Differential effects of macrophage subtype‐specific cytokines on fibroblast proliferation and endothelial cell function in co‐culture system

Macrophages are involved in several critical activities associated with tissue repair and regeneration. Current approaches in regenerative medicine are focusing on leveraging the innate immune response to accelerate tissue regeneration and improve long-term healing outcomes. Of particular interest in this regard are the currently known, four main M2 macrophage subtypes: M2^{interleukin (IL)-4,IL-13}, M2^IC, M2^IL-10, M2^{non-selective adenosine receptor agonists (NECA)} (M2^IL-4,IL-13 → M2^NECA). In this study, rat bone marrow-derived macrophages (M₀) were polarized to each of the four subtypes M2^IL-4,IL-13 → M2^NECA and cultured for 72 h in vitro. Luminex assay results highlighted increased production of tissue inhibitor of metalloproteinases-1 (TIMP-1) for M2^IL-4,IL-13, higher amounts of transforming growth factor-beta 1 (TGF-β1) for M2^IL-10, and elevated vascular endothelial growth factor A (VEGF-A) from M2^NECA. Co-culture experiments performed with M2^IL-10 macrophages and L929 fibroblasts highlighted the increased production of soluble collagen within the media as well as higher amounts of collagen in the extracellular matrix. Human umbilical vein endothelial cells (HUVECs) were co-cultured with M2^NECA macrophages, which demonstrated an increase in intercellular adhesion molecule (ICAM) and platelet endothelial cell adhesion molecule (PECAM), as well as increased formation of endothelial tubes. The findings of this study emphasize a critical demand for further characterization and analyses of distinct M₂ subtypes and careful selection of specific macrophage populations for regeneration of specific tissue types. The current, broad classification of "M₂" may be sufficient in many general tissue engineering applications, but, as conditions are constantly in flux within the microenvironment in vivo, a higher degree of specificity and control over the initial M₂ subtype could result in more consistent long-term outcomes where macrophages are utilized as part of an overall regenerative strategy.