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Comparative Study
. 2009 Oct;9(11):1313-22.
doi: 10.1016/j.intimp.2009.07.015. Epub 2009 Aug 9.

Polysaccharides derived from Yamoa (Funtumia elastica) prime gammadelta T cells in vitro and enhance innate immune responses in vivo

Jill C Graff  1 , Emily M Kimmel, Brett Freedman, Igor A Schepetkin, Jeff Holderness, Mark T Quinn, Mark A Jutila, Jodi F Hedges
Affiliations
Comparative Study

Polysaccharides derived from Yamoa (Funtumia elastica) prime gammadelta T cells in vitro and enhance innate immune responses in vivo

Jill C Graff et al. Int Immunopharmacol. 2009 Oct.

Abstract

Yamoa (ground bark of Funtumia elastica tree) is marketed and sold as a dietary supplement with anecdotal therapeutic effects in the treatment of asthma and hay fever. We determined that Yamoa and Yamoa-derived polysaccharides affected innate immunity, in part, by priming gammadelta T cells. Gene expression patterns in purified bovine gammadelta T cells and monocytes induced by Yamoa were similar to those induced by ultrapure lipopolysaccharide (uLPS). In the presence of accessory cells, Yamoa had priming effects that were similar to those of LPS on bovine and murine gammadelta T cells, but much more potent than LPS on human gammadelta T cells. The bioactive component of Yamoa was delineated to a complex polysaccharide fraction (Yam-I). Intraperitoneal injection of Yamoa and Yam-I in mice induced rapid increases in peritoneal neutrophils directed by changes in chemokine expression. In support of a unique agonist found in Yam-I, similar peritonitis responses were also observed in TLR4- and MyD88-deficient mice. Therapeutic treatment with Yam-I resulted in decreased bacterial counts in feces from mice with Salmonella enterica serotype typhimurium (ST)-induced enterocolitis. This characterization of the immune stimulatory properties of polysaccharides derived from Yamoa suggests mechanisms for the anecdotal positive effects of its ingestion and that these polysaccharides show potential for application in innate protection from disease.

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Figures

Figure 1
Figure 1
Microarray analysis of gene changes in bovine γδ T cells induced by Yamoa™, ultrapure LPS and PBS. Lines in Figure 2 represent the average relative gene expression levels of genes that changed at least 1.5-fold in Yamoa™- stimulated (right) bovine γδ T cells. The average was based on expression in treated cells from 3 different calves and compared to their expression after uLPS (middle) and PBS (left) treatment. Lines are shaded based on expression levels induced by Yamoa™ (darker shades represent greater change).
Figure 2
Figure 2
γδ T cells are primed by agonists in Yamoa™. A. Human cells were stained with CFSE, cultured with 20 ng/ml IL-15 and treated with Yamoa ™, ultrapure LPS or media only control. After 5 days, the cells were analyzed by two-color flow cytometry. Yamoa™ was effective at priming human γδ T cells (top panel), and not other lymphocytes (lower panel), to proliferate as evidenced by a reduction in CFSE staining on the x-axis at a wide range of effective concentrations, whereas LPS had very little effect. Representative of at least 3 experiments with different donors. B. Mouse spleen cells were stained with CFSE, and treated with Yamoa ™, Salmonella LPS, or media only control for 48 hours then IL-2 (1 ng/ml) was added for 72 hours and the cells were analyzed by flow cytometry. Yamoa™ was effective at priming mouse γδ T cells (front panel), to proliferate as evidenced by a reduction in CFSE staining on the x-axis, at a wide range of effective concentrations, similar to LPS. Percentage values represent live lymphocytes having divided at least once. Representative of at least 3 experiments.
Figure 3
Figure 3
Isolation and characterization of Yamoa™-derived polysaccharides. A. Beginning with the NaCl buffer elution fraction from the ion-exchange DEAE-cellulose column (Yam-I), three major fractions were eluted from a Sepharose-6B column by SEC and designated as Yam-I-1, −2 and −3. These fractions were analyzed by HPLC and approximate molecular weights were determined as follows: Yam-I-1, ~404,000Da, Yam-I-2,~35,000Da, Yam-I-3~5,900Da. B. Each resulting fraction was applied to bovine PBMC cultures for 24 hours. Cells were stained and analyzed by flow cytometry for IL2Rα surface expression.
Figure 4
Figure 4
Neutrophil trafficking induced by Yamoa™-derived polysaccharides. A. BALB/c (WT) and CXCR2-deficient mice (at least 3 per group) were treated by intraperitoneal injection with PBS, 250 µg of Yamoa™ or 30 µg Yam-I per mouse, and the number of neutrophils that accumulated in the peritoneum (top panel) and blood (bottom panel) was measured after 3 hours. Error bar denotes SEM, *p<0.01. B. Yam-I contains a concentrated source of the active component of Yamoa™. Yamoa™ (250 µg) and lower concentrations of Yam-I were tested for induction of peritonitis in mice (at least 3 per group) by analysis of neutrophil recruitment (top panel, error bar denotes SEM) and serum CXCL1/KC by ELISA (error bar denotes SD), 3 hours after intraperitoneal injection.
Figure 5
Figure 5
Responses of TLR-deficient mice to Yamoa™-derived polysaccharides. A. In a peritonitis assay, C3H:HeJ (TLR4-deficient) and C3H:HeOuJ (TLR4-competent) mice (at least 3 per group) were injected i.p. with Yamoa™ (250 µg) and polysaccharide fractions derived from it (30, 3 and 0.3 µg Yam-I, or 30 µg Yam-II or YamPS). After 3 hours, neutrophil recruitment into the peritoneum was measured by flow cytometry. Error bars represent SD. B. Yam-I was treated with two rounds of treatment with PMB beads, 2.5 and 25 µg doses of pre- and post- treatment Yam-I were injected into C3H:HeJ and C3H:HeOuJ mice (5 mice per group), and peritonitis was similarly measured. Thin bar indicates mean, thick error bars represent SEM. C. Spleen cells from C3H:HeJ mice were treated with the 3 sub-fractions of Yam-I in a priming assay, and proliferation was measured using CFSE staining in flow cytometry. Histograms show cells gated based on forward and side scatter (lymphocytes) and γδ T cell staining. D. Comparison of the percent of γδ T cells that proliferated in a priming assays described for 5C using spleen cells from C3H:HeOuJ or C3H:HeJ mice (at least 3 per group). Error bars represent SD. E. The fold change in % neutrophil in MyD88-deficient mice (on a C57/BL6 background) was compared to C57/BL6 mice in the peritonitis assay (as in 5A) after injection of 5 µg Yam-I or sterile saline. (3 mice per treatment, error bar denotes SD, *p<0.01).
Figure 6
Figure 6
Therapeutic treatment with Yam-I of mice with ST-induced enterocolitis reduced ST CFU counts in the feces. BALB/c mice (3–6 mice per treatment group) were infected with ST (1×106 CFU/mouse) and 8 hours later injected with the indicated doses of Yam-I, or vehicle only (PBS). At 24 hours after infection, fecal pellets were collected for assessment of bacterial load on LB agar containing 50 µg/ml streptomycin (CFU/g feces). Three separate experiments are shown, thin bar indicates mean, thick error bars represent SEM.
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