Nasally Administered Lactobacillus rhamnosus Accelerate the Recovery of Humoral Immunity in B Lymphocyte-Deficient Malnourished Mice

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DOI: 10.3945/jn.112.165811 · Source: PubMed
Abstract
The ability of nasally administered Lactobacillus rhamnosus CRL1505 to accelerate the recovery of respiratory B cell-mediated immunity against pneumococcal infection in replete malnourished mice was evaluated. Weaned mice were malnourished after consumption of a protein-free diet for 21 d. Malnourished mice were fed a balanced conventional diet (BCD) for 7 d (BCD group) or a BCD for 7 d with supplemental L. rhamnosus CRL1505 by the nasal route during the last 2 d (BCD+Lr group). Nonreplete malnourished and normal mice were used as the malnourished (MNC) and the well-nourished (WNC) control groups, respectively. Mice were challenged with Streptococcus pneumoniae at the end of each dietary treatment. The immune response was studied before the challenge and at different times postinfection. The MNC mice had less resistance to pneumococcal infection, fewer mature and immature B cells in lung and spleen, and a reduced production of specific antibodies compared with WNC mice. The BCD treatment did not induce a complete normalization of the number B cell populations and antibody amounts. However, the BCD+Lr group had normal numbers of spleen and lung B cells. Moreover, the BCD+Lr mice had a significantly lower susceptibility to S. pneumoniae infection and higher amounts of anti-pneumococcal antibodies. Although further studies are necessary to clarify the effect of malnutrition and nasally administered lactobacilli in other immune cell populations involved in the protection against respiratory pathogens, this work gives evidence of the importance of using nasal priming with probiotics to accelerate the recovery of respiratory immunity in immunocompromised malnourished hosts.

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Available from: Natalia Barbieri, Jan 28, 2016
The Journal of Nutrition
Nutritional Immunology
Nasally Administered Lactobacillus rhamnosus
Accelerate the Recovery of Humoral Immunity in
B Lymphocyte-Deficient Malnourished Mice
1–3
Natalia Barbieri,
4
Julio Villena,
4
Matias Herrera,
4
Susana Salva,
5
and Susana Alvarez
4,5
*
4
Reference Centre for Lactobacilli (CERELA-CONICET), Tucum´
an,Argentina;and
5
National University of Tucuman,
Tuc um ´
an, Argentina
Abstract
The ability of nasally administered Lactobacillus rhamnosus CRL1505 to accelerate the recovery of respiratory B cell-
mediated immunity against pneumococcal infection in replete malnourished mice was evaluated. Weaned mice were
malnourished after consumption of a protein-free diet for 21 d. Malnourished mice were fed a balanced conventional diet
(BCD) for 7 d (BCD group) or a BCD for 7 d with supplemental L. rhamnosus CRL1505 by the nasal route during the last 2 d
(BCD+Lr group). Nonreplete malnourished and normal mice were used as the malnourished (MNC) and the well-nourished
(WNC) control groups, respectively. Mice were challenged with Streptococcus pneumoniae at the end of each dietary
treatment. The immune response was studied before the challenge and at different times postinfection. The MNC mice
had less resistance to pneumococcal infection, fewer mature and immature B cells in lung and spleen, and a reduced
production of specific antibodies compared with WNC mice. The BCD treatment did not induce a complete normalization
of the number B cell populations and antibody amounts. However, the BCD+Lr group had normal numbers of spleen and
lung B cells. Moreover, the BCD+Lr mice had a significantly lower susceptibility to S. pneumoniae infection and higher
amounts of anti-pneumococcal antibodies. Although further studies are necessary to clarify the effect of malnutrition and
nasally administered lactobacilli in other immune cell populations involved in the protection against respiratory pathogens,
this work gives evidence of the importance of using nasal priming with probiotics to accelerate the recovery of respiratory
immunity in immunocompromised malnourished hosts. J. Nutr. 143: 227–235, 2013.
Introduction
The respiratory pathogen Streptococcus pneumoniae is responsible
for most cases of meningitis and pneumonia in young children and
of otitis media in infants (1). An increased frequency and severity
of infections by S. pneumoniae and other encapsulated bacteria
(Neisseria meningitidis,Haemophilus influenzae) is the first and
most important symptom of primary B cell immunodeficiency and
a sign of AIDS progression in HIV-infected children (2). In addition,
pneumococcal diseases are 20–100 times more frequent in
individuals with asplenia, splenectomy, and sickle-cell disease
(3). Moreover, despite appropriate therapies, mortality due to
the different pneumococcal pathologies remains high in immu-
nocompromised malnourished children; ;1 million children die
every year from pneumococcal diseases, mainly in developing
countries (4,5).
Malnutrition suppresses immune function and confers a
higher susceptibility to infectious diseases. Indeed, nutritional
deprivation induces atrophy of lymphoid tissues such as spleen
and thymus and decreases the number of circulating Tand B cells
(6). In this sense, we recently reported that protein malnutrition
induces a significant reduction in bone marrow (BM)
6
cell
compartments, which is reflected in a decrease of B cells (7).
Moreover, when we investigated the effect of nutritional
deprivation on B cell populations in BM, we observed that the
number of B220
+
cells (the whole B cell compartment) was
reduced in the BM of malnourished mice (7). In parallel with the
total B cell decrease, the proportion of the different B cell
subsets was markedly altered in malnourished mice. We ob-
served that pro-B/pre-B (B220
interm
IgM
2
) and immature B cell
(B220
interm
IgM
+
) numbers were lower in feed-deprived mice. The
reduction of immature B cells was accompanied by an increase
in the percentage of mature B cells (B220
high
IgM
+
) but not by
1
Supported by grants from PIP-632-2009, CIUNT-26D/403, and PICT-2010-1381.
2
Author disclosures: N. Barbieri, J. Villena, M. Herrera, S. Salva, and S. Alvarez,
no conflicts of interest.
3
Supplemental Figures 1–6 are available from the ‘‘Online Supporting Material’’
link in the online posting of the article and from the same link in the online table of
contents at http://jn.nutrition.org.
* To whom correspondence should be addressed. E-mail: salvarez@cerela.org.
ar.
6
Abbreviations used: BAL, bronchoalveolar lavage; BCD, balanced conventional
diet; BCD+Lr, balanced conventional diet plus nasally administered Lactobacillus
rhamnosus CRL1505; BM, bone marrow; LAB, lactic acid bacteria; MNC,
malnourished control; PFD, protein-free diet; p.i., postinfection; WNC, well-nourished
control.
ã2013 American Society for Nutrition.
Manuscript received June 26, 2012. Initial review completed July 6, 2012. Revision accepted November 11, 2012. 227
First published online December 26, 2012; doi:10.3945/jn.112.165811.
by guest on October 21, 2015jn.nutrition.orgDownloaded from
1.DCSupplemental.html
http://jn.nutrition.org/content/suppl/2013/02/08/jn.112.16581
Supplemental Material can be found at:
changes in the total number of mature B cells. These observations
suggest that nutritional deprivation leadsto the alteration of B cell
development in the BM (7).
During the last few decades, a large body of literature
established strong links among nutrition, immune function, and
infectious diseases. It was demonstrated that one of most
important strategies for the prevention of infectious diseases is to
improve healthy nutrition. Lactic acid bacteria (LAB) can be
used for this strategy. LAB strains able to modulate the immune
system (immunobiotics) (8,9) have been used to improve
intestinal and respiratory immunity. In our laboratory, several
Lactobacillus strains isolated from goatÕs milk were evaluated
according to their capacity to modulate respiratory immunity
and we found that Lactobacillus rhamnosus CRL1505, admin-
istered by the oral route at the proper dose, was able to increase
S. pneumoniae clearance rates in lung and blood, reduce lung
injuries, and increase the survival of infected mice (10). We also
demonstrated that the protective effect of the CRL1505 strain
can be achieved in immunocompromised malnourished mice
and that it was related to an upregulation of both innate and
specific immune responses in the respiratory tract (11). To
elucidate the immunological mechanisms involved in the
increased resistance to pneumococcal infection induced by
L. rhamnosus CRL1505, we performed studies of B cell popu-
lations in BM. We observed that the alteration ofB lineage cells in
the BM of malnourished mice was reverted by the treatment with
L. rhamnosus CRL1505 (7). A remarkable finding of our work
was that oral administration of L. rhamnosus CRL1505 was able
to normalize the number of immature B cells (7).
Considering that nasally administered antigens can induce
respiratory and systemic immune responses superior to those
obtained using oral stimulation (12), researchers more recently
focused on the ability of nasal stimulation with immunobiotics
to improve respiratory immune responses (13). Some studies
demonstrated that nasal administration of LAB is able to
improve respiratory immunity and significantly increase the
resistance of immunocompetent mice against influenza virus
(14), S. pneumoniae (15), and lethal pneumovirus infections
(16). Moreover, we have evaluated whether the nasal adminis-
tration of heat-killed immunobiotics during recovery of mal-
nourished mice could improve respiratory immunity. Our results
showed for the first time, to our knowledge, that nasal
administration of heat-killed Lactobacillus casei CRL431 sig-
nificantly increases the resistance of malnourished mice against
respiratory pathogens (17).
The ability of viable LAB strains when nasally administered
to immunocompromised mice to stimulate respiratory immunity
has not, to our knowledge, been studied before. Thus, the aims
of the present work were to deepen the knowledge of the effect
of malnutrition on systemic and respiratory B lymphocyte
populations and to evaluate the effectiveness of nasal adminis-
tration of L. rhamnosus CRL1505 to enhance B cell-mediated
immunity and the humoral immune response to pneumococcal
infection in replete, malnourished, immunocompromised mice.
Materials and Methods
Microorganism. Lactobacillus rhamnosus CRL1505 was obtained
from the CERELA culture collection. Lactobacilli (stored at 270°C) was
activated and cultured for 12 h at 37°C (final log phase) in Man-Rogosa-
Sharpe broth. The bacteria were harvested by centrifugation and washed
with sterile 0.01 mol/L PBS, pH 7.2 (7). Capsulated Streptococcus
pneumoniae was isolated from the respiratory tract of a patient from the
ChildrenÕs Hospital (Tucuman-Argentina).
Mice and treatment procedures. Male, 3-wk-old, Swiss-albino mice
were obtained from CERELA. Weaned mice were fed a protein-free diet
(PFD) for 21 d and the mice that weighed 45–50% less than the well-
nourished mice were selected for the experiments (18). Malnourished
mice were divided into 2 groups for treatments: mice were fed for 7 d
with a balanced conventional diet (BCD; BCD group) or BCD for 7 d;
during the last 2 d, the mice received L. rhamnosus CRL1505 (10
8
cells mouse
21
d
21
) by the nasal route (BCD+Lr group) (Fig. 1). The
dose of L. rhamnosus was chosen on the basis of preliminary experi-
ments (J. Villena, S. Salva and S. Alvarez, unpublished results). A third
group of malnourished mice was used as the malnourished control group
(MNC). The MNC mice received only a PFD during experiments.
Normal mice were used as the well-nourished control (WNC) group. The
WNC mice consumed ad libitum only the BCD during experiments. The
compositions of the BCD and PFD diets were previously described (18).
Experiments with mice were approved by the CERELA Ethical Com-
mittee of Animal Care (protocol BIOT-CRL-10).
Cellular recovery. Following thoracotomy, a right heart catheterization
was performed and the pulmonary circulation was perfused with saline-
EDTA to remove intravascular cells. Lungs were removed, minced, and
incubated in digestion medium for 1 h at 37°C. The digestion medium
consisted of RPMI-1640 supplemented with 5% FBS and 140 kU/L
collagenase type I (Sigma). Subsequently, the samples were homogenized
through a tissue strainer with RPMI 1640 with 5% FBS. Finally, samples
were subjected to RBC lysis (Tris-ammonium chloride, BD PharMingen)
washed in FACS buffer (PBS with 2% FBS, Gibco) and passed through a
50-mm cell-strainer.
Spleens were collected and tissue was homogenized through a tissue
strainer with RPMI 1640 with 2% FBS, followed by incubation with
lysis buffer to eliminate erythrocytes (7). Isolated cells were suspended in
FACS buffer, counted on a hemocytometer, and kept on ice until
immunofluorescent labeling. Viability of the cells was assessed through
Trypan blue exclusion.
Flow cytometry. Spleen or lung cells were preincubated with anti-
mouse CD32/CD16 monoclonal antibody (Fc block) and stained
with the following antibodies from BD PharMingen: fluorescein
isothiocyanate-labeled anti-mouse IgM, fluorescein isothiocyanate-
labeled anti-mouse CD19, PE-labeled anti-mouse CD24, biotinylated
anti-mouse B220, and biotinylated anti-mouse IgD antibodies.
Following incubation with biotinylated primary mAbs, the labeling
was revealed using streptavidin-Peridinin Chlorophyll-a Protein (SAv-
PerCp). In all cases, cells were then acquired on a BD FACSCaliburTM
flow cytometer (BD Biosciences) and data were analyzed with FlowJo
software (TreeStar). The number of cells in each population was
determined by multiplying the percentages of subsets within a series of
marker negative or positive gates by the total cell number determined for
each tissue.
Pneumococcal infection. Challenge with S. pneumoniae was carried
out on the day after the end of each treatment (d 8) by dropping 25 mLof
the inoculum containing 10
5
log-phase cells of S. pneumoniae in PBS into
each nostril (17,18). Survival of the infected mice was monitored for 15 d.
Bacterial cell counts in lung were performed on d 2, 5, 10, and 15
postinfection (p.i.) as previously described (17,18). Results were
expressed as log of CFU/g of lung. In addition, whole-lung samples
from control and infected mice were excised and immersed in 4%
paraformaldehyde and processed by standard histological techniques
(17,18). Samples were stained with hematoxylin-eosin for light micros-
copy examination.
Serum and broncho-alveolar lavages antibodies. Anti-pneumococcal
antibodies (IgA, IgM, and IgG) were determined by ELISA on d 10 p.i
(18). In brief, plates were coated with a 1:100 dilution of heat-killed S.
pneumoniae overnight at 4°C and blocked with PBS containing 5%
nonfat dry milk. Appropriate dilutions of the samples [serum 1:20;
bronchoalveolar lavage (BAL) 1:2] were incubated for 1 h at 37°C.
Peroxidase conjugated anti-mouse IgG, IgA, or IgM (1:500) (Sigma-
Aldrich) was added and incubated for 1 h at 37°C. The reaction was
228 Barbieri et al.
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developed with TMB Substrate Reagent (Sigma-Aldrich). The concen-
tration was measured with reference to standard curves using known
amounts of the respective murine Ig (Sigma-Aldrich).
Functional activity of antibodies. The opsonophagocytic activity of
BAL and serum antibodies was determined by measuring the killing
of live pneumococci by peritoneal macrophages in the presence of
antibodies and complement as previously described (19). For the
measurement of IgG, IgA, and IgM antibody avidity the ELISA-
NaSCN technic was used (19).
Number and activity of phagocytes. The number of blood and BAL
leukocytes was determined by using a hemocytometer as previously
described (19). The myeloperoxidase activity of blood and BAL
neutrophils was carried out using a cytochemical method with benzidine
as a myeloperoxidase chromogen. Cells were graded as negative or
positive weak, moderate, or strong and were used to calculate the score
(15). The phagocytic bactericidal activity (oxidative burst) of macro-
phages and neutrophils was measured using the nitroblue tetrazolium
reduction test (Sigma) (19).
Statistical analysis. Experiments were performed in duplicate and the
results were expressed as mean 6SD. Statistical analysis was conducted
using MINITAB software (version 15 for Windows). Two-factor
ANOVA was used to test the effects of experimental group, time (d 0
and d p.i), and their interaction. TukeyÕs post hoc test was used to test for
differences between the mean values. Significance was set at P< 0.05.
Survival statistics were performed using the log-rank test to compare
different Kaplan-Meier curves.
Results
L. rhamnosus CRL1505 accelerates the recovery of B cell
populations. Malnutrition significantly reduced spleen weight
and cellularity (Tabl e 1). The number of spleen lymphocytes and
CD19
+
B220
+
cells (the whole B cell compartment) in MNC
mice were lower than those observed in the WNC group. The
BCD group had higher numbers of spleen lymphocytes and
CD19
+
B220
+
cells compared with the MNC mice; however, the
amounts of cells did not reach the values of the WNC group. The
number of CD19
+
B220
+
cells was greater in the BCD+Lr group
than in the BCD group (Table 1; Supplemental Fig. 1). In addition,
B cell subpopulations were studied with CD19, B220, CD24,
IgM, and IgD antibodies (Fig. 2;Supplemental Fig. 2). The MNC
mice had lower numbers of both mature (CD19
+
B220
high
C-
D24
low
) and immature (CD19
+
B220
low
CD24
high
) spleen B cells
than the WNC mice. Both BCD and BCD+Lr treatments
augmented the number of spleen CD19
+
B220
high
CD24
low
B
cells, but they were unable to normalize this cell population.
CD19
+
B220
low
CD24
high
B cell numbers were higher in both the
BCD and BCD+Lr mice compared with the MNC group;
however, only BCD+Lr mice had values similar to those ob-
served in the WNC group (Fig. 2A). The spleen is a hematopoietic
organ in adult mice (20,21). Immature B cells (IgM
+
B220
low
)exit
the BM and develop and mature into ‘‘transitional’’ T1 B cells
(CD21
2
B220
+
CD24
high
IgD
2
IgM
high
), T2 B cells (CD21
+
B220
+
CD24
high
IgD
+
IgM
high
), and mature (CD21
+
B220
+
CD24
low
IgD
+
IgM
low
) B cells within spleen (3,22). A significant reduction of all
the B cell populations studied (mature, immature, transitional,
and pro/pre-B cells) was observed in MNC mice (Fig. 2B,C). The
BCD and BCD+Lr groups had more mature (B220
high
IgM
+
CD24
low
and IgD
+
IgM
low
CD24
low
) B cells compared with MNC
mice. In addition, both the BCD and BCD+Lr groups had more
B220
low
IgM
+
CD24
high
immature B cells, but this population was
normalized only in the BCD+Lr group (Fig. 2B). Moreover, only
BCD+Lr mice had similar amounts of transitional T1 B cells
(B220
+
IgD
2
IgM
high
CD24
high
) to the WNC group (Fig. 2C). The
BCD+Lr treatment significantly increased pro/pre-B cells
(B220
low
IgM
2
CD24
high
) to higher values than those in the WNC
group (Fig. 2B). Therefore, the effect of L.rhamnosus nasal
treatment was more remarkable in the earlier stages of B cell
development.
MNC mice had significantly fewer lung lymphocytes and
CD19
+
B220
+
cells compared with the WNC group. Both the
BCD and BCD+Lr groups had higher amounts of lung
CD19
+
B220
+
cells than the MNC mice; however, only the
BCD+Lr group had values similar to those in the WNC group
(Table 1; Supplemental Fig. 1). As expected, the majority
of lung B cells displayed the mature phenotype CD19
+
B220
high
CD24
low
IgD
+
IgM
2/+
, whereas few lung B cells
were immature CD19
+
B220
low
CD24
high
IgM
+
cells (Fig. 2;
Supplemental Fig. 3). The MNC mice had fewer lung mature
and immature B cells compared with the WNC mice. The
BCD and BCD+Lr mice normalized the number of lung
immature B cells (Fig. 2D,E), whereas the number of lung
mature B cells was normalized only in the BCD+Lr group
(Fig. 2DF).
L. rhamnosus CRL1505 enhances resistance against
pneumococcal infection. After the S. pneumoniae challenge,
the survival of the MNC mice was 85%, whereas none of the
WNC mice died during the experiment. This difference confirms
that MNC mice are more susceptible to pneumococcal infection
than WNC mice, as we previously demonstrated (P= 0.02) (18).
The BCD mice had a similar survival to that found in the MNC
group (88%) and it differed from the WNC mice (P= 0.04). The
FIGURE 1 Different feeding protocols used in this
work. BCD, balanced conventional diet; BCD+Lr,
balanced conventional diet plus nasally administered
Lactobacillus rhamnosus; MNC, malnourished con-
trol; WNC, well-nourished control.
Recovery of humoral immunity by probiotics 229
by guest on October 21, 2015jn.nutrition.orgDownloaded from
survival of BCD+Lr mice (91%) did not differ from the WNC (P=
0.08) or MNC (P= 0.38) mice (Fig. 3A). In addition, the
pathogen was detected in lung samples from WNC and MNC
mice throughout the 15-d period, but the MNC mice had higher
bacterial cell counts. The BCD group had lower lung pathogen
counts compared with the MNC group. The BCD+Lr mice had a
greater resistance to the pneumococcal challenge compared with
the BCD group, as they had lung pathogen counts similar to those
in the WNC group (Fig. 3B). As previously described (17,18),
histopathological studies have revealed significant lung injury in
infected MNC mice. An intense inflammatory response and
hemorrhage together with a marked reduction of alveolar spaces
was observed in lungs of MNC mice. Lung tissue injuries in
infected WNC mice were less severe than those observed in the
MNC group. The BCD mice had histological signs intermediate
to those of the WNC and MNC groups and the histological
characteristics of lungs of BCD+Lr mice were similar to those of
the WNC group for the entire period assayed (Supplemental Fig.
4). Although we found differences between the BCD and BCD+Lr
groups in the resistance against pneumococcal infection, no
differences were observed in food intake or body weight between
the 2 groups (data not shown).
L. rhamnosus CRL1505 enhances humoral immunity.
Pneumococcal infection increased the number of spleen lym-
phocytes and CD19
+
B220
+
cells in all the experimental groups
compared with basal amounts; however, infected MNC mice
had fewer lymphocytes and CD19
+
B220
+
cells than infected
WNC mice. Both the BCD and BCD+Lr treatments increased
spleen lymphocytes and CD19
+
B220
+
cells, but only the BCD
+Lr group had values that were similar to those in infected WNC
mice (Table 2). In addition, the MNC mice had significantly
reduced numbers of all spleen B cell subpopulations. The
numbers of mature, immature, T1, and T2 B cells were higher in
TABLE 1 Total cell and lymphocyte numbers in spleen and lung of WNC, MNC, BCD, and
BCD+Lr mice
1
Group Weight
Spleen Lung
Total cell counts Lymphocytes CD19
+
B220
+
cells Total cell counts Lymphocytes CD19
+
B220
+
cells
mg 10
6
cells/spleen 10
5
cells/lung
WNC 142 611.9
b
57.4 66.34
a
38.4 64.00
a
12.2 62.01
a
21.9 62.09
a
7.65 61.10
a
1.59 60.30
a
MNC 50.6 610.1
c
14.4 62.84
b
9.67 61.42
c
1.82 60.43
d
11.5 61.41
c
3.02 60.46
c
0.40 60.05
c
BCD 201 613.6
a
49.0 64.44
a
26.2 64.20
b
6.71 60.97
c
19.1 61.81
b
4.94 60.63
b
0.71 60.14
b
BCD+Lr 184 622.0
a
52.3 65.36
a
26.7 64.11
b
8.80 61.35
b
22.8 61.82
a
6.85 60.53
a
1.46 60.11
a
1
Values are means 6SD, n= 6–8. Means in a column without a common letter differ, P,0.05. BCD, balanced conventional diet; BCD+Lr,
balanced conventional diet plus nasally administered Lactobacillus rhamnosus; MNC, malnourished control mice; WNC, well-nourished
control mice.
FIGURE 2 Flow cytometry study of B cell populations in spleen (A–C) and lung (D–F) of WNC, MNC, BCD, and BCD+Lr mice. Values are
means 6SD, n = 6–8. Means without a common letter differ, P ,0.05. BCD, balanced conventional diet; BCD+Lr, balanced conventional diet
plus nasally administered Lactobacillus rhamnosus; MNC, malnourished control; T1, transitional T1 B cell; T2, transitional T2 B cell; WNC, well-
nourished control.
230 Barbieri et al.
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BCD mice than in the MNC group; however, BCD administra-
tion did not normalize these cell populations. On the contrary,
BCD+Lr mice had similar numbers of mature, immature, and
transitional B cells to those found in infected WNC mice (Table
2; Supplemental Fig. 5).
Infection reduced the number of lung lymphocytes in all
groups without affecting the total number of B cells (Table 3).
CD19
+
B220
+
cells in lungs of infected WNC mice were similar
to those in the uninfected WNC group. However, a detailed
study of lung B cell subpopulations showed that mature IgM
2
B
cells decreased after infection, whereas mature IgM
+
B cells
increased in WNC mice. The MNC group had fewer mature
IgM
2
and IgM
+
B cells than the WNC group. The BCD group
had a similar number of lung mature IgM
2
B cells to those in
WNC mice; however, there were significantly fewer mature
IgM
+
B cells than in controls. Both lung mature IgM
2
and IgM
+
B cells were similar to WNC mice in the BCD+Lr group (Table 3;
Supplemental Fig. 6). In addition, pneumococcal infection
increased the immature B cells in all the experimental groups;
however, MNC mice had fewer of these cells compared with the
WNC group. Both BCD and BCD+Lr treatments enhanced the
numbers of immature B cells, but only the BCD+Lr group had
values similar to those found in WNC mice (Table 3; Supple-
mental Fig. 6).
Antibody-mediated opsonization is an important host de-
fense mechanism against encapsulated bacteria like S. pneumo-
niae (23). Therefore, we next evaluated the amounts of IgA-,
IgM-, and IgG-specific antibodies in the respiratory tract and
serum. There were fewer BAL anti-pneumococcal antibodies in
the MNC than in the WNC mice. The BCD treatment enhanced
IgG, IgA, and IgM antibodies; the amounts of IgG were similar
to those in the WNC group and IgA values were located between
the amounts of the MNC and WNC groups. BCD+Lr treatment
normalized IgG amounts and enhanced IgA values in BAL. In
fact, the anti-pneumococcal IgA antibodies in BCD+Lr mice
were higher than those in the WNC group (Table 4). MNC mice
had less serum IgG and IgM compared with the WNC group.
BCD treatment enhanced IgM amounts, which were similar to
those in the WNC group. However, BCD treatment was did not
increase serum IgG antibodies. The BCD+Lr treatment increased
the production of serum IgG, but these anti-pneumococcal
antibodies were not as plentiful as in the WNC group. In
addition, the amounts of serum IgA in the BCD+Lr mice were
significantly higher than those in the WNC group (Table 4). The
opsonophagocytic activity of BAL and serum antibodies was
also impaired in MNC mice. Both BCD and BCD+Lr treatments
FIGURE 3 Survival (A) and bacterial cell counts on d 2, 5, 10, and 15
after challenge with Streptococcus pneumoniae (B) in lungs of WNC,
MNC, BCD, and BCD+Lr mice. In A, the numbers of mice on d 0 are
given. Percentages on d 15 without a common letter differ, P,0.05.
In B, values are means 6SD, n= 6. Means at a time without a
common letter differ, P,0.05. Time points for a group without a
common symbol differ, P,0.05. BCD, balanced conventional diet;
BCD+Lr, balanced conventional diet plus nasally administered Lacto-
bacillus rhamnosus; MNC, malnourished control mice; WNC, well-
nourished control mice.
TABLE 2 Total cell and lymphocyte numbers on d 10 after the challenge with Streptococcus
pneumoniae in spleens of WNC, MNC, BCD, and BCD+Lr mice
1
Pvalue
WNC MNC BCD BCD+Lr Group Time Group 3time
10
6
cells/spleen
Total cell counts 86.8 611.5
a*
31.2 69.72
b*
81.3 610.4
a*
88.6 613.2
a*
,0.001 ,0.001 0.012
Lymphocytes 55.2 612.3
a*
16.5 64.91
c*
43.0 65.70
b*
49.4 67.14
a,b*
,0.001 ,0.001 ,0.001
CD19
+
B220
+
22.0 62.70
a*
5.40 61.61
c*
16.3 62.50
b*
21.0 62.20
a*
,0.001 ,0.001 ,0.001
CD19
+
B220
H
CD24
L
8.54 62.21
a*
3.39 61.29
c*
5.69 60.88
b*
7.03 61.00
a*
,0.001 ,0.001 0.007
CD19
+
B220
L
CD24
H
15.4 62.12
a*
1.64 60.71
c*
11.1 61.25
b*
14.9 62.00
a*
,0.001 ,0.001 ,0.001
B220
H
IgM
+
CD24
L
8.71 61.50
a*
2.56 60.60
c*
6.02 61.00
b*
8.13 60.92
a*
,0.001 ,0.001 ,0.001
B220
L
IgM
+
CD24
H
10.3 61.80
a*
0.73 60.37
c
6.86 60.85
b*
9.36 61.13
a*
,0.001 ,0.001 ,0.001
B220
L
IgM
2
CD24
H
1.52 60.49
b
0.25 60.11
c
4.92 60.88
b*
5.16 61.00
a*
,0.001 ,0.001 ,0.001
IgD
+
IgM
L
CD24
L
7.00 61.00
a*
2.60 60.54
c*
5.44 60.59
b*
6.80 60.88
a*
,0.001 ,0.001 0.001
IgD
+
IgM
H
CD24
H
3.50 60.44
ab
0.60 60.05
c
2.67 60.35
b*
4.15 60.57
a*
,0.001 ,0.001 ,0.001
IgD
2
gM
H
CD24
H
6.52 60.76
a*
0.50 60.07
c
4.70 60.68
b*
7.15 61.13
a*
,0.001 ,0.001 ,0.001
1
Values are means 6SD, n= 6–8. Means in a row without a common letter differ, P,0.05. Asterisks indicate significantly higher values
compared with values of d 0 before challenge (shown in Table 1 and Fig. 2), P,0.05. BCD, balanced conventional diet; BCD+Lr, balanced
conventional diet plus nasally administered Lactobacillus rhamnosus; MNC, malnourished control mice; WNC, well-nourished control mice.
Recovery of humoral immunity by probiotics 231
by guest on October 21, 2015jn.nutrition.orgDownloaded from
enhanced the opsonophagocytic activity of BAL and serum
antibodies; however, only the BCD+Lr group had similar values
to those in WNC mice (Table 4). The avidity of anti-pneumococcal
IgG, IgA, and IgM in serum and BAL was also evaluated. In all the
experimental groups, antibody avidity was <60% and the groups
did not differ (data not shown).
L. rhamnosus CRL1505 increases number and functionality
of phagocytic cells. The number and activity of blood and BAL
phagocytes were enhanced on d 2 p.i in all the experimental groups
compared with basal amounts (Fig. 4). The MNC mice had
significantly fewer and less activity of phagocytes than the WNC
group. The number of leukocytes was enhanced in the BCD group
(Fig. 4A,B); however, this treatment did not increase the phago-
cytesÕactivity (Fig. 4C,D). On the contrary, the BCD+Lr mice had
more phagocytes with greater activity compared with the WNC
mice (Fig. 4).
Discussion
In adults, B cells are generated in the BM and migrate to the
periphery at the transitional B cell stage, when they are still short
lived and functionally immature. Transitional B cells are
transported to the spleen, where they develop into mature B
cells that recirculate among the lymph nodes. Mature B cells
play a major role in the adaptive immune response when they
produce antibodies after they have been stimulated, expanded,
and selected in the germinal centers in the presence of T cell help
(3). It was reported that the impairment of humoral immune
response in malnourished hosts relates to the number and
activity of both B and T cells (24). Lymphoid atrophy, evidenced
by the reduced size and cellularity of the thymus and secondary
lymphoid organs, significantly contributes to the alteration of
humoral and cellular immunity in malnourished individuals.
Along these lines, we reported a reduction of blood lymphocytes
and BM lymphoid lineage cells in our malnutrition model (25).
Moreover, we demonstrated that protein malnutrition impairs
the B cell population in BM without affecting their capacity to
produce Igs (7). In this study, we extend these findings by
demonstrating that malnutrition decreases mature and imma-
ture B cells in spleen and lung.
Antibody responses are activated in the respiratory tract after
exposure to pathogens. The concentration and type of respira-
tory antibodies is dependent on the site of exposure. Upper
airway exposure results in IgA production, which is able to
protect the host against the colonization of pathogens (26).
Mucosal IgA antibody production is impaired in malnourished
hosts (27,28). Malnutrition remarkably reduces the number of
IgA
+
cells associated with the lamina propria of the small
intestine (28) and has the same effect on the airway mucosa,
TABLE 3 Total cell and lymphocyte numbers on d 10 after the challenge with Streptococcus
pneumoniae in lungs of WNC, MNC, BCD, and BCD+Lr mice
1
Pvalue
WNC MNC BCD BCD+Lr Group Time Group x time
10
5
cells/lung
Total cell counts 23.7 62.58
a*
10.7 61.26
b
22.2 62.70
a*
21.2 61.79
a
,0.001 0.046 ,0.001
Lymphocytes 6.62 60.64
ay
1.90 60.18
dy
3.70 60.39
cy
4.80 60.61
by
,0.001 ,0.001 0.06
CD19
+
B220
+
1.61 60.21
a
0.30 60.06
c
0.95 60.15
b
1.40 60.20
a
,0.001 0.28 0.007
CD19
+
B220
H
CD24
L
1.11 60.13
ay
0.27 60.04
c
0.70 60.11
b
0.93 60.11
ay
,0.001 ,0.001 ,0.001
CD19
+
B220
L
CD24
H
0.48 60.10
a*
0.03 60.01
c
0.26 60.07
b*
0.50 60.09
a*
,0.001 ,0.001 ,0.001
B220
H
IgM
2
CD24
L
0.40 60.06
ay
0.16 60.04
b
0.34 60.06
ay
0.38 60.06
ay
,0.001 ,0.001 ,0.001
B220
H
IgM
+
CD24
L
0.55 60.09
a*
0.10 60.02
c
0.34 60.04
b
0.44 60.09
ab*
,0.001 ,0.001 0.017
B220
L
IgM
+
CD24
H
0.40 60.06
a*
0.04 60.01
c
0.22 60.05
b*
0.34 60.06
a*
,0.001 ,0.001 ,0.001
IgD
+
IgM
2
CD24
L
0.50 60.09
ay
0.15 60.02
b
0.34 60.09
ay
0.49 60.08
ay
,0.001 ,0.001 ,0.001
IgD
+
IgM
+
CD24
L
0.44 60.05
a*
0.09 60.01
c
0.23 60.06
b*
0.37 60.07
a*
,0.001 ,0.001 ,0.001
1
Values are means 6SD, n= 6–8. Means in a row without a common letter differ, P,0.05. Asterisks and crosses indicate significantly
higher or lower values, respectively, compared with values of d 0 before challenge (shown in Table 1 and Fig. 2), P,0.05. BCD, balanced
conventional diet; BCD+Lr, balanced conventional diet plus nasally administered Lactobacillus rhamnosus; MNC, malnourished control
mice; WNC, well-nourished control mice.
TABLE 4 Amounts and opsonophagocytic activity of anti-pneumococcal antibodies on d 10 after the
challenge with Streptococcus pneumoniae in BAL and serum of WNC, MNC, BCD, and BCD+Lr mice
1
Group
BAL Serum Opsonophagocytic activity
IgG IgA IgM IgG IgA IgM BAL Serum
mg/L titer
WNC 1.74 60.19
a
2.66 60.20
b
3.24 60.43
a
25.7 63.90
a
10.9 62.31
b
65.7 63.58
a
35.0 68.37
a
140 630.0
a
MNC 1.18 60.18
b
1.94 60.30
c
1.78 60.31
b
13.5 63.41
c
9.39 63.52
b
61.1 64.89
b
16.7 65.16
b
36.0 68.94
c
BCD 1.53 60.26
a
2.45 60.34
cb
2.62 60.42
a
16.1 63.01
c
12.9 64.82
b
66.2 63.19
a
35.0 65.48
a
60.0 616.0
b
BCD+Lr 1.57 60.24
a
3.16 60.40
a
1.67 60.39
b
21.1 63.30
b
18.7 64.70
a
58.7 62.01
b
35.0 65.48
a
100 630.0
ab
1
Values are means 6SD, n= 6–8. Means in a column without a common letter differ (P,0.05). BAL, bronchoalveolar lavage; BCD,
balanced conventional diet; BCD+Lr, balanced conventional diet plus nasally administered Lactobacillus rhamnosus; MNC, malnourished
control mice; WNC, well-nourished control mice.
232 Barbieri et al.
by guest on October 21, 2015jn.nutrition.orgDownloaded from
because we found fewer IgA
+
cells in the bronchus-associated
lymphoid tissue of MNC mice (29). In the present and previous
studies, we observed a significant impairment of the respiratory
humoral immune response against S. penumoniae infection in
MNC mice, which was evidenced by the lower amounts of BAL
IgA anti-pneumococcal antibodies (18,30). On the other hand,
when S. pneumoniae reaches the alveolar space in the deep lung,
there is a differentiation and expansion of IgG antibody-
secreting plasma cells (31,32). These antibodies are important
in the protection against pneumococcal infection, because
opsonizing IgG antibodies allow complement fixation and
enhance macrophage microbicidal activity. Humoral immune
activation in the lung also induces the production of antibodies
at the systemic level that are responsible for preventing the
passage of S. pneumoniae into the blood (33). Here, we
demonstrated that malnutrition reduced the production of
serum and BAL antipneumococcal IgG antibodies. Moreover,
the opsonophagocitic activity of IgG antibodies was significantly
reduced in MNC mice. These findings indicate that protein
malnutrition significantly affects B cell populations in BM,
spleen, and lungs, inducing an impairment of the humoral
immune response and increasing the susceptibility of malnour-
ished hosts to respiratory pathogens such as S. pneumoniae.
Considering the immunomodulatory capacity of certain
LAB, our laboratory has proposed their use as supplements in
treatments aimed to recover the immunosuppression associated
with malnutrition. Our previous studies in immnunocompetent
mice demonstrated that nasally administered LAB induce
systemic and respiratory immune responses superior to those
obtained using oral stimulation (13). Therefore, in this work, we
evaluated the effect of nasally administered L. rhamnosus
CRL1505 to improve respiratory defenses during recovery in
malnourished mice. The use of nongenetically modified LAB
nasally given to stimulate respiratory immunity and prevent lung
infections has scarcely been studied. Hori et al. (14) reported
that nasal treatment of immunocompetent adult mice with heat-
killed L. casei Shirota stimulated respiratory tract cellular
immunity and increased the resistance against influenza virus
infection. Our laboratory studied the effect of L. lactis NZ9000
nasal administration to adult mice and showed that this
treatment enhanced the resistance against S. pneumoniae by
upregulating innate and specific immune responses in both
respiratory and systemic compartments (15). More recently, it
was shown that nasally administered LAB are highly effective at
reducing inflammation induced by viral infection and protecting
against lethal disease. Specifically, adult mice stimulated via
intranasal inoculation with heat-killed or live L. reuteri or L.
plantarum were protected against the infection with the virulent
rodent pathogen pneumonia virus. Lactobacilli administration
resulted in reduced virus recovery and prolonged survival (16).
To our knowledge, no other reports on protection against
respiratory pathogens induced by nasally administered LAB
have been published. In the 3 studies mentioned above, adult
immunocompetent mice were used; therefore, the effect of a
nasal treatment with LAB in immunocompromised hosts has
been less extensively studied. The first report, to our knowledge,
suggesting an improvement of respiratory immunity in immu-
nocompromised hosts induced by nasally administered LAB was
developed in our laboratory (17). We demonstrated that the
nasal treatment of malnourished mice with heat-killed L. casei
CRL431 increased their resistance to pneumococcal infection.
In the present work, we demonstrated that nasal administration
of L. rhamnosus CRL1505 induces the recovery of splenic
immature B cells and lung mature B lymphocytes. Moreover,
malnourished mice treated with L. rhamnosus CRL1505 were
able to mount a normal immune response against the respiratory
infection. In fact, higher amounts of respiratory and serum IgA
and IgG were found in the BCD+Lr mice compared with the
FIGURE 4 Number of BAL (A) and blood leukocytes (B), bactericidal
function of BAL phagocytic cells (C) and blood neutrophils peroxidase
activity (D) on d 0 (before challenge) and d 2 after the challenge with
Streptococcus pneumoniae in WNC, MNC, BCD, and BCD+Lr mice.
Values are means 6SD, n= 6. Means at a time without a common
letter differ, P,0.05. Time points for a group without a common
symbol differ, P,0.05. BAL, bronchoalveolar lavage; BCD, balanced
conventional diet; BCD+Lr, balanced conventional diet plus nasally
administered Lactobacillus rhamnosus; MNC, malnourished control
mice; NBT, nitroblue tetrazolium; WNC, well-nourished control mice.
Recovery of humoral immunity by probiotics 233
by guest on October 21, 2015jn.nutrition.orgDownloaded from
MNC and BCD groups. Our results also showed that BCD+Lr
treatment had no effect on IgM production. The probable cause
for not observing an effect on IgM amounts is that the
determinations of antibodies were performed on d 10 p.i. At
this time point, the amounts of serum IgG and BAL IgA are more
relevant.
In adult mice, 2 distinct types of cells constitute the B cell
defense system against infection: B1 and mature B cells. Mature
B cells produce high-affinity antibodies in collaboration with T
cells and are able to control and clear bacterial and viral
infections. On the other hand, B1 B cells represent the first-line
defense against infections produced by encapsulated bacteria
through their capacity to produce antibodies without T cell help
(3). The early recovery of B cell populations would be of great
importance for preventing infections in an immunocompro-
mised, malnourished host. Our previous work showed that the
oral administration of L. rhamnosus CRL1505 for 5 d to
malnourished mice induced the recovery of B cells in BM and
spleen (7). However, only 2 d of nasal administration of this
lactobacillus was enough to achieve the normalization of B cells
in spleen. Moreover, L. rhamnosus CRL1505 nasal treatment
increased the number and functionality of respiratory B cells,
which would be of great importance considering the high
incidence of respiratory infections in immunocompromised
hosts, including those associated to malnutrition states. Some
works evaluated the effect of probiotics on the gut-associated
lymphoid tissue and the stimulation of the common mucosal
immune system to improve immunity against respiratory path-
ogens and for the recovery of lung immunity in malnourished
hosts (7,13). We showed that other important inductive sites for
mucosal B cell recovery using immunobiotics are bronchus-
associated lymphoid tissue and naso-pharynx-associated lym-
phoid tissue. According to our results, it is conceivable that the
oral route of administration is not optimal for regulating local
responses to infection with respiratory pathogens. As such, we
and others considered the possibility that targeting live LAB to
the respiratory mucosa might result in a more effective immu-
nomodulatory response, similar to the benefits observed when
the intestinal mucosa is exposed directly to immunobiotics (15–
17).
The improvement of B cells and antibody production induced
by L. rhamnosus CRL1505 administration could explain the
reduction of lung pathogen counts and the inflammatory
damage of lung tissue after d 10 p.i. However, differences in
survival and lung tissue damage observed between the groups
during the early days after pneumococcal challenge would be
explained by the effect of L. rhamnosus CRL1505 on innate
immunity. It is known that both innate and adaptive immune
responses are essential to avoid the invasive pneumococcal
disease (34). Alveolar macrophages and recruited neutrophils
represent the first phagocytic defense in the lungs against
pneumococci (35). Our experiments showed that BCD+Lr
mice had an improved local and systemic innate immune
response evidenced by the greater number and activity of BAL
and blood leukocytes even when compared with the WNC
group. Although the recovery of innate immunity induced by
L. rhamnosus CRL1505 is important during the early days of
pneumococcal infection, once the pathogen overcomes this barrier,
adaptive immunity is key for the resolution of infection. In this
sense, the recovery of B cells is important for the protection of
the host through the production of specific antibodies and their
antiinflammatory capacity.
In conclusion, we demonstrated that malnutrition induces
a marked hypoplasia of lymphoid organs and prominent
lymphopenia, which also induces a significant reduction of
lymphocytes in other organs such as the lung. The impairment
of lymphocyte populations explains at least in part the higher
susceptibility to infections found in malnourished hosts. In
addition, this study demonstrates that priming of the respira-
tory mucosa of immunocompromised malnourished mice with
L. rhamnosus CRL1505 significantly improves the number
and activity of phagocytic cells and the number of B cells in
spleen and lungs. Moreover, administration of live lactobacilli
directly to the respiratory mucosa resulted in a diminished
susceptibility to S. pneumoniae infection and an improved
innate and humoral immune response against the respiratory
pathogen. Although further studies are necessary to clarify the
effect of malnutrition and immunobiotics in other immune cell
populations involved in the protection against respiratory
pathogens, this work gives evidence of the importance of using
nasal immunobiotics to accelerate the recovery of respiratory
immunity of immunocompromised malnourished hosts.
Acknowledgments
N.B., J.V., M.H., S.S., and S.A. conceived and designed the
experiments; N.B., M.H., and S.S. performed the experiments;
N.B., J.V., and S.S. analyzed the data; N.B., J.V., S.S., and S.A.
wrote the paper and had primary responsibility for the final
content. All authors read and approved the final manuscript.
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by guest on October 21, 2015jn.nutrition.orgDownloaded from
    • "It would be shortsighted to dismiss the potential of the low-protein stunting model as a laboratory tool with which to probe immunological plasticity, but focusing on a detail of dietary composition rather than on details of a pathophysiology has produced a type of animal model bearing no clear relationship to any human pathology. By contrast with stunting models, appropriately crafted protocols that produce a negative nitrogen balance in rodents, albeit by means of dietary nitrogen levels uncharacteristic of human consumption patterns, nevertheless reproduce the diagnostic features of kwashiorkor in weanlings (e.g., [16]) and produce the depressed inflammatory immune competence (e.g., [9,[17][18][19][20][21][22][23]) and susceptibility to opportunistic and other infections [17,19,21,[23][24][25][26][27]that characterize the human pathology. Endocrinological considerations lead to the same conclusion regarding low-protein weanling rodent models, a point discussed elsewhere [11] in relation to the blood thyroid hormone profile. "
    [Show abstract] [Hide abstract] ABSTRACT: Inflammatory incompetence is characteristic of acute pediatric protein-energy malnutrition, but its underlying mechanisms remain obscure. Perhaps substantially because the research front lacks the driving force of a scholarly unifying hypothesis, it is adrift and research activity is declining. A body of animal-based research points to a unifying paradigm, the Tolerance Model, with some potential to offer coherence and a mechanistic impetus to the field. However, reasonable skepticism prevails regarding the relevance of animal models of acute pediatric malnutrition; consequently, the fundamental contributions of the animal-based component of this research front are largely overlooked. Design-related modifications to improve the relevance of animal modeling in this research front include, most notably, prioritizing essential features of pediatric malnutrition pathology rather than dietary minutiae specific to infants and children, selecting windows of experimental animal development that correspond to targeted stages of pediatric immunological ontogeny, and controlling for ontogeny-related confounders. In addition, important opportunities are presented by newer tools including the immunologically humanized mouse and outbred stocks exhibiting a magnitude of genetic heterogeneity comparable to that of human populations. Sound animal modeling is within our grasp to stimulate and support a mechanistic research front relevant to the immunological problems that accompany acute pediatric malnutrition.
    Full-text · Article · Apr 2016
    • "Research shows marked differences in the skin microbiota or rural and urban residents [56]. The intra-nasal administration of non-harmful microbes can also influence systemic immune signaling575859. The inhalation of phytoncide is known to reduce physiological stress [60] , and how that might impact intestinal microbiota in the context of the emerging gutbrain-microbe research is unclear. "
    [Show abstract] [Hide abstract] ABSTRACT: Recent advances in research concerning the public health value of natural environments have been remarkable. The growing interest in this topic (often housed under terms such as green and/or blue space) has been occurring in parallel with the microbiome revolution and an increased use of remote sensing technology in public health. In the context of biodiversity loss, rapid urbanization, and alarming rates of global non-communicable diseases (many associated with chronic, low-grade inflammation), discussions of natural vis-a-vis built environments are not merely fodder for intellectual curiosity. Here, we argue for increased interdisciplinary collaboration with the aim of better understanding the mechanisms—including aerobiological and epigenetic—that might help explain some of the noted positive health outcomes. It is our contention that some of these mechanisms are related to ecodiversity (i.e., the sum of biodiversity and geodiversity, including biotic and abiotic constituents). We also encourage researchers to more closely examine individual nature relatedness and how it might influence many outcomes that are at the interface of lifestyle habits and contact with ecodiversity.
    Full-text · Article · Jan 2016
    • "Our previous studies also suggest that lactic acid bacteria (LAB) with immunomodulatory capacities (immunobiotics) are able to accelerate the recovery of the respiratory immune system and improve resistance against bacterial respiratory infection in malnourished hosts [5][6][7][8]. We have demonstrated that the supplementation of balance conventional diet (BCD) with nasally or orally administered viable immunobiotics, during the recovery of malnourished mice, is able to improve respiratory innate immune response [5], emergency granulopoiesis [8] , and humoral anti-pneumococcal response [7]. Our studies also suggest that non-viable immunobiotics are effective in the immunomodulation of the systemic and respiratory immune system in malnourished hosts under repletion treatments [9]. "
    [Show abstract] [Hide abstract] ABSTRACT: The effect of non-viable Lactobacillus rhamnosus CRL1505 and its cell wall and peptidoglycan on respiratory immunity in malnourished mice was studied. Weaned mice were malnourished with a protein-free diet for 21d and received BCD during 7d (BCD) or BCD with nasal non-viable L. rhamnosus CRL1505 (BCD + UV) or its cell wall (BCD + CW) or peptidoglycan (BCD + PG) supplementation during last 2d of the treatment. Malnourished mice without treatment (MNC) and well-nourished mice (WNC) were used as controls. Mice were infected nasally with Streptococcus pneumoniae after treatments. Resistance against pneumococci was reduced in MNC mice. Repletion with BCD reduced lung and blood bacterial cell counts when compared to MNC mice but the counts did not reach the levels of the WNC group. However, when malnourished mice received BCD + UV, BCD + CW or BCD + PG, pneumococci was not detected in lung or blood samples. Pneumococcal infection increased the levels of TNF-α, IL-1β, IL-6, and IL-10 in the respiratory tract, however the values were lower in MNC than in WNC mice. BCD + UV and BCD + PG groups showed values of phagocytes, IL-1β and IL-6 that were similar to WNC mice, while TNF-α was significantly higher in those groups when compared to WNC mice. Moreover, BCD + UV and BCD + PG treatments improved levels of respiratory IL-10, reaching values that were superior to those observed in WNC mice. The work demonstrates for the first time that non-viable probiotic bacteria or their cellular fractions could be an interesting alternative as mucosal immunomodulators, especially in immunocompromised hosts in which the use of live bacteria might be dangerous.
    Full-text · Article · Mar 2015
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