探討EB病毒產物在不朽化之類淋巴母芽細胞株中透過RLRs路徑對IL-10表現之影響

The effects of Epstein-Barr virus products on IL-10 expression through RLRs signaling pathway in lymphoblastoid cell lines

蔡函育(Han-Yu Tsai)

指導教授 ： 蔡錦華

國立臺灣大學/醫學院/微生物學研究所/碩士(2021年)

https://doi.org/10.6342/NTU202102668

摘要

EB病毒 (Epstein-Barr virus, EBV) 隸屬於人類皰疹病毒科 (Herpesviridae) 中的γ皰疹病毒亞科，並且是第一個被定義與人類腫瘤密切相關的病毒。其感染與多種人類惡性腫瘤及淋巴細胞增生疾病具高度相關性，包含鼻咽癌 (nasopharyngeal carcinoma, NPC)、巴氏淋巴瘤 (Burkitt’s lymphoma)、霍杰金氏症 (Hodgkin’s disease) 及移植後淋巴組織增生症 (post-transplant lymphoproliferative disease, PTLD) 等。在體外，EB病毒感染人類B淋巴球細胞後，使其不朽化成具持續增生能力之類淋巴母芽細胞株 (lymphoblastoid cell line, LCL)。
EB病毒為了潛伏於B淋巴球細胞中，會調控許多宿主基因的表現以躲避免疫系統的攻擊。實驗室先前利用cDNA微陣列及細胞激素微陣列分析B淋巴球細胞在EB病毒感染後基因表現及細胞激素變化情形，其中IL-10的表現在EB病毒感染後顯著上升。已知IL-10具有抗發炎反應及促進B淋巴球細胞生長的特性，並且移植病人血清中IL-10表現的提高可作為EB病毒相關之PTLD早期指標，顯示IL-10對於EB病毒相關之PTLD的發生亦扮演重要的角色。本研究欲於PTLD體外模型LCL中探討EB病毒感染B淋巴球細胞後誘導IL-10表現之機制。
在先前的報導中，Dr. Takada等人根據於Akata細胞株之實驗結果提出EBERs經由RIG-I及其下游IRF3誘導IL-10表現之假說。本研究欲探討辨識dsRNA的Retinoic acid-inducible gene-I-like receptors (RLRs) 在LCL中對於IL-10表現之影響，實驗結果發現EB病毒的感染雖不影響RIG-I的表現，但能促進MDA5的表達。進一步以shRNA分別降解RIG-I或MDA5 mRNA後，LCL中IL-10的基因轉錄有下降的趨勢，並且皆能抑制下游訊息傳遞分子TBK1的活化，顯示RIG-I及MDA5的存在對於LCL中IL-10的表現皆是重要的，並可能透過活化下游訊息傳遞路徑促進IL-10之表現。
欲釐清RIG-I及MDA5之活化並誘導IL-10的表現是否由EBERs所引起，在不帶有EB病毒之人類霍杰金氏症細胞株KMH2中轉導EBER1或EBER2皆能促進IL10基因的轉錄，並且於LCL中剔除EBERs基因的表達可抑制IL-10的表現，由此確認了EBERs誘發IL10基因表現的能力。然而，在KMH2表現EBER1及EBER2或是於LCL中剔除EBERs基因的表達對TBK1及IRF3的影響皆不明顯，在本次的實驗中未能證實EBERs誘導IL-10表現之分子機制，EBERs是否為LCL中造成RLRs活化並誘導IL-10表現之分子仍待釐清。
綜合以上實驗結果，本研究發現RIG-I及MDA5對於LCL中IL10基因的表達都是重要的，EB病毒的感染可以誘導MDA5的表現或活化RLRs下游訊息傳遞而可能促進IL-10的表現 ; 另外，我們再次驗證EBERs促進IL-10表現的能力，然而其是否為誘發LCL中RLRs活化以促進IL-10表現之分子仍需要更多驗證。

關鍵字

EB病毒 ； EBERs ； RIG-I ； MDA5 ； IL-10

並列摘要

Epstein-Barr virus (EBV) is a member of the human Herpesviridae family and belongs to the Gammaherpesvirinae subfamily. As the first defined human oncogenic virus, EBV infection is associated with a wide spectrum of human malignancies and lymphoproliferative diseases, such as nasopharyngeal carcinoma, Burkitt’s lymphoma, Hodgkin’s disease and post-transplant lymphoproliferative disease, etc. In vitro, EBV can immortalize human primary B cells into lymphoblastoid cell line (LCL) which can proliferate infinitely.
To establish a latent infection in primary B cells, EBV may manipulate host gene expression so that it can escape immune surveillance. In our previous study, lab members used cDNA microarray and cytokine antibody arrays to analyze the cellular gene expression and cytokine production of primary B cells post EBV infection. Accordingly, they found that the expression and production of IL-10 are upregulated significantly after EBV infection. IL-10 is an anti-inflammatory cytokine and involves in regulating the B cells growth. In addition, increase of IL-10 in the serum of transplanted patients can be used as an early indicator of EBV-related PTLD, showing that IL-10 has an important role in the occurrence of EBV-related PTLD. This study wants to further explore the mechanism of how EBV induces IL-10 in LCL, the perfect in vitro model of PTLD.
In previous reports, Dr. Takada lab proposed the hypothesis that EBERs induce IL-10 through RIG-I-mediated IRF-3 signaling in Akata cell line. In this study, we investigated the effects of retinoic acid-inducible gene-I-like receptors (RLRs), which sense dsRNA, on the IL-10 expression in LCL. Although there is no difference in the expression of RIG-I between primary B cells and LCLs, the significant induction of MDA5 is observed. Knockdown of RIG-I or MDA5 with shRNA both suppress the transcription of IL-10 mRNA and the activation of downstream signaling molecules TBK1 in LCLs. These data implied that RIG-I and MDA5 are activated in LCLs, moreover, these receptors can mediate the expression of IL-10.
Next, we intended to clarify whether EBERs are the molecules that activate RLRs and induce IL-10 expression. We revealed that IL-10 mRNA transcription is up-regulated in EBERs transfected KMH2, EBV-negative Hodgkin’s disease cell lines, and is suppressed in LCL immortalized with EBERs knockout EBV compared to wild type. These results suggested that EBERs are able to promote IL-10 expression. However, the effects of transfected-EBERs in KMH2 and knockout-EBERs in LCL on the activation of TBK1 and IRF3 are insignificant. In this study, the mechanism of EBERs-induced IL-10 expression was not confirmed, and whether EBERs are the molecules that activate RIG-I and MDA5 to induce IL-10 in LCL remains to be discussed.
To sum up, the study revealed that both RIG-I and MDA5 are important in the expression of IL-10 in LCL. EBV infection induces the expression of MDA5 and activates the RLRs signaling pathways, which may up-regulate the transcription of IL-10 in LCL. Furthermore, we verified that EBERs promote the expression of IL-10; however, whether they are the molecules that activate RLRs to induce IL-10 in LCL remains to be investigated.

並列關鍵字

EBV ； EBERs ； RIG-I ； MDA5 ； IL-10

參考文獻

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