Simple Hydrophobic Modification of bPEI Confers Potent, STING-Dependent Innate Immunostimulatory Activity

Area of Science:

• Immunology and Polymer Science
• Innate Immune Response Modulation

Background:

• Macromolecular agonists, like branched polyethylenimine (bPEI) conjugated with nitrogen heterocycles, are known immunomodulators that activate the STING pathway.
• Previous studies highlighted the importance of branching in bPEI conjugates for STING pathway activation.

Purpose of the Study:

• To investigate whether nitrogen is indispensable for STING pathway activation by bPEI conjugates.
• To explore the role of non-nitrogenous ligands in modulating the immune response via the STING pathway.

Main Methods:

• Synthesis of a library of bPEI conjugates with non-nitrogenous ligands (heterocycles, nonheterocycles, aliphatic chains).
• Assessment of IFN-β secretion, STING clustering, and TBK1/IRF3 phosphorylation across multiple cell types.
• Genetic ablation of STING and cGAS to confirm pathway involvement.
• Physicochemical analyses including docking, logP, and time-resolved uptake profiling to establish structure-activity relationships.

Main Results:

• Over 60% of bPEI conjugates with non-nitrogenous ligands induced robust IFN-β secretion, indicating nitrogen is not essential.
• Representative conjugates promoted STING clustering and downstream signaling (TBK1/IRF3 phosphorylation), dependent on STING and cGAS.
• Conjugation strategies mitigated PEI cytotoxicity and reduced pro-inflammatory NF-κB cytokines, while physicochemical properties dictated STING binding and cellular uptake.

Conclusions:

• Nitrogen is not a critical component for STING pathway activation by macromolecular agonists.
• Simple hydrophobic modifications on a macromolecular scaffold, like bPEI, are sufficient to induce a potent type I interferon response.
• This finding offers a broader scope for designing novel immunomodulatory macromolecular agents.