Developing an in vitro model to study trained immunity

Abstract

Novel discoveries have proven that the innate immune system has the capacity to adapt a memory response, termed “trained immunity”, providing broad non-specific protection against pathogens. To date, trained immunity has been studied in vivo, and ex vivo using human peripheral blood mononuclear cells (PBMCs) and bone marrow derived macrophages (BMDMs) from mice. However, we aim to develop and characterize an in vitro cell model to study trained immunity. J774A.1 macrophages and THP-1 monocytes were evaluated as murine and human models, respectively, to study trained immunity in vitro. THP-1 monocytes were differentiated from monocytes to macrophages with 72-hour phorbol 12-myristate-13-acetate (PMA) stimulation. Cells were trained for 24 hours with muramyl dipeptide (MDP), lipopolysaccharide (LPS), glucopyranosyl lipid adjuvant (GLA), or a novel delivery system containing cationic mannosylated liposome (CL) containing muramyl dipeptide (MDP) and glucopyranosyl lipid adjuvant (GLA). After a 2 days’ rest, cells were restimulated with MDP, LPS, Dukoral or influenza virus for 24 hours. Subsequently, trained immunity was evaluated in terms of tumor necrosis factor D (TNFD) and interleukin-10 (IL-10) production, and cell surface expression of complement domain 14 (CD14) and complement domain 16 (CD16). We demonstrated that both J774A.1 cells and PMA differentiated THP-1 cells had a trained immunity cytokine profile, as defined by an increase of TNFα but not IL-10, following restimulation with various non-specific pathogens. Next, we developed a delivery system comprised of MDP and GLA entrapped in cationic liposome (CL). The delivery system significantly increased TNFD production following non-specific restimulation in both the murine and human in vitro models, indicating the ability to train the cells. By way of flow cytometry, we discovered the delivery system increased the expression of CD14 and decreased the expression of CD16 on PMA differentiated THP-1 cells To determine the method of training induced by the delivery system, we used two cell signaling inhibitors, CLI-095 and Gefitinib, and deduced that the delivery system was inducing training partially through toll-like receptor 4 (TLR4) and nucleotide-binding oligomerization domaincontaining protein 2 (NOD2), respectively. Finally, complement domain 3/ complement domain 4 (CD3/CD28) activated Jurkat T cells co-cultured with trained THP-1 macrophages produced significantly higher levels of interleukin 2 (IL-2) upon subsequent non-specific restimulation, suggesting trained innate immune cells have the potential to influence adaptive immune responses. On the whole this research has contributed to the development of an in vitro bioassay model that will allow scientists in all fields of immunology to further explore the phenomenon of trained immunity. Additionally, these in vitro models can act as tools to demonstrate the potential of trained immunity as a novel therapeutic strategy. At last, our delivery system proved to be a promising trained immunity stimulant that warrants further investigations