Introduction: Indoleamine 2,3-dioxygenase (IDO) is an intracellular enzyme that catalyzes the essential amino acid tryptophan into kynurenine and has been shown to play a role in immune suppression and tolerance. Previous work from our group has shown that IDO can be delivered extracellularly and maintain its immunomodulatory function, making IDO a promising candidate therapeutic for autoimmune disease and other inflammatory conditions. However, systemic delivery of enzyme therapeutics is often challenged by broad bodily distribution and rapid clearance by renal filtration, leading to off-target side effects, and limited accumulation within target tissues. Furthermore, systemic administration of immunosuppressants often leave patients susceptible to opportunistic infections and other complications. Localized delivery of immunosuppressants could improve efficacy, reduce off-target effects, and lower possible dosing.
Hydrogels can be used for localized delivery of therapeutics. In this work, the hydrogel system we will use is CATCH. The CATCH (Co-Assembly Tags based on Charge complementarity) platform was designed to create supramolecular protein-peptide gels. CATCH consists of a pair of oppositely charged -sheet fibrilizing peptides. Separately, the peptides do not aggregate due to electrostatic repulsion, but once combined, they co-assemble into nanofiber-based gels. Recombinant fusion of a CATCH tag to a protein of interest allows for integration of the protein into the gel structure. By recombinantly fusing IDO to CATCH, CATCH-IDO gels can be created for localized delivery. In this work, we will confirm that CATCH-IDO gels are enzymatically active and determine if they can suppress dendritic cell activation.
Materials and
Methods: • Protein Expression/Purification: CATCH-IDO was expressed in an E. coli host system and purified via nickel affinity and size exclusion chromatography.
• CATCH Hydrogel Fabrication: CATCH peptides were dissolved in aqueous buffer and combined in equimolar ratios, with the addition CATCH-IDO, to peptide concentration of 12mM.
• CATCH-IDO Gel Biochemical Activity: CATCH-IDO gels and blank gels were incubated with tryptophan, catalase, and the necessary electron donors, methylene blue and ascorbic acid. N-formyl-kynurenine (NFK), the immediate precursor to kynurenine, was measured at 321nm over the course of an hour. Specific activity of CATCH-IDO was determined based on the rate of conversion of tryptophan to NFK.
• Dendritic cell isolation: Dendritic cell precursors were isolated from C57BL/6 mouse bone marrow and cultured with GMCSF for 10 days. The purity and viability of the dendritic cells were validated by flow cytometry (Biolegend, CD11b, CD11c, MHC II).
• CATCH-IDO gel Biological Activity: The bone marrow derived dendritic cells were cultured at 0.25x10^6 cells/mL with either soluble CATCH-IDO (30ug), blank gels, or CATCH-IDO gels (30ug) for 24 hours and then stimulated with lipopolysaccharide (LPS) (1 ug/mL) overnight. Dendritic cell activation and production of IL-12p70 was measured by enzyme-linked immunosorbent assay (ELISA) (BD Biosciences). Production of IL-6 and IL-10 will also be measured by ELISA. RNA will be isolated from the dendritic cells (Qiagen), converted to cDNA (Fisher), and amplified for genes of interest by qPCR (QuantStudio) (IL-12, IL-6, IL-10).
Results, Conclusions, and Discussions: The data demonstrates that IDO remains active immobilized within the CATCH gel, as evidenced by generation of kynurenine. Dendritic cells derived for this work were found to be 92% viable and 85% CD11b+ and CD11c+, which are common dendritic cell surface markers. When CATCH-IDO gels were cultured with dendritic cells and stimulated with LPS, they suppressed inflammatory activation. CATCH-IDO gel pretreatment decreased LPS-induced IL-12 secretion, whereas blank gels had no effect. The CATCH-IDO gels decreased IL-12 to a greater degree than freely diffusing soluble IDO, possibly due to increased enzyme stability from the gel immobilization. Based on these results, the CATCH gel is a promising vehicle for IDO. Further characterization is ongoing for IL-6, IL-10, and inflammatory gene expression. Moving forward, we will determine if CATCH-IDO gels can locally modulate inflammation and pain behavior in vivo.
