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 metabolomic approaches, we found that systemic increases in kynurenine secondary to either exogenous supplementation or chronic inflammation are associated with the formation of a novel signaling-active kynurenine-derived electrophile. This mediator, potently inhibits TLR4-dependent NF-κB signaling in primary macrophages and attenuates inflammatory responses in endotoxin-challenged mice. In addition, the novel kynurenine-derived electrophile engages AhR signaling with 50-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 this novel derivative in the context of other kynurenine pathway metabolites in activated and non-activated myeloid leukocytes as well as the elucidation of uptake and export mechanisms. The kynurenine-derived electrophile is a charged amino acid at physiological pH, thus a cell-permeable alkyl-esterified analogue will be synthesized to further define its signaling mechanisms and therapeutic potential in the absence of the rate-limiting effects of active cellular transport and potential competition by other amino acids present in the extracellular milieu. Primary cells derived from pathway-specific knock-out animals and novel bio-orthogonal labeling strategies will be harnessed to define the mechanistic basis of the anti-inflammatory actions of the kynurenine-derived electrophile both in terms of the modulation of specific signaling pathways and its effects on inflammation-elicited changes in energy metabolism. Finally, the potential of the kynurenine-derived electrophile to promote tolerogenic responses will be established assessing in vitro T cell polarization and in vivo models of endotoxin resistance and tolerance. This Research Plan will reveal specific immunomodulatory actions of the kynurenine pathway and may potentially lead to the development of related pharmacological interventions for dysregulated immune responses such as chronic inflammation, autoimmune diseases, cancer, and allograft rejection.