探討第一型單純疱疹病毒經皮感染表現IL-15異構細胞激素突變鼠後的先天性免疫反應

Investigation of innate immune response in HSV-1 zosteriform infection of ENU-mutagenized mice that express IL-15 alternative splice variant

莊哲銘(Che-Ming Chuang)

指導教授 ： 顧家綺

國立臺灣大學/醫學院/免疫學研究所/碩士(2012年)

https://doi.org/10.6342/NTU.2012.01707

摘要

Interleukin-15 (IL-15)是一種多效性的細胞激素，表現在許多不同類型的細胞。藉由發炎反應的進行調節先天性免疫反應的作用並且具有促進自然殺手細胞發育、維持記憶性CD8 細胞的恆定等功能，因此在對抗病毒感染中扮演至關重要的角色。中研院基因突變鼠動物模式核心實驗室 (MMPCF)利用ENU的致突變機制產生的191品系突變鼠 (以下稱P191), 體內可表現顯著的IL-15選擇性剪接異構體mRNA (簡稱IL-15_ASE7)。雖然離體實驗曾被證明IL-15_ASE7可能具有抑制原型 IL-15生物活性的能力，它在動物體內的功能與作用仍然不清楚。本論文利用 HSV-1 感染小鼠的腹背側皮膚模式，比較抗 HSV-1感染的先天性免疫反應在該ENU 突變鼠與野生鼠的差異之處，以探討IL-15_ASE7在皮膚中調節抗病毒的先天性反應的可能角色。
本論文的實驗結果顯示，與B6相較之下，P191小鼠皮膚在 HSV-1 感染之後產生較大和嚴重的疱疹傷口，且皮膚損傷修復的時間延遲，病毒溶斑形成定量分析結果也顯示P191小鼠皮膚中的病毒量在感染後第3和5天均較高於 B6 小鼠。雖然P191 與B6 小鼠皮膚感染HSV-1後的病理現象類似，例如組織間細胞體積變大、表皮組織增生變厚、細胞崩解、壞死細胞的染色質聚集在一起呈現許多不規則的團塊狀以及皮層細胞之間空泡的形成，但是免疫細胞浸潤現象在P191小鼠卻大幅減少。進一步利用免疫組織化學染色分析發現感染HSV-1後的浸潤細胞組成，B6的皮膚疱疹中從初期(第1-3天)的中性白血球與中、後期 (第5-7天) 所出現的單核球與淋巴球，在P191小鼠都大幅下降並且達到統計的顯著差異 (p<0.01)。更重要的是在感染後 12和24 小時， 即時定量聚合酶分析結果顯示雖然MCP-1、 IL-1b 和TNF-a 的 mRNA表現量在 B6 與 P191 小鼠兩者的疱疹皮膚中差異不大， P191 小鼠皮膚中嗜中性白血球吸引趨化因子CXCL1 (KC) 和 IL-6 的mRNA表現量則比B6小鼠顯著下降。特別的是，HSV-1感染B6與P191小鼠皮膚後分別誘增IL-15與IL-15_ASE7 mRNA的表現。這些結果說明IL-15調節傳遞的訊號路徑對於造成小鼠皮膚特定細胞激素和趨化因子 mRNA 的表現有尚未被釐清的角色。 IL-15 如何調控皮膚對抗病毒感染的先天性免疫反應的機制值得未來作進一步的探討。

關鍵字

介白質-15 ； 第一型單純疱疹病毒 ； 皮膚

並列摘要

Interleukin-IL-15 (IL-15) is a pleiotropic cytokine expressed by a broad range of cell types. Not only it functions as a proinflammatory cytokine during innate immune response but also supports the development of natural killer cells and homeostasis of memory CD8 T cells. Therefore, it has been recognized as an important cytokine against viral infection. An ENU-mutagenized pedigree 191 (P191) generated at the Mouse Mutagenesis Program Core Facility (MMPCF) predominantly expresses an alternative splice IL-15 mRNA isoform called IL-15_ASE7. Studies from in vitro experiments demonstrated IL-15 splice variant protein inhibited IL-15 dependent cell proliferation; however, its function in vivo remains unclear. Using HSV-1 zosteriform mouse model, immunological consequences from HSV-1 infection were compared between B6 and P191 mice to clarify the role for IL-15 alternatively spliced variant in regulating innate immune response in skin.
Results from these experiments showed that HSV-1 created larger and severe skin lesions and delayed kinetics of skin lesion repair in P191 mice compared with B6 mouse skin. Furthermore, viral titers recovered from P191 lesional skin determined by plaque assay were higher than those from B6 lesional skin on days 3 and 5 post infection. Characteristics of HSV-1 resulted skin pathology including cell ballooning, reticular degeneration, chromosome condensation and vesicle formation in the epidermis were similar between B6 and P191 mice. However, levels of immune cell infiltration in the dermal layer were much reduced in P191 lesional skin. In addition, immunohistochemical analysis also confirmed that the influx of Gr1+ neutrophils on day 1 to day 2 and substantial infiltrations of lymphocytes and monocytes at day 3 to day 7 in B6 mice after infection were overall reduced in P191 skin (p<0.01). Importantly, real-time q-PCR analysis showed that the expression levels of MCP-1, IL-1b and TNF-a mRNA were comparable between B6 and P191 skin; however, the transcripts for neutrophil-attracting chemokine CXCL1 (KC) and IL-6 were marginally induced at 12 hr but dramatically down-regulated at 24 hr in HSV-1 infected P191 skin as opposed to elevated levels in B6 skin. The differential expression patterns of inflammatory cytokine and chemokine genes in skin between wild type and P191 mice after HSV-1 infection have implicated a yet defined role for IL-15 mediated signaling. The molecular mechanism by which IL-15 signaling controls innate immune response in skin against viral infection will be further explored.

並列關鍵字

IL-15 ； HSV-1 ； skin

參考文獻

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