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Background: Immunoregulatory capacity of mesenchymal stem cells (MSC) is triggered by the inflammatory environment, which changes during tissue repair. Macrophages are essential in mediating the inflammatory response after injury and can adopt a range of functional phenotypes, exhibiting pro-inflammatory and anti-inflammatory activities. An accurate characterization of MSC activation by the inflammatory milieu is needed for improving the efficacy of regenerative therapies. In this work, we investigated the immunomodulatory functions of MSC primed with factors secreted from macrophages polarized toward a pro-inflammatory or an anti-inflammatory phenotype. We focused on the role of TNF-α and IL-10, prototypic pro-inflammatory and anti-inflammatory cytokines, respectively, as priming factors for MSC. Methods: Secretion of immunoregulatory mediators from human MSC primed with media conditioned by human macrophages polarized toward a pro-inflammatory or an anti-inflammatory phenotype was determined. Immunomodulatory potential of primed MSC on polarized macrophages was studied using indirect co-cultures. Involvement of TNF-α and IL-10 in priming MSC and of PGE2 in MSC-mediated immunomodulation was investigated employing neutralizing antibodies. Collagen hydrogels were used to study MSC and macrophages interactions in a more physiological environment. Results: Priming MSC with media conditioned by pro-inflammatory or anti-inflammatory macrophages enhanced their immunomodulatory potential through increased PGE2 secretion. We identified the pro-inflammatory cytokine TNF-α as a priming factor for MSC. Notably, the anti-inflammatory IL-10, mainly produced by pro-resolving macrophages, potentiated the priming effect of TNF-α. Collagen hydrogels acted as instructive microenvironments for MSC and macrophages functions and their crosstalk. Culturing macrophages on hydrogels stimulated anti-inflammatory versus pro-inflammatory cytokine secretion. Encapsulation of MSC within hydrogels increased PGE2 secretion and potentiated immunomodulation on macrophages, attenuating macrophage pro-inflammatory state and sustaining antiinflammatory activation. Priming with inflammatory factors conferred to MSC loaded in hydrogels greater immunomodulatory potential, promoting anti-inflammatory activity of macrophages Conclusions: Factors secreted by pro-inflammatory and anti-inflammatory macrophages activated the immunomodulatory potential of MSC. This was partially attributed to the priming effect of TNF-α and IL-10. Immunoregulatory functions of primed MSC were enhanced after encapsulation in hydrogels. These findings may provide insight into novel strategies to enhance MSC immunoregulatory potency., This work was supported by grants from Instituto Salud Carlos III (ISCIII)-Fondo Europeo de Desarrollo Regional (FEDER), MINECO-AES (PI15/00752 and PI15/ 01118) and Comunidad Autónoma de Madrid (CAM) (S2013/MIT-2862). LS is supported by a Miguel Servet contract from ISCIII-MINECO-AES-FEDER-FSE. NV is supported by Program I2 from CAM.

Mesenchymal stem cells (MSC) modulate the macrophage-mediated inflammatory response through the secretion of soluble factors. In addition to its classical effects on calcium homeostasis, 1,25-dihydroxyvitamin D3 (1,25D3) has emerged as an important regulator of the immune system. The present study investigates whether 1,25D3 modulates the paracrine interactions between MSC and macrophages. 1,25D3 stimulated MSC to produce PGE

Mesenchymal stem cells (MSC) modulate the macrophage-mediated inflammatory response through the secretion of soluble factors. In addition to its classical effects on calcium homeostasis, 1,25-dihydroxyvitamin D3 (1,25D3) has emerged as an important regulator of the immune system. The present study investigates whether 1,25D3 modulates the paracrine interactions between MSC and macrophages. 1,25D3 stimulated MSC to produce PGE2 and VEGF and regulated the interplay between macrophages and MSC toward reduced pro-inflammatory cytokine production. Conditioned media (CM) from co-cultures of macrophages and MSC impaired MSC osteogenesis. However, MSC cultured in CM from 1,25D3-treated co-cultures showed increased matrix maturation and mineralization. Co-culturing MSC with macrophages prevented the 1,25D3-induced increase in RANKL levels, which correlated with up-regulation of OPG secretion. MSC seeding in three-dimensional (3D) substrates potentiated their immunomodulatory effects on macrophages. Exposure of 3D co-cultures to 1,25D3 further reduced the levels of soluble factors related to inflammation and chemotaxis. As a consequence of 1,25D3 treatment, the recruitment of monocytes toward CM of 3D co-cultures decreased, while the osteogenic maturation of MSC increased. These data add new insights into the pleiotropic effects of 1,25D3 on the crosstalk between MSC and macrophages and highlight the role of the hormone in bone regeneration.