Effects of Stromal Cell Conditioned Medium and Anti-purinergic Treatment on Macrophage Phenotype

The immunomodulatory capacity of the human mesenchymal stromal cell (MSC) secretome has been a critical driver for the development of cell-free MSC products, such as conditioned medium (CM), for regenerative medicine applications. This is particularly true as cell-free MSC products present several advantages over direct autologous or allogenic MSC delivery with respect to safety, manufacturability, and controlled potency. Recently, significant effort has been placed into creating novel MSC CM formulations with an immunomodulatory capacity tailored for specific regenerative contexts. For instance, the immunoregulatory nature of MSC CM has previously been tuned through a number of cytokine priming strategies. Herein, we propose an alternate method to tailor the immunomodulatory "phenotype" of cytokine-primed MSC CM through coupling with the pharmacological agent, suramin. Suramin interferes with the signaling of purines including extracellular ATP, which plays a critical role in the activation of the innate immune system following injury. Toward this end, human THP-1 derived macrophages were activated to a proinflammatory phenotype and treated with: 1) unprimed (native) MSC CM, 2) IFNγ/TNFα-primed MSC CM, 3) suramin alone, or 4) primed MSC CM and suramin. Markers of key macrophage functions - cytokine secretion, autophagy, oxidative stress modulation, and activation/migration - were assessed. Consistent with previous literature, primed CM elevated macrophage secretion of several proinflammatory and pleiotropic cytokines relative to native CM; whereas addition of suramin imparted consistent shifts in terms of TNFα (↓), IL-10 (↓), and HGF (↑) irrespective of CM. In addition, both primed CM and suramin, individually and combined, increased reactive oxygen species production relative to native CM, and addition of suramin to primed CM shifted levels of CX3CL1, a factor involved in ATP-associated macrophage regulation. Varimax rotation assessment of the secreted cytokine profiles confirmed that primed CM/suramin resulted in a THP-1 phenotypic shift away from the LPS-activated proinflammatory state that was distinct from that of primed CM or native CM alone. This altered phenotype may prove beneficial for healing in certain regenerative contexts. These results may inform future work into coupling anti-purinergic treatments with MSC-derived therapies in regenerative medicine applications.