Molecular signaling in the regulation of dendritic cell function

*树突状细胞(DC)在免疫反应的协调中起着关键作用。DC成熟状态对T细胞功能有深远影响。成熟DC(MDCs)可以诱导有效的T细胞反应，而不成熟DC(IDCs)和半成熟DC(SmDC)则促进和维持T细胞耐受。驱动DC发挥免疫原性或调节功能的分子机制尚不完全清楚。我们已经从NOD和Balb/c小鼠的骨髓中培养出DC，无论是低剂量的GM-CSF还是GM-CSF+IL-4。我们发现GM-CSF诱导的DC具有半表型，产生高水平的IL-10，但产生较少的IL-12，对CD8+T细胞的激活能力降低。由GM-CSF+IL-4诱导的内毒素激活的DC已完全成熟，可产生大量IL-12，但产生较少IL-10，并可诱导CD8+T细胞的强烈增殖。Western印迹分析显示MAP激酶ERK 1/2在smDC中持续激活，而在mDCs中不表达。我们还发现ERK1/2的激活参与了IL-10的产生，而不影响DC的表型。对核因子p52、p65和RelB的分析表明，p52/p65在SMDC中优先激活，而p52/RelB在MDCS中优先激活。这些结果提示，NF？B复合体中缺乏RelB和ERK1/2的激活参与了抗成熟DC的形成及其产生IL-10的增加。对CREB、AP1和C/EBP促进共刺激分子表达的结合能力及其与NF-kB组分的相互作用的分析表明，C/EBP？亚型在SMDC中被激活，而在MDCS中不被激活。这些结果提示Rel/B和C/EBP？在控制DC成熟阶段中起关键作用。C/EBP？转录因子可能参与巨噬细胞的分化和激活。例如，来自C/EBPç-KO小鼠的巨噬细胞产生的IL-12较少，并且不能诱导Th1反应。此外，我们的初步数据支持这一观点，即在SMDC中激活的C/EBP？异构体在调节性smDC的生成中起关键作用。*假设C/EBP？转录因子异构体及其调节因子触发前体DC(PDC)选择性地分化为调节性SMDC或效应MDCs。*目的：*1)剖析C/EBP？在smDC或MDCS的生成中的相对作用。*2)确定触发pDC分化为SMDC和MDCS的C/EBP？亚型伙伴。*3)研究C/EBP？LAP或/和LAP*的激活剂亚型或/和LAP*或其抑制亚型LIP是否调节mDC和mDCs的表型和功能。*意义：了解DC分化为调节性smDC或效应mDCs的机制将提高我们对基础免疫学的认识，并可能有助于设计新的靶向治疗自身免疫性疾病和病毒感染的药物。**

***Dendritic cells (DCs) are key players in the orchestration of the immune response. DCs maturation status has a profound effect on T cell functions. Mature DCs (mDCs) are known to induce an effective T cell response whereas immature (iDCs) and semimature DCs (smDCs) promote and maintain T cell tolerance. The molecular mechanisms that drive DCs to immunogenic or to regulatory functions are not fully known. We have generated DCs from bone marrow of NOD and Balb/c mice either with low doses of GM-CSF or with GM-CSF+IL-4. We found that DCs generated with GM-CSF had a semimature phenotype, produced high amounts of IL-10 but less IL-12, and had a decrease capacity to activated CD8+ T cells. LPS-activated DCs generated with GM-CSF+IL-4 were fully mature, produced large amounts of IL-12 but less IL-10 and induce strong proliferation of CD8+ T cells. Western blot analysis revealed a sustained activation of MAP kinase ERK 1/2 in smDCs but not in mDCs. We also found that ERK1/2 activation is involved in IL-10 production without affecting DCs phenotype. The analysis of NF-?B components p52, p65 and RelB, showed that p52/p65 is preferentially activated in smDCs whereas p52/RelB is preferentially activated in mDCs. These results suggest that the absence of RelB in NF?B complex and that the activation of ERK1/2 are involved in the development of maturation-resistant DCs and their increased IL-10 production. The analysis of the binding capacity of CREB, AP1 and C/EBP which are known for their contribution to increase the expression of costimulatory molecules, and their interaction with NF-kB component, revealed that C/EBPß isoform is activated in smDCs but not in mDCs. These results suggest that Rel/B and C/EBPß played a key role in the control of maturation stage of DCs.***Rational The C/EBPß transcription factors are known to be involved in differentiation and activation of macrophages. For example, macrophages from C/EBPß-KO mice produced less IL-12 and could not induce a Th1 response. Furthermore, our preliminary data support the notion that C/EBPß isoforms, which were activated in smDC, played a critical role in the generation of regulatory smDCs.***Hypothesis C/EBPß transcription factor isoforms and their regulators trigger the selective differentiation of precursor DCs (pDCs) into regulatory smDC or effector mDCs.***Objectives:***1) to dissect relative role of C/EBPß in the generation of smDcs or mDCs. ***2) to identify C/EBPß isoforms partners that trigger differentiation of pDCs into smDC and mDCs. ***3) to investigate whether the activator isoforms of C/EBPß LAP or/and LAP* or its inhibitor isoform LIP regulate smDCs and mDCs phenotypes and functions. ***Significance: Understanding the mechanisms that control differentiation of DCs into regulatory smDCs or effector mDCs will improve our knowledge of basic immunology and may help to design new targeted therapeutics for the treatment of autoimmune diseases and viral infections. **