Rapid inflammation-resolving polarization of monocytes/macrophage using cytokines

Chan, Mable Wing Yan (1, 2), Kamal, Ahmad (1, 2), Viswanathan, Sowmya (1, 2, 3, 4)

(1) Arthritis Program, Krembil Research institute, University Health Network

(2) Institute of Biomaterials and Biomedical Engineering, University of Toronto

(3) Cell Therapy Program, University Health Network(4) Division of Hematology, Department of Medicine, University of Toronto

(4) Division of Hematology, Department of Medicine, University of Toronto

Hypothesis: Monocytes/macrophages are immune cells that can have pro-inflammatory or inflammation-resolving functionality depending on microenvironmental cues. They are key players in immunoregulation and wound healing; their dysregulation is implicated in the pathobiology of many inflammatory disorders. Although monocytes/macrophages have mixed phenotypes in vivo, they can be ex vivo polarized towards distinct inflammation-resolving subtypes. 7-day ex vivo polarized macrophages are currently in clinical trials for liver cirrhosis and renal transplant rejection. We hypothesize that abbreviated <48-hour treatment with cytokines can produce monocytes/macrophages with stable inflammation-resolving functionality.

Materials and Methods: Primary peripheral blood monocytes are isolated by density centrifugation and CD14 magnetic separation, then treated with a cytokine cocktail (IL-10+TGF-beta) for 24-hour and 48-hour time points. Treatment groups are compared against naïve cells, pro-inflammatory (IFN-gamma+lipopolysaccharide) polarization, and 7-day clinical trial-based differentiation (M-CSF). Monocytes/macrophages characterized using surface marker flow cytometry, phagocytic capacity, and gene expression.

Results: Both 48-hour IL-10+TGF-beta cytokine-polarized and 7-day M-CSF polarized monocytes/macrophages have high surface expression of CD163 and CD206 with low expression of CD86 and HLA-DR while upregulating expression of anti-inflammatory IL-10. 48-hour cytokine-polarized monocytes/macrophages maintain their surface marker profile after co-culture with osteoarthritic explants for up to 7 days.

Conclusion: Abbreviated 48-hour cytokine treatment polarizes monocytes/macrophages towards an inflammation-resolving phenotype similar to 7-day M-CSF differentiation. This new finding has applications towards ex vivo therapeutic polarization of monocyte/macrophage therapies for inflammatory indications, such as osteoarthritis.