Nasal Acai Polysaccharides Potentiate Innate Immunity
to Protect against Pulmonary Francisella tularensis and
Burkholderia pseudomallei Infections
Jerod A. Skyberg1*, MaryClare F. Rollins1, Jeff S. Holderness1, Nicole L. Marlenee2,3, Igor A. Schepetkin1,
Andrew Goodyear2,3, Steven W. Dow2,3, Mark A. Jutila1, David W. Pascual1
1Department of Immunology and Infectious Diseases, Montana State University, Bozeman, Montana, United States of America, 2Rocky Mountain Regional Center for
Excellence in Bioterrorism and Emerging Infectious Diseases, Colorado State University, Fort Collins, Colorado, United States of America, 3Department of Microbiology,
Immunology, and Pathology, Colorado State University, Fort Collins, Colorado, United States of America
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-c. 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-c by NK cells in the lungs of F. tularensis-infected mice, and
neutralization of IFN-c 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-c responses by NK and cd T cells in the lungs, while neutralization of IFN-c 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.
Citation: Skyberg JA, Rollins MF, Holderness JS, Marlenee NL, Schepetkin IA, et al. (2012) Nasal Acai Polysaccharides Potentiate Innate Immunity to Protect
against Pulmonary Francisella tularensis and Burkholderia pseudomallei Infections. PLoS Pathog 8(3): e1002587. doi:10.1371/journal.ppat.1002587
Editor: Frank R. DeLeo, National Institute of Allergy and Infectious Diseases, National Institutes of Health, United States of America
Received July 28, 2011; Accepted January 30, 2012; Published March 15, 2012
Copyright: ? 2012 Skyberg et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work is supported by the Rocky Mountain Regional Center for Excellence in Bioterrorism and Emerging Infectious Diseases (NIH U54AI-65357),
along with NIH P01AT-04986, and NIH Contract HHSN2662004000009/N01-AI40009. The funders had no role in study design, data collection and analysis, decision
to publish, or preparation of the manuscript.
Competing Interests: I have read the Journal’s policy and we have the following conflicts: MAJ holds shares in LigoCyte Pharmaceuticals, which together with
Montana State University, held a National Institutes of Health contract that partially funded this work. LigoCyte Pharmaceuticals had no monetary or intellectual
input toward the research nor the interpretation of this report. A provisional patent application of the therapeutic use of the Acai-derived polysaccharides has
been submitted on behalf of JAS, JSH, IAS, MAJ, and DWP. Acai extract is contained in many commercial products. This does not alter the authors’ adherence to all
the journal policies on sharing data and materials. All other authors have no financial conflict of interest.
* E-mail: firstname.lastname@example.org
Francisella tularensis is a highly infectious, Gram-negative faculta-
tive intracellular bacterium that causes the zoonotic infection
tularemia.F. tularensis infections canoccurviainsectbites,cutaneous
contact with infected animal carcasses, ingestion of contaminated
food and water, or inhalation of viable organisms . The type and
severity of tularemia depends on the strain, dose, and route of
infection . F. tularensis subspecies tularensis (type A) and holarctica
(type B) cause the majority of human cases, with subspecies tularensis
being more virulent . Cutaneous tularemia is the most common
form of human disease, but is rarely fatal . Inhalation of F.
tularensis results in respiratory or pneumonic tularemia and is most
common in people in endemic areas who perform tasks that
predispose them to infectious aerosols . Untreated respiratory
forms of disease have mortality rates of .30% , while antibiotic
treatment can decrease this number to approximately 2% .
Pulmonary tularemia can present from a mild pneumonia to an
acute infection with high fever, malaise, chills, cough, delirium, and
pulse-temperature dissociation . The high infectivity (10–50
microorganisms)  and mortality of F. tularensis infections have led
to the weaponization of the organism, including the introduction of
antibiotic resistance, by several nations . Due to these concerns,
F. tularensis has been determined to be a Category A Bioterrorism
agent by CDC. No vaccines are currently licensed to prevent
tularemia. Although a live vaccine strain (LVS) derived from F.
tularensis subspecies holarctica was created over 50 years ago,
questions remain regarding its efficacy and possible reversion to
virulence,anditisnot licensedforhumanuse . LVSisattenuated
in humans, but remains virulent for mice, although it is not as
virulent as wild-type A and B strains. As LVS causes a disease in
mice that mimics tularemia in humans, it has been studied
PLoS Pathogens | www.plospathogens.org1March 2012 | Volume 8 | Issue 3 | e1002587
extensively as a model intracellular pathogen  and is utilized here
as model to assay the efficacy of agonists to enhance resistance to
Francisella in vitro, while our in vivo studies employ the fully virulent
SchuS4 strain of type A F. tularensis.
Burkholderia pseudomallei and B. mallei are gram-negative faculta-
tive intracellular bacterial pathogens. B. pseudomallei is the etiologic
agent of melioidosis and is endemic in parts of southeast Asia and
northern Australia.Theclinical manifestations ofmelioidosisare
protean and may vary from acute sepsis to chronic focal pathology
and latent infection, which can reactivate decades later from an, as
yet, unknown tissue reservoir . Melioidosis can also mimic other
infections such as glanders, typhoid fever, bacterial sepsis, and TB,
depending on whether the disease is acute or chronic [8–10].
Community-acquired infection with melioidosis is mostlikely dueto
exposure to bacteria in soil or water through cuts or skin abrasions
or via inhalation or ingestion . No licensed prophylactic or
therapeutic vaccine exists for Burkholderia infections, and B.
pseudomallei is intrinsically resistant to a wide range of antimicrobial
agents. In addition, prolonged antibiotic therapy (up to 6 months) is
required to treat Burkholderia infections, and 10–15%of patients may
relapse when antibiotic therapy is withdrawn [8,11].
Due to the lack of efficacious vaccines and concerns about F.
tularensis acquiring resistance to antibiotics via natural or illicit
means and the intrinsic antimicrobial resistance of B. pseudomallei,
we hypothesized that alternative immune or natural therapeutic-
based intervention strategies could prove beneficial to augment
current treatment regimens. Activation of the innate immune
system can enhance resistance to a variety of bacterial and viral
infections [6,11–14]. Immunotherapeutics may be particularly
beneficial against diseases caused by intracellular pathogens since
the antibiotics often recommended for treatment of these diseases,
such as gentamicin, poorly penetrate host cells and therefore fail to
reach the etiological agent of disease . In situations where the
etiological agent of disease is unknown, stimulation of innate
immunity may also be useful since these immune responses are
often capable of providing protection against a broad range of
pathogens [6,14]. To achieve this goal several natural agonists,
including apple polyphenols (APP), amphotericin B (AmpB),
securinine, Yamoa PS, and Acai PS were tested for their ability
to enhance immunity to F. tularensis since each of these agonists has
been previously shown to exhibit proinflammatory properties
Herein, we showed polysaccharides isolated from the Acai berry
(Acai PS) enhanced clearance of F. tularensis from human
macrophages upon co-culture with autologous natural killer
(NK) cells. Mucosal administration of Acai PS also conferred
both prophylactic and therapeutic protection against pulmonary F.
tularensis and B. pseudomallei infections. The immunological basis for
Acai PS-mediated protection both in vitro and in vivo is elucidated in
Acai PS and Yamoa PS enhance RAW264.7 macrophage
clearance of F. tularensis LVS in vitro
An initial screen of natural agonists for their ability to enhance
macrophage resistance to F. tularensis infection was conducted.
RAW264.7 cells, a murine macrophage-like cell line , were
treated overnight prior to F. tularensis LVS infection. LPS (E. coli
0:55, B5) was also included in our screen as a positive control for
macrophage activation. Both intracellular bacterial burden and
NO2accumulation were measured (Figure 1). While amphotericin
B (Amp B), Apple Polyphenol (APP; ), LPS, Acai PS, and
Yamoa polysaccharides (Yamoa PS ) all enhanced nitric oxide
(NO) production by RAW264.7 cells (Figure 1B), only LPS, Acai
PS, and Yamoa PS significantly enhanced macrophage resistance
to F. tularensis LVS (Figure 1A) at the indicated doses. Preliminary
in vivo experiments indicated that only Acai PS was able to provide
protection against pulmonary LVS challenge (data not shown).
Yamoa PS previously was shown to induce strong reactivity to the
Limulus Amebocyte Lysate (LAL) assay  and therefore was
eliminated from further study. However, Acai PS has low amounts
of endotoxin reactivity as measured by LAL assay, and its
immunomodulatory effects are resistant to polymyxin B treatment
; therefore, it was selected for further evaluation.
Acai PS upregulates macrophage surface activation
molecule expression and stimulates proinflammatory
cytokine production by mock- and LVS-infected
To assess the immunomodulatory effects upon surface activa-
tion molecule expression by Acai PS treatment, RAW264.7 cells
(originally derived from BALB/c mice) were treated with varying
doses of Acai PS overnight prior to mock- or LVS- (Multiplicity of
Infection [MOI],300) infection. RAW264.7 macrophages were
then cultured for an additional 20 h prior to assessment of changes
in surface activation molecule expression by flow cytometry, and
cytokine and NO production were also measured in cell culture
supernatants. Acai PS alone markedly stimulated CD40, CD80,
and CD86 (Table S1). Subsequent LVS infection, Acai PS
enhanced surface expression of CD11b, CD40, CD80, CD86,
TLR2, and MHC class II in a dose-dependent fashion, while
TLR4 expression was downregulated in both mock- and LVS-
infected macrophages (Table S1). Acai PS also enhanced
generation of NO, TNF-a, and IL-6 in a dose-dependent manner
by RAW264.7 cells (Table S2) and also induced trace amounts of
IL-1b and IL-12p40 (,300 pg/ml, data not shown).
Acai PS mediates clearance of type A F. tularensis in
RAW264.7 cells, but not in primary murine bone marrow-
derived macrophages (BMDMs) via NO
To investigate the mechanism by which Acai PS enhances
RAW264.7 cell resistance to F. tularensis infection, RAW264.7 cells
Activation of the innate immune system offers an
alternative and effective means to counter infection,
particularly, in cases when the etiologic agent is unknown,
such as a potential bioterrorism attack or when the agent
is resistant to antibiotics. Here we report that a natural
polysaccharide extract derived from the Acai berry (Acai
PS) has potent abilities to counter infection when applied
as a mucosal immunotherapeutic. Acai PS diminishes the
replication of F. tularensis in human macrophages co-
cultured with NK cells in vitro. In addition, nasal treatment
of mice, prophylactically or therapeutically, with Acai PS
results in significant protection against morbidity and
mortality against pulmonary infection with virulent F.
tularensis or B. pseudomallei. Of particular interest is that
Acai PS utilizes the same mechanism of protection by
enhancing Th1 cell immunity by both human and murine
cells. Since an optimal Th1-type response is required for
protection against a wide variety of infectious diseases,
Acai PS represents a novel immunotherapeutic that could
augment antibiotic therapy against a broad range of
Acai Polysaccharides Enhance Pulmonary Immunity
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and BMDMs derived from BALB/c mice were treated with varying
amounts of Acai PS before infection with F. tularensis SchuS4.
Pretreatment of RAW264.7 cells with as little as 10 mg/ml of Acai
PS reduced SchuS4 replication, while the greatest protection was
obtained using a 100 mg/ml dose (Table 1). Although the addition
of 400 mM NG-Methyl-L-arginine (L-NMA), an inhibitor of NO
production , did not totally abrogate NO production by
RAW264.7 cells prestimulated with 100 mg/ml Acai PS (Table 2),
L-NMA treatment did significantly diminish Acai PS-mediated
resistance to F. tularensis SchuS4, while having no effect on
unstimulated cells (Table 1); similar results were obtained using F.
tularensis LVS (Figure S1). While Acai PS reduced intracellular
replication of F. tularensis SchuS4 in RAW264.7 cells in an NO-
dependent manner, Acai PS did not induce NO or enhance the
clearance of F. tularensis SchuS4 from murine BMDMs (Table 1),
which highlights the limitations of using cell lines as surrogates for
primary cells. However, pretreatment of BMDMs with Acai PS did
enhance phagocytosis of F. tularensis SchuS4 (Table 1). In addition,
while infection of macrophages with strains of Francisella that do not
cause disease in humans, such as F. novicida, results in rapid
activation of the inflammasome and cell death , we did not find
type A F. tularensis infection, or Acai PS to induce robust cytotoxicity
of murine BMDMs or primary human macrophages at 20 h post-
infection under the conditions tested (Table S3). This is in
concordance with other studies that show type A F. tularensis does
not vigorously activate the inflammasome in human dendritic cells
Acai PS treatment enhances the clearance of F. tularensis
LVS from primary human macrophages co-cultured with
autologous NK cells
While Acai PS was unable to restrict the replication of F.
tularensis in primary BMDMs, Acai PS previously was found to
activate a variety of human leukocytes . Therefore, we
adopted a co-culture system in which primary human macro-
phages were infected with F. tularensis and co-cultured with
autologous NK cells. Briefly, CD14+cells were sorted and
differentiated prior to Acai PS overnight treatment. Macrophages
were infected with F. tularensis LVS and then cultured with or
without purified autologous NK cells, some of which were also
prestimulated with varying amounts of Acai PS overnight. CFU
determinations were performed 20 h after infection, and total
RNA was isolated from the NK cells at the same time. As little as
1 mg/ml of Acai PS was able to reduce LVS replication in
macrophages co-cultured with autologous NK cells (Figure 2A).
When Acai PS-treated macrophages were cultured without
autologous NK cells, Acai PS-mediated protection occurred only
at elevated concentrations ($100 mg/ml) and varied from donor
to donor (data not shown). While Acai PS was not found to
Figure 1. Natural agonists restrict replication of F. tularensis LVS in RAW264.7 macrophages. RAW264.7 macrophages (106/well, 3 wells/
treatment) were stimulated overnight (,14 h) with 50 ng/ml LPS, 40 mM securinine, 40 mg/ml APP, 500 ng/ml amphotericin B, 10 mg/ml Acai PS, or
10 mg/ml Yamoa PS prior to infection with F. tularensis LVS (MOI,300). After 20 h of infection, A) macrophages were lysed, and supernatants were
diluted for CFU enumeration, and B) nitrite levels in the supernatants were determined. Error bars represent SD, * P,0.05, as compared to untreated
cells. Data are representative of two independent experiments.
Table 1. Acai PS enhances the clearance of type A F. tularensis from RAW264.7 cells, but not murine BMDMs via NO.
SchuS4-infected RAW264.7 cells (CFU burden)a
SchuS4-infected BMDMs (CFU burden)a
Acai PS concentration Acai PS concentration
Media10 mg/ml100 mg/mlMedia10 mg/ml100 mg/ml
0 hr 4.64 (0.14)4.66 (0.11) 4.35 (0.14)4.23 (0.26)4.45 (0.06) 5.55 (0.11)*
4 hr 5.1 (0.05)5.15 (0.09)4.75 (0.06)*4.92 (0.09) 5.09 (0.11)5.82 (0.17)*
20 hr 6.12 (0.13)‘
5.55 (0.16)* 3.89 (0.18)*‘
6.25 (0.14)6.31 (0.02) 6.62 (0.20)
20 hr+L-NMA 6.01 (0.03) ND5.72 (0.05)*6.13 (0.11)ND 6.47 (0.19)
Cells were treated with Acai PS 16 hr prior to infection F. tularensis SchuS4 (MOI,30), some wells were also pre-treated with 400 mM L-NMA.
aLog10CFU/well from three wells/treatment shown; standard deviation in parentheses; results are representative of two independent experiments.
*p,0.05 as compared to the same cell type not treated with Acai PS at the same time point.
‘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.
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augment IFN-c mRNA expression by NK cells in the absence of
infected macrophages (data not shown), Acai PS did enhance IFN-
c mRNA expression by NK cells co-cultured with F. tularensis LVS-
infected macrophages (Figure 2B) in a manner inversely correlated
with intracellular replication of LVS. Acai PS also augmented
TNF-a mRNA by NK cells co-cultured with F. tularensis LVS-
infected macrophages (Figure 2B); however, this effect was not
consistent amongst all donors (data not shown). Acai PS was not
found to consistently enhance mRNA’s characteristic of cytotoxic
activity (granzyme B, perforin, TRAIL) or the expression of IL-17
and IL-21 by NK cells co-cultured with F. tularensis LVS-infected
macrophages (Figure 2B).
Acai PS impairs F. tularensis SchuS4 replication in human
primary macrophages co-cultured with NK cells via IFN-c
Since Acai PS enhanced the resistance of human primary
macrophages co-cultured with NK cells to F. tularensis infection,
subsequent studies addressed the relevance of IFN-c to this
protection. Macrophages were prestimulated with Acai PS
(100 mg/ml) overnight, infected with wild-type F. tularensis SchuS4
(MOI,30), and then cultured with or without purified, autologous
NK cells, some of which had been prestimulated with Acai PS
(100 mg/ml) overnight. CFU determinations were performed at
20 h after macrophage infection. Similar to what was observed
with LVS, Acai PS treatment of human macrophages alone had
no effect on intracellular bacterial burden, while Acai PS
treatment of macrophage/NK cell co-cultures reduced intracellu-
lar bacterial burden .100 fold without affecting phagocytosis
(Figure 3A). Neutralization of IFN-c completely ablated the
protective capacity of Acai PS, while neutralization of IFN-c in the
absence of Acai PS or NK cells had no effect on intracellular
bacterial replication (Figure 3B). The addition of 400 mM L-NMA
to co-cultures treated with Acai PS had no effect upon bacterial
replication, and NO was not detected via the Griess Reaction,
Table 2. Acai PS induces TNF-a and NO in type A F. tularensis-infected RAW264.7 cells, but not in murine BMDMs.
SchuS4-infected RAW264.7 cells (NO) or (TNF-a) SchuS4-infected BMDMs (NO) or (TNF-a)
Acai PS concentrationAcai PS concentration
Media10 mg/ml100 mg/mlMedia 10 mg/ml100 mg/ml
2.82 (0.19)9.94 (0.38)* 58.6 (1.9)*2.27 (0.19) 2.05 (0.16) 4.29 (0.38)*
2.1 (0.80)4.1 (0.32)* 9.6 (1.5)*000
Cells were treated and infected as in Table 1. 3/wells treatment at 20 hr post-infection shown; standard deviation in parentheses; results are representative of two
aMean NO (mM) or
bTNF-a (ng/ml) production.
*p,0.05 as compared to the same cell type not treated with Acai PS.
‘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.
Figure 2. Acai PS enhances LVS clearance from in human primary macrophages and enhances NK cell IFN-c mRNA. Human primary
macrophages (16104cells/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 b-actin (control) and IFN-c, TNF-a, IL-17A, IL-21, granzyme B, perforin, and TRAIL. Results are representative of
independent experiments from five different blood donors.
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indicating that the protective effect of IFN-c induced by Acai PS is
independent of NO production (data not shown).
Acai PS enhances innate immunity to pulmonary type A
F. tularensis infection
We previously found Acai PS to induce immunomodulatory
effects when instilled into the lungs of naı ¨ve mice . In
particular, Acai PS was shown to induce IL-12, which is protective
against F. tularensis LVS infection . To assess whether Acai PS
could confer protection against pulmonary infection with virulent
F. tularensis SchuS4, C57BL/6 mice were treated nasally with 10,
100, or 1000 mg of Acai PS 24 h prior to aerosol infection with F.
tularensis SchuS4, and changes in body weight and morbidity were
recorded over time for up to 28 days after infection. Treatment of
mice with 100 mg of Acai PS led to 80% survival, while 10 or
1000 mg Acai PS doses exhibited less potency (Figure 4A).
Importantly, mice treated with Acai PS that survived infection
showed negligible weight loss (Figure 4B) and clinical symptoms
(data not shown); indicating Acai PS confers protection against
both morbidity and mortality induced by virulent F. tularensis
infection. Since the 100 mg dose of Acai PS was found to be
optimal against aerosol challenge, in subsequent experiments mice
were treated with 100 mg Acai PS at various time points after
infection with F. tularensis SchuS4. When delivered by the
intranasal (i.n.) route immediately after aerosol infection, Acai
PS conferred 70–80% survival upon treated mice (Figure 4C),
while all vehicle-treated animals succumbed to infection. Sixty
percent of mice treated i.n. with Acai PS 24 h after aerosol
challenge with F. tularensis SchuS4 survived, and even when Acai
PS was given 48 h after infection, 33% of animals still survived
(Figure 4C). As described above for prophylactic therapy, animals
treated with Acai PS after aerosol infection that survived challenge
displayed negligible weight loss and clinical symptoms (data not
shown). Oral treatment of animals with Acai PS also conferred
some level of protection against type A F. tularensis infection;
however, this effect was variable (Table S4).
Acai PS enhances a protective IFN-c response during F.
To determine the mechanism by which Acai PS confers
protection against F. tularensis infection, expression of intracellular
IFN-c by pulmonary leukocytes was assayed by flow cytometry.
These studies utilized a 1000 mg Acai PS pretreatment, which we
found to be optimal to protect against intranasal F. tularensis
SchuS4 challenge (data not shown). The finding that a 1000 mg
Acai PS dose was optimal against i.n. F. tularensis SchuS4 infection,
while a 100 mg Acai PS dose was optimal against aerosol F.
tularensis SchuS4 infection may reflect variations in the aerosol
versus i.n. challenge models used in this study. We found i.n.
pretreatment of mice enhanced intracellular expression of IFN-c
by NK T cells within two days after F. tularensis SchuS4-infection
(Figure 5A). In addition, while Acai PS reduced bacterial burdens
in the lungs and spleens of F. tularensis SchuS4, neutralization of
IFN-c abrogated this effect (Figure 5B–C).
Acai PS enhances innate immunity to B. pseudomallei
pulmonary infection and reduces bacterial replication
Stimulation of innate immunity with an immunotherapeutic
such as Acai PS would be particularly valuable in situations where
the etiological agent of disease is unknown, such as a bioterrorist
attack, as induced innate immune responses are often capable of
providing protection against a broad range of organisms. In
addition, immunotherapy could be of particular benefit to counter
infections due to bacteria that are intrinsically resistant to
antibiotics, such as B. pseudomallei, a CDC Category B Bioterrorism
agent. As Acai PS augmented immunity to F. tularensis infection,
along with enhancing the expression of IFN-c, which is crucial for
protection from B. pseudomallei infection , we tested the effects
of Acai PS against B. pseudomallei infection to assay whether Acai
PS has potential as a broad spectrum therapeutic to combat
pulmonary infections. C57BL/6 mice were treated i.n. with Acai
PS prior to, or immediately after, i.n. infection with 36103CFUs
of B. pseudomallei 1026b. Body weights and clinical scores were
recorded. I.n. treatment of mice with 100 or 1000 mg Acai PS 24 h
prior to, or immediately after, B. pseudomallei infection resulted in
significantly diminished weight loss and clinical scores (Figure 6A–
B). Treatment of mice with #10 mg of Acai PS or treatment of
mice with Acai PS (10–1000 mg) $24 h after B. pseudomallei
infection did not result in significant protection (data not shown).
Next, to determine the effects of Acai PS on bacterial colonization
and dissemination, mice were treated i.n. with 100 or 1000 mg of
Figure 3. Acai PS reduces type A F. tularensis from primary human macrophages co-cultured with NK cells via IFN-c. A) and B) Primary
human macrophages (105/well, 3 wells/treatment) and NK cells were isolated and treated separately with 100 mg/ml Acai PS 16 h prior to infection
with F. tularensis SchuS4 (MOI,30). Neutralizing anti-IFN-c 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-c. 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.
Acai Polysaccharides Enhance Pulmonary Immunity
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Acai PS 24 h prior to i.n. infection with 36103CFUs of B.
pseudomallei 1026b. Bacterial burdens were determined in the lungs,
spleens, and livers 72 h after infection. Treatment of mice with
either dose of Acai PS reduced B. pseudomallei replication in the
lungs by ,10,000-fold (Figure 6C). Treatment of mice with Acai
PS also reduced dissemination into peripheral tissues. B.
pseudomallei CFUs were below the limit of detection (,33 CFUs)
in the spleens of 80% of animals treated with either dose of Acai
PS, while no bacteria were recovered from the livers of any
animals treated with either dose of Acai PS (Figure 6C). In
addition, all mice treated prophylactically with 100 or 1000 mg
Acai PS (n=20) survived nasal infection with 36103CFUs of B.
pseudomallei 1026b; however, the lethality of this dose in control
animals varied from 60–100% in different experiments (8/10
control animals succumbed to infection). While 100 and 1000 mg
Acai PS doses conferred similar protection against challenge with
36103CFUs of B. pseudomallei, a 1000 mg Acai PS provided the
best protection against high dose i.n. challenge (16104CFUs) with
B. pseudomallei (Figure S2A–B). These results indicate that an
elevated dose of Acai PS may be required against a high dose
bacterial challenge in order to protect the host against a more
Acai PS enhances IFN-c responses by NK cells and cd T
cells during pulmonary Burkholderia infection
To assess the mechanism of protection mediated by Acai PS on
innate lymphocytes during pulmonary infection, the B. thailandensis
(BSL-1 strain) model of Burkholderia infection  was used.
C57BL/6 mice were given Acai PS i.n. 24 h prior to i.n. infection
with 56105CFUs of B. thailandensis E264. Pulmonary NK and cd
T cells were then assayed 24 h after infection by flow cytometry
for the intracellular expression of IFN-c. Acai PS enhanced IFN-c
expression by both NK and cd T cells in B. thailandensis-infected
mice (Figure 7); indicating Acai PS can augment the IFN-c
responses of innate lymphocytes during pulmonary Burkholderia
Figure 4. Nasal administration of Acai PS confers prophylactic and therapeutic protection against pulmonary Type A F. tularensis
infection. Female C57BL/6 mice (5/group) were treated with PBS or with 10, 100, 1000 mg of Acai PS by the intranasal (i.n.) route one day prior to
aerosol infection with F. tularensis 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 mg of Acai PS immediately after, one day after, or two days after aerosol infection with F. tularensis 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.
Figure 5. Acai PS enhances IFN-c by innate leukocytes during pulmonary type A F. tularensis infection. 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 F. tularensis SchuS4. Some mice were also depleted of IFN-c two days
prior to infection. A) Intracellular expression of IFN-c 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.
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Acai PS requires IFN-c and NK cells to confer protection
against pulmonary B. pseudomallei infection
As Acai PS was found to enhance the pulmonary Th1-type
response, which is critical for control of Burkholderia infections
[11,26,28], the role of Th1-type responses in Acai PS-mediated
protection against B. pseudomallei infections was further investigat-
ed. For these studies, mice were treated i.n. with 1000 mg of Acai
PS 24 h prior to infection. Some mice were also depleted of IFN-c
or NK cells via neutralizing antibody 24 h prior to Acai PS
treatment (control animals received rat IgG). While the survival
conferred by Acai PS in control animals was suboptimal against a
high-dose challenge, Acai PS-mediated survival was totally ablated
in IFN-c-depleted mice and partially reduced in mice depleted of
NK cells (Figure 8). In addition, while Acai PS mitigated clinical
symptoms in B. pseudomallei-infected mice, this effect was abrogated
in the absence of IFN-c (data not shown). These results indicate
that, similar to what was observed in vitro and in vivo with F.
tularensis; Acai PS requires IFN-c and possibly NK cells for
protection against pulmonary infection with B. pseudomallei.
Enhancing innate immunity by agonist therapy could poten-
tially augment resistance to infection and could also complement
traditional vaccination and antibiotic strategies for treating
infectious diseases [6,11,12,14,15]. In this study, the abilities of
several natural agonists with immunomodulatory capabilities:
APP, AmpB, securinine, Yamoa PS, Acai PS, and LPS [12,15–
18] were assayed for their ability to potentiate macrophage
resistance to F. tularensis LVS infection. While APP, AmpB, Yamoa
PS, and Acai PS each enhanced NO production by LVS-infected
RAW264.7 macrophages, only Yamoa PS and Acai PS conferred
significant resistance to LVS replication at the doses tested. LPS
also enhanced NO production and LVS clearance, which was not
surprising, as TLR4 agonists have been shown to increase
resistance to infection with F. novicida, a strain of Francisella that
is virulent for mice, but rarely causes disease in humans .
Yamoa PS was not further examined due to concerns about
possible endotoxin contamination, presumably due to endophytic
bacteria residing in bark , the source of this polysaccharide. In
contrast, Acai PS contains low amounts of endotoxin (,0.01 EU/
mg), has MyD88-independent effects, and has immunomodulatory
effects resistant to polymyxin B treatment . In addition, Acai
PS is non-toxic to lymphocytes at concentrations up to 500 mg/ml
 and is not found to have direct antibacterial (cytotoxic) effects
against Francisella in PBS or cell culture media (data not shown).
Acai PS is derived from the berry of the palm tree Euterpe oleracea
Mart. indigenous to the Amazon River basin in South America.
This fruit is commonly used to make beverages and food additives
and is used as a herbal medicine [30–34]. Biochemical studies
reveal Acai contains numerous compounds, particularly anthocy-
anins, proanthocyanidins, and other flavonoids . While many
studies have focused on the antioxidant properties of Acai [33,35–
38] presumably attributable to its polyphenols and related classes
of compounds, here we concentrated on the activities of Acai PS as
the polysaccharide fraction, rather than the polyphenol fraction of
Acai, induces a proinflammatory response . We previously
demonstrated that Acai PS stimulates both cd T cells and myeloid
cells in vitro and incites the recruitment of neutrophils and activates
DCs/macrophages to the lung in vivo . Therefore, since Acai
Figure 6. Nasal administration of Acai PS confers protection against pulmonary B. pseudomallei infection. Female C57BL/6 mice (n=5/
group) were treated i.n. with PBS or with 100–1000 mg of Acai PS one day prior to, or immediately after, intranasal infection with 36103CFUs of B.
pseudomallei 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.
Figure 7. Acai PS augments IFN-c responses by cd T cells and
NK cells during pulmonary infection. C57BL/6 mice (n=5/group)
were treated by the i.n. route with 1 mg of Acai PS one day prior to i.n.
infection with 56105CFUs of B. thailandensis E264. One day after
infection, cells were harvested from lungs, and intracellular IFN-c
expression by cd T cells and NK cells was determined by flow cytometry.
Error bars depict SEM. *P,0.05 as compared to the same cell type from
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PS has potent immunomodulatory activities and is effective at
restricting the replication of F. tularensis LVS in RAW264.7 cells, it
was investigated for its potential as an innate immune agonist.
In addition to augmenting the clearance of F. tularensis LVS and
SchuS4 in RAW264.7 cells via NO, Acai PS also enhanced cell
surface expression of CD11b, CD40, CD80, CD86, MHC class II,
and TLR2 in a dose-dependent manner in both mock- and F.
tularensis LVS infected-macrophages; however, TLR4 expression
was downregulated. TLR4 expression has been shown to be
downregulated following LPS stimulation , and while Acai PS
is low in endotoxin (,0.01 EU/mg), is resistant to polymyxin B
neutralization, and has MyD88-independent effects , it is
possible Acai PS may still signal through TLR4 via an alternative
mechanism such as TRIF .
While Acai PS was able to reduce the intracellular replication of
F. tularensisinRAW264.7cells,Acai PSwasnotfound to induceNO
or restrict the replication of F. tularensis in primary human
macrophages or murine BMDMs. This finding is presumably due
to the fact that primary cellsand, inparticularhumanmacrophages,
do not produce NO as readily as do macrophage cell lines , and
such findings stress that cell lines are not always a suitable surrogate
for primary cells. While Acai PS did not enhance the clearance of F.
tularensis in macrophages alone, Acai PS can also activate innate
lymphocytes in addition to macrophages . Therefore, we
adapted a co-culture system in which we tested the effect of Acai PS
treatment onhuman monocyte-derivedmacrophages(culturedwith
or without autologous NK cells) infected with F. tularensis. While
Acai PS was not able to directly stimulate human primary
macrophages for clearance, a ,100–1000-fold reduction in
replication occurred when macrophages were co-cultured with
autologous NK cells. Others have shown murine NK cells
stimulated in vivo could impair intracellular replication of F. tularensis
LVS in vitro , and depletion of NK cells reduces the time to
lethality during pulmonary infection . Of particular interest is
that NK cells are a major source of IFN-c in pulmonary tularemia
. RT-PCR analysis revealed Acai PS stimulated human NK
cells co-cultured with LVS-infected macrophagespossessedelevated
levels of IFN-c mRNA, while neutralization of IFN-c in vitro
diminished the protective effect of Acai PS in macrophages infected
withF. tularensis LVSorSchuS4.NOwasnotdetected incellculture
supernatants from our human macrophage/NK cell co-cultures,
and iNOS inhibition had no effect on replication, indicating the
protection conferred by Acai PS-induced IFN-c in the human co-
culture model is NO-independent, similar to what others have
described for IFN-c treated macrophages infected with F. tularensis
SchuS4 . Treatment of NK cells in the absence of Francisella-
infected macrophages did not result in robust induction of IFN-c
mRNA, indicating there may be a synergetic effect of Acai PS and
infection upon NK cells. Our previous finding that Acai PS induces
IL-12 in vivo may indicate the macrophage is responsible for IL-12
production, which in turn induces IFN-c mRNA by the NK cell.
Indeed, neutralization of IL-12 in vitro did reduce IFN-c mRNA by
NK cells in some co-cultures treated with Acai PS (data not shown).
While it is known human NK cells can enhance the clearance of
intracellular organisms, such as Brucella in autologous macrophages
via contact-dependent, cytotoxic mechanisms , the effect of
Acai PS on NK cell mediated-cytoxicity may be minimal, since
marked differences in the mRNA expression for perforin, granzyme
B, or TRAIL were not observed by Acai PS-treated NK cells co-
cultured with autologous LVS-infected macrophages.
As IFN-c was induced by Acai PS in vitro, and is essential for
Acai PS could confer protection against in vivo challenge by
employing an aerosol model of type A F. tularensis infection thought
to most mimic human disease . We utilized F. tularensis SchuS4
for all our in vivo infections, because, while F. tularensis LVS is widely
used as a model organism to study immunological responses ,
emerging evidence suggests the in vivo immune response differs
between SchuS4 and LVS [24,47,49], and immunotherapeutic
strategies that confer potent protection against pulmonary LVS
infection only confer partial or negligible protection against
pulmonary infection with SchuS4 [6,50,51]. Since Acai PS
enhanced the clearance of Francisella in murine macrophages and
in human macrophages co-cultured with NK cells, and because
Acai PS had potent immunomodulatory effects in the lung ,
Acai PS was tested as a mucosal immunotherapeutic to treat
pulmonary type A F. tularensis infections. It was found that i.n.
pretreatment of mice with Acai PS conferred up to 80% protection
against F. tularensis-induced mortality, which, to our knowledge, is
the highest degree of protection demonstrated by an immunother-
Figure 8. Acai PS requires IFN-c and NK cells for optimum protection against B. pseudomallei infection. Two days prior to infection,
C57BL/6 mice received rat IgG, anti-IFN-c, 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 16104CFUs of B. pseudomallei 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-c.
Acai Polysaccharides Enhance Pulmonary Immunity
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apeutic and also represents the first mucosal immunotherapeutic to
confer significant survival against pulmonary type A F. tularensis
infection. Importantly, Acai PS provided significant protection
when administered i.n. within 48 h after pulmonary infection and
thus is the first immunotherapeutic demonstrating post-exposure
protection of any kind against pulmonary type A F. tularensis
infection. Acai PS was also able to reduce bacterial burdens in the
lungs and spleens of mice infected with F. tularensis SchuS4. In
addition, similar to what was observed in vitro in human cells, Acai
PS augmented IFN-c expression by NK cells in the lungs of treated
animals infected with F. tularensis SchuS4, while neutralization of
IFN-c abrogated the protective effect of Acai PS. The finding that
Acai PS is able to protect against infection even when administered
one or two days after infection, at which time SchuS4 is already
presentin the spleen and liver, is intriguing. Mucosaladministration
of therapeutics can have systemic effects, and compounds delivered
that mucosal administration of Acai PS enhances serum levels of
TNF-a (Holderness et al, manuscript in preparation). As TNF-a is
protective against tularemia, it is possible that nasal administration
of Acai PS also has an effect against systemic replication of
Francisella, which may account for the post-exposure protection
conferred by Acai PS observed here. Acai PS is also heat-resistant,
and wehave found ittohavepotentprotective effects after shipment
at ambient temperature, as demonstrated by the protection
observed in Figure 6A–C. Therefore, since Acai PS does not
require refrigeration (cold-chain management) and adapts a needle-
free mucosal method of administration, it offers a practical strategy
during emergencies, such as pandemics or bioterrorist attacks, when
expeditious treatments of the affected populace would be required
The downregulation of TLR4 by Acai PS observed by flow
cytometry indicated Acai PS may signal at least partially through
TLR4. However, work by others indicates TLR4 stimulation
alone is an insufficient method to protect against experimental
tularemia, particularly, when administered after infection. Nasal
administration of a TLR4 agonist prior to, but not after,
pulmonary infection with F. novicida could confer protection ,
while intraperitoneal administration of a TLR4 agonist could
confer some level of protection when given 48 h before pulmonary
infection with type A F. tularensis ; however, this effect is
diminished when the TLR4 agonist was given only at the time of
infection. In addition, others have demonstrated that pulmonary
administration of LPS has minimal effects upon the immune
response when given 24 h after type A F. tularensis infection ,
indicating that type A F. tularensis infection actively suppresses
TLR4 signaling. Since we found Acai PS has potent protective
effects when given $24 h after infection, it would appear that Acai
PS also signals through a receptor in addition to TLR4, and the
low levels of LPS present in Acai PS are not responsible for the
observed protection. Botanical polysaccharides are known to
signal through a variety of receptors, including TLRs and
carbohydrate receptors . Work on the receptors utilized by
Acai PS is ongoing in a separate study, but Acai PS appears to
require both TLR4/TRIF along with carbohydrate receptors
(Holderness et al, manuscript in preparation) to mediate its effects.
Therefore, future studies of the receptors required for Acai PS-
mediated signaling and protection could reveal receptors to be
targeted for immunotherapy against F. tularensis and other diseases.
Stimulation of innate immunity with an immunotherapeutic
such as Acai PS would be particularly valuable in situations where
the etiological agent of disease is unknown, as induced innate
immune responses are often capable of providing protection
against a broad range of organisms . In addition, immunother-
apy could be of particular benefit to counter bacterial infections
intrinsically resistant to antibiotics. To this end, we tested Acai PS
against pulmonary infection with B. pseudomallei, an organism
intrinsically resistant to antibiotics, to determine if Acai PS has
potential as a broad spectrum immunotherapeutic. We found Acai
PS enhanced immunity to B. pseudomallei when given prior to, or
immediately after, infection. Acai PS also potently restricted B.
pseudomallei replication within the lungs and dissemination to
To assess the effects of Acai PS on innate leukocytes during
infection, we assayed the expression of IFN-c in leukocytes from
the lungs of mice infected with B. thailandensis. We found Acai PS
augmented the expression of IFN-c in both NK and cd T cells
from B. thailandensis-infected animals, indicating an enhanced Th1-
type response of these cell types. Since Acai PS also enhanced the
IFN-c response of human and murine NK cells during in vitro and
in vivo models of F. tularensis infection, the role of NK cells and IFN-
c in Acai PS-mediated protection against B. pseudomallei was
assessed. As a result, IFN-c was entirely responsible for Acai PS-
mediated protection, while NK cells were also required to some
extent. These results demonstrate Acai PS mediates protection
against infection in human cells in vitro and in in vivo murine models
in a similar manner as NK cells and IFN-c are required for
protection in both systems, indicating our protective effects in vivo
with mice have relevance to humans. As neutralization of NK cells
did not entirely ablate protection against B. pseudomallei in vivo, it is
possible the effects of Acai PS on other cells are also required for
protection. We have previously found Acai PS to stimulate human
cd T cells in vitro , and here we show Acai PS augments the
Th1-type responses of cd T cells in infected lungs; therefore, as cd
T cells are known to confer protection against a number of
intracellular pathogens such as Brucella and Listeria [55,56], future
studies will investigate the role of cd T cells in Acai PS-mediated
protection. In a clinical setting, an immunotherapeutic such as
Acai PS would most often be used in conjunction with antibiotics.
Recent studies have demonstrated immunotherapy can synergize
with antibiotic therapy of bacterial infections, including Burk-
holderia ; therefore, additional studies will assess the effects of
Acai PS in combination with antibiotic therapy.
In summary, we show immunotherapy with natural agonists
such as Acai PS is an effective means to confer protection against
bacterial infection. In fact, Acai PS appears to be the most potent
immunotherapeutic reported to date to combat pulmonary type A
F. tularensis infections and is also the first one demonstrated to
confer significant survival when given mucosally or after infection.
Of particular interest is Acai PS was also able to confer protection
against pulmonary infection with both F. tularensis and B.
pseudomallei, as previous studies demonstrated immunotherapeutics
that induce potent protection against B. pseudomallei may only
confer partial or negligible protection against type A. F. tularensis
[6,11,50], indicating Acai PS has broad spectrum immunothera-
peutic potential to combat intracellular bacterial infections. Acai
PS also enhanced the Th1 cell response of innate leukocytes
during infection both in vivo and in human cells. As optimal Th1
cell immunity is required for protection against a broad range of
infections, Acai PS should be investigated as a possible
immunotherapy that could augment or complement traditional
antibiotic and vaccination strategies against a range of pathogens.
Materials and Methods
All animal care and procedures were in accordance with the
recommendations in the Guide for the Care Use of Laboratory
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Animals of the National Institutes of Health. All animal protocols
were approved by Institutional Animal Care and Use Committees
at Montana State University (protocol approval: 2009-27, 2011-
25) or Colorado State University (protocol approval 09-001) and
all efforts were made to minimize suffering. Human subjects
testing was performed in accord with the Institutional Review
Board of Montana State University (protocol approval: JS072809),
and written, informed consent was obtained from all individuals.
Bacterial strains, culture conditions and mice
F. tularensis SchuS4 or LVS was cultured in modified Mueller–
Hinton (MMH) broth (0.025% ferric pyrophosphate, 2% Iso-
VitaleX and 0.1% glucose) at 37uC with constant shaking
overnight, aliquotted into 1 ml samples, frozen at 280uC, and
thawed just before use, as previously described . Frozen stocks
were titrated by enumerating viable bacteria from serial dilutions
plated on MMH agar (0.025% ferric pyrophosphate, 2%
IsoVitaleX, 0.1% glucose, and 0.025% fetal bovine serum). The
numbers of viable bacteria in frozen stock vials varied by less than
5% over a 10 month period. These stocks were used to generate
cultures for F. tularensis SchuS4 or LVS infection studies. Frozen
stocks of B. pseudomallei of known titers were prepared from cultures
grown in Luria-Bertani (LB) broth (BD Biosciences, San Jose, CA)
by freezing the cultures in LB medium containing 20% glycerol.
Inocula for in vivo infection with B. pseudomallei were prepared by
thawing and diluting frozen stocks in sterile PBS . All
experiments with F. tularensis SchuS4 or B. pseudomallei 1026b
were performed in biosafety level 3 facilities at Montana State
University or Colorado State University. Burkholderia thailandensis
E264 was acquired from ATCC (Manassas, VA). Prior to
infection, B. thailandensis were grown from frozen glycerol stock
in LB at 37uC overnight and freshly diluted 1:100 into 100 ml of
LB. The bacteria were grown to an optical density (OD600) of 1.9
(,16109cfu/ml) and diluted in PBS prior to infection .
Six-week-old female C57BL/6 or BALB/c mice were pur-
chased from Charles River Laboratories. All mice were housed in
sterile microisolater cages in the laboratory animal resources
facility at Montana State University or the Biohazard Research
Building BSL-3 facility at Colorado State University and were
provided with sterile water and food ad libitum.
Acai PS preparation
Acai fruit pulp was obtained from Acai Berry Pure (Acai Berry
Pure Bulk; Carlsbad, CA). Polysaccharides were isolated from this
powdered Acai, as described previously [15,19]. Briefly, 1500 g of
Acai powder was extracted with 8 liters boiling distilled H2O for
1 h. The aqueous extract was then centrifuged at 2,0006 g for
15 min, and a 4-fold volume of ethanol was added to the
supernatant to precipitate polysaccharides overnight at 4uC. The
precipitate was pelleted by centrifugation, re-dissolved in distilled
H2O and centrifuged at 2,0006 g for 15 min. The supernatant
fluid (crude polysaccharide extract) was fractionated using ion-
exchange chromatography on a DEAE-cellulose column equili-
brated with 0.05M Tris-HCl buffer (pH 8.0). Bound material was
sequentially eluted with 0.05M Tris-HCl buffer and 2M NaCl.
The presence of polysaccharides in the unbound fraction, eluted
with 0.05M Tris-HCl buffer, was minimal (,0.1% of total bound
fraction). The Acai-PS fraction was generated from this prepara-
tion after concentration in an Amicon concentrator with a 10 kDa
Amicon PM10 membrane (Millipore; Billerica, MA). This
preparation yields a fraction that is .99% carbohydrate and
.92% polysaccharides. Monosaccharide analysis reveals that Acai
PS consists primarily of arabinose, galacturonic acid, and galactose
. Endotoxin levels were determined using the LAL assay, as
described . Endotoxin levels for the Acai PS used in this study
were ,0.01 EU/mg.
Generation of bone marrow-derived macrophages
Bone marrow-derived macrophages (BMDM) were generated
by flushing the bone marrow from the femurs of BALB/c mice
with RPMI 1640 media. Freshly collected bone marrow cells were
cultured overnight in complete media (CM; RPMI 1640, 10%
fetal bovine serum [Atlanta Biologicals, GA], 10 mM HEPES
buffer, 10 mM nonessential amino acids, 10 mM sodium
pyruvate) containing 5 ng/ml recombinant murine M-CSF
(Peprotech, Rocky Hill, NJ). The non-adherent cells were then
collected and cultured for an additional six days in CM with
30 ng/ml M-CSF to generate macrophages.
Infection of RAW264.7 cells and murine BMDMs
Murine BMDMs or RAW264.7 macrophages were seeded at
16106cells/well in CM without antibiotics in 24-well microtiter
plates (BD Labware, Franklin Lakes, N.J.) at 37uC/5% CO2prior
to infection. Macrophages were infected with Francisella tularensis
LVS at an MOI of ,300 or F. tularensis SchuS4 at an MOI of ,30
for two h at 37uC. Cells were then washed once with PBS, and
then fresh CM containing 50 mg/ml gentamicin were added to
each well, and cells were incubated for 30 min at 37uC to kill
extracellular bacteria. Cells were then washed twice with PBS, and
then fresh complete media without antibiotics were added to the
wells for the remainder of the experiment (this is considered the
‘‘0 hour’’ time point). For time points of .8 h, gentamicin was
added to the wells for the last 45 min of incubation. To enumerate
intracellular bacteria, cells were washed three times with PBS and
then lysed with sterile deionized water. Serial logarithmic dilutions
of macrophage lysates were then performed and plated in
triplicate onto MMH agar for incubation at 37uC/5% CO2for
2–3 days. In some cases, macrophages were stimulated at various
time points before or after infection with varying concentrations of
agonist. In addition, L-NMA (Sigma-Aldrich, St. Louis, MO) was
added to selected wells to inhibit NO production. Supernatants
were collected and frozen until analysis by cytokine ELISA or the
Cytotoxicity, cytokine, and NO22production assays
Supernatants from Francisella-infected RAW264.7 and human
macrophages were collected at various time points and measured
for cell death, production of cytokines and, the oxidized product of
NO. Cell death was determined by measuring lactate dehydro-
genase LDH release using a cytotoxicity detection kit according to
manufacturer’s instructions (Roche, Indianapolis, IN). Cytokine-
specific ELISAs were performed, as described previously [58,59].
All NO22detection chemicals were obtained from Sigma-Aldrich.
Aliquots of 50 ml of cell culture supernatant were reacted with
equal volumes of Griess reagent (1% sulfanilamide, 0.1%
naphthylenediamine dihydrochloride, 2.5% H3PO4) at room
temperature (RT) for 10 min. Sodium nitrite was used to generate
a standard curve for NO22production, and peak absorbance was
measured at 550 nm with a Thermomax microplate reader
(Molecular Devices, Sunnyvale, CA). Cell-free medium contained
,1.5 mM NO22.
Flow cytometry analysis of cell surface molecule
activation by RAW264.7 cells
RAW264.7 cells were detached from 24-well culture plates,
resuspended, and washed. Immunofluorescent staining for cell
surface molecule expression was performed using the following
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fluorochrome-labeled mAbs from eBioscience (San Diego, CA),
Biolegend (San Diego, CA), or BD Biosciences: CD11b (clone
M1/70), CD80 (clone 16-10A1), CD40 (clone 3/23), TLR4 (clone
MT5510), CD86 (clone GL1), TLR2 (clone T2.5) and MHC-II
(clone AMS-32.1). Fluorescence was acquired on FACSCaliber,
LSRII, or Canto (BD Biosciences). FlowJo (Tree Star, Ashland,
OR) software was used for analysis.
Isolation and infection of human macrophages co-
cultured with autologous NK cells
Heparinized human peripheral blood was subjected to Histo-
paque 1077 (Sigma-Aldrich) density gradient centrifugation. The
collected mononuclear cell fraction was collected, and monocytes
were isolated with CD14 microbeads (Miltenyi Biotec, Auburn,
CA) according to manufacturer’s instructions. Monocytes (.95%
purity,104–105/well) were then seeded into 48-well microtiter
plates in CM without antibiotics, supplemented with 10 ng/ml
GM-CSF (Peprotech, Rocky Hill, NJ) for 4–5 days at 37 Cu/5%
CO2 to generate macrophages. Human macrophages were
(MOI,30) in the same manner as described above for murine
macrophages. One day prior to macrophage infection, autologous
‘‘untouched’’ NK cells were isolated from human PBMCs using an
NK cell isolation kit from Miltenyi Biotec according to
manufactures instructions. Isolated NK cells (.95% purity) were
cultured overnight in complete media at 37 Cu/5%CO2with or
without agonist stimulation. NK cells were washed with fresh CM
prior to being added to wells containing infected autologous
macrophages (,2–20 NK cell/macrophage).
To inhibit the effects of IFN–c in vitro, a neutralizing mAb (IFN–
c [clone B27, 1 mg/ml] in a no azide/low endotoxin (NA/LE)
format was purchased from BD Biosciences and added to selected
wells containing Francisella-infected macrophages with or without
NK cells .
(MOI,300) or SchuS4
Extraction of RNA and RT-PCR analysis of human NK cells
Human NK cells cultured with or without LVS-infected
macrophages and/or Acai PS were centrifuged and resuspended
in RNAlater reagent (Qiagen, Valencia, CA) until RNA
extraction. Cells were then centrifuged and resuspended in
Qiagen RLT buffer prior to lysis on a Qiashredder Column
(Qiagen) and RNA extraction with an RNeasy Mini Kit (Qiagen).
cDNA was generated using the Superscript III First Strand
Synthesis System (Invitrogen). Primers for immune-related genes
(TNF-a, IFN-c, IL-17A, IL-21, IL-22, granzyme B, perforin, and
TRAIL), along with b-actin (endogenous control), were designed
using the PrimerQuest application from IDTDNA.com. The
reference sequences used to generate these primers are listed below
(paragraph ‘‘Accession numbers’’). Amplicons were visualized
under UV illumination on a 2% agarose gel containing GelRed
(Biotium, Hayward, CA).
Mouse infection, agonist treatment, in vivo
neutralization, and CFU determination
Mice were infected with F. tularensis SchuS4 at Colorado State
University via a whole-body low-dose aerosol, as previously
described [53,61]. Conscious mice within a stainless steel basket
were exposed to the SchuS4 strain of F. tularensis by aerosol
exposure in a Glascol Inhalation Exposure System (Glas-Col, Inc.,
Terre Haute, IN, USA). Prior to exposure, the nebulizer was
loaded with bacteria diluted in PBS to a concentration of
,56106CFU/ml. Mice were exposed to a total of ,46107
bacteria, aerosolized into a volume of 5 cubic feet over a period of
30 min, followed by a 20 min period of cloud decay in which
airflow was maintained without introducing additional bacteria.
This inoculum method generally delivers ,50 CFUs of F. tularensis
to the lungs of exposed mice and routinely results in 100%
mortality and a mean time to death of 5–6 days following infection
. Mice infected with F. tularensis SchuS4 at Montana State
University were infected with a 20 ml nasal volume (50 CFUs)
placed onto the anterior nares following anesthesia induced by
intraperitoneal (i.p.) injection of 100 ml of ketamine (12.5 mg/
ml)+xylazine (3.8 mg/ml). For survival experiments, mice were
monitored for morbidity and mortality twice daily for up to 28
days, at which time survivors were euthanized. Mice were treated
with varying doses of Acai PS (in PBS) before or after infection.
Mice were treated nasally under anesthesia (10 ml/nare induced
by i.p. injection with ketamine/xylazine cocktail. For oral
treatments, mice received 200 ml volume via gavage. Control
mice were inoculated with PBS. For in vivo neutralization studies,
mice were treated with 500 mg of mAb i.p. to neutralize IFN-c
(clone XMG 1.2) on day 22, while control mice received rat IgG
. In some experiments, mice were sacrificed 2 days post post-
infection for CFU determination in lungs and spleens. Mouse
organs were homogenized in sterile PBS, and homogenates were
serially diluted and plated on MMH plates, which were then
incubated at 37uC for 48 h, at which time CFUs were
For B. pseudomallei infection, mice under ketamine/xylazine-
induced anesthesia were infected with i.n. (10 ml/nare) with 36103
or 16104CFUs of B. pseudomallei 1026b. Clinical scores were
graded as 0=normal; 1=slightly ruffled; 2=ruffled, sick looking;
3=hunched posture and obviously ill; 4=moribund; 5=eutha-
nized. For in vivo neutralization studies, mice received 500 mg of
mAb i.p. to neutralize IFN-c (clone XMG 1.2) or NK cells (clone
PK136) on day 22, while control mice received rat IgG [62,63].
Mice were sacrificed at 3 days post-infection for CFU determi-
nation in lungs, livers and spleens. Mouse organs were
homogenized in sterile PBS, and homogenates were serial diluted
and plated on Tryptic Soy Agar (BD Biosciences) plates, which
were then incubated at 37uC for 48 h, at which time CFUs were
Pulmonary leukocyte activation assay
For Francisella studies, C57BL/6 mice were nasally treated with
Acai PS 24 h prior to i.n. infection with 50 CFUs of F. tularensis
SchuS4. Forty-eight h after infection, lung tissue was minced
followed by digestion for 1 h at 37uC in CM containing 200 U/ml
collagenase, (Sigma) and 0.08 U/ml DNAse (Promega, Madison,
WI). The resulting cell suspensions were filtered through 35 mm
NitexH nylon mesh (Sefar America; Depew, NY) to remove tissue
debris, washed in CM, resuspended in 30% Percoll (Pharmacia,
Uppsala, Sweden) and layered onto 70% Percoll, and subjected to
density gradient centrifugation. Mononuclear cells were removed
from the interface layer, washed, resuspended in CM, and
cultured for 4 h in the presence of 12-myristate 13-acetate
(PMA; 50 ng/ml), 500 ng/ml ionomycin, and 10 mg/ml brefeldin
A. Cells were then analyzed by FACS analysis using conventional
methods [64,65]. Cells were stained for extracellular markers with
fluorochrome-conjugated mAbs (Becton Dickinson or eBioscience,
San Diego, CA): anti-NK1.1 (clone PK136); prior to fixation with
2% paraformaldehyde. Cells were then permeabilized with 0.2%
saponin and stained for intracellular expression of IFN-c (clone
XMG1.2). Stained leukocytes were analyzed using an LSRII flow
cytometer (BD Biosciences) and analyzed using FlowJo software
(Tree Star Inc., Ashland, OR).
Acai Polysaccharides Enhance Pulmonary Immunity
PLoS Pathogens | www.plospathogens.org 11March 2012 | Volume 8 | Issue 3 | e1002587
For Burkholderia studies, C57BL/6 mice were i.n. treated with
Acai PS 24 h prior to i.n. infection with 56105CFUs of B.
thailandensis E264. Twenty-four h after infection, lung tissue was
processed, and cells were cultured and stained as described above.
Statistical differences between two groups were determined
using a Student’s t test with the significance set at P,0.05. For
comparison between three or more groups, analysis was done by
one-way ANOVA followed by Tukey’s multiple comparisons test
with significance determined at P,0.05. For in vivo studies,
significance in survival was assessed using log-rank analysis with
significance set at P,0.05.
The GenBank (http://www.ncbi.nlm.nih.gov) accession numbers
for DNA sequences utilized to generate primers are as follows:
NM000594 (TNF-a), NM000619 (IFN-c), NM002190 (IL-17A),
NM021803 (IL-21), NM020525 (IL-22), NM004131 (granzyme B),
FJ555237 (perforin), BC032722 (TRAIL), and NM001101 (b-actin).
RAW264.7 cells. A–C) RAW264.7 cells (106/well, 3 wells/
Acai PS before or after infection and/or treated with L-NMA
(400 mM), an iNOS inhibitor. Twenty h after infection, cells were
lysed and intracellular bacteria were enumerated A) and C). B)
Nitrite levels in cell culture supernatants were measured; error bars
represent SD.*P,0.05as compared to untreated wells,‘P,0.05 as
compared to overnight pretreatment with 100 mg Acai PS (-13 h) in
A–B), and Acai PS given 8 h after infection in C). Results are
representative of two independent experiments. NA=not applica-
ble. D) RAW264.7 cells (106/well, 3 wells/treatment) were infected
with LVS. Some wells were stimulated with Acai PS (100 mg/ml)
immediately after infection. At 4, 8, and 20 h after infection, cells
were lysed, and intracellular bacteria were enumerated. E–G) NO
bars represent SD* P,0.05 as compared to untreated wells.Results
are representative of two independent experiments.
Acai PS confers time-, dose-, and NO-
optimal for protection against high dose B. pseudomallei
with 100 or 1000 mg of Acai PS one day prior to i.n. infection with
16104CFUs of B. pseudomallei 1026b. A) Survival and B) clinical
scores were monitored over time. Error bars depict SEM. *P,0.05
as compared to PBS group. **** indicates that *P,0.05 for all Acai
PS-treated groups relative to the PBS group at the same time point.
Prophylactic Acai PS immunotherapy is
surface activation molecules in both mock- and LVS-
infected RAW264.7 cells. RAW264.7 macrophages (106/well,
3 wells/treatment) were stimulated overnight (,16 h) or not with
Acai PS prior to infection with F. tularensis LVS (MOI,300). After
20 h of infection, Median fluorescence intensity (MFI) mean from
three wells/treatment was determined via flow cytometry.
Standard error in parentheses; results are representative of two
independent experiments. *P,0.05 as compared to cells not
treated with Acai PS within same infection treatment.
Acai PS induces up-regulation of macrophage
RAW264.7 cells. RAW264.7 macrophages (106/well, 3 wells/
to infection with F. tularensis LVS (MOI,300). After 20 h of infection,
the production of cytokines and NO was determined by ELISA or the
Griess reaction. Standard error in parentheses; results are represen-
tative of two independent experiments. *P,0.05 as compared to cells
not treated with Acai PS within same infection treatment.
Acai PS induces production of proinflamma-
cytokines inboth mock-and LVS-infected
and human macrophages infected with type A F.
tularensis. RAW264.7 cells, murine BMDM, or human macro-
tularensis SchuS4 (MOI,30). Cytotoxicity was measured by LDH
release at 20 hr after infection and expressed as a percentage of LDH
release by Triton X-100 detergent. Standard deviation in parentheses.
Acai PS does not induce cytotoxicity in murine
variable protection against aerosol infection with F.
tularensis SchuS4. C57BL/6 mice were treated orally with PBS
or Acai PS before or after aerosol infection with F. tularensis
SchuS4, and survival was monitored over time.
Oral administration of Acai PS confers
We thank Dr. Catherine M. Bosio, (NIH/NIAID Rocky Mountain
Laboratories, Hamilton, MT) for kindly providing the F. tularensis LVS and
SchuS4 strains used in these studies; Dr. Herbert Schweizer (Colorado
State University) for kindly providing the B. pseudomallei 1026b strain used
in these studies; and Ms. Nancy Kommers for her assistance in preparing
Conceived and designed the experiments: JAS MAJ DWP. Performed the
experiments: JAS MFR JSH NLM. Analyzed the data: JAS MAJ DWP.
Contributed reagents/materials/analysis tools: JAS JSH IAS AG SWD.
Wrote the paper: JAS DWP.
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