Adjuvant Properties of Thermal Component of Hyperthermia Enhanced Transdermal Immunization: Effect on Dendritic Cells

Hyperthermia enhanced transdermal (HET) immunization is a novel needle free immunization strategy employing application of antigen along with mild local hyperthermia (42°C) to intact skin resulting in detectable antigen specific Ig in serum. In the present study, we investigated the adjuvant effect of thermal component of HET immunization in terms of maturation of dendritic cells and its implication on the quality of the immune outcome in terms of antibody production upon HET immunization with tetanus toxoid (TT). We have shown that in vitro hyperthermia exposure at 42°C for 30 minutes up regulates the surface expression of maturation markers on bone marrow derived DCs. This observation correlated in vivo with an increased and accelerated expression of maturation markers on DCs in the draining lymph node upon HET immunization in mice. This effect was found to be independent of the antigen delivered and depends only on the thermal component of HET immunization. In vitro hyperthermia also led to enhanced capacity to stimulate CD4+ T cells in allo MLR and promotes the secretion of IL-10 by BMDCs, suggesting a potential for Th2 skewing of T cell response. HET immunization also induced a systemic T cell response to TT, as suggested by proliferation of splenocytes from immunized animal upon in vitro stimulation by TT. Exposure to heat during primary immunization led to generation of mainly IgG class of antibodies upon boosting, similar to the use of conventional alum adjuvant, thus highlighting the adjuvant potential of heat during HET immunization. Lastly, we have shown that mice immunized by tetanus toxoid using HET route exhibited protection against challenge with a lethal dose of tetanus toxin. Thus, in addition to being a painless, needle free delivery system it also has an immune modulatory potential

Dipeptidyl Peptidase IV Inhibition Activates CREB and Improves Islet Vascularization through VEGF-A/VEGFR-2 Signaling Pathway

Substitution of pancreatic islets is a potential therapy to treat diabetes and it depends on reconstitution of islet’s capillary network. In this study, we addressed the question whether stabilization of Glucagon-Like-Peptide-1 (GLP-1) by inhibiting Dipeptidyl Peptidase-IV (DPP-IV) increases β-cell mass by modulating vascularization. Mouse or porcine donor islets were implanted under kidney capsule of diabetic mice treated with DPP-IV inhibitor sitagliptin. Grafts were analyzed for insulin production, β-cell proliferation and vascularization. In addition, the effect of sitagliptin on sprouting and Vascular Endothelial Growth Factor (VEGF)-A expression was examined ex vivo. The cAMP response element-binding (CREB) and VEGF-A/ Vascular Endothelial Growth Factor Receptor (VEGFR)-2 signaling pathway leading to islet vascularization was explored. Sitagliptin increased mean insulin content of islet grafts and area of insulin-positive tissue as well as β-cell proliferation. Interestingly, sitagliptin treatment also markedly increased endothelial cell proliferation, microvessel density and blood flow. Finally, GLP-1 (7-36) stimulated sprouting and VEGF expression, which was significantly enhanced by sitagliptin- mediated inhibition of DPP-IV. Our in vivo data demonstrate that sitagliptin treatment phosphorylated CREB and induced islet vascularization through VEGF-A/VEGFR-2 signaling pathway. This study paves a new pathway for improvement of islet transplantation in treating diabetes mellitus

Serum anti-TT Abs in mice primed by HET.

<p>Mice were immunized by soluble TT alone (i/m), TT with alum (i/m) or TT via HET. Unimmunized animal served as control. Booster of soluble TT (i/m) was given on Day 28 and mice were bled on week 5. Serum anti TT IgG1 (A1), IgG2a (A2) and IgG (A3) were estimated by ELISA. Recall response was evaluated by estimating the proliferative index by <i>in vitro</i> restimulation of splenocytes from immunized animals with TT antigen (B). Each dot represents individual animal and bars represent the geometric mean of each group.</p

Migration of DCs from epidermis upon local Hyperthermia.

<p>Mice were given local hyperthermia by placing hyperthermia patched on shaved skin. Epidermal sheets were prepared from control (A) and treated (B) mice 24 hours after treatments and stained for CD11c (PE). Nuclear staining was done using DAPI. Images were taken at 10× and are representative of three independent experiments.</p

Boosting upon HET immunization and toxin challenge.

<p>Mice were immunized via a single dose of HET patch or multiple doses of HET patch on day 0, 14, 28 and 42. The positive control was needle immunized with TT+alum on day 0 and 28. Toxin challenge was given one week after the completion of immunization schedule.</p

Migration of EpCAM+ epidermal DCs to the lymph node.

<p>Mice were immunized by TT, with and without HET. Unimmunized (PBS without HET) animals served as control. Cell suspension from draining and non-draining lymph nodes were prepared after 24 h and stained with Langerin (CD207)-PE and EpCAM-Alexa Flour 488 antibodies. Stained cells were analyzed by flow cytometry, Langerin+ cells were gated and the expression of EpCAM was analyzed. Number on the selected area indicates percent of EpCAM+ cells among Langerin+ cells in total lymph node cells. The bar graph shows average percentages of three experiments.</p

<i>In vitro</i> hyperthermia differentially regulates the expression of maturation markers on BMDCs.

<p>Immature or LPS (1 µg/ml; 6 hours) matured BMDCs were either maintained at 37°C or exposed to hyperthermia for 42°C for 30 minutes and analyzed by flow cytometry at indicated recovery periods, after staining with anti-CD80, anti-CD86, anti-MHC-II, anti-CD40, anti-CCR5 and anti-CCR7 mAbs. Results are shown as dot plots indicating the percentage of cells expressing the various markers and are representative of three independent experiments.</p

<i>In vitro</i> hyperthermia affects the secretion of IL-10 by BMDCs.

<p>Immature or LPS matured (1 µg/ml; 6 hour) BMDCs were cultured at either 37°C or 42°C for 30 minutes followed by recovery at 37°C. Supernatant were collected after 24 hours and cytokine levels were determined by sandwich ELISA. Results are plotted as Mean ±SD of a representative of three experiments.</p

A. <i>In vitro</i> hyperthermia enhances the T cell proliferative capacity of BMDCs in allo MLR.

<p>BMDCs from Balb/c mice (stimulator; 0.1 M) were treated at 37°C or 42°C for 30 minutes and co-cultured with either lymph node cells or purified CD 4+ T cells from spleen. B. Cytokines secreted in supernatant of allo MLR as estimated by ELISA.</p

Migration of LCs from epidermis upon local Hyperthermia vs. needle immunization.

<p>Mice (n = 3) were immunized with TT by either conventional i/m (NI) or HET with (H+TT) or without (H) antigen. Unimmunized animals served as control. Cell suspension from draining lymph nodes were prepared after 24 h and analyzed by flow cytometry after staining with Langerin (CD207)–PE antibody. Results are expressed as % of positive cells in total draining lymph node population.</p