The Effect Of Vitamin E Supplementation On Immune Response To Tetanus Toxoid Immunisation In Normal Human Volunteers And Mice

Abstract

Vitamin E is a major lipid-soluble anti-oxidant, which scavenges free radicals in biological membrane and protects the cellular structure against oxidative damage. Several studies have shown that vitamin E, both tocopherol and tocotrienol supplementation induces a favourable effect on animal and human immune system and has been implicated in the reduced risk for several immune and inflammatory diseases. In this study, We first examined the immunoinodulatory effects of orally administered tocotrienols and tocopherols in the mouse model upon an immunogenic challenge, and then we investigated the effect of tocotrienol rich fraction (TRF) supplementation on immune modulation in normal healthy volunteers Whose immune system was challenged With a booster tetanus toxoid (TT) vaccine. In the mouse model, 20 female BALB/c mice were divided into four groups of five mice each and were orally gavaged with on-tocopherol (α-T), TRF and δ-tocotiienol (δ-T3) respectively while the control animals were not given any supplements. Two Weeks after the supplementation, mice from all four groups received a single dose of TT vaccine (4 flocculation units (Lf)) injected intramuscularly in the left hind leg quadriceps of the mice. This was followed by a second and third booster doses at intervals of two-weeks. Sera from mice were collected via retro-orbital bleeding on day 0, 28 and 56. All experimental animals were orally-gavaged with the respective supplements for two months until they were sacrificed on day 56. Blood, spleen and adipose tissue were harvested from the sacrificed mice. At autopsy, splenocytes from control and experimental mice were isolated and cultured in vitro in the presence of Concanavalin-A (ConA), TT or Lipopolysaccharide (LPS). The cytokine produced by the cultured splenocytes and the amount of anti-TT antibodies generated were quantified using the enzyme linked immunosorbent assay (ELISA) while the proliferation of splenic lymphocytes was measured using the methyl thiazole tetrazolium (MTT) assay. Results from the animal study demonstrate that vitamin E-treated mice showed a significant (P < 0. 05) increase in splenocytes proliferation as compared to untreated animals after TT vaccination. However, no differences in splenocytes proliferation were observed among groups treated with the different types of vitamin E. Interferon-gamma (IFN-ƴ) and Interleukin-4 (IL-4) productions by Con A or TT-stimulated splenocytes also significantly (P < 0. 05) increased in the vitamin E supplemented animals compared to control animals. The level of IFN-ƴ was highest in δ-T3 supplemented animals and this was followed by TRF and α-T fed mice. However, there was no significant (P > 0.05) difference observed in the levels of IL-4 produced within the vitamin E-treated groups. Secretion of Tumour Necrosis Factor-alpha (INF-α) was suppressed in the vitamin E-treated group compared to the untreated animals. The production of anti-TT antibodies was significantly (P < 0.05) augmented in δ-T3 and TRF supplemented animals compared to α-T fed mice. In the human study, 100 healthy female volunteers aged between 18-25 years were recruited to participate ill a randomised, placebo-controlled, double- blinded clinical trial. The volunteers were given either 400 mg vitamin E (TRF) or placebo daily for 2 months. On day 28, all volunteers were vaccinated with the TT vaccine (20 Lt) intramuscularly. Blood samples were obtained from all volunteers on day 0, 28, and 56 for flow cytometry analysis, blood leucocyte culture and analysis of plasma vitamin E as well as anti-TT antibody levels. Plasma vitamin E was determined by high performance liquid chromatography (HPLC) while plasma levels of anti-TT antibody i.e. Immunoglobulin G (IgG) was quantified by ELISA. The amount of IFN-ƴ, IL-4, Interleukin-10 (IL-10) and Interleukin-6 (IL-6) produced by mitogen or TT-stimulated leucocyte culture was determined using ELISA. The results from the clinical trial showed that TRF supplementation significantly increased total vitamin E levels ill the plasma of the TRF supplemented group as compared to placebo, indicating overall compliance. Volunteers supplemented with TRF showed a significant (P < 0.05) enhanced production of IFN-ƴ and IL-4 by the mitogen or TT-stimulated leucocytes as compared to control group. However, no differences were observed in the levels of IL-10 produced between the TRF and placebo supplemented groups. Volunteers supplemented with TRF produced significantly (P < 0.05) lower amounts of IL-6 as compared to the control group. Anti-TT IgG production was also significantly (P < 0.05) augmented in the TRF supplemented group compared to placebo group. In contrast, no significant changes were obse1ved in the proportion of total T-lymphocytes, B-lymphocytes as well as NK cells between volunteers who received TRF and placebo supplementation. The results from the clinical trial concur with the findings form the animal study. These results demonstrate the efficacy of TRF and 8-T3 for modulating an immune response towards a mixed TH1/TH2 type upon an immunogenic challenge. Therefore; we conclude that TRF and 8-T3 from palm oil have immunostimulatory effects and potential clinical benefits to enhance immune response to vaccines and probably, infectious agents.