent apoptosis remain unknown. In the future, by identifying the molecules associated with the SP110 isofroms and by analyzing other anti-apoptosis proteins, we can further elucidate the mechanism of SP110 isoforms in regulating innate immune homeostasis and apoptosis. Key words : Nuclear protein; Macrophage; Tuberculosis.