Menstrual Blood-Derived Mesenchymal Stromal Cell Secretome Modulates Macrophage Polarization in a Preconditioning-Dependent Manner

María de los Ángeles De Pedro^1,2María Pulido¹Ana María Marchena^1,2Verónica Álvarez Pérez¹Francisco Manuel González-Nuño¹Witold Szymanski^3,4Johanna Pörschke⁵Silke Reinartz⁶Johannes Graumann^3,4Elke Pogge von Strandmann^5,7Francisco Miguel Sanchez Margallo^1,2María Gómez-Serrano^5*Esther López^1,2

• ¹Stem Cell Therapy Unit, Jesús Usón Minimally Invasive Surgery Centre, Cáceres, Spain
• ²RICORS-TERAV Network, ISCIII, Madrid, Spain
• ³Institute of Translational Proteomics, Biochemical/Pharmacological Center, Philipps University, Marburg, Germany
• ⁴Core Facility Translational Proteomics, Philipps University, Marburg, Germany
• ⁵Institute for Tumor Immunology, Center for Tumor Biology and Immunology (ZTI), Philipps University, Marburg, Germany
• ⁶Translational Oncology, Center for Tumor Biology and Immunology, Philipps University, Marburg, Germany
• ⁷EV-iTEC Core Facility, Philipps University, Marburg, Germany

Background: The effects of menstrual blood-derived mesenchymal stromal cell secretome (S-MenSC) on macrophage polarization remain unclear. This study studied the impact of secretomes from basal MenSCs (S-bMenSCs) and those preconditioned with IFNγ and TNFα (S-pMenSCs) on human monocytes and macrophages in vitro. Methods: S-MenSCs were used to assess their effects on three stages of monocyte-derived cell maturation: (i) monocyte differentiation; (ii) polarization of monocyte-derived macrophages (MDMs) toward M1-like or M2-like phenotypes; and (iii) reprogramming of pre-polarized M1 or M2 macrophages. Surface markers were analyzed by flow cytometry and cytokine gene expression by RT-qPCR. In addition, a proteomic profiling was performed to identify proteins involved in the observed effects. Results: Our results confirmed the capacity of S-MenSCs of modulating innate immune response and in particular macrophage polarization. More concretely, the in vitro experiments showed that: (i) both secretomes partly promoted monocyte differentiation into an M1-like phenotype; (ii) during macrophage polarization, S-bMenSCs partially limited the shift to an M1 phenotype, whereas treatment with S-pMenSCs boosted it; and, (iii) in the pre-polarized macrophages, S-bMenSCs reinforced M1 traits, whereas S-pMenSCs promote partial phenotype switching. Finally, proteomic analysis revealed significant differences in the composition of both secretomes, comprising key proteins associated with macrophage polarization. Conclusions: These findings extend the knowledge on the immunomodulatory capacity of the S-MenSC, supporting that MenSCs, particularly when preconditioned, may play a significant role in regulating macrophage polarization, and, thus, modulating the inflammatory response.