Phosphorothioate antisense oligonucleotide induced innate immune activation is attenuated by tryptophan oxidation products.

Phosphorothioate antisense oligonucleotides (PS ASOs) are a clinically validated therapeutic modality, yet their capacity to activate innate immunity through Toll-Like Receptors, specifically Toll-Like Receptor 9 (TLR9), remains a challenge. While the molecular mechanisms governing TLR9 activation, including PS content, CpG motifs, 2' modifications, sequence composition, and protein interactions, are well defined, far less is understood about the mechanisms that resolve these responses. Here, we investigate how PS ASO-mediated innate immune activation interfaces with tryptophan metabolizing enzymes, indoleamine 2,3-dioxygenase 1 (IDO1) and interleukin-4-induced-1 (IL4I1), which generate immunomodulatory tryptophan oxidation products. Across lymphoid and myeloid cell systems, we demonstrate that PS ASOs differing in TLR9 agonist potency and kinetics elicit differential induction of IDO1/IL4I1, accompanied by proportional changes in downstream metabolites. Complementary IDO1 knockdown and IL4I1 overexpression experiments alter TLR9-dependent signaling, indicating that these enzymes actively constrain PS ASO-driven immune activation. Finally, exogenous kynurenine and indole pathway metabolites attenuate PS ASO-induced signaling, supporting their role as negative-feedback regulators. Together, these findings solidify a mechanistic link between PS ASO innate immune activation and tryptophan catabolism, revealing enzymatic and metabolite feedback mechanisms that attenuate innate immune signaling, which may be leveraged to enhance the tolerability of immunostimulatory PS ASOs.