Therapeutic Protein Nanoparticles That Subvert Intracellular Pathways for Immunomodulation As a Treatment of IBD

Certain bacterial pathogens use needle-like structures, known as the type III secretion system (T3SS) to inject effector proteins directly into the cytoplasm of host cells. These effectors have evolved to subvert a variety of eukaryotic signaling pathways, including MAPK and NF-κB, and hence, modulate immune signaling and fate of the host cell. We have identified one of these effectors, acetyltransferase-A (ATA), as a potential therapeutic agent for acute and chronic inflammation such as that observed in inflammatory bowel disease (IBD). However, significant challenges remain to deliver ATA in vivo without bacterial T3SS in order to enable its use as an immunotherapeutic.

In prior work, we established that ATA decreases immune and inflammatory signals in vitro. However, delivering ATA to cells in the gut is difficult due to the low solubility of ATA and the harsh conditions of the gastrointestinal system and the mucosal barrier. In the present study, we replace the T3SS delivery mechanism by producing protein nanoparticles made directly from ATA and enhanced-green fluorescent protein. We have demonstrated nanoparticle size control through modification of multiple desolvation parameters including imidazole concentration, protein concentration, cross-linker type and cross-linking time. Tuning the size of these particles is critical for facilitating penetration and crossing of the mucosal layer, as well as endocytosis by intestinal epithelial or immune cells.

We observe that ATA nanoparticles are internalized in vitro at significantly higher rates than soluble protein and that ATA in the nanoparticles retains its bioactivity when delivered. Furthermore, we demonstrate anti-inflammatory activity in two different in vivo models. In a murine peritonitis model of acute inflammation, ATA nanoparticle pretreatment decreases neutrophil influx. In a mouse colitis model, we observe decreased inflammation measured by clinical parameters and histological indices. Therefore, this bacterial effector protein nanoparticle is a novel alternative that can replace the T3SS of bacterial pathogens for immunomodulation as a treatment for IBD, and potentially inflammatory and autoimmune diseases in general.