探討第一型單純疱疹病毒在表現IL-15選擇性剪接異構體ENU 突變鼠的皮膚與背根神經節的感染力

Abstract

單純疱疹第一型病毒 (HSV-1)是一種雙股DNA病毒。其可以感染黏膜表皮細胞，並在中樞神經中建立潛伏性感染，再次活化的病毒可以造成免疫力下降的宿主新的病徵。 HSV-1從皮膚感染小鼠之後，會在感染的局部 (初級感染區)先進行複製，之後以反向軸突運輸的方式進入背根神經節。當病毒再次從神經細胞以順向軸突運輸的方式突現時，會在該神經分布的皮膚上觀察到新的病毒傷痂 (稱為次級感染)。 IL-15ΔE7是IL-15 pre-mRNA通過選擇性剪接機制產生的異構體。我的論文研究和先前的實驗結果均發現， ENU突變P191品系小鼠(P191)皮膚會表現相當數量的IL-15ΔE7及IL-15 mRNA。感染HSV-1後，可以觀察到P191小鼠比野生型B6小鼠形成較嚴重而且持續較長時間的病毒傷痂。因此本論文的主要目標是希望進一步探討，皮膚在IL-15ΔE7調節的免疫反應情況下對於HSV-1在皮膚以及背根神經節的感染力的影響。 實驗結果顯示，雖然HSV-1感染野生型小鼠皮膚48小時後可以誘導IL-15 mRNA 的表現， P191小鼠皮膚感染HSV-1後，反而容易誘導IL-15ΔE7的表現量，增加的情形比IL-15 mRNA 提高5倍。對病毒傷痂皮膚進行病理分析的結果發現，HSV-1感染野生型小鼠皮膚48小時後會在表皮觀察到合胞體細胞、染色質邊緣化等細胞病變，可以觀察到免疫細胞浸潤現象；感染後的表皮細胞在第五到七天時大量死亡同時伴隨新的角質細胞增生，免疫細胞浸潤程度更為顯著；第十天時原來的傷痂已經癒合。反觀P191小鼠，感染後的表皮細胞在48小時內即出現顯著的細胞死亡及大範圍的組織破壞，但是不若野生型小鼠皮膚有免疫細胞浸潤的現象。因HSV-1感染導致細胞死亡及組織破壞可以延伸至真皮層，感染後第十天雖然在病毒傷痂處的表皮有新增生的角質細胞，表皮卻未癒合修復。從病毒溶斑分析的結果發現，野生型小鼠皮膚在感染後48小時病毒量達到最高，在感染後第四天病毒量開始減少。相較之下，P191小鼠皮膚受HSV-1感染後，病毒量同樣在48小時達到最多，在感染第四天些微減少後卻在皮膚維持一定的病毒量。這些實驗結果說明P191小鼠皮膚感染HSV-1後容易發生細胞死亡、免疫細胞浸潤減少、表皮細胞修復慢等因素，可能是造成P191小鼠HSV-1傷痂在外觀上比野生型小鼠更加嚴重的原因。以即時定量PCR檢測小鼠背根神經節中的病毒量的實驗結果發現，與野生型小鼠相較下，HSV-1可以更早在P191小鼠的背根神經節增殖而且延長在該組織中擴增的結果，這極有可能是造成P191小鼠的腹側出現病毒傷痂持續時間較長的因素。 總體來說，我的論文研究發現IL-15 的表現對於提高皮膚對HSV-1感染的抵禦能力相當重要。表皮角質細胞對HSV-1感染的抗性增加不僅維持皮膚的完整性，而且還引發對HSV-1的適當免疫反應以達到更好和更早的表皮恢復。而當HSV-1感染P191小鼠皮膚主要是誘發IL-15ΔE7的表現時，感染的表皮細胞的大量且快速死亡除了阻礙免疫細胞浸潤到皮膚的路徑與作用，角質細胞增生慢、延遲皮膚傷痂修復時間，除了提高HSV-1進入背根神經節，周邊皮膚免疫功能下降也可能促使病毒在神經細胞複製和擴增。IL-15ΔE7容易促使角質細胞感染HSV-1後死亡的分子機制將值得作進一步的探討。

Herpes simplex virus-1 (HSV-1) is a double-stranded DNA virus. It can infect mucoepithelial cells and establish latent infection in the central neuron system. Reactivation of HSV-1 from latently infected ganglia of immune-reduced host may cause new disease. After infection via flank skin, HSV-1 first replicates locally (primary infection site), and then conveys to the dorsal root ganglia (DRG) by retrograde axonal transport pathway. When HSV-1 re-emerges from infected neurons by anterograde axonal transport, new viral lesion can be observed along the innervating dermatome (secondary infection site). IL-15ΔE7 isoform is produced by an alternatively splicing mechanism from IL-15 pre-mRNA. Our previous studies have found that the skin of an ENU-mutagenized pedigree 191 (P191) mice expressed comparable levels of IL-15ΔE7 and IL-15 mRNAs. Infection of P191 flank skin with HSV-1 caused a more severe and prolonged HSV-1 lesion compared to wild type C57BL/6 mice (WT). The overall goal of my thesis is to further investigate the effect of IL-15ΔE7 modulated-skin immune response on the infectivity of HSV-1 in skin and dorsal root ganglia in P191 mice. Results from these experiments showed that HSV-1 infection upregulated transcription of IL-15 mRNA in WT skin at 48 hours post infection. In contrast, a greater transcriptional level of IL-15ΔE7 than IL-15 mRNA was induced by HSV-1 in P191 skin. While cytopathology such as syncytia and chromatin marginalization were observed in HSV-1 infected epidermal keratinocytes within 48 hours following by evident cell death between 5-7 days post infection in WT mice, prominent cell death was found in the epidermis of P191 mice as early as 24 hours post infection. Less immune cell infiltration to leasional skin was found in P191 compared to WT mice during primary HSV-1 infection. Viral lesion was healed by newly proliferated keratinocytes by day 10 post infection in WT mice. However, the epidermis was not completely healed at day 10 in P191 mice. HSV-1 replication was evaluated by measuring viral yields from lesioanl skin by plaque forming assays. Virus titers were peaked at 48 hours, starting to decline at day 5 and continued to decrease at day 7 post infection in WT mice. While HSV-1 replication was comparable between WT and P191 at day 2, virus titers remained at a stable level until day 7 in P191 skin. Accumulated HSV-1genomes in the innervating DRG of P191 were higher in the quantity as well as in the duration compared to those of WT mice after HSV-1 primary infection. Results from my thesis research found that induced expression of IL-15ΔE7 expression by HSV-1 enhances cytopathology in both epidermis and the dermis, decreasing anti-viral immune responses and prolongs viral replication in the skin after primary HSV-1 infection. Enhancement of viral replication in the skin may facilitate HSV-1 to replicate in the innervating DRGs for a longer period and therefore increase HSV-1 reactivation from the infected neurons. Altogether, this research has emphasized an important role for IL-15 in mediating HSV-1 pathogenesis in the skin and the DRGs. The increased resistance of epidermal keratinocytes in response to HSV-1 infection not only maintains the integrity of the skin which initiates appropriate immune responses to HSV-1 but also attenuates HSV-1 replicative capability in the DRGs. The molecular mechanism by which IL-15ΔE7 promotes the death of keratinocytes after HSV-1 infection is worth to further investigate.