- 學位論文
探討CLEC5A參與巨噬細胞引發的發炎反應及相關疾病
The roles of CLEC5A in the inflammatory responses in macrophages and associated diseases
摘要
CLEC5A是先天免疫受體中C型凝集素(C-type lectin)受體的一員，並且廣泛的表現在骨髓細胞。過去的研究顯示CLEC5A在登革熱病毒與日本腦炎病毒的感染、類風溼性關節炎和抽菸造成的肺部發炎扮演著重要的角色。我們使用CLEC5A基因剔除的老鼠來研究CLEC5A在盲腸穿刺的敗血症疾病模式和葡聚醣硫酸鈉(DSS)引起的結腸炎扮演的角色。同時我們使用CLEC5A基因剔除老鼠的骨髓衍生的巨噬細胞來探討CLEC5A在TLR4訊息傳遞、TLRs引發的發炎基因表現、M1和M2巨噬細胞極化所扮演的角色。在盲腸穿刺的敗血症疾病模式中，我們發現CLEC5A基因剔除的老鼠有較低的死亡率、較溫和的臨床症狀、血清中較低的白細胞介素1β (IL-1β)和腫瘤壞死因子α (TNF-α)，並且肺臟與肝臟的促炎細胞因子的基因表現也較WT低。在葡聚醣硫酸鈉引起的結腸炎的動物疾病模式中，CLEC5A基因剔除的老鼠的腸道出血、腹瀉、體重減輕與腸道縮短與野生型(WT)的老鼠相比較不嚴重，且CLEC5A基因剔除老鼠的大腸中促炎細胞因子的基因表現相較於野生型老鼠也減少。在CLEC5A基因剔除骨髓衍生的巨噬細胞中，脂多醣 (LPS;TLR4 配體)、pam3 (TLR2配體) 和CpG (TLR9配體)引發的促炎細胞因子在誘發的晚期(約6到12小時)較野生型老鼠低，除了腫瘤壞死因子α的基因表現在兩小時即已明顯降低。我們也發現脂多醣刺激所引發的IKK 和 MAPKs的訊息活化在CLEC5A基因剔除的巨噬細胞中並未與野生型巨噬細胞有所不同。值得注意的是，缺乏CLEC5A有利於巨噬細胞傾向於M2的極化，並且在脂多醣/干擾素γ和白細胞介素-4 (IL-4) 的訊息傳遞有相反的效果。綜上所述，我們認為CLEC5A與腸道微生物所引發的敗血症和腸道發炎有關，而其原因可能歸因於CLEC5A對於TLRs誘導的發炎反應和對巨噬細胞M1極化反應具有正向調控的功能。
並列摘要
CLEC5A is an innate immunity receptor belonging to C-type lectin receptors and widely expressed on myeloid cells. Previous studies showed that CLEC5A plays an important role in the pathology of dengue virus and Japanese encephalitis virus infection, rheumatoid arthritis and cigarette smoke-induced lung inflammation. In this study we used CLEC5A-/- mice to determine the roles of CLEC5A in the pathological severity of cecal ligation and puncture (CLP)-induced sepsis and dextran sodium sulfate (DSS)-induced colitis. In vitro we also used CLEC5A-/- bone marrow-derived macrophages (BMDM) to explore the roles of CLEC5A in TLR4 signaling, TLRs-induced inflammatory cytokine gene expression, and M1/M2 polarization. In CLP-induced polymicrobial bacterial peritonitis, we found CLEC5A-/- mice displayed lower responses of lethality, clinical symptoms, serum levels of IL-1β and TNF-α, and the gene expression of proinflammatory cytokines in lung and liver. In DSS-induced colitis model, intestinal bleeding, diarrhea, body weight loss, shorten of colon and proinflammatory cytokines expression in colon were reduced in CLEC5A-/- mice as compared to WT control. In TLR ligands-activated BMDM, we found that the proinflammatory cytokines mRNA induced by LPS (TLR4 ligand), pam3 (TLR2 ligand) and CpG (TLR9 ligand) at late phase around 6-12 h were attenuated in CLEC5A-/- cells. Nevertheless, TNF-α gene expression was reduced at 2 h. We also found that LPS-induced signaling activation of IKK and MAPKs were unaltered in CLEC5A-/- cells. Notably, CLEC5A deficiency favors macrophage polarization to M2 status, and exerts opposite effects on LPS/IFNγ and IL-4 signaling. Taken together, we suggest that CLEC5A is involved in microbiota-induced sepsis and gut inflammation, and these actions might be ascribed to the functions of CLEC5A in positive regulation of TLRs-induced inflammation responses and M1 macrophage polarization.