Development of an abbreviated protocol for the polarization and characterization of therapeutic inflammation-resolving monocytes/macrophages

Background & Aim Monocytes/macrophages (MΦs) are innate immune cells that exist on a spectrum of pro-inflammatory to inflammation-resolving phenotypes. Their polarization is mediated in vivo through the local microenvironment or ex vivo through exogenous factors. Such ex vivo polarized cells have been used in early clinical trials for a variety of immunosuppressive or reparative indications (e.g. spinal cord injury, liver cirrhosis, transplant rejection). Manufacturing protocols have been variable between studies with similarities in 7-day duration and surface marker characterization. We aim to develop an abbreviated (i.e. Methods, Results & Conclusion Peripheral blood MΦs are isolated by density centrifugation and CD14 magnetic beads, then treated with in-house cytokine cocktail or small molecule inhibitors for 24-48h. Polarized MΦs are co-cultured with late stage osteoarthritic explants to test phenotypic stability. MΦs are characterized through: flow cytometry (surface markers, viability, dextran endocytosis), qRT-PCR and/or immunoassay (inflammatory/chemotactic/catabolic mediators), and T cell proliferation. Treatment phenotype is compared against 7-day treatment with M-CSF and against naive 48h MΦs. 48h polarized MΦs demonstrate a similar phenotypic profile to 7-day polarized MΦs. Gene expression of anti-inflammatory IL-10 is upregulated in both groups relative to naive controls. Surface expression of scavenger receptors CD163 and CD206, markers of an inflammation-suppressive phenotype, are higher in both the 48h and 7d groups relative to control. Pro-inflammatory markers CD86 and HLA-DR are not highly expressed in either group. 48h polarized MΦs maintain a similar surface marker profile after co-culture with osteoarthritic explant tissue for up to 7 days. Preliminary results demonstrate that the 48h polarization protocol yields a similar phenotypic profile to 7d polarized MΦs. With further functional assay and protocol optimization using small molecule agents, we aim to develop an efficient, well-characterized inflammation suppressive MΦ phenotype that can be easily translated for clinical use.