Functions and Mechanisms of NF-kB Factors and their Regulators

本项目的重点是鉴定NF-kB转录因子及其调控因子在健康和疾病中的生理关键功能和作用机制。NF-kB是一个相关的二聚体转录因子家族，在先天和适应性免疫反应中作为主要的细胞内介质。此外，重要的是，NF-kB的异常调节在炎症和自身免疫性疾病以及许多肿瘤中起着重要作用。因此，了解单个NF-kB因子及其调节因子的功能和作用机制是必要的，因为这将需要制定适当的治疗干预策略，旨在以精确靶向的方式减少异常调节的NF-kB。为了确定生理作用和机制，我们利用缺乏NF-kB转录因子家族或其调节因子成分的小鼠模型，以及NF-kB因子可以选择性激活的模型。我们的工作重点是所谓的替代，但也经典激活NF-kB，但我们特别研究调节因子Bcl-3和IkBzeta。另一种NF-kB激活途径通常由TNF受体亚群启动。Bcl-3和IkBzeta是不典型的IkB家族成员，其功能是NF-kB活性的核调节因子。我们之前发现Bcl-3在树突状细胞适当诱导T细胞在应答抗原中增殖的能力中起关键作用，从而启动对病原体的保护性适应性免疫反应，例如弓形虫，这是免疫功能低下患者的严重健康风险。在树突状细胞中缺乏Bcl-3的情况下，小鼠死于这种感染。Bcl-3对上皮细胞也有重要的功能，比如在角质形成细胞中，它有助于界定过敏反应。特别有趣的是，我们之前发现T细胞需要Bcl-3来驱动T细胞依赖性自身免疫性疾病，包括实验性自身免疫性脑脊髓炎（多发性硬化症模型）和T细胞转移诱导的结肠炎（炎症性肠病模型）。此外，我们最近发现，与其在B细胞中的促肿瘤作用相反，它在肠上皮细胞中具有肿瘤抑制作用。在结肠炎炎症诱导的结肠癌模型中，这些细胞中缺乏Bcl-3的小鼠表现出更高的肿瘤率，突出了该调节因子的环境特异性功能。在2019财年，我们在确定Bcl-3的环境依赖功能、其作用机制和转录靶点的长期目标方面取得了重大进展。首先，我们从CD4-T细胞转移诱导的结肠炎小鼠中收集了野生型和Bcl-3缺陷的CD4 T细胞，并对它们进行了大量的RNAseq分析，目前正在验证Bcl-3的潜在关键靶点。我们还建立了急性LCMV病毒感染模型来阐明Bcl-3的作用。使用这个模型，我们确定Bcl-3似乎在CD8记忆T细胞发育中起重要作用，我们正在分析单细胞RNAseq实验。这为了解Bcl-3以细胞类型特异性方式控制的整体功能、靶基因和途径打开了大门。了解Bcl-3控制CD8 T细胞记忆形成的基因在肿瘤免疫治疗中也至关重要，CD8记忆样T细胞，而不是效应T细胞，被认为在长期肿瘤抑制中特别有效。为了更好地了解Bcl-3的作用机制，我们在鉴定相互作用蛋白方面取得了重大进展。在2019财年，我们成功地培育和使用了Bcl-3携带一个小标签的小鼠，该标签可以以特定细胞类型的方式条件附着。对于这些小鼠，我们目前正在通过MassSpec进行共免疫沉淀实验，以鉴定T细胞中的相互作用蛋白，特别是在实验诱导的T细胞依赖性自身免疫性疾病的背景下。在2019财年，我们还在LCMV病毒感染模型的T细胞和咪喹莫特诱导的银屑病炎症模型的角质形成细胞中培养了条件性IkBz缺失的小鼠。最后，我们产生了通过替代途径激活的NF-kB因子RelB可以被全局或有条件地消融的小鼠；我们用这些小鼠来证明这种NF-kB蛋白在多发性硬化症小鼠模型EAE中的重要作用。结果表明，RelB对成熟少突胶质细胞的存活有负面影响，从而加剧了发病机制。

This project is focused on the identification of physiologically critical functions and mechanisms of action of NF-kB transcription factors and their regulators in health and disease. NF-kB is a family of related dimeric transcription factors that serve as primary intracellular mediators during innate and adaptive immune responses. In addition, and importantly, aberrant regulation of NF-kB plays a major role in inflammatory and autoimmune diseases as well as in numerous tumors. It is thus imperative to understand the functions and mechanisms of action of individual NF-kB factors and their regulators, as this will be required to devise appropriate strategies for therapeutic interventions aimed at curtailing aberrantly regulated NF-kB in a precisely targeted manner. To identify physiologic roles and mechanisms we make use of mouse models engineered to lack components of the NF-kB transcription factor family or their regulators, as well as models in which the NF-kB factors can be selectively activated. Our work is focused on so-called alternatively, but also classically activated NF-kB, but we are especially investigating the regulators Bcl-3 and IkBzeta. The alternative NF-kB activation pathway is normally initiated by a subset of TNF receptors. Bcl-3 and IkBzeta are atypical IkB family member that function as nuclear regulators of NF-kB activity. We previously discovered a critical role for Bcl-3 in the ability of dendritic cells to properly prime T cells to proliferate in response antigen, and thus to initiate a protective adaptive immune response to pathogens, such as to Toxoplasma gondii, a serious health risk in immune-compromised patients. In the absence of Bcl-3 in dendritic cells, mice succumb to this infection. Bcl-3 also has critical functions epithelial cells, such as in keratinocytes, where it helps to delimit hypersensitivity reactions. Of particular interest is our previous discovery that Bcl-3 is required in T cells to drive T-cell dependent autoimmune diseases, including experimental autoimmune encephalomyelitis, a model for Multiple Sclerosis, and T cell transfer-induced colitis, a model for Inflammatory Bowel Disease. In addition, we recently discovered that in contrast to its pro-tumorigenic role in B cells, it has a tumor-suppressive role in gut epithelial cells. Mice lacking Bcl-3 in these cells exhibited an increased tumor rate in a colitis inflammation-induced colon cancer model, highlighting the context-specific functions of the regulator. In FY2019 we have made significant progress towards our long-term goal to identify context-dependent functions of Bcl-3, its mechanisms of actions and its transcriptional targets. First, we collected wild-type and Bcl-3-deficient CD4 T cells from CD4-T cell transfer induced colitis mice and subjected them to bulk RNAseq analyses and are presently verifying potentially critical targets of Bcl-3. We also established an acute LCMV viral infection model to elucidate the role of Bcl-3. Using this model, we determined that Bcl-3 appears to have an important role in CD8 memory T cell development and we are in the process of analyzing single-cell RNAseq experiments. This opens the door towards understanding the overall functions, target genes and pathways controlled by Bcl-3 in a cell-type specific manner. Understanding what genes Bcl-3 controls in CD8 T cell memory formation is also vital in the context of tumor immunotherapy, where CD8 memory-like T cells, rather than effector T cells are thought to be specifically efficacious in long-term tumor suppression. In order to better understand the mechanisms of action of Bcl-3 we have made significant progress in identifying interacting proteins. In FY2019 we succeeded in generating and using mice in which Bcl-3 carries a small Tag that can be conditionally-attached in a cell-type specific manner. With these mice we are presently performing co-immunoprecipitation experiments to identify interacting proteins in T cells via MassSpec, specifically in the context of experimentally induced, T cell-dependent autoimmune diseases. In FY2019 we have also generated mice with conditional depletion of IkBz in T cells in the LCMV viral infection model and in keratinocytes in the Imiquimod-induced psoriatic inflammation model. Finally, we have generated mice in which the NF-kB factor RelB which is activated via the alternative pathway can be globally or conditionally ablated; we have used these mice to demonstrate a significant role for this NF-kB protein in EAE, a mouse model for Multiple Sclerosis. RelB turned out to have a negative effect on survival of mature oligodendrocytes, thus exacerbating pathogenesis.