In vitro evaluation of immune responses to bacterial hydrogels for the development of living therapeutic materials

Abstract

In living therapeutic materials, organisms genetically programmed to produce and deliver drugs are encapsulated in porous matrices or hydrogels acting as physical barriers between the therapeutic organisms and the host cells. The therapeutic potential of such constructs has been highlighted in in vitro studies, but the translation to in vivo scenarios requires evaluation of the immune response to the presence of the encapsulated, living organisms. In this study, we investigate the responses of human peripheral blood mononuclear cells (PBMCs) exposed to a living therapeutic material consisting of engineered E. coli encapsulated in Pluronic F127-based hydrogels. The release of inflammation-related cytokines (IL-2, IL-4, IL-6, IL-10, IL-17A, TNF and IFN{gamma}) and cytotoxic proteins (granzyme A, granzyme B, perforin, granulysin, sFas, and sFasL) in response to the bacterial hydrogels, as well as the subsets of natural killer cells and T cells after exposure to the bacterial hydrogel for up to three days were examined. In direct contact with PBMCs, both E. coli and its endotoxin-free variant, ClearColi, induce apoptosis of the immune cells and trigger IL-6 release from the surviving cells. However, we found that encapsulation of the bacteria in Pluronic F127 diacrylate hydrogels considerably lowers their immunogenicity and practically abolishes apoptosis triggered by ClearColi. In comparison with E. coli, free and hydrogel-encapsulated ClearColi induced significantly lower levels of NK cell differentiation into the more cytolytic CD16dim subset. Our results demonstrate that ClearColi-encapsulated hydrogels generate low immunogenic response and are suitable candidates for the development of living therapeutic materials for in vivo testing to assess a potential clinical use. Nevertheless, we also observed a stronger immune response in pro-inflammatory PBMCs, possibly from donors with underlying infections. This suggests that including anti-inflammatory measures in living therapeutic material designs could be beneficial for such recipients.