Real time imaging of tolerance induction by mucosal DCs

描述(申请人提供)：拒绝细胞(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，但它们也与诱导和维持耐受性有关。DC的这些不同功能在胃肠道中最为明显，在胃肠道中，需要保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。近来研究表明，表达于粘膜中的树突状细胞的常驻Fractalkine受体(CX3CR1)通过跨上皮树突状突起的延伸直接采样肠腔内容物，并在稳态条件下表达耐受性表型。然而，目前尚不清楚这些树突状细胞是否直接参与诱导T细胞对它们所采集的管腔抗原的耐受。由于无法在体外模拟这些DC在黏膜道内遇到的复杂环境来研究它们的功能，因此很难用传统技术解决其中的许多问题。这一建议利用多光子显微镜的深层组织成像来直接原位评估CX3CR1树突状细胞的功能。荧光标记的DC和抗原特异性T细胞在感染重组抗原表达细菌(E.coli-OVA)后的抗原提呈将被用来确定CX3CR1 DC是否直接参与诱导耐受性T细胞反应。这项建议的第二个目标是确定CX3CR1树突状细胞的功能在病原体感染后诱导的炎症反应中是否发生改变。我们将使用一种与致病性鼠伤寒沙门氏菌和卵清蛋白在大肠杆菌中的表达受限的混合感染模型来解决这个问题。这样的模型将允许在炎症条件下跟踪对“共生膜样”抗原的抗原特异性反应。由于CX3CR1树突状细胞不断采样共生菌和食物抗原，它们在炎症反应期间功能的可塑性可能在口腔耐受性崩溃中发挥作用。 公共卫生相关性：目前的研究将揭示常驻CX3CR1 DC是否构成维持口服耐受性的粘膜固有免疫细胞机制的重要功能臂，这对于防止针对食物抗原和共生体的不良免疫反应至关重要。此外，我们将确定CX3CR1 DC功能反应的可塑性是否有助于炎症条件下口服耐受性的崩溃，并有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.