Influenza virus infection may activate dendritic cells (DCs) and trigger the pro-inflammatory and adaptive immune responses in hosts. In this study, we prepared recombinant hemagglutin (HA) proteins of mouse-adapted H1N1 (WSN), swine-origin 2009 pandemic H1N1 (Texas), and high-pathogenic avian influenza H5N1 (KAN-1) viruses using baculovirus/insect cell expression system and purified through Ni-NTA resin chromatography. These purified H1 and H5 HA proteins were treated with mouse bone marrow-derived dendritic cells (BMDCs) and measured for cytokine production using FACS analysis and ELISA. Our results showed that TNF-□ and IL-12 p70 production by DCs treated with H1 or H5 HA proteins were all increased dose-dependently. However, no significant differences were found in the cytokine production between the treatments of H1 and H5 HA proteins. Proteinase K treatment of H1 and H5 HA proteins demonstrated the increase of TNF-□ production in DCs was due to the HA protein stimulation. Higher levels of CD40 and CD86 expression as well as the reduce of endocytosis capability were also observed on H1 and H5 HA-treated DCs, suggesting the DCs were activated and maturated through H1 and H5 HA proteins stimulation. In addition, the activated pathways by H1 and H5 HA proteins were examined in DCs obtained from TLR4 mutant and MyD88-/- mice. The data showed that DCs can be activated through TLR4-MyD88 dependent pathway. The results implied that HA proteins could promote T-cell differentiation into TH1 cells and trigger CD8+ cytotoxic T lymphocytes activation in adaptive immune responses. These findings provide some clues to understand how the activation and maturation of dendritic cells response to influenza virus infection, which may be applied for new cross-protective vaccine design against influenza virus infection in the future.

流行性感冒(Influenza)是全球普遍的感染性疾病，每年導致高或低的發病率及致死率，甚至對全球經濟造成衝擊。早期研究指出流感病毒血球凝集素hemagglutinin (HA)被視為調控流行性感冒病毒毒性、致病性的重要標的物；受到流感病毒感染後會刺激促發炎(pro-inflammatory)因子的生成，並進一步活化樹突狀細胞(dendritic cells)和引發後天性(adaptive) 的免疫反應。然而，目前對於血球凝集素如何參與調控流行性感冒及其對免疫反應的影響機制仍不清楚。此篇研究主要是利用桿狀病毒昆蟲表現系統(baculovirus/insect cell expression system)分別產生mouse-adapted H1(WSN)、2009年爆發的豬起源(swine-origin)流行性感冒 H1 (Texas)和禽流感H5(KAN-1)的重組血球凝集素蛋白，並用 Ni-NTA resins將其有效地純化出來並用西方墨點法確認HA特性。接著將這些重組蛋白加入老鼠骨髓來源的樹突狀細胞(bone marrow-derived dendritic cells(BMDCs))中，並利用流式細胞儀(FACS)和酵素結合免疫吸附分析(ELISA)偵測細胞激素TNF-□□和IL-12□p□□的產量。結果顯示此三種不同品系(strains)的流感病毒蛋白HA皆可以促進TNF-□和IL-12p70的生成，活化DCs，但三者之間並無顯著的差異性。另外，利用proteinase K，進一步證明DCs的活化是由於HA蛋白的刺激所致。除此，藉由DCs表面上輔助刺激分子(co-stimulatory molecule) CD40和CD86表現量的提升及其內嗜作用能力(endocytosis capability)降低的結果，可知當DCs被流感病毒HA蛋白刺激後，會活化且進一步成熟，由以上結果推判DCs可完整呈現抗原並進一步活化TH1細胞分化和/或刺激胞殺性T淋巴球(CTL)後天性免疫反應。另外，利用TLR4 mutant和MyD88-/-基因剔除鼠(knock-out mice)進一步證明DCs的活化途徑和TLR4¬-MyD88路徑相關。藉由上述結果可讓我們對於DCs在受流行性感冒病毒感染時，其所參與調控HA的機制更為了解，這些資訊可提供重要的參考依據，對日後高致病性(pathogenicity)或高傳染性(transmission)流行性感冒的疫苗設計或是專一性的藥物標靶治療將有所助益。

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