Nanoemulsion Adjuvant Augments Retinaldehyde Dehydrogenase Activity in Dendritic Cells via MyD88 Pathway

Abstract

Mucosal surfaces are the primary point of entry for many infectious agents and mucosal immune responses serve as the primary defense to these pathogens. In order to mount an effective mucosal immune response, it is important to induce T cell homing to mucosal surfaces. Conventional vaccine adjuvants induce strong systemic immunity but often fail to produce mucosal immunity. We have developed an oil-in-water nanoemulsion (NE) adjuvant that provides mucosal immunity and efficient protection against mucosal pathogens when administered as part of an intranasal vaccine. In the present study, we demonstrate that intranasal immunization with NE indirectly activates the retinaldehyde dehydrogenase (RALDH) activity in dendritic cells through epithelial cell activity leading to SIgA as well as potent cellular responses and expression of α4β7 and CCR9 gut homing receptors on T cells. Confirming these findings, ex-vivo stimulation of splenocytes from NE nasally immunized animals showed increase in Th1/Th17 cytokines while suppressing Th2 responses. In examining mechanisms underlying this activation NE activated RALDH via MyD88 dependent pathways in DCs but did not activate the retinoic acid receptor directly. These results suggest that RALDH immune activities can be achieved by epithelial activation without direct RAR activation, which has significant implications for understanding mucosal immunity and the design of mucosal vaccines.

Keywords: MyD88 pathway; dendritic cells; epithelial cells; gut homing; nanoemulsion; retinaldehyde dehydrogenase.

Figure 1

Nanoemulsion adjuvant treated epithelial cells indirectly activate retinaldehyde dehydrogenase activity in co-cultured dendritic cells. (A) Monolayers of epithelial cells were incubated for 6 h with the indicated treatment conditions and washed. BMDCs were then added at 1 × 10⁶ per well-seeding density. After 24 h incubation, co-cultures were stained for CD11c and aldefluor assay to examine RALDH activity. (B) In second set of experiment, BMDCs (1 × 10⁶) were directly treated with NE. After 6 h incubation, NE was washed off from the culture. RALDH activity was measured in BMDCs after 24 h. Representative mean ± s.e.m of two independent experiments. Statistically significant differences using One-way ANOVA (Kruskal-Wallis multiple comparisons test) ^*p < 0.05 compared to untreated control group.

Figure 2

Nanoemulsion adjuvant increases expression of gut homing receptors on naïve T cells in vitro. NE pretreated epithelial cells were co-cultured with BMDCs for 16 h. Naïve T cells were added to co-cultures to study expression of α4 (A), β7 (B), and CCR9 (C). mRNA was isolated from primary T cells after 48, 72, and 96 h in co-cultures. Gene expression was normalized to β-actin and fold change was calculated over the untreated control. Expression of gut homing receptors on naïve T cells was also analyzed by flow cytometry after 5 days in co-culture. Samples were stained with anti-CD3 and anti-α4β7 (D) or anti-CCR9 (E) antibodies. Representative mean ± s.e.m from two independent experiments, each performed in triplicate (D,E). Statistically significant differences using Two-way-ANOVA (Tukey's multiple comparisons test) and One-way ANOVA (Kruskal-Wallis multiple comparisons test)^§p < 0.0001 #p < 0.0005, ^**p < 0.005, ^*p < 0.05 compared to control group.

Figure 3

Immunization with nanoemulsion vaccine increases retinaldehyde dehydrogenase activity in dendritic cells and induces expression of gut homing receptors in lymph node cells. (a) Intra-nasal (i.n.) immunization schedule for in vivo experiments. Kinetics of RALDH activity on CD11c+CD103+ cells and expression of gut homing marker's expression in ex vivo stimulated cells in CLNs on day 3 (b) and in MLNs on day 10 (c) in immunized animals. Data is representative mean ± s.e.m. of two independent experiments. Statistically significant differences using One-way ANOVA (Kruskal-Wallis multiple comparisons test) and Two-way-ANOVA (Tukey's multiple comparisons test) #p < 0.0005, ^*p < 0.05 compared to PBS OVA immunized animals.

Figure 4

Mucosal immunization with nanoemulsion vaccine induces OVA-specific sIgA and cytokine response. (A) mucosal IgA levels determined by ELISA at the time of euthanasia. (B–G) Splenocytes isolated from immunized animals were stimulated ex vivo with 200 μl OVA (20 μg/mL). mRNA was collected after 24 h in culture. Gene expression was normalized to β-actin and fold change was calculated over unstimulated cells for every immunization group. Data is representative of two experiments mean ± s.e.m. Statistically significant differences using One-way ANOVA (Kruskal-Wallis multiple comparisons test) ^*p < 0.05, ^§p < 0.0001 compared to PBS OVA immunized animals.

Figure 5

Nanoemulsion induces retinaldehyde dehydrogenase activity independent of retinoic acid, TLR 2 or TLR 4 signaling in co-cultured dendritic cells. NE treated epithelial cells were co-cultured with BMDCs isolated from (A) CD11c-RAR^−/−, (B) TLR2^−/−, and (C) TLR4^−/− animals. Co-cultures were washed and stained for CD11c and RALDH activity with Aldefluor kit. Representative mean ± s.e.m of two independent experiments performed in technical triplicates.

Figure 6

Nanoemulsion mediates retinaldehyde dehydrogenase activity in dendritic cells via MyD88 pathway. Nanoemulsion treated epithelial cells were co-cultured with BMDCs from WT and MyD88^−/− animals. RALDH activity was measured in CD11c cells with aldefluor kit after 24 h. (A) Representative data of experiments performed in technical duplicates. (B–G) Co-cultures were harvested after 16 h and CD11c cells were separated using CD11c positive isolation kit (Stem cell technologies). Gene expression was analyzed by qRT-PCR. β-actin was used as a housekeeping gene and fold change was calculated over the WT untreated control. Data presented here is mean ± s.e.m. pool of two independent experiments done in technical duplicates. Statistically significant differences using One-way ANOVA (Kruskal-Wallis multiple comparisons test) ^*p < 0.05 compared to untreated control group.