The aim of the present

The aim of the present study was to assess the effect of different inflammatory stimuli on eBM-MSCs immunoregulatory ability and immunogenicity, studying the expression of immunogenic and immunomodulation-related molecules. Firstly, the influence of allogeneic inflammatory SF on eBM-MSCs was investigated, and subsequently, the effect of priming eBM-MSCs with a combination of the two pro-inflammatory molecules IFNγ and TNFα, was tested at two different doses. SF and CK inflammatory conditions were chosen according to previous studies (Ren et al., 2008; Leijs et al., 2012; van Buul et al., 2012; Vézina Audette et al., 2013; Zimmermann and McDevitt, 2014). This work contributes to understand the effects of inflammatory exposure on eBM-MSCs, as a previous step to enhance their use in vivo for equine joint diseases.

Materials and methods

Results

Discussion
The cultured SEA0400 showed capacity for attachment to plastic and the ability of differentiation into osteoblast, adipocyte and chondrocyte, as criteria established to define human MSCs (Dominici et al., 2006). Equine BM-MSCs displayed a gene and cell surface expression pattern similar to previous reports for this species and showed a normal growth pattern, with a proliferation rate and viability similar to other studies (Ranera et al., 2011). Inflammatory conditioned media were prepared based on recent publications on this field. Inflammatory SF was obtained from an allogeneic donor and added at a concentration considered likely to act as an enhancer of the immunoregulatory potential of MSCs (Leijs et al., 2012). SAA and Hp were used to determine the inflammatory status of the SF. Both APP were elevated with regard to described ranges (Basile et al., 2013; Jacobsen et al., 2006) in agreement with the expected changes for these proteins (Jacobsen and Andersen, 2007). For CK-conditioned media, the synergy displayed by the pro-inflammatory cytokines TNFα and IFNγ (Zimmermann and McDevitt, 2014) supported the decision of using them to stimulate the eBM-MSCs. The induction of immunoregulatory properties of BM-MSCs by synergistic cytokine priming is not previously reported in horses. Equine BM-MSCs has been stimulated with 100ng/ml of IFNγ alone in some studies (Paterson et al., 2014; Schnabel et al., 2014), but their gene and surface expression of immunoregulation-related molecules were not studied after the stimulation. Therefore, the tested doses (20ng/ml and 50ng/ml) were chosen according to previous studies in human and mouse MSCs showing induction of immunoregulatory factors expression or secretion (Ren et al., 2008; Waterman et al., 2010; Hegyi et al., 2012; van Buul et al., 2012) to determine if the same conditions also operate similar changes in the behaviour of MSC from equine species. The available volume of inflammatory SF did not allow a time course to be performed and thus a single time-point of 72h was chosen to maximize potential effects of exposure to SF. This was longer than previous studies on the effects of inflammatory SF (Leijs et al., 2012; Vézina Audette et al., 2013) but longer exposure to individual cytokines has been reported (Paterson et al., 2014) and immunosuppressive effects of IFNγ-activated MSCs have been shown to depend on the exposure time to IFNγ (Chan et al., 2006). Our results are similar to previous reports in other species using shorter times of cytokine stimulation, as it will be further discussed. However, the exposure of eBM-MSCs to SF did not produce remarkable effects despite of using an exposition time longer than the previously described (Leijs et al., 2012; Vézina Audette et al., 2013).
Several mechanisms have been proposed to participate in the immunoregulatory function of MSCs, including the participation of chemokine axis, adhesion molecules and soluble factors (Ma et al., 2014). The co-culture of MSCs in the presence of T cells triggers the expression of VCAM-1 (Ren et al., 2010), according to the results obtained in our CK20 conditions. This finding supports the implication of cell-to-cell contact in the MSC immunoregulatory mechanism. Adhesion molecules are also related to MSC migration, a mechanism which could be critical for the recruitment of MSCs into wound sites for tissue regeneration. This process is complex, especially in inflammatory environments, and whereas some studies describe the enhancement of MSC migratory property under inflammatory stimulation (Ries et al., 2007; Shi et al., 2007), others report a decrease in this property depending on the time of exposure (Waterman et al., 2010). The MFI of the hyaluronan receptor CD44, related to cell migration through the extracellular matrix (Zhu et al., 2006), was increased under CK50 conditions, indicating an enhancement in the expression level of this marker, despite the number of positive cells remained similar. However, the gene expression of the chemokine receptor CXCR4, also implied in the MSC migration (Honczarenko et al., 2006), was decreased in both CK Experiments, suggesting that MSC migration might be diminished in the tested inflammatory conditions.