探討表現IL-15選擇性剪接異構體突變鼠感染第一型?疹病毒後在皮膚樹突細胞的活化及分化

Abstract

單純?疹病毒第一型(HSV-1)是感染人類常見的病原體，其感染始於黏膜組織並且可以於初次感染後終生潛伏在背根神經節(DRG)。藉由皮膚或周邊組織中的樹突細胞將HSV-1病毒抗原呈現給位於淋巴結中的初始T細胞以產生HSV-1抗原特異性T細胞是控制HSV-1感染所不可或缺的。已知IL-15具有促進樹突細胞活化、遷移與分化抗原特異性CD8＋ T細胞的作用。ENU誘變191譜系小鼠(P191)體內主要表達於IL-15基因外顯子7中具有部分缺失的一種選擇性剪接異構體，稱為IL-15ΔE7。我們先前的研究中發現，與野生型B6小鼠相比，主要表現IL-15ΔE7的P191小鼠在皮膚感染HSV-1後產生的gB-抗原特異性CD8＋ T細胞明顯減少其免疫功能亦有缺損。此外P191小鼠不僅募集至引流淋巴結的皮膚來源性樹突細胞較少，引流淋巴結中的周邊CD8α+ 樹突細胞與野生型小鼠相比也較少。初步實驗結果說明P191小鼠中表現較多的IL-15ΔE7對於樹突細胞的募集、存活甚至是HSV-1特異性CD8+ T細胞的增生及分化有抑制的作用。藉由免疫組織化學染色，我發現野生型小鼠在皮膚感染HSV-1後，其皮膚處的整體CD11c+ 樹突細胞在感染後第七天數量較P191小鼠少。流式細胞儀的分析結果顯示，P191小鼠皮膚處的朗格漢斯細胞及CD103+CD207+ 真皮層樹突細胞於HSV-1感染後第七天數量均較野生型小鼠多。野生型小鼠引流淋巴結中整體CD11c+ 樹突細胞數量在皮膚感染HSV-1後第七天到達高峰，而P191小鼠在感染後第七天，引流淋巴結中整體CD11c+ 樹突細胞較野生型小鼠少。進一步分析CD11c+樹突細胞亞群發現減少的數量主要源自皮膚募集的朗格漢斯細胞及週邊CD8α+ DC，意味此兩群樹突細胞亞群最可能受到IL-15ΔE7的影響。我們進一步研究並發現，P191小鼠不論是皮膚處或引流淋巴結的CD4+和CD8+ T 細胞在皮膚感染HSV-1後，數量均較野生型小鼠少。其中P191小鼠引流淋巴結中CD8+ T細胞CD44活化標記及IL-15受體CD122的表現量均較野生型小鼠少。P191小鼠皮膚感染HSV-1後減少的朗格漢斯細胞及常駐CD8α+ DC與增長較少的CD4+和CD8+ T 細胞，兩者的關聯可揭示IL-15ΔE7在產生有效的抗病毒免疫反應中具有負調控的角色。更詳細的機制則需要更多的實驗加以說明。

Herpes simplex virus type 1 (HSV-1) is a common human pathogen. Primary infection is initiated at mucosal tissues and may establish life-long latency in the dorsal root of ganglia (DRG). Presentation of peripheral HSV-1 viral antigen by skin-derived and regional dendritic cells (DCs) is essential to prime nave CD8+ T cells in draining lymph nodes to develop HSV-1 specific CD8+ T cell immunity for HSV-1 control. IL-15 is reportedly to play critical roles both in the activation and migration of DC and helping the priming of antigen-specific CD8+ T cells. The ENU-mutagenized mice pedigree 191 (P191) is prevalent in expressing an alternatively spliced IL-15 that has a partial deletion in the exon 7, IL-15ΔE7. Results from our previous experiments found that HSV-1 skin infection of P191 mice resulted in reduced number of gB-specific CD8+ T cells compared to wild type (WT) B6 mice. Effector functions of P191 CD8+ T cells were also impaired. Not only skin-migratory DCs were less recruited to draining lymph nodes, the population size of regional CD8α+ DCs was reduced in P191 compared to WT mice. These results have led us to hypothesize that increased expression of IL-15ΔE7 in P191 mice may negatively regulate the recruitment and survival of DCs and may further affect the proliferation and differentiation of HSV-1 specific CD8+ T cells. Using immunohistochemical staining, I found that total number of CD11c+ DC was decreased in the skin of WT mice on day 7 after HSV-1 skin infection compare with P191. Analysis of flow cytometry demonstrated that the number of the Langerhans cells and CD103+CD207+ dDCs were increased in the lesional flank skin of P191 mice on day 7 after HSV-1 infection compare with WT mice. The number of CD11c+ DC in WT draining lymph nodes was peaked on day 7 after HSV-1 infection in comparison with decreased CD11c+ DC in P191 mice. Further analysis of subsets in CD11c+ DC found that the decrease was largely attributed by the number of skin-recruiting Langerhans cells and regional CD8α+ DC, implicating both DC subsets were most likely affected by IL-15ΔE7. We further investigated and found that the number of CD4+ and CD8+ T cells were reduced both in the lesional flank skin and draining lymph nodes of P191 mice after HSV-1 infection. The expression level of activation marker CD44 and IL-15 receptor CD122 were decreased in the CD8+ T cells from the draining lymph nodes of P191 mice compare with WT mice after HSV-1 infection. The correlation between reduced cell numbers of Langerhans cells and resident CD8α+ DC and the decreased expansion of CD4+ and CD8+ T cells in P191 mice after HSV-1 skin infection has suggested a negative role of IL-15ΔE7 in generating effective anti-viral immune responses. More experiments are needed to carry out to delineate detail mechanisms.