The Journal of Nutrition
Nasally Administered Lactobacillus rhamnosus
Accelerate the Recovery of Humoral Immunity in
B Lymphocyte-Deficient Malnourished Mice1–3
Natalia Barbieri,4Julio Villena,4Matias Herrera,4Susana Salva,5and Susana Alvarez4,5*
4Reference Centre for Lactobacilli (CERELA-CONICET), Tucum´ an, Argentina; and5National University of Tucuman,
Tucum´ an, Argentina
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+Lrgroup). Nonreplete malnourished and normal micewere usedas 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.
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
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
Malnutrition suppresses immune function and confers a
higher susceptibility to infectious diseases. Indeed, nutritional
deprivation induces atrophy of lymphoid tissues such as spleen
andthymus 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)6cell
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 (B220intermIgM2) and immature B cell
(B220intermIgM+) numbers were lower infeed-deprived mice.The
reduction of immature B cells was accompanied by an increase
in the percentage of mature B cells (B220highIgM+) but not by
1Supported by grants from PIP-632-2009, CIUNT-26D/403, and PICT-2010-1381.
2Author disclosures: N. Barbieri, J. Villena, M. Herrera, S. Salva, and S. Alvarez,
no conflicts of interest.
3Supplemental 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: email@example.com.
6Abbreviations 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
ã 2013 American Society for Nutrition.
Manuscript received June 26, 2012. Initial review completed July 6, 2012. Revision accepted November 11, 2012.
First published online December 26, 2012; doi:10.3945/jn.112.165811.
by guest on October 21, 2015
Supplemental Material can be found at:
changes in the total number of mature B cells. These observations
suggestthat nutritionaldeprivationleadstothealteration ofB 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 goat?s 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 of B 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
Children?s 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 (108
cells ? mouse21? d21) 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.
Normalmicewere usedasthe well-nourishedcontrol(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
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
Pneumococcal infection. Challenge with S. pneumoniae was carried
out on the day after the end of each treatment (d 8) by dropping 25 mL of
the inoculumcontaining105log-phasecells of S. pneumoniae inPBS 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-
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
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