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

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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.