探討IL-15異構蛋白對HSV-1皮膚疱疹與神經侵襲力作用的動物模式

Abstract

第 15 號細胞激素 (IL-15)具有維持記憶性 CD8、自然殺手以及自然殺手 T 細 胞的發育和細胞數恆定的功能。IL-15 同時也具有驅化免疫細胞、血管新生以及 增強發炎激素生成的作用。更多的證據指出組織中表現過量的 IL-15 與多種人類 發炎疾病與癌化的相關。正常小鼠體內除了表現 IL-15 之外,也會經轉錄後修飾 作用使 Exon 7 部分剔除形成異構體 IL-15。由於表現該異構體 IL-15 的 ENU 突變 鼠,發生記憶性 CD8 細胞缺失的結果,因此異構體 IL-15 命名為 DM-IL-15。本 實驗室過往的研究結果發現,經過表現 DM-IL-15 DNA 質體的小鼠皮膚,對於引 起皮膚發炎的各式刺激例如:機械性刮擦、化學物質刺激或是免疫製劑處理都比未 處理組皮膚呈現較不敏感的反應。在我的論文中主要的研究目的為以動物模式探 討 DM-IL-15 對於HSV-1 皮膚疱疹與神經細胞侵襲力的影響。利用主成份分析法 比較 179 個與發炎反應相關基因的表現在皮膚表現 DM-IL-15 實驗組和空質體控 制組的皮膚受 HSV-1 感染後的實驗結果發現:表現 DM-IL-15 的皮膚受 HSV-1 感 染後,促進嗜中性白血球趨移的趨化激素基因(CXCL1、CXCL2、CXCL3、 CXCL5)和促發炎基因(IL-1β、IL-6 及 TNFSF14)表現量也都低於對照組皮膚,顯 示 DM-IL-15 可能藉由調控角質細胞中的 NF-κB 活化路徑,使宿主發炎反應活性 降低的功能;除此之外,DM-IL-15 轉染的皮膚會增加 IL-4、IL-23A 及 LTA 的表 現量,顯示 T 細胞活化相關的免疫反應可能受到影響。正常小鼠皮膚受 HSV-1 感 染後第 2 天開始形成疱疹傷痂,但於 10 天開始癒合,但是表現過 DM-IL-15 的皮 膚經過 HSV-1 後感染後第 14 天依然有嚴重的傷痂,且從 H&E 染色結果可看到嗜 中性白血球的浸潤。在 25 天的連續觀察實驗中,與對照組小鼠相比,皮膚表現 DM-IL-15 增加 HSV-1 感染後疱疹傷痂嚴重度和範圍大小,且傷痂出現的時間更 早,持續時間更久。降低 HSV-1 接種量後,可看到表現 DM-IL-15 的皮膚中 HSV-1 轉錄因子(ICP4)和病毒結構蛋白(Glycoprotein B)的 mRNA 表現量高於對照 組,顯示 DM-IL-15 可能在 HSV-1 感染前已對皮膚的免疫環境造成影響而提高病 毒複製的結果。進一步分析背根神經節中的 HSV-1 病毒活性,表現 DM-IL-15 的 皮膚感染 HSV-1 後第七天與第十天,在背根神經節中 ICP4 和 gB 基因表現量同樣 都高於對照組。以上結果顯示 DM-IL-15 對於皮膚組織和神經細胞對抗 HSV-1 感染的能力具有重要的影響,將來進一步釐清 DM-IL-15 主要作用於何種免疫細胞 族群與調控因子。

Interleukin-15 (IL-15) plays an important role in maintaining the homeostasis and the development of memory CD8 T cells, NK cells and NKT cells. IL-15 functions to increase immune cells infiltration, angiogenesis and pro-inflammatory cytokine production. Clinical researches show that overexpression of IL-15 in tissues is correlated with human inflammatory diseases and carcinogenesis. ENU mutant C57BL/6 mice that predominantly expressed an alternatively spliced IL-15 isoform generated from partial deletion in exon 7 of the IL-15 gene are deficient in memory CD8+ T cells. The IL-15 isoform is thus named DM-IL-15. Our previous study demonstrated that ectopic expression of DM-IL-15 in the wild type mouse skin resulted in the modulated proinflammatory response in skin after various types of stimulation by abrasion, chemical compound and immunologic agent. The overall goal of my thesis was to investigate the effects of DM-IL-15 on Herpes simplex virus-1 (HSV-1) zosteriform and neuroinvasiveness in mouse model. Principal component analysis of the transcriptional levels of 179 inflammation related genes demonstrated that they were differentially expressed in wild type C57BL/6 (B6) mouse skin expressed with empty vector (WT/pEV) or a plasmid expressing DM-IL-15 (WT/pDM-IL-15) after HSV-1 infection. Forced expression of DM-IL-15 reduced the gene expression levels of neutrophil chemokines (CXCL1, CXCL2, CXCL3 and CXCL5) and pro-inflammatory cytokines (IL-1β, IL-6 and TNFSF14) in HSV-1 infected skin tissue, suggesting that DM-IL-15 might regulate NF-κB signaling in skin after HSV-1 infection. DM-IL-15 expressing skin also expressed a higher level of IL-4, IL-23A and LTA than control skin after HSV-1 infection, implicating DM-IL-15 was likely involved in regulating the development of T cell immunity against HSV-1 infection. While HSV-1 skin lesion was evident on day 2 and resolved on day 10 post infection, HSV-1-infected DM-IL-15 expressing mouse skin showed severe lesion until day 14 after HSV-1 infection accompanied with evident neutrophil infiltration by H&E analysis. In a 25-day time course experiment, daily documentation on the development of HSV-1 lesions observed that an earlier onset of new lesion and prolonged skin lesion at the non-inoculated ventral side occurred in DM-IL-15 expressing compared with pEV-treated mouse skin. The higher transcriptional levels of ICP4 gene encoding HSV-1 transcriptional factor and gB gene encoding structural glycoprotein B expressed in DM-IL-15 expressing mouse skin after infected with low dose of HSV-1 have suggested that expression of DM-IL-15 before HSV-1 inoculation may modulate host cell response and promote viral infection in skin. Moreover, the levels of transcription of ICP4 and gB in the dorsal root ganglion (DRG) isolated from correspondent animals at days 7 and 10 were higher in DM-IL-15 expressed than in control group. These results indicate that DM- IL-15 plays an important role in modulating HSV-1 pathogenesis in skin and the neuron. Cells or mediators that are targeted by DM-IL-15 require further thorough investigations.