Canine mesenchymal stem cells treated with TNF-alpha and IFN-gamma enhance anti-inflammatory effects through the COX-2/PGE(2) pathway

Mesenchymal stem cells (MSCs) have been used in studies on treatment of various diseases, and their application to immune-mediated diseases has garnered interest. Various methods for enhancing the immunomodulation effect of human MSCs have been used; however, similar approaches for canine MSCs are relatively unexplored. Accordingly, we evaluated immunomodulatory effects and mechanisms in canine MSCs treated with TNF-alpha and IFN-gamma. Lipopolysaccharide (LPS)-stimulated RAW 264.7 cells were incubated with the conditioned media (CM) from canine MSCs for 48 h. Expression of RNA was assessed by quantitative reverse transcription PCR (qRTPCR), and protein levels were assessed by western blot. Expression of inducible nitric oxide synthase (iNOS), IL-6 and IL-1 beta was significantly (one-way ANOVA) decreased in LPS-stimulated RAW 264.7 cells incubated with CM from canine MSCs compared to that in LPS-stimulated RAW 264.7 cells alone. Furthermore, anti-inflammatory effects of TNF-alpha- and IFN-gamma-primed canine MSCs were significantly increased compared with those of naive canine MSCs. Expression of cyclooxygenase 2 (COX-2) and prostaglandin E-2 (PGE(2)) were likewise significantly increased in primed canine MSCs. The level of iNOS protein in LPS-stimulated RAW 264.7 cells incubated with CM from the primed canine MSCs was decreased, but it increased when the cells were treated with NS-398(PGE(2 )inhibitor). In conclusion, compared with naive canine MSCs, cells primed with TNF-alpha and IFN-gamma cause a greater reduction in release of anti-inflammatory cytokines from LPS-stimulated RAW 264.7 cells; the mechanism is upregulation of the COX-2/PGE(2) pathway.