Nasal Acai Polysaccharides Potentiate Innate Immunity to Protect against Pulmonary Francisella tularensis and Burkholderia pseudomallei Infections

Pulmonary Francisella tularensis and Burkholderia pseudomallei infections are highly lethal in untreated patients, and current antibiotic regimens are not always effective. Activating the innate immune system provides an alternative means of treating infection and can also complement antibiotic therapies. Several natural agonists were screened for their ability to enhance host resistance to infection, and polysaccharides derived from the Acai berry (Acai PS) were found to have potent abilities as an immunotherapeutic to treat F. tularensis and B. pseudomallei infections. In vitro, Acai PS impaired replication of Francisella in primary human macrophages co-cultured with autologous NK cells via augmentation of NK cell IFN-γ. Furthermore, Acai PS administered nasally before or after infection protected mice against type A F. tularensis aerosol challenge with survival rates up to 80%, and protection was still observed, albeit reduced, when mice were treated two days post-infection. Nasal Acai PS administration augmented intracellular expression of IFN-γ by NK cells in the lungs of F. tularensis-infected mice, and neutralization of IFN-γ ablated the protective effect of Acai PS. Likewise, nasal Acai PS treatment conferred protection against pulmonary infection with B. pseudomallei strain 1026b. Acai PS dramatically reduced the replication of B. pseudomallei in the lung and blocked bacterial dissemination to the spleen and liver. Nasal administration of Acai PS enhanced IFN-γ responses by NK and γδ T cells in the lungs, while neutralization of IFN-γ totally abrogated the protective effect of Acai PS against pulmonary B. pseudomallei infection. Collectively, these results demonstrate Acai PS is a potent innate immune agonist that can resolve F. tularensis and B. pseudomallei infections, suggesting this innate immune agonist has broad-spectrum activity against virulent intracellular pathogens

Acai PS requires IFN-γ and NK cells for optimum protection against <i>B. pseudomallei</i> infection.

<p>Two days prior to infection, C57BL/6 mice received rat IgG, anti-IFN-γ, or anti-NK1.1 mAb. Mice were treated i.n. with PBS (n = 5–10/group) or 1 mg of Acai PS (n = 10/group) one day prior to i.n. infection with 1×10⁴ CFUs of <i>B. pseudomallei</i> 1026b. Survival was monitored over time. *P<0.05 as compared to animals receiving PBS and IgG. ∧P<0.05 as compared to animals receiving Acai PS and anti-IFN-γ.</p

Acai PS induces TNF-α and NO in type A <i>F. tularensis</i>-infected RAW264.7 cells, but not in murine BMDMs.

<p>Cells were treated and infected as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002587#ppat-1002587-t001" target="_blank">Table 1</a>. 3/wells treatment at 20 hr post-infection shown; standard deviation in parentheses; results are representative of two independent experiments.</p>a<p>Mean NO (µM) or</p>b<p>TNF-α (ng/ml) production.</p>*<p>p<0.05 as compared to the same cell type not treated with Acai PS.</p>∧<p>p<0.05 as compared to the same cell type, with the same Acai treatment, treated with L-NMA at 20 hr post-infection. ND = not done.</p

Acai PS enhances the clearance of type A <i>F. tularensis</i> from RAW264.7 cells, but not murine BMDMs via NO.

<p>Cells were treated with Acai PS 16 hr prior to infection <i>F. tularensis</i> SchuS4 (MOI∼30), some wells were also pre-treated with 400 µM L-NMA.</p>a<p>Log₁₀ CFU/well from three wells/treatment shown; standard deviation in parentheses; results are representative of two independent experiments.</p>*<p>p<0.05 as compared to the same cell type not treated with Acai PS at the same time point.</p>∧<p>p<0.05 as compared to the same cell type, with the same Acai treatment, treated with L-NMA at 20 hr post-infection. ND = Not done.</p

Nasal administration of Acai PS confers protection against pulmonary <i>B. pseudomallei</i> infection.

<p>Female C57BL/6 mice (n = 5/group) were treated i.n. with PBS or with 100–1000 µg of Acai PS one day prior to, or immediately after, intranasal infection with 3×10³ CFUs of <i>B. pseudomallei</i> 1026b. A) Body weights and B) clinical scores were recorded daily, and C) on day 3, CFU determinations were performed in the lungs, spleens, and livers. Error bars depict SEM. *P<0.05 as compared to PBS group. **** indicates that *P<0.05 for all Acai PS-treated groups in relation to PBS-treated group at this time point. Data depicted in A–B) are representative of two independent experiments. The dashed line in C) indicates the limit of bacterial CFU detection.</p

Acai PS enhances LVS clearance from in human primary macrophages and enhances NK cell IFN-γ mRNA.

<p>Human primary macrophages (1×10⁴cells/well, 3 wells/treatment) were derived from PBMCS and infected with LVS (MOI∼300). One day prior to macrophage infection, autologous NK cells were also isolated via magnetic sorting. Macrophages and NK cells were treated separately with varying amounts of Acai PS 16 h prior to macrophage infection. After infection of the macrophages, fresh media with or without Acai PS or fresh media containing NK cells (∼20 NK cells/macrophage) with or without Acai PS were then added to the macrophage containing wells. A) Twenty h after infection, NK cells (non-adherent) were removed, macrophages were lysed, and intracellular bacteria enumerated. Error bars represent standard error. *P<0.05, as compared to untreated macrophages. B) Total RNA was extracted from NK cells co-cultured with LVS-infected macrophages (with or without Acai PS), and RT-PCR was performed for β-actin (control) and IFN-γ, TNF-α, IL-17A, IL-21, granzyme B, perforin, and TRAIL. Results are representative of independent experiments from five different blood donors.</p

Acai PS enhances IFN-γ by innate leukocytes during pulmonary type A <i>F. tularensis</i> infection.

<p>C57BL/6 mice (n = 5/group) were treated i.n. with 1 mg of Acai PS one day prior to i.n. infection with 50 CFUs of <i>F. tularensis</i> SchuS4. Some mice were also depleted of IFN-γ two days prior to infection. A) Intracellular expression of IFN-γ was determined for lung NK cells by flow cytometry, and two days after infection, and B) lung and splenic bacterial burdens were determined. Data are representative of two independent experiments. Error bars depict SEM. *P<0.05 as compared to PBS-treated animals receiving the same antibody treatment.</p

Nasal administration of Acai PS confers prophylactic and therapeutic protection against pulmonary Type A <i>F. tularensis</i> infection.

<p>Female C57BL/6 mice (5/group) were treated with PBS or with 10, 100, 1000 µg of Acai PS by the intranasal (i.n.) route one day prior to aerosol infection with <i>F. tularensis</i> SchuS4. A) Mice were monitored for morbidity and mortality twice daily for a period of 14–28 days, at which time survivors were euthanized, and B) body weights were monitored. C) Female C57BL/6 mice (n = 15–20/group) were i.n. treated with PBS or with 100 µg of Acai PS immediately after, one day after, or two days after aerosol infection with <i>F. tularensis</i> SchuS4. Mice were monitored for morbidity and mortality. *P<0.05 as compared to PBS group. Error bars depict S.D. Data depicted in C) are pooled from two independent experiments.</p

Acai PS reduces type A <i>F. tularensis</i> from primary human macrophages co-cultured with NK cells via IFN-γ.

<p>A) and B) Primary human macrophages (10⁵/well, 3 wells/treatment) and NK cells were isolated and treated separately with 100 µg/ml Acai PS 16 h prior to infection with <i>F. tularensis</i> SchuS4 (MOI∼30). Neutralizing anti-IFN-γ mAb, and/or autologous NK cells (∼5 NK cells/macrophage) were also added to some wells. Either immediately after (0 hr) or twenty h after infection, NK cells (non-adherent) were removed and A) macrophages were lysed and intracellular bacteria enumerated. Error bars represent standard deviation. *P<0.05 as compared to untreated macrophages. B) ∧P<0.05 as compared to Acai PS stimulated co-cultures neutralized of IFN-γ. Results are representative of independent experiments from three different blood donors. Similar results were obtained in macrophages infected with LVS (data not shown). ND = not determined.</p