囊蟲症動物模式之建立及豬囊尾蟲抑制宿主Th1型肉芽腫發炎反應之探討

Abstract

本研究首先建立有鉤絛蟲 (Taenia solium) 與亞洲絛蟲 (T. saginata asiatica) 在近交品系小鼠的感染模式，以作為人類囊蟲病的動物模式 (animal model)。結果顯示，在測試的三種近交品系小鼠 －C3H/HeN(以下簡稱C3H)、C57BL/6與BALB/cAnN中，C3H與C57BL/6小鼠可成功地感染囊蟲，但以前者之蟲體回收率較高。C3H小鼠在感染囊蟲後4到6週時血清中IgG1總量升高，明顯呈現Th2型免疫反應。在C3H小鼠感染豬囊尾蟲6週後，以cDNA微陣列晶片分析比較活囊蟲周邊的組織與未感染的小鼠同部位組織，基因表現之差異，發現在囊蟲的周邊組織中，與發炎或Th1免疫型反應相關的細胞激素下降，而與Th2型反應相關或抑制發炎的因子上升，顯示囊蟲周邊的發炎反應被抑制而且免疫反應傾向Th2型。其中調控Th1型免疫反應的造骨素(osteopontin, OPN) 基因表現更明顯地降低，進一步以RT-PCR半定量分析的結果亦驗證OPN的表現降低。但以藥物praziquantel治療殺死囊蟲而導致其周邊組織發炎，則會大幅的提高OPN基因的表現，顯示活的囊蟲在宿主組織內具有降低OPN基因表現的能力。進一步的以體外細胞培養實驗證實，正常C3H小鼠之白血球 (blood leukocytes)與活囊蟲或囊蟲之排泌性抗原共同培養，白血球的OPN基因表現會降低，並使得IL-12 與IFN-γ亦隨之降低。以ELISA檢測感染囊蟲之C3H小鼠的血液單核球經囊蟲排泌性抗原刺激後細胞激素之產量，發現單核球產生的OPN與IL-12蛋白質的總量會下降，證實囊蟲所產生的排泌性物質會降低單核球OPN的表現，並抑制免疫反應傾向Th1型。再者，若將rmOPN(重組小鼠OPN) 或抗OPN抗體直接注射於移植囊蟲周邊的小鼠組織，接受rmOPN注射的小鼠，其移植囊蟲在四週後全部鈣化，而注射抗OPN抗體的小鼠，其囊蟲周邊之發炎反應則遠低於接受PBS注射的對照組，確認了OPN在囊蟲的肉芽腫發炎或鈣化反應中扮演重要的角色。整體而言，囊蟲可能藉抑制OPN基因之表現降低Th1發炎反應，並藉此機制逃避宿主免疫系統的傷害，因而延長囊蟲在宿主體內存活的時間。

In this study, we tried to use three inbred mice stains, C3H/HeN, C57BL/6 and BALB/cAnN established an animal model of Taenia solium and T. saginata asiatica metacestode infection for human cysticercosis. Worm recovery results demonstrated that C3H/HeN and C57BL/6 strains were able to be infected by injection of oncospheres. Moreover, C3H strain mice exhibited higher worm recovery than C57 mice. Immune response of mice was trended to Th2-type from 4 to 6 weeks post-infection, because the increase in total serum IgG1 level of C3H mice. Microarray analysis of C3H mice tissue surrounding 6-weeks-old viable T. solium cysticerci and tissue at similar location without cysticercus revealed that metacestodes infection led to the decrease in expression of inflammation- and Th1-type response related cytokines, and enhanced anti-inflammatory factors and Th2-type response cytokine expression. Of special interest was the drastic down-regulation in the expression of Osteopontin (OPN) gene. Furthermore, inflammation and granuloma formation in human neurocysticercosis has been attributed to Th1-type immune responses of the host. In the present murine model, over 94% of Taenia solium metacestodes were viable and elicited no granulomatous inflammation whereas parasites killed by praziquantel treatment elicited rapid granuloma formation that calcified within two weeks. OPN is a Th1-related cytokine that is up-stream of IL-12 and which may play an essential role in granuloma formation and calcification. OPN mRNA expression was down-regulated in tissues surrounding viable cysticerci, but was up-regulated in inflammatory tissues surrounding degenerating cysticerci. Moreover, co-culture with a viable cysticercus or excretory/secretory (ES) products from these metacestodes led to a decrease in OPN, IFN-γ, and IL-12 expression whereas co-culture with somatic protein enhanced OPN expression by leukocytes. Addition of recombinant mouse OPN (rmOPN) was able to counteract the down-regulation of IL-12 and IFN-γ mRNA expression, but not OPN mRNA expression in leukocyte cultures. Furthermore, injection of rmOPN into the tissues surrounding implanted cysticerci enhanced inflammatory responses while similar injection of anti-rmOPN antibody reduced inflammation. Suppression of host Th1-type granulomatous inflammation by ES products from T. solium metacestodes is related to down-regulation of OPN gene expression, confirming that OPN plays an important role in inflammation and granuloma formation. In conclusion, metacestodes inhibit Th1-type inflammation via decreasing expression of OPN gene and utilize this mechanism to escape damages caused by host immune response.