Real time imaging of tolerance induction by mucosal DCs

描述（由申请人提供）：树突细胞（dc）在启动针对病原体的保护性免疫反应中起着至关重要的作用，但它们也涉及诱导和维持耐受性。dc的这些不同功能在胃肠道中最为明显，在胃肠道中需要维持对共生菌群和食物抗原的低反应性，同时产生针对病原体的有效免疫反应。然而，这些功能共存的机制目前尚不清楚。在粘膜道中表达dc的常驻fractalkine受体（CX3CR1+）最近被证明通过上皮树突的延伸直接参与肠腔内容物的采样，并在稳态条件下表达耐受性表型。然而，尚不清楚这些dc是否直接参与诱导T细胞对它们所取样的腔内抗原的耐受性。无法模拟这些dc在粘膜道内遇到的复杂环境，在体外研究它们的功能，使得传统技术难以解决许多这些问题。本研究利用多光子显微镜深层组织成像直接原位评估CX3CR1+ DCs的功能。通过荧光标记的dc和抗原特异性T细胞在感染了模拟共生菌群的重组抗原表达细菌（大肠杆菌- ova）后的抗原呈递，将用于确定CX3CR1+ dc是否直接参与诱导耐受性T细胞反应。本提案的第二个目的是确定CX3CR1+ DCs的功能是否在病原体感染后诱导的炎症反应中发生改变。致病性鼠伤寒沙门氏菌和大肠杆菌的共感染模型，其中卵清蛋白的表达仅限于大肠杆菌，将用于解决这个问题。这种模型将允许在炎症条件下追踪抗原对“共生样”抗原的特异性反应。由于CX3CR1+ dc持续采样共生细菌和食物抗原，其在炎症反应中功能的可塑性可能在口腔耐受的破坏中发挥作用。

DESCRIPTION (provided by applicant): Dendiritc cells (DCs) play a vital role in initiating protective immune responses against pathogens, but they are also implicated in the induction and maintenance of tolerance. These diverse functions of DCs are most apparent within the gastrointestinal tract where hypo-responsiveness to commensal flora and food antigens needs to be maintained, while generating efficient immune responses against pathogens. However the mechanisms by which these functions coexist are currently unclear. Resident fractalkine receptor (CX3CR1+) expressing DCs in the mucosal tract have recently has been shown to be involved in directly sampling the contents of the intestinal lumen through extension of transepithelial dendrite and express a tolerogenic phenotype under steady state conditions. However, it is not known if these DCs are directly involved in inducing T cell tolerance to the luminal antigens they sample. The inability to mimic the complex milieu that these DCs encounter within the mucosal tract, in vitro to study their function, makes it difficult to address many of these questions by conventional techniques. This proposal utilizes deep tissue imaging by multiphoton microscopy to directly assess the function of CX3CR1+ DCs in situ. The antigen presentation by fluorescently tagged DCs and antigen-specific T cells following infection with recombinant antigen-expressing bacteria that model commensal flora (E.coli-OVA) will be used to determine if CX3CR1+ DCs are directly involved in the induction of tolerogenic T cell responses. A second objective of this proposal is to determine if the function of CX3CR1+ DCs is altered during an inflammatory response induced following pathogen infection. A co-infection model with pathogenic S. typhimurium and E.coli Ova where ovalbumin expression is limited to E.coli will be used to address this question. Such a model will allow tracking of antigen specific responses to a "commensal- like" antigen during inflammatory conditions. As CX3CR1+ DCs are continually sampling commensal bacteria and food antigens, plasticity in their function during an inflammatory response could play a role in the breakdown of oral tolerance. PUBLIC HEALTH RELEVANCE: The current studies will reveal if the resident CX3CR1+ DC form a functionally important arm of the mucosal innate immune cell machinery that maintains oral tolerance, which is vital for preventing adverse immune responses against food antigens and commensals. In addition, we will determine if plasticity of the CX3CR1+ DC functional response contributes to the breakdown of oral tolerance under inflammatory conditions, potentially identifying them as targets for future therapeutics to treat food allergies and inflammatory bowel diseases.