探討致病性長鏈非編碼核糖核酸IRAK3-3與VP1特異性擴增的T細胞受體庫在71型腸病毒感染中之影響

Abstract

71型腸病毒為一致命性病毒，好發於亞太地區。71型腸病毒感染會造成典型手足口症、腦脊髓炎、肺水腫、類小兒麻痺症癱瘓或其他神經系統壞損症狀。截至目前，並無有效之藥物可對抗71型腸病毒。因此，徹底瞭解71型腸病毒感染後宿主之防禦機制以及病毒之致病機轉，對於開發抗病毒治療方式是有其必要性與迫切性。研究指出，71型腸病毒可透過調控宿主的轉錄物質，如mRNA與miRNA，進而關閉宿主蛋白質合成機制，逃脫宿主的免疫攻擊，並促進病毒自身的複製。除了mRNA及miRNA的變化，宿主於病毒感染過程中亦會改變長片段非編碼核糖核酸lncRNAs。長片段非編碼核糖核酸為一長度大於200個核苷酸之非編碼核糖核酸。71型腸病毒感染會抑制長片段非編碼核糖核酸lnc-IRAK3-3表現，而此長片段非編碼核糖核酸參與於宿主細胞凋亡機制。lnc-IRAK3-3可捕取微核糖核酸miR-891b，以增進GADD45β表現，進而加速宿主細胞產生凋亡。反之，受71型腸病毒感染之宿主細胞藉由抑制lnc-IRAK3-3，導致受lnc-IRAK3-3捕取之miR-891b得以釋放，進而抑制GADD45β表現以減少細胞凋亡，避免宿主細胞產生嚴重損傷。此外，免疫反應是人體中相當重要的防禦系統，特別於病原體感染。T細胞受器在辨識細胞內病原體和清除受病原體感染的細胞扮演關鍵性角色。利用所建立之71型腸病毒小鼠感染模式，可觀察受病毒感染之實驗小鼠腦幹T細胞受器庫(TCR repertoire)於病毒感染過程之角色。以次世代定序技術結合生物資訊分析，找尋71型腸病毒感染過程，於實驗小鼠腦幹中過度擴增之TCR 群落。71型腸病毒感染所造成之過度擴增TCR，或可作為免疫治療之標的。 本論文針對宿主長片段非編碼核糖核酸與T細胞受器庫於71型腸病毒感染過程之角色進行研究。第一部分的研究指出，宿主細胞可調控lnc-IRAK3-3表現量，啟動lnc-IRAK3-3/miR-891b/GADD45β分子調控路徑以減緩71型腸病毒感染所造成之細胞凋亡。在第二部分研究中，利用71型腸病毒小鼠感染模式，觀察到病毒感染會改變宿主腦幹T細胞受器庫β (TCRβ)，並且找尋到受病毒蛋白 VP1 (Viral protein 1)過度激活之特異性T細胞受器β群落。承上之研究，可對71型腸病毒致病機轉更臻了解。

Enterovirus 71 (EV71) is a life-threatening pathogen particularly in the Asia-Pacific region and EV71 infection causes typical hand-foot-and-mouth disease, encephalomyelitis, pulmonary edema, poliomyelitis-like paralysis or even neurologic and psychiatric effects. At present, there is no antiviral drug available against EV71. Understandings of viral pathogenesis and host defense mechanisms in EV71 infection thoroughly are urgent for antiviral therapies development. It was reported that EV71 can govern host protein synthesis, escape host immune attacks and further facilitate viral propagation by regulating host transcripts such as mRNAs and miRNAs. Besides both RNAs, host cells also regulated long non-coding RNA (lncRNA) expressions in response to EV71 infection. lncRNA is one kind of non-protein coding RNAs with longer than 200 nucleotides. lnc-IRAK3-3 was down-regulated in EV71 infection, which involved in host cells apoptosis. lnc-IRAK3-3 has the ability capturing miR-891b to enforce GADD45β expression and eventually promotes apoptosis. On the contrary, host cells suppress lnc-IRAK3-3 to relieve lnc-IRAK3-3-sequestered miR-891b, restrain GADD45β and attenuate apoptosis in EV71 infection that prevent host cells from severe damages. On the other hand, the immune response is the most important defense system particularly on pathogens infections. T-cell receptor (TCR) plays a critical role in recognizing intracellular pathogens and initiating the destruction cascade of pathogen-infected cells. We characterized the TCR repertoire in the mice brainstems by using mouse-adapted EV71 (mEV71) model. Through integration of next-generation sequencing (NGS) technology and bioinformatics analysis, we discovered the expanded specific TCR clone clusters from mice brainstems in mEV71 infection. The expanded specific TCR clones might be considered as the target for immunotherapy. In this thesis, I investigated the regulations of lncRNA and characterized the role of TCR repertoire in EV71 infection. In the first part, I discovered a new molecular mechanism by which host cells governed lnc-IRAK3-3 expression and further counteracted EV71-induced apoptosis through the lnc-IRAK3-3/miR-891b/GADD45β axis. Secondly, we observed that mEV71 infection shaped host TCRβ repertoire and presumably expanded VP1-specific TCRβ cluster in mEV71-infected mouse brainstem. It can better the understanding on EV71 pathogenesis in accordance with these findings.