Mef2c轉錄因子調控漿狀樹突細胞發育

Abstract

樹突狀細胞 (Dendritic cell, DC)主要分為傳統樹突細胞以及漿狀樹突細胞，而他們兩者的功能是連結先天免疫以及適應免疫，因此對於免疫的調節極為重要。雖然我們已知樹突細胞對於免疫調節的重要性，但相關於樹突細胞的分化途徑以及其調控的機轉仍然不清楚。而在這裡我們利用shRNA去靜默造血幹細胞與前期細胞株 (immortalized stem and progenitor cell line, iHSPC)的基因表現量，以此實驗方法去作為篩選策略，研究這些基因對於樹突細胞發育的影響。我們總共篩選了14個在漿狀樹突細胞中相較於傳統樹突細胞有較高量表現的轉錄因子，接著我們發現Mef2c跟Tcf12這兩個基因對於漿狀樹突細胞的生成有非常明顯的影響，因此我們以這兩個基因作為我們主要研究的目標。我們抑制了這兩個基因在iHSPC細胞株裡的表現量，並且利用MS-5滋養層細胞去進行培養，也發現漿狀樹突細胞的生成有減少的情形。有關於機制層面的研究則顯示了在此兩基因被抑制的iHSPC細胞株發育過程中Tcf4 (目前已知影響漿狀樹突細胞最重要的轉錄因子)的表現量有降低的傾向。並且我們利用報導基因系統分析，當Mef2c被過度表現的時候其Tcf4的活性相較於控制組增加了近一倍。最後，我們分析了Mef2c基因剔除的小鼠裡樹突細胞的分群，並且發現漿狀樹突細胞在骨髓、脾臟、淋巴結都有明顯減少的情形，而且在脾臟以及淋巴結內的減少比骨髓更顯著，暗示著漿狀樹突細胞可能有遷徙的缺陷。而我們利用慢病毒感染小鼠骨髓細胞，以進行體外剔除基因的方式，也觀察到漿狀樹突細胞生成的減少。除此之外，Mef2c也會正向調控Flt3受器的表現，我們在靜默Mef2c的iHSPC細胞株內也觀察到Flt3表現量下降的情形。藉由這些機制層面，我們認為Mef2c對於漿狀樹突細胞的調節機轉可能有二個層面分別是透過Tcf4/Flt3來控制生成及未知因素來調控細胞自骨髓遷移到周邊。

Dendritic cells (DC) can classified into two subsets, namely conventional dendritic cells (cDC) and plasmacytoid dendritic cells (pDC), both of which play important roles in bridging the innate and adaptive immunity. Although the functions of DCs are critical for immunomodulation, the regulatory mechanisms of DC developmental process still unclear. Here, we performed a powerful screening strategy and identified Mef2c as a transcription factor which regulates DC development. Through shRNA-mediated knockdown of target genes in immortalized hematopoietic stem and progenitor (iHSPC) cell line, we have screen 14 transcription factors that are preferentially expressed in pDC versus cDC, we identified two transcription factors Mef2c and Tcf12, which significantly affected pDCs generation in feeder free culture system. Knockdown of Mef2c and Tcf12 in iHSPC cell line decrease pDC generation in MS-5 feeder system. Mechanistically, expression of Tcf4 (encodes E-2.2), a master regulation of pDC development, was reduced in iHSPC stably express shMef2c and shTcf12. Reporter assay also showed the Tcf4 reporter activity was up-regulated by overexpression of Mef2c. We analyzed the DC populations from Mef2cf/f Tie2-Cre mice and proved that Mef2c deficiency indeed alter DCs generation, it reduce pDCs generation from bone marrow, spleen to lymph node. Also, in vitro deletion of Mef2c in primary bone marrow cell decreased pDCs frequency compared to control which show coherence to ex vivo results. Moreover, Mef2c also positively regulate Flt3 receptor expression. Mef2c knockdown decreased Flt3 expression in iHSPC. These results suggest Mef2c may regulate pDC development through control the expression of Tcf4.