AH carried out ELISA assay. experimental conditions (Additional file 1: Figure S1). Expression of GM-CSF in mucosal and systemic sites of vaccinated micePrior to assessing the contribution of GM-CSF on the mucosal adjuvant activities of the cationic liposomes, we first examined the expression of GM-CSF at the site of injections. As shown in Fig.?1a, intranasal administration with OVA and DOTAP/DC-chol liposomes significantly exerted the expression of GM-CSF in nasal areas (2.3- to 3.3-fold expression compared to mice that received either vehicle or OVA alone). On the other hand, immunization with OVA and DOTAP/DC-chol liposomes did not induce any GM-CSF expression in the spleen. Additionally, since it is known that T cells activated via TCR signalling are capable of producing a large amount of GM-CSF [32, 33], we investigated antigen-specific GM-CSF secretion in splenocytes by re-stimulating them with OVA in vitro. As shown in Fig.?1b, splenocytes from OVA and DOTAP/DC-chol liposome-vaccinated mice produced higher levels of GM-CSF than those from OVA-only administered mice when re-stimulated with OVA. These results indicated that nasally administered DOTAP/DC-chol liposomes were polarized to induce the expression of GM-CSF. Open in a separate window Fig.?1 mRNA expression of granulocyteCmacrophage colony-stimulating factor (GM-CSF) in nasal and spleen tissues (a) and antigen-specific production of GM-CSF in splenocytes from mice administered intranasally with OVA plus the liposomes. a Nasal tissue and spleens were collected 6?h after the last immunization. mRNA expression was measured using qPCR. b Spleens were collected 1?week after the last immunization, and then harvested splenocytes were cultured for 72?h in the presence of OVA (10?g/mL). After culture, concentrations of GM-CSF were determined using ELISA. The values are the mean??SD of technical duplicates from three biologically independent experiments. Significance was assessed using unpaired not significant as evaluated using the KruskalCWallis test with Dunns post hoc test Discussion In this study, we demonstrated the following: (1) intranasal administration with DOTAP/DC-chol liposomes induced the expression of GM-CSF at the site of injections; (2) recombinant GM-CSF showed the mucosal adjuvant effect when nasally administered in mice; and (3) GM-CSF expression in nasal area induced by DOTAP/DC-chol liposomes was not required for the mucosal Asarinin adjuvant activities. The development of safe and efficient mucosal adjuvants is needed to prevent fatal infectious diseases. To accomplish this, understanding the mechanism(s) underlying mucosal adjuvant induction of immune responses to antigenic proteins is essential. Generally, adjuvants show their activities through the depot effect, with the gradual release of antigen at the site of infection and increase in antigen uptake by APCs. Resent research has focused on the role of APCs in activating innate immunity [34]. In particular, the cytokine/chemokine milieu induced by external stimuli, including adjuvants, determines the immune response to antigenic proteins, including the production of antibody to the antigen [35C37]. Many studies on the immunomodulating activities of GM-CSF have been reported. For instance, GM-CSF has been shown to stimulate the maturation and function of APCs, such as DCs and macrophages. GM-CSF is also a strong inducer of interleukin-6 (IL-6), which promotes germinal center development and B cell growth and differentiation in these centers [38, 39]. We found that intranasal administration of DOTAP/DC-chol Rabbit Polyclonal to COMT liposomes induced IL-6 expression in the nasal mucosa, and that this cytokine was critical for Asarinin the induction of antigen-specific IgA by the cationic liposomes (unpublished results). Therefore, local GM-CSF expression likely plays a role in enhancing humoral responses to the cationic liposomes. Furthermore, intranasal co-administration of antigens with a GM-CSF-expressing plasmid has been shown to increase OVA-specific mucosal IgA and serum IgG titers, suggesting Asarinin that GM-CSF plays an essential role in the induction of humoral immune responses to antigenic proteins in both mucosal and systemic compartments [10, 18, 19, 40]. We investigated the role of GM-CSF on the mucosal adjuvant activities of the cationic liposomes in this study and found that GM-CSF blocking did not affect their activities, clearly indicating that other soluble factors control the mucosal adjuvant activities of the cationic liposomes. Further experiments are required to clarify the molecular mechanism(s) underlying the induction of humoral immune responses.