Kynurenine-dependent redox signaling at the interface between innate and adaptive immunity

犬尿氨酸途径主要通过以下作用分解代谢超过95%的所有色氨酸： 肝细胞中的色氨酸2，3-双加氧酶2（TDO 2）和骨髓中的吲哚胺2，3-双加氧酶1（IDO 1） 白细胞树突状细胞（DC）中继发于IDO 1上调的犬尿氨酸合成增加强烈地抑制了IDO 1的表达。 与通过促进抗炎信号传导产生致耐受性表型相关，调节性T细胞 （Treg）极化和免疫耐受，由免疫细胞引起的减弱的炎症反应， 反复暴露于TLR配体。然而，虽然这一途径的病理生理相关性很好- 尽管犬尿氨酸的免疫调节作用已经建立，但犬尿氨酸的免疫调节作用背后的机制仍然不清楚。使用 半靶向代谢组学方法，我们最近发表了犬尿氨酸水平的全身性增加， 继发于外源性补充或慢性炎症与形成的 新型半胱氨酸反应性犬尿氨酸衍生的亲电体Kyn-CKA。Kyn-CKA促进Nrf 2依赖性信号传导， 抑制TLR 4依赖的NF-κB通路并减轻内毒素攻击小鼠的炎症反应 以氧化还原依赖的方式。此外，Kyn-CKA参与AhR信号传导的效力比其高20倍。 犬尿氨酸前体，表明在DC和T细胞中的潜在促耐受性作用。特别设计的国家- 最先进的LC-MS/MS测定将能够在其他犬尿氨酸途径的背景下定量Kyn-CKA 活化和非活化髓系白细胞中的代谢物，以及阐明限速细胞 吸收和出口机制。原代巨噬细胞、树突状细胞和T细胞来源于通路特异性 基因敲除动物将与新的生物正交标记策略和同位素- 跟踪代谢通量分析，以确定Kyn-CKA抗炎作用的机制基础， 在特定信号通路的调节及其对炎症引起的变化的影响方面， 能量代谢将通过评估Kyn-CKA促进致耐受性应答的能力， 其对常规DC亚群的成熟和活化的作用，其对T细胞极化的影响， 以及其在体内调节内毒素抗性和耐受性的能力。小鼠表达或缺乏关键 Kyn-CKA形成的介质和细胞特异性隔室中的作用将用于获得机制 在体内的洞察力。总之，该研究计划解决了迄今未被重视的氧化还原依赖性 犬尿氨酸途径的免疫调节作用的组成部分，通过形成 亲电Kyn-CKA。如果成功，我们的工作将使潜在的开发新的Kyn-CKA为基础的 用于失调的免疫应答如慢性炎症、自身免疫 疾病、癌症和同种异体移植排斥。

The kynurenine pathway catabolizes over 95% of all tryptophan primarily through the actions of tryptophan 2,3-dioxygenase 2 (TDO2) in hepatocytes and indoleamine 2,3-dioxygenese 1 (IDO1) in myeloid leukocytes. Increased kynurenine synthesis in dendritic cells (DC) secondary to IDO1 upregulation is strongly linked with the generation of a tolerogenic phenotype by promoting anti-inflammatory signaling, regulatory T cell (Treg) polarization and immune tolerance, an attenuated inflammatory response instigated by immune cells that are repeatedly exposed to TLR ligands. However, while the pathophysiologic relevance of this pathway is well- established, the mechanism behind the immunomodulatory effects of kynurenine remain poorly defined. Using semi-targeted metabolomic approaches, we recently published that systemic increases in kynurenine levels secondary to either exogenous supplementation or chronic inflammation are associated with the formation of the novel cysteine-reactive kynurenine-derived electrophile Kyn-CKA. Kyn-CKA promotes Nrf2-depedent signaling, inhibits TLR4-dependent NF-κB pathways and attenuates inflammatory responses in endotoxin-challenged mice in a redox-dependent manner. In addition, Kyn-CKA engages AhR signaling with 20-fold higher potency than its kynurenine precursor, suggesting a potential pro-tolerogenic role in DC and T-cells. Specifically designed state- of-the-art LC-MS/MS assays will enable the quantification of Kyn-CKA in the context of other kynurenine pathway metabolites in activated and non-activated myeloid leukocytes, as well as the elucidation of rate-limiting cellular uptake and export mechanisms. Primary macrophages, dendritic, and T cells derived from pathway-specific knock-out animals will be harnessed in conjunction with novel bio-orthogonal labeling strategies and isotope- tracing metabolic flux analyses to define the mechanistic basis of the anti-inflammatory actions of Kyn-CKA both in terms of the modulation of specific signaling pathways and its effects on inflammation-elicited changes in energy metabolism. The ability of Kyn-CKA to promote tolerogenic responses will be established by assessing its effect on the maturation and activation of conventional DC subpopulations, its impact on T cell polarization, and its ability to modulate endotoxin resistance and tolerance in vivo. Mice expressing or lacking critical mediators of Kyn-CKA formation and action in cell-specific compartments will be used to obtain mechanistic insights in vivo. In summary, the Research Plan addresses a hitherto unappreciated redox-dependent component of the immunomodulatory actions of the kynurenine pathway, mediated by the formation of electrophilic Kyn-CKA. If successful, our work will enable the potential development of novel Kyn-CKA based pharmacological interventions for dysregulated immune responses such as chronic inflammation, autoimmune diseases, cancer, and allograft rejection.