Real time imaging of tolerance induction by mucosal DCs

描述（由申请人提供）：树突状细胞（DC）在启动针对病原体的保护性免疫应答中起重要作用，但它们也涉及耐受性的诱导和维持。DC的这些不同功能在胃肠道内最明显，在胃肠道中需要维持对肠道植物群和食物抗原的低应答性，同时产生针对病原体的有效免疫应答。然而，这些功能共存的机制目前尚不清楚。在粘膜道中表达驻留的Fractalkine受体（CX 3CR 1+）的DC最近已被证明通过跨上皮树突的延伸参与肠腔内容物的直接取样，并且在稳态条件下表达致耐受性表型。然而，尚不清楚这些DC是否直接参与诱导T细胞对它们采样的管腔抗原的耐受。不能模拟这些DC在粘膜道内遇到的复杂环境，在体外研究它们的功能，使得难以通过常规技术解决许多这些问题。该建议利用多光子显微镜的深层组织成像来直接评估原位CX 3CR 1 + DC的功能。在用模拟肠道植物群的重组抗原表达细菌（大肠杆菌-OVA）感染后，荧光标记的DC和抗原特异性T细胞的抗原呈递将用于确定CX 3CR 1 + DC是否直接参与诱导致耐受性T细胞应答。该建议的第二个目的是确定在病原体感染后诱导的炎症反应期间CX 3CR 1 + DC的功能是否改变。建立了致病性S.鼠伤寒沙门氏菌和大肠杆菌Ova，其中卵清蛋白表达限于大肠杆菌，将用于解决这个问题。这样的模型将允许在炎性病症期间追踪对“类脑膜炎”抗原的抗原特异性应答。由于CX 3CR 1 + DCs持续采样肠道细菌和食物抗原，因此在炎症反应期间其功能的可塑性可能在口服耐受性的破坏中发挥作用。 公共卫生相关性：目前的研究将揭示常驻CX 3CR 1 + DC是否形成维持口服耐受性的粘膜先天免疫细胞机制的功能重要臂，这对于预防针对食物抗原和唾液的不良免疫应答至关重要。此外，我们将确定CX 3CR 1 + DC功能反应的可塑性是否有助于炎症条件下口服耐受的破坏，可能将其确定为未来治疗食物过敏和炎症性肠病的靶点。

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.