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Nutritional Immunology

Lactobacillus casei Improves Resistance to Pneumococcal Respiratory Infection in Malnourished Mice¹

Julio Villena^*, Silvia Racedo, Graciela Agüero^*, Elena Bru, Marcela Medina^*, and Susana Alvarez^*,,2

^* Facultad de Bioquímica, Química y Farmacia, Universidad Nacional de Tucumán, CP 4000, San Miguel de Tucumán, Tucumán, Argentina and Centro de Referencia para Lactobacilos (CERELA-CONICET), Chacabuco 145, 4000 Tucumán, Argentina

²To whom correspondence should be addressed. E-mail: salvarez{at}cerela.org.ar.

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
We studied the effect of Lactobacillus casei CRL 431 used as a supplement in a repletion diet on the resistance to Streptococcus pneumoniae respiratory infection in malnourished mice. Weaned mice were malnourished after they consumed a protein-free diet (PFD) for 21 d. Malnourished mice were fed a balanced conventional diet (BCD) with or without supplemental L. casei for 7, 14, or 21 consecutive days, or BCD for 7 d with L. casei supplementation on d 6 and 7 (7dBCD+2dLc). The malnourished control (MNC) group was fed only the PFD, whereas well-nourished control (WNC) mice consumed the BCD ad libitum. Mice were challenged with S. pneumoniae at the end of each dietary treatment. Lung colonization and bacteremia were significantly greater in MNC than in WNC. Normalization of the immune response occurred in malnourished mice fed the BCD for 21d. L. casei supplementation reduced the time required for a normal response from 21 to 7 d. Mice administered the 7dBCD+2dLc repletion treatment had a more effective pathogen clearance from blood and significantly lower lung damage than MNC. This treatment improved both the number of leukocytes and neutrophils in blood and bronchoalveolar lavages (BAL) and the bactericidal function of phagocytic cells to levels that did not differ from those of WNC. In the 7dBCD+2dLc mice, antipneumococcal IgA in BAL was higher than in WNC, whereas antipneumococcal IgG in serum and BAL did not differ. This study suggests that the addition of L. casei to the repletion diet has a beneficial effect because it accelerates the recovery of the innate immune response and improves the specific immune mechanisms against an S. pneumoniae respiratory infection in malnourished mice.

KEY WORDS: • Lactobacillus casei • malnourished mice • lung infection • Streptococcus pneumoniae

Streptococcus pneumoniae is one of the major causes of bacterial pneumonia, meningitis, bacteremia, and otitis media. Despite the availability of antibiotics, mortality and morbidity rates remain high, especially in high-risk groups (1). Although polysaccharide vaccines can elicit a protective immune response against pneumococcal infection in adults, they have little effect on young children, the elderly, or immunodeficient patients (2).

Nutrition is a critical determinant of the immune response; consequently, malnutrition is the most common cause of immunodeficiency throughout the world. The reasons for malnutrition are multiple and complex; infection is a common precipitating factor. Conversely, malnutrition is also an important factor in the occurrence of infection, so that the 2 interact (3). Protein-energy malnutrition is associated with impaired cell-mediated immunity, phagocyte function, the complement system, secretory immunoglobulin A antibody concentrations, and cytokine production (4). It is not surprising that protein deficiency is so consistently observed to interfere with resistance to infection because most immune mechanisms depend on cell replication or on the production of active protein compounds (3).

The idea that gut microflora play a very important role in the maintenance of health and well-being is acquiring worldwide acceptance (5). Lactic acid bacteria (LAB)³ are present in the intestine of most animals. The beneficial role played by these microorganisms in humans and other animals has been reported extensively (6–8). One of the most effective ways of ensuring the predominance of beneficial bacteria in the extremely competitive environment of the gastrointestinal tract is to include live bacteria in the diet. Several studies investigated the role of immune-modulating probiotics in the control of microbial enterpathogens using rodent infection-challenged models (5,7). Although most research concerning probiotic-mediated enhanced immune protection is focused on gastrointestinal tract pathogens, a few recent studies tested whether probiotics might sufficiently stimulate the common mucosal immune system to provide protection to other mucosal sites as well (9). Lactobacillus casei CRL 431 was reported to induce both a secretory immune response and to protect against enteric infections in well-nourished (10) and malnourished (11) mice. The oral administration of this bacterial strain was also shown to increase the number of IgA⁺ B lymphocytes that enter the IgA cycle and repopulate the bronchus (8). This is important because the oral ingestion of L. casei could protect the respiratory mucosa against pathogens. We reported previously that the oral administration of L. casei CRL 431 to young mice enhances the phagocytic activity of alveolar macrophages and the lung clearance of Pseudomonas aeruginosa (12). Similarly, other investigators found that the oral administration of an immunomodulator containing lipopolysaccharides restores the number of IgA cells in bronchus-associated lymphoid tissue in malnourished rats (13). The aim of this work was to study the effect of the oral administration of L. casei CRL 431 on the resistance to a S. pneumoniae respiratory infection in malnourished mice.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Animals

Male 6-wk-old Swiss albino mice were obtained from the closed colony kept at the bioterio of CERELA. They were housed in plastic cages at room temperature. Each assay was performed in groups consisting of 25–30 mice (5–6 for each day before and after infection) that were housed individually during the experiments. Weaned mice were malnourished after they consumed a protein-free diet (PFD) (Table 1) for 21 d (Fig. 1A). At the end of this period, mice that weighed 40–55% less than well-nourished mice were selected for experiments. Well-nourished control mice consumed ad libitum a balanced conventional diet (BCD) (Table 1) and were used to compare lung infection with malnourished mice after repletion. Experiments were approved by the Ethical Committee for animal care at CERELA.

View larger version (17K): [in this window] [in a new window]   FIGURE 1 Different feeding protocols used in this work. (A) Preparation of malnourished mice and well-nourished and malnourished infected controls. Repletion of malnourished mice: (B) feeding for 7 d; (C) feeding for 14 d; (D) feeding for 21 d.

Lactobacillus casei CRL 431 was obtained from the CERELA culture collection. The culture was kept freeze-dried and then rehydrated using the following medium: peptone, 15.0 g; tryptone, 10.0 g; meat extract, 5.0 g; and distilled water, 1 L, pH 7. It was cultured for 8 h at 37°C (final log phase) in Man-Rogosa-Sharpe broth (MRS, Oxoid). The bacteria were harvested through centrifugation at 3000 x g for 10 min and washed 3 times with sterile 0.01 mol/L PBS, pH 7.2. Capsulated pneumococcus was isolated from the respiratory tract of a patient from the Department of Clinical Bacteriology of the Niño Jesús Children’s Hospital in San Miguel de Tucumán, Argentina. The pathogen strain belongs to the 14 serotype, 1 of the 10 most frequent serotypes isolated in pneumococcal infections in Argentina (serotypification was performed in Administración Nacional de Laboratorios e Institutos de Salud-ANLIS "Dr. Malbran," Buenos Aires, Argentina).

Feeding procedures

To compare the influence of L. casei administration on the susceptibility to S. pneumoniae infection, malnourished mice were fed the BCD with or without supplemental L. casei [10⁹ cfu/(mouse · d)] for 7 (Fig. 1B), 14 (Fig. 1C), or 21 (Fig. 1D) consecutive days. Because administration of L. casei (Lc) for 2 d is the optimal dose to provide protection against enteropathogens (10) and S. pneumoniae (unpublished data) in well-nourished mice and against P. aeruginosa respiratory infection in young mice (12), a third group of mice was added. These mice were fed the BCD for 7 d with L. casei supplementation on d 6 and d 7 (7dBCD+2dLc) (Fig. 1B). The malnourished control (MNC) group was fed only the PFD, whereas well-nourished control (WNC) mice consumed the BCD ad libitum (Fig. 1A).

Experimental infection

S. pneumoniae were first grown on blood agar for 18 h; freshly grown colonies were suspended in Todd Hewitt broth (Oxoid) and incubated at 37°C overnight. The pathogen was harvested through centrifugation at 3000 x g for 10 min at 4°C and then washed 3 times with sterile PBS. Cell density was adjusted to 4 x 10⁴ cfu/L. The size of the inoculum was confirmed by serial dilutions and quantitative subcultures on blood agar. The infecting dose was chosen on the basis of bacterial cell counts recovered from the blood of mice suffering from severe pneumonia (unpublished data).

Challenge with S. pneumoniae was performed on the day after the end of each dietary treatment (d 8, 15, or 22) (Fig. 1). Mice were infected by dropping 25 µL of the inoculum containing 10⁵ log-phase cfu of S. pneumoniae in PBS into each nostril and allowing it to be inhaled. To facilitate migration of the inoculum to the alveoli, mice were held in a head-up vertical position for 2 min. WNC and MNC mice were infected in the same way (Fig. 1A). Then, mice were killed on d 0 (before infection) and on d 1, 2, 5, 10, or 15 postinfection. During the 15-d postinfection period, all mice were fed only the BCD, with the exception of MNC that were fed the PFD.

Body weight determination

Body weight was determined before and after each dietary treatment. The experiment was performed on 10 mice/group to validate the statistical results.

Bacterial cell counts in lung homogenates and blood

Mice were killed 1, 2, 5, or 10 d after challenge with pneumococcus and their lungs were excised, weighed, and homogenized in 0.005 L of sterile peptone water. Homogenates were diluted appropriately, plated in duplicate on blood agar, and incubated for 18 h at 37°C. S. pneumoniae was identified by standard techniques (14) and the results were expressed as log of cfu/g of organ.

Progression of bacterial growth to the bloodstream was monitored by sampling blood obtained through cardiac puncture with a heparinized syringe and plating on blood agar. Bacteremia was reported as negative or positive hemocultures after incubation for 18 h at 37°C.

Determination of total and differential number of leukocytes in blood and bronchoalveolar lavages (BAL)

Blood samples were obtained through cardiac puncture at the end of each dietary treatment (d 0) and 1, 2, 5, and 10 d after challenge and collected in heparinized tubes. The total number of leukocytes was determined with a hemocytometer. Differential cell counts were performed by counting 200 cells in blood smears stained with May Grünwald-Giemsa (MGG). BAL samples were obtained according to the technique of Bergeron et al. (15) modified as follows: the trachea was exposed and intubated with a catheter, and 2 sequential bronchoalveolar lavages were performed in each mouse by injecting 5 x 10^–4 L of sterile PBS; the recovered fluid was centrifuged for 10 min at 900 x g; the pellet was used to make smears that were stained with MGG; and the fluid was frozen at –70°C for subsequent antibody analyses.

Antibodies from serum and BAL

An ELISA was developed to measure antipneumococcal antibodies (IgA and IgG) in serum and BAL 1, 2, 5, 10, and 15 d after challenge; basal levels were determined on d 0, before infection. BAL samples were obtained as described above.

Each plate was coated with 200 µL of a 1:100 dilution of S. pneumoniae vaccine (NEUMO 23 polyvalent vaccine, Aventis Pasteur S.A) in a sodium carbonate-bicarbonate buffer (pH 9.6). After overnight incubation at 4°C, plates were washed 5 times with PBS containing 0.05% (v:v) Tween 20 (PBS-T). Nonspecific protein binding sites were blocked with PBS containing 50 g/L nonfat dry milk for 30 min at room temperature. After the addition of 200-µL portions of the appropriate dilutions of the samples with PBS-T (serum 1:20; BAL 1:2), plates were incubated for 60 min at 37°C. After the plates were washed 5 times with PBS-T, peroxidase-conjugated goat anti-mouse IgA or IgG (anti- chain specific # A4700, anti- chain specific # A3673 Sigma-Aldrich) was diluted 1:500 in PBS-T and 200 µL was added to each well. The plates were incubated at 37°C for 60 min and then washed 5 times with PBS-T. Plates were subsequently poured with 200 µL of a substrate solution [3–3', 5–5'-tetramethylbenzidine (# T2885, Sigma-Aldrich)] in citrate-phosphate buffer (pH 5, containing 0.05% H₂O₂). After incubation for 30 min at room temperature, the reaction was stopped by the addition of 50 µL of 1 mol/L H₂SO₄. Readings were carried out at 493 nm (VERSAmax Tunable microplate reader) and the antibody concentration (g/L) in each unknown sample was determined from a standard curve made with commercial mouse IgA (Catalog # M-1421, Sigma-Aldrich) and mouse IgG (Catalog # I-5381, Sigma-Aldrich).

Phagocytic cell activation

    Washburn test. Measurement of myeloperoxidase activity of blood and BAL neutrophils was carried out using a cytochemical method, with benzidine as a myeloperoxidase chromogen (16). Cells were graded as negative or weakly, moderately, or strongly positive and were used to calculate the score.

    Nitroblue tetrazolium (NBT) test. The phagocytic bactericidal activity (oxidative burst) of macrophages and neutrophils was measured using the NBT reduction test (# 840-W, Sigma-Aldrich) in the pellet of BAL. NBT was added to each sample with (positive control) or without addition of the bacterial extract; then samples were incubated at 37°C for 20 min. In the presence of oxidative metabolites, NBT (yellow) is reduced to formazan, which forms a blue precipitate (17). Smears were prepared and, after staining, samples were examined under a light microscope for blue precipitates. A hundred cells were counted and the percentage of NBT positive (+) cells was determined.

Histopathology

At prechosen intervals, whole-lung samples from control and infected mice were excised and washed with PBS 0.01 mol/L, pH 7.2. Then, tissues were immersed in 4% (v:v) formalin saline solution. Once fixed, samples were dehydrated and embedded in Leica Histowax (Leica Microsystems Nussloch GmbH) at 56°C. Finally, lungs were cut into 4-µm serial sections and stained with hematoxylin-eosin for light microscopy examination.

Statistical analysis

Experiments were performed in triplicate and results were expressed as means ± SD. For body weight gain, 1-way ANOVA was used. For all other determinations, 2-way ANOVA was used. Tukey’s test (for pairwise comparisons of the means of the different groups) was used to test for differences between the groups. Differences were considered significant at P < 0.05.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Body weights. All repletion diets given to malnourished mice significantly increased body weights to the same extent (Table 2). Mice reached normal weight on d 14 of dietary treatment.

View larger version (13K): [in this window] [in a new window]   FIGURE 2 Streptococcus pneumoniae cfu counts in lung of malnourished mice replete with BCD with or without supplemental Lactobacillus casei for 7 (A), 14 (B), or 21 (C) d and challenged at the end of each dietary treatment. The cfu counts in lung of malnourished (MNC) and well-nourished (WNC) infected control mice are presented in A, B, and C. Results are expressed as means ± SD, n = 6. *Different from the MNC mice, P < 0.05. **Different from the MNC and WNC mice, P < 0.05.

    BAL differential white cell counts. The number of leukocytes, neutrophils, macrophages, and lymphocytes in BAL was determined in 7dBCD+2dLc and 7dBCD replete mice and control groups (WNC and MNC).

Alveolar macrophages were the predominant resident leukocytes in the alveolar spaces of noninfected mice. WNC mice showed an important recruitment of neutrophils (Table 5) and macrophage emigration into alveoli, resulting in high leukocyte counts in BAL during the infection. MNC mice showed a significant impairment in the recruitment of neutrophils and macrophages (Table 5). However, 7dBCD+2dLc treatment was enough to normalize the response to the infection, with values of leukocytes, neutrophils, and macrophages similar to those in WNC mice. The mice treated with 7dBCD continued to have lower values than WNC mice.

View larger version (145K): [in this window] [in a new window]   FIGURE 3 Lung architecture of well-nourished control mice (A), malnourished control (B), malnourished mice fed 7dBCD (C), and malnourished mice fed 7dBCD+2dLc (D) 10 d after challenge with Streptococcus pneumoniae. Light micrographs, original magnification X200.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Effective pulmonary host defense against respiratory pathogens is thought to be mediated mainly via phagocytosis by alveolar macrophages and recruited neutrophils (20,21). If pneumococci overcome these defenses and gain entry to the blood stream, systemic protection is afforded by anticapsular antibodies (22). In this work, a progressive increase in white blood cells was observed in WNC mice after infection, and differential cell counts indicated that this phenomenon involved mainly neutrophils. MNC mice showed a mild leukopenia before the infection, with all cell populations affected. A moderate increase was observed after challenge with pneumococci, but there was a decrease of leukocytes on d 5 postinfection, probably because the cell release from the bone marrow compartment was affected (23). Alveolar macrophages were the predominant resident leukocytes in the alveolar spaces of noninfected mice, and only a few neutrophils could be recovered from that site. Infection rapidly triggered neutrophil recruitment into the alveoli of WNC mice, resulting in increased cell counts in BAL. There were significantly fewer neutrophils in BAL in MNC mice compared with the WNC group at the same period postinfection. Treatment with BCD with or without supplemental L. casei increased leukocytes and neutrophils in blood and BAL. However, mice administered the 7dBCD+2dLc treatment had significantly more leukocytes and greater phagocytic activity than those in the 7dBCD group. Consequently, L. casei was able to induce an earlier recovery of the innate immune response.

Malnutrition produces a remarkable decrease in the number of IgA-secreting cells associated with the lamina propria of the small intestine (24), and it probably has the same effect on other mucosal tissues. This would explain the significantly lower concentration of BAL IgA found in MNC mice in this work. The oral administration of an appropriate dose of L. casei CRL 431 was reported to enhance the number of IgA⁺ cells in the intestine of malnourished mice (24) and to induce a significant increase in the levels of BAL IgA, IgM, and IgG after a P. aeruginosa infection in young mice (12,25,26). In the present work, when 7dBCD+2dLc treatment was administered, BAL antipneumococcal IgA reached higher levels than those in WNC mice. The IgA antibodies can bind the antigen and minimize its entry with a consequent reduction in inflammatory reactions, which prevents a potentially harmful effect on the tissue (8). The immune exclusion and elimination of the pathogen at the mucosal surfaces by secretory IgA is crucial in preventing pneumococcal bacteremia (27).

In addition, this report showed that 7dBCD+2dLc treatment increased serum and BAL antipneumococcal IgG, which reached levels similar to those in WNC mice. It was suggested that the most important factor contributing to protection and septicemia prevention might be the specific antipneumococcal serum IgG (22,28). Although the levels of antibodies that offer protection against pneumococcal disease have not been clearly defined, the response of serum antibodies and their opsonophagocytic activity represent the major defense mechanism of the host against pneumococcal bacteremia (2,29).

In the present study, histopathological examination of lung showed that tissue damage was less in mice treated with L. casei, despite the more intense inflammatory response. Alveolar macrophages play an essential role in the regulation of the lung inflammatory response during pneumococcal pneumonia (30). L. casei administration induces macrophage activation (12), which could partially explain the anti-inflammatory effect elicited by this strain. Moreover, it was reported that administration of L. casei CRL 431 for 2 d can induce anti-inflammatory cytokines such as interleukin-4 or -10 (31), and Blum et al. suggested that lactic acid bacteria could protect tissues from the deleterious effect of an ongoing inflammatory process (32).

Administration of L. casei for 2 d was a more effective in improving mucosal immune response than administration of this strain for 7 or 14 d. Therefore, even though the administration of L. casei has beneficial effects on the protection against S. pneumoniae, there would be a stage at which it would provoke an exaggerated inflammatory response as a consequence of the entrance into the gut of a large number of bacterial antigens. This effect would be counteracted by the appearance of suppressor T cells, which would prevent an undesirable effect in the gut but would provoke a decrease in immune stimulation. The increase in the mucosal immune response obtained with the 21dBCD+Lc treatment would be a consequence of the feeding period rather than of a stimulation of the lactic acid bacteria because no differences were found between the 21dBCD+Lc and the 21dBCD treatments.

The results of this study suggest that the addition of L. casei CRL 431 to the repletion diet fed to malnourished mice has a beneficial effect because it significantly accelerates the recovery of the defense mechanisms against an S. pneumoniae respiratory infection. However, much remains to be learned about the cellular and molecular mechanisms involved in the interaction of lactic acid bacteria and the mucosal immune system, especially if they are to be used as supplements for repletion diets or as vaccine vectors in immunosuppressed hosts.

	FOOTNOTES

 
¹ Supported by grants from FONCyT PICT 99 # 5–7206 and CIUNT 26 D/202.

³ Abbreviations used: BAL, bronchoalveolar lavages; BCD, balanced conventional diet; BCD+Lc, balanced conventional diet with supplemental Lactobacillus casei; LAB, lactic acid bacteria; Lc, Lactobacillus casei; MGG, May Grünwald-Giemsa; MNC, malnourished control; NBT, nitro blue tetrazolium; PFD, protein-free diet; WNC, well-nourished control; 7dBCD (14dBCD, 21dBCD), mice fed 7 (14, 21) d with BCD; 7dBCD+Lc (14dBCD+Lc, 21dBCD+Lc), mice fed 7 (14, 21) d with BCD+Lc; 7dBCD+2dLc, mice fed 7 d with BCD with supplemental Lc on d 6 and d 7.

Manuscript received 29 September 2004. Initial review completed 22 October 2004. Revision accepted 17 February 2005.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

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