探討小鼠在一次和二次登革感染後T細胞反應作用

Abstract

登革是世界上最為流行的疾病之一。依照其病徵可分為：登革熱(DF)，出血性登革熱(DHF)和登革熱休克(DSS)。出血性登革熱(DHF)目前診斷的依據為血漿滲出(plasma leakage)，血小板過低(throbocytopenia)和外部流血(bleeding)。感染登革病毒可能引發出血性登革熱(DHF)，而研究顯示出血病例多數發生在登革二次感染的病人身上。目前對於登革二次感染，宿主體內的T細胞反應仍不甚清楚。 本實驗中，我利用實驗室建立的老鼠出血模式，觀察到老鼠感染後T細胞的活化。而且登革二次感染無論是同型或不同型病毒，比起登革一次感染都較多比例的細胞活化並且有較多產生細胞激素的T細胞產出現。老鼠二次感染後，腋窩淋巴結內CD4+ 和 CD8+ T細胞會表現CD69+， CD44hi 和 CD62Llow。此外腋窩淋巴結內的CD4+ 和 CD8+ T細胞也表現了CLA和CCR4，讓T細胞可以準備進入皮膚。實驗中我觀察到這些活化的現象主要出現在二次感染後第1天到第1.5天，而這些T細胞也在感染後第1.5天時，表現皮膚趨化因子CLA和CCR4。 在體外實驗中，我利用anti-CD3抗體刺激來觀察腋窩淋巴結內CD4+ 和 CD8+ T細胞表現細胞激素的能力。實驗結果顯示，二次感染老鼠有能力產生TNF-α的CD4+ 和 CD8+ T細胞都顯著高於一次感染老鼠，而二次感染老鼠產生IFN-γ的CD4+ T細胞雖顯著高於一次感染，但IFN-γ的CD8+ T細胞卻無顯著差異。 總結這些結果，我觀察到老鼠在皮下注射感染登革病毒後，腋窩淋巴結內T細胞會活化。而T細胞的活化主要發生在登革二次感染後第1天到第1.5天。在二次感染後第1.5天，病灶淋巴結內T細胞會表現皮膚趨化因子。另外，二次感染後老鼠病灶淋巴結內的T細胞比起一次感染有較多能產生TNF-α 和IFN-γ。這篇論文是首次使用登革出血老鼠模式研究病灶淋巴結T細胞表現皮膚趨化因子，或許這些結果可此解釋為何在皮下感染登革後，皮膚常為出血患處的原因。

Dengue is one of the most widely spread infectious diseases in the world. Dengue disease manifests different clinical symptoms which are categorized as: dengue fever (DF), dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Dengue hemorrhagic fever is diagnosed as having plasma leakage, thrombocytopenia and hemorrhage manifestations including bleeding from nose and gum, skin hemorrhage and blood in vomits in addition to common DF symptoms. Although hemorrhage can develop in patients with primary DENV infection, greater percentage of patients who develop DHF experience secondary infection. The contribution of T cells to host response to DENV infection in secondary infection is still not well understood. In this study, utilizing the hemorrhage mouse model our laboratory developed, I investigated T cell activation and cytokine production in mice infected with homotypic as well as heterotypic secondary DENV infections and compared to mice with primary infection. CD4+ and CD8+ T cell expressions of CD69+, CD44hi and CD62Llow were analyzed. In addition, CD4+ and CD8+ T cell expressions of CLA and CCR4, skin homing receptors, were also studied. Here, I found that CD4+ and CD8+ T cells in the brachial lymph nodes expressed activation phenotypes at as early as days 1-1.5 after secondary infection and the activated T cells expressed skin homing receptors. With plate-bound anti-CD3 antibody to restimulate brachial lymph node cells in vitro, I analyzed CD4+ and CD8+ T cell cytokine response. The results showed that there were higher percentages of TNF-α-producing CD4+ and CD8+ T cells in the lymph nodes of mice with secondary infection than that in mice with primary infection. There were higher percentages of IFN-γ-producing CD4+ but not CD8+ T cells in the lymph nodes of mice with secondary infection than that in mice with primary infection. In summary, the results together showed that draining lymph node T cells are activated after intradermal inoculation of DENV. T cell activation and expansion occur as early as days 1-1.5 after secondary infection. T cells in the draining lymph nodes also express skin homing receptors after secondary infections and higher percentages of them produced TNF-α and IFN-γ than in mice with primary infection. This is the first study utilizing hemorrhage mouse model to show that T cells in the draining lymph nodes express skin homing receptors, which could possibly explain why skin is the most prevalent site of hemorrhage manifestations after intradermal DENV inoculation.