Retinoic acid and polyriboinosinic:polyribocytidylic acid stimulate robust anti-tetanus antibody production while differentially regulating type 1/type 2 cytokines and lymphocyte populations

Abstract

Retinoic acid (RA), a bioactive retinoid, and polyriboinosinic:polyribocytidylic acid (PIC) are known to promote immunity in vitamin A-deficient animals. In this study, we hypothesized that RA, PIC, and the combination can provide significant immunoadjuvant activity even in the vitamin A-adequate state. Six-week-old C57BL/6 mice were immunized with tetanus toxoid (TT) and treated with RA and/or PIC at priming in three independent studies of short and long duration. RA and PIC differentially regulated both primary and secondary anti-TT IgG isotypes, whereas the combination of RA + PIC stimulated the highest level of anti-TT IgG production and, concomitantly, a ratio of IgG1 to IgG2a similar to that of the control group. The regulation of Ab response was strongly associated with type 1/type 2 cytokine gene expression. Whereas RA reduced type 1 cytokines (IFN-gamma and IL-12), PIC enhanced both type 1 and type 2 cytokines (IL-4 and IL-12) and cytokine-related transcription factors. Despite the presence of PIC, the IL-4:IFN-gamma ratio was significantly elevated by RA. In addition, RA and/or PIC modulated NK/NKT cell populations and the level of expression of the costimulatory molecules CD80/CD86, evident 3 days after priming. Notably, the NKT:NK and CD80:CD86 ratios were correlated with the IL-4:IFN-gamma ratio, indicative of multiple converging modes of regulation. Overall, RA, PIC, and RA + PIC rapidly and differentially shaped the anti-tetanus Ig response. The robust, durable, and proportionate increase in all anti-TT IgG isotypes induced by RA + PIC suggests that this combination is promising as a means to enhance the Ab response to TT and similar vaccines.

Figure 1

RA and PIC synergistically enhance primary anti-TT IgG, but differentially regulate IgG isotypes. Six-week-old mice were immunized with TT ± RA and/or PIC, followed by an additional 10-day treatment with RA or oil (control). Plasma anti-TT IgM (a) and IgG (b), including IgG isotypes (c–f), were measured by ELISA on day 7 (IgM) and day 10 (IgGs) after priming. The Ab titers were calculated based on a standard curve, in which 1 U was defined as the dilution fold that gave 50% of OD_ma. Bars show mean ± SE, n = 12–16 mice/group. Different letters above bars within panels indicate significant differences (p < 0.05, a < b < c < d). Results of two-way ANOVA for each factor (RA, PIC, and interaction) are also shown in each panel.

Figure 2

RA and PIC differentially regulate mRNA levels of type 1/type 2 cytokines. Six-week-old mice were immunized with TT ± RA and/or PIC, followed by additional 10-day treatment with RA or oil. On day 11 after priming, the mice were rechallenged with TT ± RA and/or PIC. Spleens were collected 24 h later, and RNA was extracted. The mRNA levels of IL-4 (a), IL-10 (b), IL-12 (c), and IFN-γ (d) were quantified by ³³P-labeled PCR, normalized, and then shown as fold induction relative to control. The ratio of IL-4 mRNA to IFN-γ mRNA was calculated and shown in e. Bars shown are means ± SE, n = 12-16/group. Bands from two representative mice per group are shown for illustration below the group means. Different letters above bars within panels indicate significant differences (p < 0.05, a < b < c). Results of two-way ANOVA for each factor (RA, PIC, and interaction) are also shown in each panel.

Figure 3

RA and PIC significantly regulate mRNA levels of IL-4 and IFN-γ 3 days after priming. Six-week-old mice were immunized with TT ± RA and/or PIC, followed by additional 2-day treatment of RA or oil. Spleens were collected on day 3 after priming, and RNA was extracted. The mRNA levels of IL-4 (a) and IFN-γ (b) were quantified by ³³P-labeled PCR, normalized, and then shown as fold induction relative to control. The ratio of IL-4 mRNA to IFN-γ mRNA was calculated and shown in c. Bars shown are means ± SEM, n = 12–16/group. Bands from two representative mice per group are shown for illustration. Different letters above bars within panels indicate significant differences (p < 0.05, a < b < c). Results of two-way ANOVA for each factor (RA, PIC, and interaction) are also shown in each panel.

Figure 4

The ratio of IL4/IFN-γ is significantly associated with the ratio of NK/NKT cells, and the ratio of CD80/CD86 costimulatory molecules. Mice were treated as described in Table II and Fig. 3. The relationship between ratio of IL4/IFN-γ and ratio of NK/NKT cells (a) and ratio of CD80/CD86 (b) was assessed by simple regression analysis.

Figure 5

RA and/or PIC treatments given with the primary immunization enhance secondary anti-TT Ab IgG responses. Six-week-old mice were immunized with TT ± RA and/or PIC, followed by additional 6-day treatment of RA or oil. Five weeks later, the mice were reimmunized with TT only. Secondary anti-TT IgG (a) and IgG isotypes (b–e) in plasma were measured by ELISA 7 days after reimmunization. Different letters above bars within panels indicate significant differences (p < 0.05, a < b < c). Results of two-way ANOVA for each factor (RA, PIC, and interaction) are also shown in each panel.