A novel direct co-culture assay analyzed by multicolor flow cytometry reveals context- and cell type-specific immunomodulatory effects of equine mesenchymal stromal cells

Abstract

The immunomodulatory potential of multipotent mesenchymal stromal cells (MSC) provides a basis for current and future regenerative therapies. In this study, we established an approach that allows to address the effects of pro-inflammatory stimulation and co-culture with MSC on different specific leukocyte subpopulations. Equine peripheral blood leukocyte recovery was optimized to preserve all leukocyte subpopulations and leukocyte activation regimes were evaluated. Allogeneic labeled equine adipose-derived MSC were then subjected to direct co-culture with either non-stimulated, concanavalin A (ConA)-activated or phosphate 12-myristate 13-acetate and ionomycin (PMA/I)-activated leukocytes. Subsequently, production of the cytokines interferon-γ (IFN- γ), interleukin-1 (IL-1) and tumor necrosis factor-α (TNF-α) and presence of FoxP3 were determined in specific cell populations using multicolor flow cytometry. Prostaglandin E2 (PGE2) was measured in the supernatants. ConA-stimulation induced mild activation of leukocytes, whereas PMA/I-stimulation led to strong activation. In T cells, PMA/I promoted production of all cytokines, with no distinct suppressive effects of MSC. However, increased numbers of CD25/FoxP3-positive cells indicated that MSC supported regulatory T cell differentiation in PMA/I-activated leukocyte cultures. MSC also reduced numbers of cytokine-producing B cells and granulocytes, mostly irrespective of preceding leukocyte activation, and reversed the stimulatory effect of ConA on IFN-γ production in monocytes. Illustrating the possible suppressive mechanisms, higher numbers of MSC produced IL-10 when co-cultured with non-stimulated or ConA-activated leukocytes. This was not observed in co-culture with PMA/I-activated leukocytes. However, PGE2 concentration in the supernatant was highest in the co-culture with PMA/I-activated leukocytes, suggesting that PGE2 could still mediate modulatory effects in strongly inflammatory environment. These context- and cell type-specific modulatory effects observed give insight into the interactions between MSC and different types of immune cells and highlight the roles of IL-10 and PGE2 in MSC-mediated immunomodulation. The approach presented could provide a basis for further functional MSC characterization and the development of potency assays.

Rho/ROCK Inhibition Promotes TGF-β3-Induced Tenogenic Differentiation in Mesenchymal Stromal Cells

“…The mechanisms of action of MSC are highly contextsensitive [28,29], which include their adaptation to the local extracellular environment. This adaptation can enhance the cellular potency, as observed after targeted priming of MSC [30], but it could also compromise therapeutic mechanisms and success.…”

The immunomodulation–immunogenicity balance of equine Mesenchymal Stem Cells (MSCs) is differentially affected by the immune cell response depending on inflammatory licensing and major histocompatibility complex (MHC) compatibility

“…Mesenchymal stem cells (MSCs) are of great interest to treat several pathologies, including musculoskeletal injuries such as those affecting the horse, which is a species of remarkable value as both patient and translational models ( 1 , 2 ). The regulatory and immunomodulatory properties of MSCs are currently considered their main therapeutic mechanism and involve both direct cell-to-cell contact and contact-independent paracrine signaling, via the expression of adhesion molecules like vascular cell adhesion molecule 1 ( VCAM1 ) and the secretion of molecules such as interleukin 6 (IL6) and prostaglandin E2 (PGE 2 ) ( 3 ), respectively ( 4 ). Since the immunomodulatory properties of equine MSCs might have profound therapeutic implications in the treatment of many inflammatory-mediated processes in the horse, a growing number of studies have focused on analyzing such immune properties ( 4 ).…”

MSC in Tendon and Joint Disease: The Context-Sensitive Link Between Targets and Therapeutic Mechanisms

“…Mild inflammatory conditions increased the percentage of MSC synthesizing the anti-inflammatory IL-10, while stronger inflammatory conditions promoted the regulatory effects of MSC on T cells, possibly via prostaglandin E2. However, not all effects observed in strong inflammatory conditions were strictly anti-inflammatory ( Hillmann et al, 2019 ). With respect to joint disease, recent data has shown that synovial fluid collected from OA-affected joints influences the immunomodulatory properties of the MSC secretome and thereby promotes an anti-inflammatory subset of immune cells including an enhanced macrophage polarization into the M2-like phenotype ( Cifù et al, 2020 ).…”