Re acute GVHD was noted in those organs of vehicle-treated animals.

Re acute GVHD was noted in those organs of vehicle-treated animals. Curcumin treatment on donor splenocytes significantly improved pathologic severity scores in the liver, skin, colon and lung. To ascertain whether the protective functions of curcumin work by inhibiting the function of nuclear activator protein-1 (AP-1), the expression of its components, c-Fos and c-Jun, in the skin and intestine were assessed by immunohistochemical staining. Expression of both proteins in epithelial tissues of skin and intestine was significantly reduced in curcumin-treated acute GVHD animals (Fig. 3A, B).Results Curcumin Modulates Alloreative T Cell Responses in vitroTo assess the effects of curcumin on the K162 supplier proliferative capacity of donor CD4+ T cells in response to alloantigens following transplantation, T cell alloreactivity was measured by [3H]thymidine incorporation to assess T cell proliferation in mixed lymphocyte reactions (MLR). Proliferative responses to BALB/c T cells (allogeneic stimulator) was observed in C57BL/6 (B6) T cells (responder cells). Treatment with curcumin inhibited T cell alloreactivity in a dose-dependent manner (Fig. 1A). To ascertain whether or not the inhibitory effect on alloreactive T cell responses by curcumin was associated with the apoptosis induction. Annexin-V and propidium iodide (PI) double staining were performed and analyzed using flow 58543-16-1 cytometery (Fig. S1B). In addition, we performed MTT assays to determine whether curcumin treatment affects cell viability (Fig. S1A). The results indicated that the treatment with curcumin and DSMO at various concentrations did not induce apoptosis or affect cell viability. IFN-c and IL-17 concentrations were then measured using an enzyme-linked immunosorbent assay (ELISA). Curcumin treatment significantly reduced IFN-c and IL-17 level in culture supernatants (Fig. 1B). To determine whether in vitro curcumin treatment can regulate the Th1 and Th2 balance, B6 splenic T cells incubated with irradiated B6 splenic T cells (syngeneic stimulator) or BALB/c splenic T cells (allogeneic stimulator) in the absence of presence of curcumin (2.5 mM) were analyzed by intracellular staining for IL-4, IFN-c, IL-17, and Foxp3 (Fig. 1C). While the Th1 cell population (IFN-c+IL-42 T cells) tended to decrease upon the curcumin treatment, the Th2 cell population (IFN-c2IL-4+ T cells) tended to increase (statistically insignificant). Similarly, curcumin treatment generated reciprocal changes in Th17 and Treg cell differentiation, although the changes were not statistically significant (Fig. 1C).Curcumin Acts in the Acute GVHD Model via Reciprocal Regulation of Th1 and Treg CellsTo investigate the in vivo mechanism of curcumin in the acute GVHD murine model, the numbers of CD4+IFN-c+, CD4+IL4+, CD4+IL-17+, and CD4+CD25+Foxp3+ T cells in spleens isolated from each group were counted using confocal staining. The numbers of CD4+IFN-c+ and CD4+IL-17+ T cells in spleens were decreased in curcumin-treated GVHD animals as compared with the vehicle-treated group. On the other hand, the numbers of CD4+IL-4+ and CD4+CD25+Foxp3+ splenocytes were increased in the curcumin-treated group, although the differences between the two groups were modest (Fig. 4A). Fourteen days after BMT, lymph node cells isolated from each group were analyzed for the expression of IL-4, IL-17, and IFN-c. The number of lymph node cells expressing IFN-c was significantly decreased in the curcumin-treated group, whereas IL-4- and IL-17-expr.Re acute GVHD was noted in those organs of vehicle-treated animals. Curcumin treatment on donor splenocytes significantly improved pathologic severity scores in the liver, skin, colon and lung. To ascertain whether the protective functions of curcumin work by inhibiting the function of nuclear activator protein-1 (AP-1), the expression of its components, c-Fos and c-Jun, in the skin and intestine were assessed by immunohistochemical staining. Expression of both proteins in epithelial tissues of skin and intestine was significantly reduced in curcumin-treated acute GVHD animals (Fig. 3A, B).Results Curcumin Modulates Alloreative T Cell Responses in vitroTo assess the effects of curcumin on the proliferative capacity of donor CD4+ T cells in response to alloantigens following transplantation, T cell alloreactivity was measured by [3H]thymidine incorporation to assess T cell proliferation in mixed lymphocyte reactions (MLR). Proliferative responses to BALB/c T cells (allogeneic stimulator) was observed in C57BL/6 (B6) T cells (responder cells). Treatment with curcumin inhibited T cell alloreactivity in a dose-dependent manner (Fig. 1A). To ascertain whether or not the inhibitory effect on alloreactive T cell responses by curcumin was associated with the apoptosis induction. Annexin-V and propidium iodide (PI) double staining were performed and analyzed using flow cytometery (Fig. S1B). In addition, we performed MTT assays to determine whether curcumin treatment affects cell viability (Fig. S1A). The results indicated that the treatment with curcumin and DSMO at various concentrations did not induce apoptosis or affect cell viability. IFN-c and IL-17 concentrations were then measured using an enzyme-linked immunosorbent assay (ELISA). Curcumin treatment significantly reduced IFN-c and IL-17 level in culture supernatants (Fig. 1B). To determine whether in vitro curcumin treatment can regulate the Th1 and Th2 balance, B6 splenic T cells incubated with irradiated B6 splenic T cells (syngeneic stimulator) or BALB/c splenic T cells (allogeneic stimulator) in the absence of presence of curcumin (2.5 mM) were analyzed by intracellular staining for IL-4, IFN-c, IL-17, and Foxp3 (Fig. 1C). While the Th1 cell population (IFN-c+IL-42 T cells) tended to decrease upon the curcumin treatment, the Th2 cell population (IFN-c2IL-4+ T cells) tended to increase (statistically insignificant). Similarly, curcumin treatment generated reciprocal changes in Th17 and Treg cell differentiation, although the changes were not statistically significant (Fig. 1C).Curcumin Acts in the Acute GVHD Model via Reciprocal Regulation of Th1 and Treg CellsTo investigate the in vivo mechanism of curcumin in the acute GVHD murine model, the numbers of CD4+IFN-c+, CD4+IL4+, CD4+IL-17+, and CD4+CD25+Foxp3+ T cells in spleens isolated from each group were counted using confocal staining. The numbers of CD4+IFN-c+ and CD4+IL-17+ T cells in spleens were decreased in curcumin-treated GVHD animals as compared with the vehicle-treated group. On the other hand, the numbers of CD4+IL-4+ and CD4+CD25+Foxp3+ splenocytes were increased in the curcumin-treated group, although the differences between the two groups were modest (Fig. 4A). Fourteen days after BMT, lymph node cells isolated from each group were analyzed for the expression of IL-4, IL-17, and IFN-c. The number of lymph node cells expressing IFN-c was significantly decreased in the curcumin-treated group, whereas IL-4- and IL-17-expr.