Keeping Living Therapeutic Materials under the Radar of Neutrophils

Abstract

Living therapeutic materials (LTM) is a promising approach for long-term drug delivery in vivo. The basic principle is a bacteria encapsulated in hydrogel, engineered to produce the drug of interest. Bacterial components, such as formyl peptides and lipopolysaccharides (LPS), are potent mediators of neutrophil chemotaxis. Therefore, neutrophils sense bacteria by formyl peptide (FPR) and Toll-like receptors (TLR) that activate neutrophil chemotaxis, degranulation, the release of matrix metalloproteinases (MMPs), cytokines, reactive oxygen species (ROS), and NETs. The strong response of neutrophils can result in severe tissue damage and systemic side effects. To circumvent this, ClearColi, an E. coli strain genetically modified to produce mutated LPS, with a potentially lower capacity to activate neutrophils. Furthermore, L. plantarum was used to develop a commensal bacteria-based drug-delivery system to target inflammatory disease conditions. The study aimed to investigate the response of neutrophils, relevant immune cell population in vivo, to living therapeutic materials (LTMs). This is the first evaluation of the potential activation of neutrophils. The LTM includes hydrogel, hydrogel-encapsulated bacteria and only bacteria like E. coli and candidates derived from L. plantarum WCFS1. The investigation was performed on various assay types classified into simple and short term, intermediate complex, and more complex assays allowing long term evaluations. This includes, respectively, direct stimulation assays (DSA) which are utilized to check cytokine production resulting from direct short-term interaction of neutrophils exposed to a single condition. Antibody-dependent cell cytotoxicity (ADCC) is used to evaluate the cytotoxic effects of neutrophil interaction with various conditions influencing target cells as intermediate term technique. More complex (organotypic 3D-cultures) provide the dynamics of neutrophil migration through tissue-like culture (in vivo-like culture). We found that all hydrogel constructs could activate neutrophils strongly. Then, both E. coli and ClearColi supernatants could trigger neutrophil activation, which was higher in the case of E. coli than ClearColi, translated to the release of MMPs and cytokines, and enhancing neutrophils' ability to kill tumor cell line (A431) in ADCC. To determine the difference in activation level between both strains, LPS was applied in the same concentration (conc.) and measured in both candidates' supernatants. As expected, LPS could activate neutrophils higher than ClearColi supernatants. Then by comparing ClearColi (modified LPS), E. coli (unmodified LPS), and commercial LPS, we could prove that the higher activation level of E. coli supernatant originated from LPS, which can activate neutrophil TLR-signaling. L. planetarium is a lactic acid bacteria characterized by health-promoting and disease-curing effects. L. planetarium candidates displayed significant activation and attraction marked by neutrophil degranulation and migration. Moreover, the response of neutrophils varied at different bacterial statuses, showing there might be different signaling pathways. However, more studies are needed including neutrophil function assays in addition more modifications are required for ClearColi so as not to trigger a neutrophil response.