Nutrition80846 123..129
The Journal of Nutrition
Nutritional Immunology
Feeding a Diet Containing
a Fructooligosaccharide Mix Can
Enhance Salmonella Vaccine Efficacy in Mice1
Jalil Benyacoub,* Florence Rochat, Kim-Yen Saudan, Isabelle Rochat, Nicolas Antille, Christine Cherbut,
Thierry von der Weid, Eduardo J. Schiffrin, and Stephanie Blum
Nestle´ Research Center, CH-1000, Lausanne 26, Switzerland
Abstract
Fructooligosaccharides (FOS) are considered prebiotics because of their ability to promote growth of specific beneficial
gut bacteria, such as bifidobacteria. Some studies reported potential immune-modulating properties. The aim of this study
was to investigate the effect of FOS:inulin mix on murine response to Salmonella vaccine and evaluate the relevance
toward protection against Salmonella infection. Balb/c mice were fed a diet containing 5% FOS:inulin mix or a control diet
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1 wk before oral immunization with a suboptimal dose of live attenuated Salmonella typhimurium vaccine. Four weeks
after vaccination, mice were infected with LD100 of virulent S. typhimurium. Specific blood Salmonella immunoglobulin G
and fecal immunoglobulin A significantly increased in mice fed the diet containing prebiotics compared with control mice
4 wk postimmunization. Peritoneal macrophage phagocytic activity also significantly increased in FOS:inulin-fed mice at
1 wk postimmunization compared with control mice. No detectable effects were observed on the percentage of lymphoid
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cell subsets in the spleen. However, production of cytokines, interferon-g, interleukin-12, and tumor necrosis factor a, was
numerically increased in spleen cell cultures stimulated with mitogens from FOS:inulin-fed mice 1 and 4 wk postim-
munization. Salmonella translocation to lymphoid organs was not affected by feeding FOS:inulin. However, the improved
response to Salmonella vaccine was concomitant with an increase in the survival rate of FOS:inulin-fed mice upon
by on February 9, 2010
challenge with virulent Salmonella. No detectable effects were observed on the composition or the metabolic activity of
the microbiota. Overall, the data suggest that a diet supplemented with FOS:inulin mix stimulates mucosal immunity and
seems to improve efficacy of an oral vaccine.
J. Nutr. 138: 123–129, 2008.
Introduction
Immune-modulating effects of FOS were reported by several
Prebiotics are defined as nondigestible food ingredients that
research groups. FOS increase leukocyte numbers in the gut
beneficially affect the host by selectively stimulating the growth
mucosa and in the blood (7) and decrease pro-inflammatory
and/or activity of 1 or a limited number of bacteria in the colon
cytokines in human patients (8). Moreover, FOS were shown
and thus improve host health (1). Fructooligosaccharides (FOS)2
to indirectly enhance T cell functions, natural killer cells, and
selectively promote the growth of bifidobacteria in the colon of
phagocytic activities through modulation of gastrointestinal
humans and may decrease the count of potentially detrimental
tract lactic acid bacteria density, which protected mice from
bacteria, thus being considered as prebiotics (1–3). Besides their
enteric and systemic pathogens and tumor inducers (9).
bifidogenic effect, short-chain FOS and long-chain FOS (such as
The importance of Salmonella infections (particularly in
inulin) have additional effects, including reducing colonic pH
developing countries) (10,11), the relatively low efficacy of the
and increasing stool weight and stool bulking, justifying their
commercially available Salmonella typhy vaccine (12,13), and
classification as dietary fibers (4). Furthermore, FOS and inulin
the increasing appearance of antibiotic-multi-resistant strains
are able to increase the bioavailability of calcium (5) and
prompted the scientific community to design new vaccine strains
possibly inhibit hepatic lipogenesis (6).
(14–16). The difficulty was to obtain a strain highly attenuated,
safe, and sufficiently immunogenic to induce a significant
1
protective response (12,17).
Author disclosures: J. Benyacoub, F. Rochat, K.-Y. Saudan, I. Rochat, N. Antille,
An alternative to improving the vaccine efficacy would be to
C. Cherbut, T. von der Weid, E. J. Schiffrin, and S. Blum, no conflicts of interest.
2 Abbreviations used: CFU, colony-forming unit; PWM, pokeweed mitogen;
use nutritional supplements that could stimulate the immune
FOS, fructooligosaccharide; IFNg, interferon-g; Ig, immunoglobulin; IL-12,
system as adjuvant. In that sense, several studies have shown
interleukin-12; MLN, mesenteric lymph node; NK, natural killer cells; PP,
that different nutritional interventions, including dietary nucle-
Peyer’s patches; PWM, pokeweed mitogen; Th1, T helper 1; TNFa, tumor
otides, probiotics, and prebiotics, could likely enhance immune
necrosis factor-a.
* To whom correspondence should be addressed. E-mail: jalil.benyacoub@rdls.
responses to various vaccines in children and adults through a
nestle.com.
T helper 1 (Th1)-like-mediated adjuvant effect (7,18–21).
0022-3166/08 $8.00 ª 2008 American Society for Nutrition.
123
Manuscript received 20 August 2007. Initial review completed 19 September 2007. Revision accepted 22 October 2007.
Salmonella has been suggested to preferentially cross the
used as control for our prebiotic nutritional intervention studies (unpub-
intestinal epithelial barrier through specialized M cells, found at
lished data).
the surface of the Peyer’s patches (PP), before disseminating to
distant sites such as the liver and spleen (22,23).
Bacterial strains and cultures. The 2 bacterial strains used were
It is well documented that host resistance to Salmonella relies
derived from the same genetic background, Salmonella typhimurium
SL1344, wild-type strain (14), used as infectious strain and its derivative,
initially on the production of inflammatory cytokines leading to
Salmonella typhimurium SL1479 Daro A attenuated strain (ATCC-
the infiltration of activated inflammatory cells in the tissue, in-
39183), was used as vaccine.
cluding interferon-g (IFNg), tumor necrosis factor-a (TNFa), and
Bacteria were grown in Luria-Bertoni broth at 37°C for 18 h and then
interleukin-12 (IL-12). Thereafter, both T and B cell-mediated
concentrated in 1 mL of PBS. The number of viable cells was determined
immune responses are elicited to control primary infection and
by agar plate counting. Identification of Salmonella was conducted using
protection against secondary challenge (24,25). In that context,
the API 20E test (BioMe´rieux SA).
secretory immunoglobulin (Ig) A produced in the intestine play a
major role in the defense mechanism against these bacteria (15,26).
Vaccination and infection protocols. The first study aimed at
The aim of this exploratory study was to investigate the
evaluating the response to vaccine, 2 groups of mice were fed a control
capacity of a FOS:inulin mix to enhance murine immune
diet or a diet containing 5% FOS mix (n ¼ 20 per group). One week after
response to Salmonella vaccine and evaluate its relevance for
starting the feeding, the 2 groups of mice were immunized by oral gavage
protection against Salmonella infection.
with 5 3 107 colony-forming units (CFU) of attenuated S. typhimurium
Our working hypothesis is that a prebiotic combination such
SL1479. This dose of Salmonella vaccine was established as conferring
as the association of FOS and inulin could exert immune-
50% protection against challenge with wild-type Salmonella strain in a
previous study (unpublished data). The immune response was monitored
enhancing functions that could be useful to protect the host
during the 4 wk postimmunization that corresponds to the peak of
against pathogens and/or could act as adjuvant for vaccines.
antibody response (14–16,20) (see Fig. 1 for trial design). Two separate
subgroups of mice from each treatment group (n ¼ 5) were killed at 1 wk
Materials and Methods
and 4 wk postvaccination to evaluate immune effects at the cellular level
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(peritoneal macrophages and splenocytes) and bacterial translocation vs.
Mice and diets. Conventional 6-wk-old female Balb/c mice (18–20 g)
survival rate in lymphoid organs, respectively.
were used in all experiments (Iffa-Credo). The mice were housed (5 mice
A second study aimed at evaluating the protection against Salmonella
per cage) under specific pathogen-free conditions at the Nestle´ research
infection, mice were randomized into 4 groups (n ¼ 20 per group).
center animal facility. Mice were fed a commercial diet (Kliba 3434)
Group control were mice fed a control diet. Group control 1 vaccine
supplemented with either 5% (wt:wt incorporated in the pellets) of a
were mice fed a control diet and vaccinated with a suboptimal dose of 5
prebiotic mix (70% Raftilose P95–30% Raftiline HP wt:wt in the mixed
3 107 CFU of attenuated Salmonella SL1479 prior to infection (as
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powder preparation, both from Orafti) or cellulose, hereafter called FOS
described above). Group FOS consisted of mice fed a diet containing
mix or control, respectively. The macronutrient content as fed to mice
FOS mix, and Group FOS 1 vaccine consisted of mice fed a diet
was 17.6% protein, 4.3% fat, 4.3% crude fiber, 51.5% carbohydrate,
containing FOS mix and vaccinated prior infection (Fig. 1).
and 13.5 kJ/g metabolizable energy. The dose of prebiotics or cellulose
All groups of mice were infected 4 wk after vaccination or equiv-
corresponds to a daily intake of 8–10 mg/g of body weight. Such an
alent period (for unvaccinated mice) with 3 3 107 CFU per mouse of
by on February 9, 2010
amount of cellulose has never shown any effect on intestinal ecology or
S. typhimurium SL1344 by oral gavage. This dose of virulent Salmonella
immunity in our previous animal studies, which is why it is commonly
was established as the LD100 in a previous study (unpublished data). The
FIGURE 1
Trial design of control and treatment groups. Study 1: 2 groups were tested (mice fed a control diet or the same diet containing
FOS-mix). Study 2: four groups were tested. Control, mice fed a control diet. Control + vaccine, mice fed a control diet and vaccinated. FOS, mice
fed a diet containing FOS-mix. FOS + vaccine, mice fed a diet containing FOS-mix and vaccinated.
124
Benyacoub et al.
infection was followed daily during 3 wk, including weighing and mac-
PP, mesenteric lymph nodes (MLN), spleen, and liver were
roscopic examinations. Criteria justifying euthanasia of mice included
asceptically collected, weighted, and homogenized in 2 mL of sterile
bristled coat, loss of weight (.20%), and loss of mobility. Mice were
PBS. The number of viable bacteria in each lymphoid organ was
killed by cervical dislocation under anesthesia with isofluorane.
determined by plating 0.1 mL of processed tissue on Luria-Bertoni agar.
Salmonella O antiserum (Becton Dickinson) was used for agglutination
Assessment of antibody responses. Blood samples were collected
test to complete the identification of S. typhimurium. The minimal
from the tail vein of mice of study 1 just before vaccination and at 4 wk
amount of Salmonella detectable is 20 CFU per total organ that
postimmunization. Samples were incubated overnight at 4°C and
corresponds to 1 colony per plate. Therefore, the limit of detection
centrifuged at 10,000 3 g; 15 min. Sera were collected and frozen at
expressed in the results is log10 1.3.
220°C until analysis for IgG.
Fresh fecal samples were collected just before vaccination and at 4 wk
Analysis of microbiota. Fecal samples were collected from mice in
postimmunization. Mice were individually isolated in a clean cage and
study 1 24 h and 1 wk after immunization to analyze specifically
fecal samples were collected right after emission. Samples were homog-
Salmonella content. In addition, fecal samples from the same mice were
enized with extraction buffer (50 mmol/L EDTA, 100 mg/L soybean
collected just before immunization (1 wk) and 4 wk after to evaluate
trypsin inhibitor in PBS; Sigma). After centrifugation at 10,000 3 g;
microbiota composition. Freshly collected feces were homogenized in
15 min, the supernatants were given doses for protein contents by
Ringer medium containing 10% glycerol, and enterobacteria, lactoba-
BCA protein assay (Pierce) and stored at 220°C until analysis for IgA.
cilli, bifidobacteria, and enterococci or Salmonella, when specifically
Total antibody levels (IgA in fecal extract or IgG in sera) were
evaluated, were immediately enumerated by plating on semiselective
measured by ELISA as previously described (27). A monoclonal mouse
media following the method previously described (8).
IgA or mouse IgG was used as a standard and results were then expressed
as the means 6 SEM of IgA or IgG in mg/L (the detection limits for both
Statistical analysis. Statistical analyses were performed to evaluate dif-
antibodies were 2 mg/L). The fecal IgA data were normalized to the
ferences between treatment groups of mice (mean values for each group
protein content of each fecal extract.
represent the experimental unit for this nutritional intervention study).
The amounts of specific anti-Salmonella IgA and IgG in feces were
In study 1, differences among means (control vs. FOS) were evaluated
determined using the same ELISA except that the plates were coated with
for each variable within a sampling period using a 2-tailed Student’s
either 5 mg per well of Salmonella-LPS (Sigma) or 10 mg per well of
t test. The antibody responses were further tested for changes over time
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Salmonella-flagellin extracted as previously described (28,29). Briefly,
using a 1-way ANOVA with repeated measures. Before each analysis,
bacteria were grown overnight and flagellin was isolated by acidic
data were tested for normal distribution and homoscedasticity.
denaturation for 30 min at room temperature. Supernatant containing
In study 2, differences among survival rates at 3 wk postinfection
depolymerized flagella was clarified by centrifugation at 100,000 3 g;
(control vs. FOS vs. control 1 vaccine vs. FOS 1 vaccine) were evaluated
1 h at 4°C and equilibrated at pH 8.0. Flagellin was identified by SDS-
using a chi-square test for independence. A confidence level of 95% was
PAGE using the anti-flagellin monoclonal antibody 15D8 (IGEN
applied for all tests. Values in the text are means 6 SEM.
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International). The data were expressed as mean OD at 450 nm 6 SEM.
Assessment of phagocytic activity. Subgroups of control and FOS
Results
mix-fed mice from study 1 were killed 1 wk and 4 wk postimmunization
(n ¼ 5 for each group at each time point). Peritoneal macrophages were
Antibody responses (study 1). Neither total IgG levels in the
by on February 9, 2010
collected in ice-cold sterile PBS. Macrophages were seeded into 48-well
sera nor IgA levels in the fecal contents were influenced by FOS
microtiter plates at a concentration of 5.105 cells per well and mixed with
mix supplementation (data not shown).
fluorescent E. coli (E. coli:FITC) for 30 min (Phagotest-kit, Orpegen).
There were no differences in the specific anti-Salmonella
Fluorescent macrophages, reflecting the phagocytic activity, were analyzed
by flow cytometry according to the manufacturer’s recommendations.
antibody responses between the 2 groups at 1 wk postimmuni-
zation. The responses then significantly increased in both groups
Assessment of cell subsets, cell proliferation, and cytokine
at 4 wk postimmunization. However, this increased anti-
production. Spleen samples from subgroups of control and FOS mix-
Salmonella response was much stronger in the FOS mix group
fed mice from study 1, killed at 1 and 4 wk postimmunization (as
compared with controls. Indeed, the specific-LPS-IgA levels in
described in the section above), were analyzed (n ¼ 5 for each group at
the fecal contents were greater in the FOS mix-fed group than in
each time point). Spleens were collected, homogenized through a cell
the control group (P ¼ 0.04) 4 wk after vaccination (Fig. 2A).
strainer (70 mm) in 2 mL complete RPMI-1640 medium (Gibco), and
The amount of specific flagellin-IgG in the plasma was
centrifuged at 250 3 g; 5 min. The pellets were rapidly lysed with 1 mL
greater in the FOS mix-fed group than in the control group (P ¼
of sterile distilled water and centrifuged at 250 3 g for 5 min. The cells
0.01) 4 wk after vaccination (Fig. 2B).
pellets were then suspended in complete RPMI-1640 medium to obtain
109 cells/L. Cell subsets were quantified by 2-color flow cytometry
Stimulation of phagocytic activity (study 1). The phagocytic
according to the method previously described (27). Spleen cell prolifer-
ation assay was performed according to the method described by Humen
activity of peritoneal cells 1 wk postimmunization was higher in
et al. (30). Proliferation rates were evaluated on unstimulated cells or
the FOS mix-fed group (52.5 6 0.7%) than in the control group
cells stimulated with the following mitogens (all purchased from Sigma):
(38.1 6 3.3%; P ¼ 0.02). After 4 wk, phagocytic activity in the
2 mg/L concanavalin A (T cell stimulant), 1 mg/L pokeweed mitogen
FOS mix-fed group (40.5 6 0.4%) had returned to the level in
(PWM; B cell stimulant), or 2.5 mg/L LPS from S. typhimurium
the controls (39.3 6 0.6%).
(monocyte and B cell stimulant). After incubation at 37°C for 72 h, 0.15
mL of supernatant was collected for IL-12, INFg, and TNFa cytokine
Spleen cell subsets, cell proliferation, and cytokine pro-
content analysis using ELISA kits purchased from R&D Systems
duction (study 1). The percentage of cell subsets, including
according the manufacturer’s recommendations. As specified in the kit,
CD41, CD81, B2201, MHCII1, CD11b1, and CD11c1 cells,
the detection limits were 2.5 ng/L, 2 ng/L, and 5.1 ng/L, respectively.
did not differ between FOS mix- and control mice. However, the
mean fluorescent intensity of MHCII1 cells was greater in FOS
Quantification of Salmonella in lymphoid organs. As indicated
above in the section ‘‘Vaccination and infection protocols,’’ subgroups of
mix-fed mice (135 6 29.5) than in controls (89 6 21.4; P ¼ 0.03).
control and FOS mix-fed mice from study 1 were killed at 1 and 4 wk
Feeding the FOS mix did not significantly influence the spleno-
postimmunization, which correspond to the peak of Salmonella coloni-
cyte proliferation rates (data not shown). However, IL-12, IFNg,
zation and survival limit, respectively (31).
and TNFa cytokine concentrations in cell culture supernatants
Stimulation of vaccine response by fructooligosaccharides
125
FIGURE 2
Specific anti-LPS fecal IgA (A)
and anti-flagellin serum IgG (B) measured
1 and 4 wk postvaccination in mice (study 1).
Data are means OD 450 nm 6 SEM, n ¼
10 duplicates. Fecal samples were diluted
1:20 and serum samples, 1:200 for sera.
*Different from controls at that time, P ,
0.05. #Different from the initial value, P ,
0.05.
from FOS mix-fed mice tended to be higher than in controls
attenuated Salmonella led to 40% protection (Fig. 3). This
when stimulated with mitogens at 1 and 4 wk postimmuniza-
protection rate improved to 73% upon feeding FOS mix (P ¼
tion. IFNg and IL-12 levels were significantly enhanced in mice
0.04). In most cases, Salmonella infection leads to deaths
fed FOS mix compared with controls at both time points when
between 7 and 15 d postinfection in nonprotected animals.
splenocytes were stimulated with LPS (Table 1).
During this period, the differences between the groups were in
agreement with the ones described above at 3 wk postinfection.
Recovery of Salmonella from lymphoid organs (study 1).
The number of Salmonella recovered from PP, MLN, liver, and
spleen at 1 wk after immunization did not differ between the 2
Discussion
groups of mice with mean log10 CFU/g of 4 6 0.2 in PP, 2.7 6
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0.1 in MLN, 2.3 6 0.1 in liver, and 1.5 6 0.3 in spleen. The
This study shows that a FOS mix (FOS:inulin, 70:30 wt:wt in the
numbers of salmonella decreased by more than 1 log in all
mixed powder preparation) is able to improve the immune
organs in both groups at 4 wk postimmunization (data not
response to a Salmonella vaccine that contributes to increased
shown).
vaccine efficacy. The idea of using foods as booster and/or carrier
for human vaccine has been proposed in several studies
Microbiota analysis (study 1). There was no significant effect
[reviewed by Korban et al. (32)]. However, these approaches
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of FOS mix on microbiota composition compared with controls.
mainly consist of using genetically modified products, the use of
The fecal amounts of SCFA, including acetate, propionate, and
which may be subject to regulatory issues in some countries. In
butyrate, were not significantly different between the 2 groups
this work, the data support the concept of using nongenetically
(data not shown). Salmonella were rapidly cleared after immu-
modified food supplements containing a FOS:inulin mix to
nization with a similar ;2 log bacterial loss in both groups at
enhance vaccine efficacy.
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24 h postimmunization (log10 CFU/g of feces of 5.4 6 0.4 in con-
It was previously established that Salmonella strains mutated
trol mice and 6 6 0.1 in FOS mix group) and barely detectable
in genes involved in the aromatic biosynthetic pathway (aro
levels at 4 wk postimmunization (log10 CFU/g of feces of 3.3 6
mutants) were effective vaccines in several animal species
0.1 in control mice and 2.8 6 0.1 in the FOS mix group).
(14,33,34). Immunization with Salmonella elicits a broad range
of antigen-specific responses. In that respect, LPS and flagellin
Protection against Salmonella infection (study 2). The
have gained particular interest as potential protective antigens
survival rate in the control group was ;10% at 2 wk postin-
(35–37). Therefore, both mucosal and systemic responses
fection. This rate remained the same after 3 wk. FOS mix alone
toward LPS and flagellin were investigated in this study.
was not able to protect mice from Salmonella infection as
We used a suboptimal dose of Salmonella vaccine to measure
demonstrated by the survival rate that was similar to the control
the potential effect of the dietary intervention. The low oral dose
group (i.e. 10% at the same time point). As expected, vaccina-
used in this study was still able to elicit detectable antibody titers,
tion of mice fed a control diet with a suboptimal dose of
as observed in control mice. Specific anti-Salmonella antibody
TABLE 1
Cytokine concentrations in cell culture supernatants of splenocytes from mice fed FOS
mix or controls stimulated with different mitogens (Study 1)1
Wk 1
Wk 4
Cytokines
ConA
PWM
LPS
ConA
PWM
LPS
IL-12
ng/L
Control
24 6 5
18 6 5
47 6 9
42 6 2
12 6 5
25 6 6
FOS
35 6 10
25 6 2
51 6 7
64 6 2*
27 6 9*
26 6 9
TNFa
Control
174 6 24
371 6 40
1091 6 189
286 6 37
569 6 63
843 6 117
FOS
181 6 43
337 6 12
1209 6 102
357 6 34
722 6 61
857 6 114
IFNg
Control
3132 6 228
5608 6 476
404 6 166
3919 6 389
4822 6 588
40 6 19
FOS
4278 6 729
6507 6 39
935 6 207*
5130 6 508
5785 6 491
210 6 55*
1 Values are means 6 SEM, n ¼ 5 triplicates. *Different from controls, P , 0.05.
126
Benyacoub et al.
translocation or the survival rates of Salmonella. These data are
in contradiction with results previously published by Bruggen-
cate et al. (40) and Bovee-Oudenhoven et al. (41). The authors
suggest that feeding FOS (Raftilose P95) could increase Salmo-
nella translocation in rats in a dose-dependent manner. Whether
these discrepancies could be attributed to a difference in the
doses of prebiotics or a difference in Salmonella strains used in
the 2 studies could be questioned. We cannot exclude that
differences in macronutrient and micronutrient contents of the
diets used in the 2 studies could had differently impacted the
prebiotic effect on the host. We did not observe any signs of
immune suppression or other potential side effects in our study
upon feeding with FOS mix. The dose used in this study corre-
sponds well with the assumed dose of nondigestible carbohy-
drates consumed by humans, which is ;3–5% of daily dry food
intake (42).
The safety of FOS and the FOS:inulin mix is further sup-
ported by a few clinical trials in infants where no adverse effects
have been observed. Importantly, no Salmonellosis has been
FIGURE 3
Survival rate of mice that were fed a diet containing FOS
reported so far upon feeding with prebiotics such as FOS and/or
mix or a control diet and were vaccinated or not with an attenuated
inulin in humans (4,20,43).
Salmonella prior to an infection with virulent Salmonella at 4 wk after
We wanted to evaluate if feeding the FOS mix could either have
immunization or equivalent period for unvaccinated mice (study 2).
a direct protective effect or confer indirect protection through an
Data are expressed as the percentage of survival, n ¼ 20 for each group.
improved response to vaccine. Unvaccinated mice were not
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*Different from control 1 vaccine at that time, P , 0.05. #Different from
nonvaccinated mice, P
protected regardless of diet, suggesting that the FOS mix alone
, 0.05.
was not able to protect mice from Salmonella infection. These
results do not corroborate previous data reported by Buddington
responses were significantly enhanced in mice fed FOS mix
et al. (9) showing that feeding Raftilose P95 or inulin protected
compared with control mice, suggesting that a diet containing
mice from systemic Salmonella infection and prevented C. albicans
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FOS mix might act as a mucosal adjuvant. One previous clinical
translocation. This difference in outcomes might be explained by
trial has shown that the same FOS mix incorporated into cereals
the differences in the doses of prebiotics used. However, differ-
significantly improved the response to measles vaccine in infants
ences in the diet composition and the routes of infections could also
(20). Nevertheless, these data should be confirmed and the
explain the results in the 2 studies.
capacity of this prebiotic to trigger and boost systemic responses
Our results showed that there was an improvement in the
by on February 9, 2010
to vaccines needs further investigation.
vaccine protection rate upon feeding with the FOS mix (from 40
Uptake of Salmonella by phagocytic cells after the bacteria
to 73%). These results correlate well with the relative increase
have crossed the epithelium represents a key feature of the innate
in the Th1 cytokine production and the high vaccine-specific
response of the host to Salmonella infection (22,23). In this
immune response observed in FOS mix-fed mice at the time of
study, the percentage of peritoneal cell phagocytic activity was
challenge, i.e. 4 wk postimmunization, and are in agreement
transiently but significantly enhanced in the FOS mix-fed group
with the well-documented important role played by mucosal
compared with controls (P , 0.01 at 1 wk postimmunization).
LPS-IgA and potentially systemic flagellin-IgG in the prevention
Along with this observation, the phagocytic activity as previously
against Salmonella infection (35–37,44,45).
tested in vitro was significantly enhanced after treatment of
It is postulated that clinical benefits from consumption of
murine macrophage cell line J774 with 10 mg/L of FOS mix
prebiotics are obtained through their effect on the colonic micro-
compared with nontreated cells (P ¼ 0.02), reflecting the
biota. Improved growth of lactic acid bacteria upon prebiotic
capacity of FOS mix per se to interact with immune cells (data
feeding leads to a decrease in the pH that could contribute to the
not shown). The IL-12, IFNg, and TNFa cytokines are known to
protection against pathogens (1,2,46). It is also assumed that
play a key role in the regulation of both innate and acquired
modifications in gut microbiota induced by prebiotics such as
immunity to Salmonella (24,38). The relative increase in these
FOS may ultimately affect immune functions [reviewed by Schley
cytokine levels, mainly IL-12 and IFNg, seems to indicate an
and Field (7)].
activation of T and B cells as well as macrophages. Moreover, the
We attempted to highlight the potential mechanism by which
enhanced mean fluorescent intensity of MHCII molecules upon
the FOS mix could influence the immune system. No detectable
feeding with FOS mix suggests a stimulation of antigen presen-
effects were observed on microbiota composition or on SCFA
tation in vivo. These results reflect the capacity of FOS mix to
fecal concentrations (data not shown). The prebiotic effect of the
promote a Th1-like adjuvant effect that might have supported the
FOS:inulin mix, well studied in humans and other animals as
strong specific humoral responses observed in FOS mix-fed mice.
indicated above (1,2,4,7,20,46), could not be featured in this
The ability of S. typhimurium to translocate into lymphoid
model; however, because of limitations of the methods used in
organs is known to be one of the major steps of its pathogenicity
this study, we could not exclude it. Indeed, analysis of SCFA in
(39). In agreement with a previous publication (31), the DaroA
the fecal content only partially reflects the real colonic metab-
mutant strain SL1479 was not impaired in its ability to colonize
olism situation and the bacterial count on semiselective agar
and survive in lymphoid organs. The number of Salmonella
media presents some limitations in sensitivity. Thus, evaluation
recovered from PP, MLN, liver, and spleen at 1 wk after
of the microbial ecology with other techniques such as fluores-
immunization did not differ between the 2 groups of mice,
cent in situ hybridization or denaturing gradient gel electropho-
suggesting that FOS mix per se did not influence either the
resis might help to better study the effect of FOS mix.
Stimulation of vaccine response by fructooligosaccharides
127
The mechanism by which FOS impacts immunity still needs
19. Pickering LK, Granoff DM, Erickson JR, Masor ML, Cordle CT,
further investigation. Nevertheless, we can speculate on a pos-
Schaller JP, Winship TR, Paule CL, Hilty MD. Modulation of the
immune system by human milk and infant formula containing nucle-
sible direct interaction of the FOS mix with macrophages and/or
otides. Pediatrics. 1998;101:242–9.
dendritic cells underlying the gut mucosa either via a transfer
20. Haschke F, Firmansyah A, Meng M, Steenhout P, Carrie´ A-L.
through the epithelium or a direct sampling in the lumen by den-
Functional food for infants and children. Monatsschr Kinderheilkd.
dritic cells, that can penetrate the gut epithelial monolayer (47,48).
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