Novel insights into the regulation of CCR7 receptor expression in immune cells

Toll样受体（TLR）识别微生物分子，其导致由调节细胞因子和趋化因子表达的NF-κ B的活化引起的炎症反应的发展。病原体导致的TLR激活提供了炎症细胞到感染部位的快速募集，并激活它们以诱导一系列抗菌功能。 直到最近，只有先天性免疫细胞，如巨噬细胞和树突状细胞，和上皮细胞内衬粘膜表面被称为表达功能上有能力的TLR。然而，许多研究现在已经报道了淋巴细胞中生理相关的信号活性TLR。趋化因子CCL 19和CCL 21是CCR 7的天然配体，对于免疫细胞向淋巴结的迁移是重要的。由于TLR激活和炎症是病毒和细菌感染的标志，因此确定它们是否可以调节CCR 7依赖性迁移以更好地了解导致T细胞传播的因素至关重要。在这项研究计划中，我们试图获得新的见解分子和细胞的事件，导致CCR 7表达后TLR激活T细胞。根据我们令人兴奋的初步结果，在这个五年的研究计划中提出了以下具体目标： AIM 1.确定TLR活化是否调节T细胞中CCR 7的表达和功能。 在这个目标中，我们将监测TLR激活和炎症的T细胞后，通过实时RT-PCR的CCR 7 mRNA的诱导。使用针对CCR 7的特异性抗体的FACS分析将证实CCR 7在细胞表面的表达。将通过趋化性测定验证受体的功能性。 AIM 2.确定TLR激动剂激活的T细胞中引发的导致CCR 7表达调节的转录因子和信号传导模块。 我们建议阐明TLR诱导的CCR 7表达所必需的信号事件和转录因子。首先，我们将研究启动子活性的分子构建体含有全长或删除版本的启动子区的CCR 7。接下来，使用电泳迁移率变动和染色质免疫沉淀试验，我们将探讨转录因子以及TLR刺激的T细胞中CCR 7表达所需的信号事件。 AIM 3.确定观察到的T细胞中CCR 7表达的调节是否影响小鼠模型中的迁移。 具有人源化免疫系统的小鼠代表了在理解免疫系统方面获得重要见解的合适动物模型。在这里，我们将使用这样的模型来研究我们的假设的生理相关性。 这项创新的研究计划将首次对T细胞迁移的理解提供新的见解，以应对两种CCR 7天然配体CCL 19和CCL 21的感染。

Toll-like receptors (TLR) recognize microbial molecules, which results in the development of inflammatory reactions caused by the activation of the NF-kB regulating expression of cytokines and chemokines. Pathogen-led TLR activation provides rapid recruitment of inflammatory cells to the site of infection and activates them to induce an arsenal of antimicrobial functions. Until recently, only innate immune cells, such as macrophages and DCs, and epithelial cells lining mucosal surfaces were known to express functionally competent TLRs. However, many studies have now reported physiologically relevant signal-competent TLRs in lymphocytes. Chemokines CCL19 and CCL21, the natural ligands of CCR7 are important for immune cell migration to lymph nodes. Since TLR activation and inflammation are hallmarks of viral and bacterial infections, it is of vital importance to determine whether they can regulate CCR7-dependent migration in order to better understand factors leading to T cell dissemination. In this research program, we seek to gain novel insights into the molecular and cellular events leading to CCR7 expression following TLR activation in T cells. Based on our exciting preliminary results, the following specific aims are proposed in this five-years research program: AIM 1. To establish whether TLR activation modulate CCR7 expression and functionality in T cells. In this objective, we will monitor CCR7 mRNA induction following TLR activation and inflammation of T cells by real-time RT-PCR. FACS analysis using a specific antibody against CCR7 will confirm the expression of CCR7 at cell surface. The functionality of the receptors will be verified by chemotaxis assays. AIM 2. To determine transcription factors and signaling modules elicited in T cells activated by TLR agonists that lead to the modulation of CCR7 expression. We propose to elucidate signaling events and transcription factors essential for the TLR-induced expression of CCR7. First, we will study the promoter activity using molecular constructs containing the full-length or deleted versions of the promoter region of CCR7. Next using electrophoretic mobility shift and chromatin immunoprecipitation assays, we will explore the transcription factors as well as the signaling events required for CCR7 expression in TLR-stimulated T cells. AIM 3. To determine whether the observed modulation in CCR7 expression in T cells affects migration in a mouse model. Mice that have an humanized immune system represent a suitable animal model to gain important insights in the comprehension of the immune system. Here, we will use such model to study the physiological relevance of our hypothesis. This innovative research program will give for the first time, novel insights into the comprehension of T cell migration in response to infections in response to CCL19 and CCL21, two CCR7 natural ligands.