Phenotype and functional characteristics of mouse dendritic cells in response to cholera toxin and toll-like receptor 2 ligands

Abstract

Cholera toxin (CT) is a potent mucosal adjuvant which acts to stimulate antigen-specific secretory IgA and systemic IgG antibody responses and known to act as a mucosal adjuvant. Dendritic cells (DCs) are the major antigen-presenting cells bridging innate and adaptive immunity and play a critical role in the immune responses to vaccines. Since toll-like receptor (TLR) ligands efficiently stimulate DCs, various TLR ligands (S. aureus LTA, Pam2CSK4, CpGODN etc.) have been investigated as potential candidates of adjuvants. Thus, in order to examine if TLR2 ligands enhance the adjuvanticity of non-TLR-based adjuvant, CT, we evaluated the immunomodulatory activity of CT and TLR2 ligands using mouse bone marrow-derived DCs. Immature mouse DCs were prepared from bone marrow, collected from mouse femur and tibia, treated with mouse recombinant GM-CSF and 2-mercaptoethanol. Maturation and activation of DCs were examined by investigating surface markers, co-stimulatory molecules and phagocytic activity using flow cytometry. Cytokine profiles in response to CT together with each TLR2 ligand (S. aureus LTA, Pam2CSK4 and Pam3CSK4) were measured by ELISA. Also, to investigate the ability of DCs to induce T cell activation and differentiation, DCs stimulated with CT and TLR2 ligands were co-cultured with splenocytes and analyzed for Th17 and regulatory T cell responses using flow cytometry. We found that the combination of CT and each TLR2 ligand additively increased expression of CD80 and CD86 on DCs, but decreased expression of MHC class I. There was no difference in MHC class II expression. CT and TLR2 ligands additively induced maturation of DCs and decreased phagocytic activity of DCs. Furthermore, secretion of IL-6, IL-23 and IL-10 was increased in DCs stimulated with CT and each TLR2 ligand. However, CT decreased TLR2 ligands-mediated production of TNF-α by DCs. DCs stimulated with both CT and TLR2 ligands induced increase in IL-17 secreting T cells and decreased induction of regulatory T cell population when treated DCs were co-cultured with fresh splenocytes. In conclusion, CT and each TLR2 ligand supported maturation of DCs and led DCs to secrete IL-6 and IL-23 which are the major cytokines for Th17 responses. These results suggest that CT and each TLR2 ligand work as immunomodulators regulating DCs to control both innate and adaptive immune responses. Elicited Th17 responses can improve epithelial cell barrier function and help induction of antibodies involved for humoral immunity. Thus, Th17 responses induced by CT and each TLR2 ligand will be beneficial for mucosal immunity and also support their adjuvanticity.