探討Siglec-5和Siglec-14受體對於單核球細胞抗病毒反應的調控作用

To investigate the roles of Siglec-5 and Siglec-14 in antiviral responses in monocytes

林佩昀(Pei-Yun Lin)

指導教授 ： 張永祺

國立臺灣大學/醫學院/微生物學研究所/碩士(2019年)

https://doi.org/10.6342/NTU201902153

摘要

Siglec為辨識唾液酸的受體，其中的Siglec-5和Siglec-14表現於單核球細胞、巨噬細胞和嗜中性白血球，能夠受相同的配體活化，分別傳遞抑制性和活化性的訊息。人類已知具有SIGLEC5/14基因多型性，此基因多型性可影響個體在病原體感染後的抗菌結果及發炎程度。我們與其他的實驗室均發現Siglec-14可以促進細胞在細菌感染後的促炎性反應，但關於SIGLEC14基因多型性在病毒感染後對個體的促炎性反應或是抗病毒反應之研究則尚不清楚。A型流感病毒之感染經常伴隨細胞激素風暴發生，造成肺部組織受損。仙台病毒能引起強烈的宿主抗病毒反應，被廣泛用來探討宿主IFNs和其下游基因表現，因此我們將同時以A型流感病毒與仙台病毒對人類細胞株的感染模型來探討SIGLEC5/14基因多型性對單核球細胞抗病毒的先天免疫反應之調控。我們發現表現Siglec-14的THP-1細胞(S14/THP-1)在H5N1或SeV感染時促炎性細胞激素產生較S5/THP-1細胞多，而且H5N1感染時，S14/THP-1細胞中的p38和AKT也較S5/THP-1細胞活化，顯示Siglec-14可能透過增加p38和AKT的活化來增進細胞的促炎性反應。IAV或SeV感染時，S5/THP-1細胞製造比S14/THP-1細胞多IFN-b的mRNA，但是IFN-b蛋白表現量不易測定，而且參與調控type I IFN表現的磷酸化態TBK1和IRF3也無法在H5N1感染後被偵測到，所以我們之後會再尋找適合的實驗方法或條件得到這個部分的結論。此外，我們發現Siglec-5和Siglec-14不會影響IAV複製，表示Siglec-5和Siglec-14對於先天免疫反應不同的調控與IAV進入細胞的多寡和新病毒顆粒產生的速度無關。已知鍵結具有ITIM的Siglecs會造成細胞凋亡，因此我們也進一步探討Siglec-5和Siglec-14是否會影響單核球細胞被病毒感染後的存亡。結果顯示S14/THP-1細胞在IAV感染時的存活率較S5/THP-1細胞好，但是其調控機制還不清楚。除此之外，我們的實驗結果也顯示IAV會直接結合Siglec-5和Siglec-14，暗指IAV可能可以透過鍵結Siglec-5和Siglec-14直接影響單核球細胞抗病毒的先天免疫反應。

關鍵字

Siglec ； SIGLEC5/14基因多型性 ； 先天免疫反應 ； 病毒感染

並列摘要

Siglecs (sialic acid-binding immunoglobulin-like lectins) are receptors recognizing sialic acids. Siglec-5 and Siglec-14, expressing on monocytes, macrophages and neutrophils, can transduce inhibitory and activating signals through ITIM and ITAM, respectively, upon ligand engagement. In addition, SIGLEC5/14 polymorphism has been discovered in human population and the presence of SIGLEC14 WT allele has been linked to exaggerated host antimicrobial and inflammatory responses upon infection. We and others have demonstrated that Siglec-14 promotes proinflammatory responses of macrophages and neutrophils upon bacterial infection. However, the role of SIGLEC5/14 polymorphism in host proinflammatory and antiviral responses upon viral infection is largely unknown. Influenza A virus (IAV) infection often associates with exacerbated host inflammation (cytokine storm) and eventually leads to lung tissue destruction. Sendai virus (SeV), structurally similar to IAV, is a wildly used model to study host antiviral responses since SeV infection constantly mounts a robust host interferon (IFN) response. Thus, we applied both IAV and SeV to infect THP-1 cells expressing either Siglec-5 or Siglec-14 to explore the role of SIGLEC5/14 polymorphism in antiviral innate immune responses. We found that H5N1 or SeV infection stimulates more TNF-a and IL-1b production in S14/THP-1 cells (THP-1 cells expressing Siglec-14) compared with that produced in infected S5/THP-1 cells (THP-1 cells expressing Siglec-5). In addition, H5N1 infection caused enhanced p38 and AKT phosphorylation in S14/THP-1 cells. Our data suggest that Siglec-14 may promote proinflammatory responses through activating p38 and AKT signaling pathway upon viral infection. In contrast, reduced production of IFN-b mRNA was observed in S14/THP-1 cells infected with IAV or SeV. Neither the released IFN protein nor the activated TBK1 and IRF3 can be detected in our experimental system, we will therefore optimize experimental conditions to further clarify the role of Siglec-14 in regulating host IFN responses. Comparable IAV replication was observed in S5/THP-1 and S14/THP-1 cells, indicating that reduced IFN responses in the S14/THP-1 cells upon IAV infection were not directly affected by restricted virus entry and virion production by Siglec-14. Engagement of ITIM-bearing Siglecs have been shown to trigger cell death in various cell types, we next examined whether Sigelc-5 and Siglec-14 can modulate cell survival upon IAV infection. Our data showed that IAV-infected S14/THP-1 cells survive better than infected S5/THP-1 cells, although the detail mechanism is still unknown. Finally, we demonstrated that IAV can directly interact with Siglec-5 and Siglec-14, indicating that IAV may engage Siglec-5 or Siglec-14 expressing on the monocytes to modulate their antiviral innate immune responses.

並列關鍵字

Siglec ； SIGLEC5/14 polymorphisms ； innate immune responses ； viral infection

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