Polysaccharides isolated from Açaí fruit induce innate immune responses

Abstract

The Açaí (Acai) fruit is a popular nutritional supplement that purportedly enhances immune system function. These anecdotal claims are supported by limited studies describing immune responses to the Acai polyphenol fraction. Previously, we characterized γδ T cell responses to both polyphenol and polysaccharide fractions from several plant-derived nutritional supplements. Similar polyphenol and polysaccharide fractions are found in Acai fruit. Thus, we hypothesized that one or both of these fractions could activate γδ T cells. Contrary to previous reports, we did not identify agonist activity in the polyphenol fraction; however, the Acai polysaccharide fraction induced robust γδ T cell stimulatory activity in human, mouse, and bovine PBMC cultures. To characterize the immune response to Acai polysaccharides, we fractionated the crude polysaccharide preparation and tested these fractions for activity in human PBMC cultures. The largest Acai polysaccharides were the most active in vitro as indicated by activation of myeloid and γδ T cells. When delivered in vivo, Acai polysaccharide induced myeloid cell recruitment and IL-12 production. These results define innate immune responses induced by the polysaccharide component of Acai and have implications for the treatment of asthma and infectious disease.

Figure 1. γδ T cell stimulatory activity in Acai is concentrated in the polysaccharide fraction and effective in all species tested.

A) Aqueous extract of Acai was separated via EtOH precipitation or Kupchan fractionation. The resulting fractions were lyophilized, re-suspended in water, and tested in bovine PBMC culture for γδ T cell agonist activity. Data represent mean and SD from triplicate cultures from the same calf. EtOH precipitant (ppt.) responses are representative of cultures from three calves and three separate preparations. B) Human cells were cultured for 48 h with Acai-PS or medium prior to analysis for cell activation (CD69 expression) using flow cytometry. Values represent the average response of duplicate cultures from a single donor. Data are representative of two experiments. C) CFSE-labeled TCRα^−/− splenocytes were cultured in X-VIVO with PBS, Yam-PS (9 µg/mL), or Acai-PS (10 µg/mL) for 24 h, then medium was replaced with fresh medium containing IL-2 and cultured for an additional 72 h. Percent cell proliferation was determined as the percent of γδ T cells (lymphocyte, GL3⁺ gates) or others (lymphocyte, GL3- gates) divided at least once and are representative of two Acai-PS preparations.

Figure 2. Acai fruit does not contain polyphenol-based γδ T cell agonists.

A) PVPP-extracted Acai polyphenols were cultured with human PBMCs to detect γδ T cell activation. As a control, APP was used to induce polyphenol-based γδ T cell activation. B) Acai-PS was treated with PVPP to remove polyphenols and the resulting preparation (Acai–PS^PR) or untreated Acai-PS was cultured with human PBMCs. γδ T cell activation from the subsequent cultures was measured by FACS as induced CD69 expression. Results are from three individual donors. Error bars represent SD. Experiments were performed independently with respect to donor, experiment date, and Acai-PS^PR extraction.

Figure 3. Chromatographic characterization and fractionation of Acai polysaccharides.

Water extract of Acai was prepared and separated on DEAE-cellulose column (Acai-PS) and quantified using multiple methods: A) Acai-PS fractionation by gel chromatography on Sepharose-6B column. Three polysaccharide fractions (designated Acai-1, Acai-2, and Acai-3) were selected based on total carbohydrate and diene conjugate contents. B) High pressure gel filtration chromatography elution profile of Acai-PS with a refractive index detector. C) Synchronous fluorescence spectra of polysaccharides isolated from Acai-PS{500 µg/ml of each polysaccharide fraction in NaHCO₃ buffer (pH 8.5)}.

Figure 4. Acai fractions induce cell activation, as well as, ROS and cytokine production.

A) PBMCs were collected from three donors and cultured with indicated agonists at various concentrations (x axis). Cultures were performed in triplicate. Data represent the mean fold increase (CD69 mean fluorescence) versus medium for each agonist/concentration value. Error bars represent normalized SD. B) PBMCs were incubated with polysaccharide fractions (150 µg/mL) and ROS production was measured over 300 min. C) ROS production from PBMCs was measured as a function of dose. PBMCs were incubated with the indicated concentrations of polysaccharide fractions, LPS, or vehicle only for 24 h. ROS production was then measured for 3 h from triplicate samples. Data represent the mean ± SD total luminescence over 3 h. Values are from one experiment, representative of three independent experiments. D). An ELISA was used to measure cytokine production by human PBMCs treated with 50 µg Acai-PS. Values represent the mean fold increase versus medium control cultures from triplicate wells. Error bars represent SD. Cultures were from one subject. Production of IL-1β, IL-6, GM-CSF, and TNF-α, and as well as limited IL-8 was confirmed in PBMCs from at least one additional donor using different ELISA reagents.

Figure 5. Effect of Acai polysaccharide on TNF-α and IL-6 production in MonoMac-6 and human PBMCs.

Human PBMCs or MonoMac-6 macrophages were incubated for 24 h with the indicated concentrations of polysaccharide fractions Acai-1, Acai-1 pretreated with endotoxin-removing gel (Acai-1^ER), Acai-2, Acai-3, or 200 ng/mL LPS. Cell-free supernatants were collected, and extracellular TNF-α and IL-6 were quantified by ELISA. Values represent the mean ± SD of triplicate samples from one experiment, which is representative of at least three independent experiments.

Figure 6. Acai polysaccharides induce MyD88-independent neutrophil influx to the peritoneum.

A) BALB/c mice were injected intraperitoneally with saline, Acai, or Yam-1. After 4 h, mice were euthanized, peritoneal cells collected, and total neutrophil counts measured by flow cytometry. Data represent the average total cell count from a minimum of four mice per treatment group and error bars represent the SEM. B) C57BL/6 or MyD88^−/− mice of mixed ages (12–23 weeks) and sexes were injected i.p with Acai-PS (400 µg) or saline and neutrophil flux was measured as in A) without the use of FACS beads to estimate total cell counts. The data are representative of the mean percentage of neutrophils in the wash ± SD from a single experiment with 3–4 mice/group. p-values (Student's T test) for both figures are represented as: *<0.05, **<0.01, ***<0.005, ****<0.001.

Figure 7. Intratracheal (i.t.) treatment with Acai-PS activates lung myeloid cells and induces IL-12 production in mice.

BALB/c mice (n = 3) were treated i.t. with vehicle (dH₂0) or 500 µg Acai-PS in a volume of 100 µL. BALF and lung cells were isolated 24 h post-treatment. Cells were stained with antibodies for CD11b and CD11c and analyzed via flow cytometry for myeloid cell activation/recruitment. A) BALF alveolar macrophages were gated (autofluorescent/CD11c⁺; ovals) and activation was measured as an increase in mean CD11c-associated fluorescence within this gate. B) Cells in the lung interstitium were collected via collagenase extraction and similarly analyzed by FACS for myeloid cell recruitment/activation. The percentage of myeloid cells (rectangle gate) in relation to total live leukocytes was compared between Acai-PS and vehicle treated mice. Data from A) and B) are representative of three similar experiments and were repeated in C57BL/6 (3 experiments) and C3H/HeOuJ (2 experiments). C) BALF was collected from BALB/c mice provided varying dosages of Acai-PS i.t. Cells were removed by centrifugation, and IL-12(p70) concentration was determined in the supernatant fluid by cytokine ELISA.