Functional consequences of dynamic MAPK signalling on the host defence response of vertebrates to bacterial infections

在我们的世界里，生物体不断地相互作用。这些相互作用的结果是多样的，表现为不同的模式，如捕食、共生、同居或寄生。我对理解决定宿主对病原体反应的分子机制特别感兴趣。宿主防御细菌和真菌感染的一个关键因素是向感染部位招募中性粒细胞。因此，宿主对中性粒细胞炎症的适当调节至关重要。一旦感知到病原体，就会发出求救信号，导致中性粒细胞的招募。最近，我们已经确定了上皮细胞中的三个关键信号通路，上皮细胞是抵抗感染的第一道防线，导致中性粒细胞募集以响应革兰氏阴性菌。这三条通路分别是：TAK1-MKK3-p38a、TAK1-IKKß-NF？B和TAK1-IKKß-TPL2-MKK1-ERK2。这些途径共同协调炎症介质的合成。***尽管我们在识别细菌感染时炎症调节的重要关键信号方面取得了良好进展，但我们仍然缺少许多有助于更好地理解这一重要生物反应的细节。目前的大多数方法，包括我们的方法，使用准时探测来测量信号强度，在获取的信息中留下重要的空白。这项资助旨在开发工具来克服这一限制，以便更全面地了解由人类上皮细胞感染引发的炎症的调节。***目标1：开发炎症动态成像工具。动态评估tlr - tak1介导的MKK3-p38a、IKKß-NF？B和IKKß-TPL2-MKK1-ERK2途径，我们将使用以下技术：双分子荧光互补（BiFC），生物发光共振能量转移（BRET），福斯特共振能量转移（FRET）和荧光素酶转录报告。生成的工具将用于第二个目标，以探索信号转导调节炎症的基本机制。***目的2：阐明宿主-病原体相互作用过程中的炎症反应。在第二个目标中，我们将对培养的人上皮细胞感染期间发生的炎症进行实时成像。感染后，我们将动态测量细胞内信号通路和相关转录因子的激活。同时，使用经典方法，我们将测量细胞因子的产生和中性粒细胞的募集来评估炎症的程度。综上所述，这些研究应该为炎症调节的基本分子机制描绘出一幅更清晰的图景。* * * * * * * *

In our world, living organisms constantly interact with each other. The outcome of these interactions is diverse and represented by different modes like predation, symbiosis, co-habitation or parasitism. I am particularly interested in understanding the molecular mechanisms determining the host response to pathogens. A key element of host defense against bacterial and fungal infections is the recruitment of neutrophils to site of infections. Therefore it is vitally important for the host to properly regulate neutrophilic inflammation. Upon sensing of pathogens, distress signals are emitted leading to the recruitment of neutrophils. Recently we have identified three key signaling pathways in epithelial cells, the first line of defense against infections that lead to neutrophil recruitment in response to gram-negative bacteria. The three pathways are: TAK1-MKK3-p38a, TAK1-IKKß-NF?B and TAK1-IKKß-TPL2-MKK1-ERK2. Together, these pathways coordinate the synthesis of inflammatory mediators. ***Although we have made good progress in identifying key signals important for the regulation of inflammation in response to bacterial infection, we are still missing a lot of the details that would contribute to a much better understanding of this important biological response. The majority of current approaches, including ours, use punctual probing to measure signal intensities, leaving important gaps in the information acquired. This grant aims to develop tools to overcome this limitation in order to get a more complete understanding of the regulation of inflammation triggered by infection of human epithelial cells. Two objectives will be undertaken:***Objective 1: Developing tools to dynamically image inflammation. To dynamically assess TLR-TAK1-mediated activation of the MKK3-p38a, IKKß-NF?B and IKKß-TPL2-MKK1-ERK2 pathways, we will be using the following technologies: Bimolecular fluorescence complementation (BiFC), Bioluminescence Resonance Energy Transfer (BRET), Forster Resonance energy transfer (FRET) and luciferase transcriptional reporters. The tools generated will be used in the second objective to probe fundamental mechanisms of signal transduction regulating inflammation.***Objective 2: Shedding light on the inflammatory response during host-pathogen interactions. In this second objective, we will image inflammation in real-time occurring during infection of human epithelial cells in culture. Following infection, we will measure dynamically the activation of intracellular signaling pathways and associated transcription factors. In parallel, using classical approaches we will measure the production of cytokines and neutrophil recruitment to evaluate the magnitude of inflammation. ***Taken together, these studies should paint a much clearer picture of the fundamental molecular mechanisms underlying the regulation of inflammation. ********