A Mechanistic Study of Lysosome Infiltration of N-Dihydrogalactose Chitosan to Activate the Stimulator of Interferon Genes (STING)

In the humans, viral respiratory tract infections are the most common types of infection. Successful viral vaccination requires the development of antibodies (humoral immunity) and T cell responses (cellular immunity). Adjuvants are added to vaccines to sustain the antigen and enhance the cellular and/or humoral immunological responses to the targeted pathogen. However, current adjuvants approved for human use are not ideal for viral pathogens such as influenza and SARS viruses as they only initiate humoral and not cellular immune responses. Type I interferons (IFNs) are key to antiviral immunity and protective cellular responses. We have developed a novel biosynthetic polymer, N-dihydrogalactochitosan (GC), that drives the production of type I IFN and IL-1β responses in dendritic cells (DCs). DCs are professional antigen presenting cells (APCs) that direct cellular immune responses. To determine if GC works well as a stimulant/delivery method for vaccination against viral pathogens, we combined GC with the recombinant nucleocapsid and spike proteins from SARS-CoV-2 and vaccinated human ACE2 transgenic mice two times. Preliminary data revealed that this vaccination is highly protective against COVID-19 viral challenge, prompting us to uncover the mechanism by which GC activates DCs. In DCs, the stimulator of interferon genes (STING) is required for GC to induce type I IFNs and an inflammatory form of cell death (ICD) that activates caspase-1 to cleave IL-1β and gasdermin-D. Using live cell imaging we discovered that many DCs that phagocytose GC swell and rupture, and this is dependent on the lysosomes upstream of STING activation. We hypothesize that the lysosome digests GC into smaller fragments which them leak into the cytosol where it interacts with STING to trigger the production of type I IFNs. We further hypothesize that the strong binding of GC to STING results in STING trafficking to the lysosome to trigger lysosomal cell death, which recruits and enhances the inflammatory response of neighboring DCs. To examine whether GC is digested into smaller fragments by lysosomal enzymes, lysosomes were isolated from DC2-4 cells. Lysosomal extracts were then incubated with GC at varying concentrations and then analyzed via an SDS-Page gel to examine fragmentation of GC. To examine STING trafficking to the lysosomes, subcellular fractionation on GC stimulated DC2-4 cells was performed. Western blots were used to examine STING localization in the cytoplasm, lysosome, membrane bound organelles, and the nucleus. Our data will provide insight into the mechanism in which GC activates DCs to drive cellular immunity.