Mef2c 轉錄因子調控漿狀樹突細胞的發育及遷徙

Abstract

樹突細胞包括漿狀樹突細胞和典型樹突細胞在先天和適應性免疫反應扮演重要的橋樑。兩種樹突細胞皆能在含有Flt3 ligand的生長環境中從骨髓前驅細胞和淋巴前驅細胞發育而來。典型樹突細胞又可依照表面分子及轉錄因子而細分為cDC1和cDC2兩種，反觀漿狀樹突細胞至今仍是一個異質的混合群體。Mef2c 為一對於心血管和肌肉發四重要的轉錄因子，儘管Mef2c表現量在漿狀樹突細胞內比經典樹突細胞高，但其在樹突細胞發育扮演的角色仍是未知的。在永生化的造血前驅及幹細胞株（immortalized stem and progenitor cell line, iHSPC）中利用CRISPR-Cas9方法剔除Mef2c 只損害漿狀樹突但不影響經典樹突細胞的發育，除此之外，在Mef2c Tie2Cre異合子老鼠中，無論在骨隨、脾臟和淋巴結的漿狀樹突細胞皆減少，同時，由Mef2c Tie2Cre異合子老鼠的淋巴系前驅幹細胞 (common lymphoid progenitor, CLP) 和樹突前驅細胞 (common dendritic cell progenitor, CDP) 培養的漿狀樹突細胞亦減少，此說明Mef2c 調節漿狀細胞的發育具有劑量依賴性。另值得關注的是淋巴結漿狀樹突細胞的缺失來的比骨隨顯著，同時，也觀察到對於漿狀樹突細胞遷徙重要的趨化因子受體CCR2、CCR9和CXCR3下降，說明Mef2c 對成熟漿狀細胞的遷徙具有調節作用。除此之外，在只在後期成熟樹突細胞剔除Mef2c 的CD11cCre老鼠中，我們亦觀察到周邊更顯著漿狀樹突細胞的缺失。更重要的是，在 CCR2 基因剔除的老鼠中，我們觀察到淋巴結的漿狀樹突細胞數下降，說明了CCR2參與了漿狀樹突細胞遷徙至淋巴結的過程。機制層面上，我們發現對於分別對於漿狀樹突細胞發育和遷徙重要的轉錄因子Tcf4和Runx2的啟動子和內含子上有Mef2c的DNA結合序列，其表現量在 Mef2c 基因剔除的永生化造血前驅及幹細胞株皆下降。綜上所述，我們發現了一個全新調控漿狀樹突細胞的發育及移行轉錄因子Mef2c，而其深入的調控機制則有待進一步研究及探討。

Dendritic cells (DCs), including plasmacytoid dendritic cells (pDCs) and classical dendritic cells (cDCs) play a crucial role in bridging the innate and adaptive immunity. While cDCs have been classified into cDC1 and cDC2 based on surface markers and transcription factors during development, pDCs remain heterogeneous. Mef2c is a transcription factor that is known to be critical for cardiac morphogenesis and myogenesis and vascular development. However, despite that Mef2c is preferentially expressed in pDC compared to cDC, its role in pDC development remains unclear. Mef2c KO in an immortalized hematopoietic stem and progenitor cell line (iHSPC) impaired pDC development in vitro. More importantly, Mef2cf/+Tie2Cre heterozygous mice also showed a reduced frequency of pDC, but not cDC, in the BM, spleen and lymph nodes. An impaired pDC development was also observed using Mef2cf/+Tie2Cre CLP or CDP to do in vitro culture, suggesting a dose-dependent requirement of Mef2c for pDC development. Interestingly, the defect in the lymph nodes was even more pronounced than that in BM. Reduced expression of CCR2 CCR9 and CXCR3, indispensable chemokine receptors for egress of pDC from BM to lymph node, was observed, suggesting that Mef2c may be also involved in pDC migration. A similar but more dramatic reduction of pDC frequency was observed in the periphery CD11c-driven deletion of Mef2c in mice in later stage of DC development. Moreover, CCR2 KO also decreased pDC frequency in lymph nodes as opposed to WT control, suggesting a critical role of CCR2 in pDC migration. Mechanistically, Mef2c binding motif is found on promoter/intron of Tcf4 and Runx2, two transcription factors know to be critical for pDC development and migration, respectively. Mef2c KO iHSPCs reduced the expression of these two genes. In sum, we demonstrate that Mef2c, a novel TFs preferentially expressed in pDC, not only regulate development but also migration of pDCs.