See “Prevention of Rotavirus-induced Diarrhea” by Newburg on page 2.
Rotaviruses (RVs) cause a major health problem in infants and young children worldwide. RVs infect enterocytes in the small intestine and prevent the uptake of fluids and nutrients, as well as the expression of some digestive enzymes, causing excess fluid excretion and diarrhea (1,2), which may lead to serious dehydration and death without appropriate intervention (3–5).
Particular caution about RV infection should be taken for neonates because younger children have a significantly higher risk for severe RV disease (6). Although general measures to prevent infection, such as improvements in sanitation, decrease the incidence of diarrhea of bacterial origin, they tend to show limited benefit in the case of RV infection (7); RVs are transmitted mainly by the fecal–oral route, which is likely to be less dependent on the status of environmental sanitation, making all young children in both developed and developing countries equally vulnerable to RV infection (5). It should also be noted that the immune system of neonates, particularly the mucosal immune system, is relatively undeveloped against infection (8), and that antibodies produced by neonates have poor functional activity as compared with adults, partly because neonates have fewer somatic mutations that enhance the affinity of antibodies to viruses (9). Therefore, it is vital for neonates to be protected from severe infection by passive immunity and by their own immunity. An effective measure for this protection would be the consumption of breast milk that contains higher levels of protective antibodies. The importance of breast-feeding in preventing RV infection has been well studied (10,11) and it has been indicated that antibodies are crucial for the prevention of infection (12,13). Levels of RV-specific immunoglobulin (Ig) A antibody are high in breast milk because this antibody is synthesized in the breast tissue where IgA-producing cells have migrated from gut-associated lymphoid tissue (14).
Probiotics are known to stimulate gut-associated lymphoid tissue function (15), which helps to enhance the maternal production of protective antibodies in breast milk. Yasui et al (16) reported that oral administration of Bifidobacterium breve strain YIT4064 to mouse dams enhanced the anti-RV IgA antibody response in the mammary gland and intestine, protecting mouse pups from RV-induced diarrhea. There are, however, few other studies focusing on the effect of the administration of probiotics to dams rather than pups. In some representative studies, for example, probiotics have been given to pups: oral administration of bifidobacteria (B bifidum and B infantis) to BALB/c pups delayed the onset of RV-induced diarrhea and elevated RV-specific IgA secretion in feces (17); supplementation with milk fermented by Lactobacillus casei (strain DN-114 001) to germ-free suckling rats reduced the clinical signs of diarrhea (18); and administration of live L rhamnosus strain GG in combination with anti-RV antibodies to BALB/c mouse pups reduced the incidence of diarrhea (19).
L gasseri strain SBT2055 (LG2055) is a human intestine–originated probiotic bacterium with properties such as bile tolerance (20), ability to become established in the intestine and to improve the intestinal environment (21,22), and preventive effects on abdominal adiposity in rats (23,24) and humans (25), among others; however, whether the maternal administration of LG2055 promotes protection against RV-induced diarrhea remains to be examined. Furthermore, this administration should also be assessed in a strain-dependent manner because different probiotic bacteria have different effects (26–28).
In the present study, we examined whether the administration of heat-treated LG2055 cells to mouse dams could augment IgA levels in breast milk collected from the stomach of mouse pups and thereby reduce the incidence of diarrhea in a mouse model of RV infection. We also investigated a possible mechanism of such prevention, whereby the preferential secretion of IgA in breast milk may be attributed to the stimulation of Toll-like receptors (TLRs) by LG2055.
Six-week-old female BALB/c mice and 11-week-old male BALB/c mice were purchased from Charles River Laboratories Japan Inc (Tokyo, Japan) and used in the RV infection study. TLR-2 and TLR-4-knockout (KO) mice, and their cognate wild-type mice (6-week-old female BALB/c), were purchased from Oriental Bio Service Co (Kyoto, Japan) and used in cell culture experiments. Experimental protocols were done in accordance with the animal experimentation regulations of Shinshu University (Nagano, Japan).
RV and Host Cell
Simian RV SA-11 (group A) and MA104 cells established from fetal rhesus monkey kidney were used. MA104 cells were grown in Eagle's minimum essential medium (E-MEM) containing 10% (weight/volume) heat-inactivated fetal bovine serum (FBS), 0.75% sodium hydrogen carbonate, and 4% L-glutamine. For the proliferation and maintenance of RV, E-MEM containing 2.95% Bacto tryptose phosphate broth (BD), 0.05% yeast extract, 0.5% L-glutamine, 0.1% glucose, and 1.5% sodium hydrogen carbonate were used as a maintenance medium (MM).
MA104 cells fully grown in culture dishes were incubated in E-MEM without FBS for 1 hour and then inoculated with RV suspension treated with 10 μg/mL of acetyl trypsin (Sigma-Aldrich, St Louis, MO) at 37°C for 30 minutes, followed by incubation at 37°C for 1 hour. The MA104 cells thus adsorbed with RV were further incubated in MM for 3 days, at which point a cytopathic effect in cells was observed. The RV-infected cells were frozen and thawed 3 times to produce a suspension containing disrupted cells; the suspension was then centrifuged at 9000g for 20 minutes at 4°C to obtain supernatant containing RV. The supernatant was quantified for viral infectious units by means of a tissue culture infectious dose (TCID), being defined as 106.9 TCID50/mL, which means that when a 1-mL inoculum of a 1:106.9 dilution of RV is added to individual microplate wells containing MA104 cells, 50% of the wells become infected.
L gasseri SBT2055 (LG2055) is a bacterial strain deposited in the International Patent Organism Depository, National Institute of Advanced Industrial Science and Technology (Tsukuba, Ibaraki 305–8566, Japan) as FERM BP-10953. LG2055 cells were cultivated in deMan, Rogosa, and Sharpe medium (Difco, Detroit, MI), developed for the cultivation of Lactobacillus species, at 37°C for 18 hours (2 × 109 colony-forming units/milliliter of medium) and harvested by centrifugation at 7000g for 10 minutes at 4°C. These harvested cells were washed 3 times with water, heated at 121°C for 15 minutes, and lyophilized to make powder (1 mg of powder was obtained from 1 mL of medium).
First, female BALB/c mice were divided into 2 groups: a control group (C) in which mice were fed a commonly used MM3 diet (Funabashi Farm Co, Chiba, Japan) and a group in which mice were fed the MM3 diet containing 0.1% heat-treated lyophilized LG2055 cells. The mice were maintained on these diets for 4 weeks before being mated with male BALB/c mice and thereafter until the experiment ended. One week after mating, each group of female mice was further divided into 2 subgroups: the C group was subdivided into a group in which mice were treated with 0.5 mL of phosphate buffered saline (PBS) orally (C/C) and a group in which mice were immunized with 0.5 mL of RV suspension (106.9 TCID50/mL) orally (C/RV). The LG2055 group was also subdivided into a group treated with PBS (LG2055/C) and a group immunized with RV (LG2055/RV). Five days after birth, mouse pups were orally infected with 50 μL of the RV suspension (106.9 TCID50/mL), and 4 days later, the incidence of diarrhea in mouse pups was determined on the basis of fecal status, which was classified as either normal or diarrhea (ie, apparently exhibiting loose feces).
Extracts From Breast Milk
Coagulated breast milk collected from the stomach of mouse pups was suspended in PBS at 400 mg/mL, homogenized on ice, and centrifuged at 9000g for 20 minutes to obtain the supernatant for analyses.
RV-neutralizing Activity of Breast Milk of Stomach Origin
The RV suspension (106.9 TCID50/mL) was diluted 10-fold in MM containing 10 μg/mL of acetyl trypsin, incubated at 37°C for 30 minutes, and further diluted 5-fold in MM. This RV suspension and the above-described breast milk supernatant diluted 5-fold in MM were mixed together at a ratio of 1:1 and incubated at 37°C for 1 hour. After incubation, each mixture was further diluted in MM to yield serially diluted mixtures of RV and breast milk. These serially diluted mixtures were added to 96-well microplates (25 μL/well), in which MA104 cells had previously been incubated for 1 hour in E-MEM without FBS, in such a way that the E-MEM without FBS was replaced by the diluted mixtures. The microplates were incubated at 37°C for 1 hour to enable the MA104 cells to become infected with RV. After incubation, 200 μL/well of MM was added to the microplates, and the cells were further incubated at 37°C for 5 days; cell morphological changes were then observed under a phase-contrast microscopy to determine the highest dilution at which a clear cytopathic effect (dead cells observed floating in the medium) was maintained. Viral infectious units (10n TCID50/mL) after neutralization were calculated on the basis of the dilution.
Splenocyte and Peyer's Patch Cell Cultures
The spleen and Peyer's patches were removed from mouse dams. The spleen was mechanically disrupted and Peyer's patches were treated with 1.1 U/mL of Dispase (Gibco/Invitrogen, Grand Island, NY) (29). These cells were then suspended in RPMI 1640 cell culture medium containing 10% FBS, and filtered through a 70-μm cell strainer to obtain single-cell suspensions. Cells were washed twice, resuspended in RPMI 1640 medium, and dispensed into wells of a 96-well cell culture plate (BD Biosciences, San Jose, CA) at a density of 5 × 105 cells/well, together with 20 μg/well of LG2055 lyophilized cells, in a final volume of 0.2 mL/well. Cell culture was carried out for 7 days at 37°C in an atmosphere of 5% CO2, and then the supernatants were collected for the quantification of IgA.
The feces of mouse dams were collected immediately after the observation of diarrhea in mouse pups. The feces collected were suspended in PBS at 200 mg/mL, homogenized on ice, and centrifuged at 9000g for 20 minutes to obtain the supernatant.
Quantification of IgA
RV-specific IgA levels were quantified by an enzyme-linked immunosorbent assay (ELISA), in which an RV antigen prepared as described below was used as both an antigen for immobilization on the ELISA microtiter wells and an antigen for immunization to produce a hyperimmune antiserum to RV that was used as a standard for RV-specific IgA.
The RV antigen was prepared by purifying the above-described RV suspension using a standard cesium chloride density-gradient centrifugation method (30), which produced inner-shell and double-shell particle fractions. These fractions were pooled and frozen in PBS.
The hyperimmune antiserum to RV that was used as a standard for RV-specific IgA was prepared as described previously (16). In brief, the RV antigen (4-μg protein/mouse) with complete Freund adjuvant was injected intraperitoneally into BALB/c mice; after 2 weeks, an oral immune boost was performed 4 times every 2 weeks using the RV suspension diluted 8-fold in PBS; a further intravenous immune boost was performed 12 times every 2 weeks by injecting 0.4 μg of protein/mouse of the RV antigen into the tail vein. A 100-fold-diluted solution of this hyperimmune antiserum was defined as 1 unit.
The assay procedure for the ELISA was performed as follows. The RV antigen dissolved in carbonate buffer (10 μg/mL, pH 9.6) was immobilized on microtiter wells overnight at 4°C, and the wells were washed with PBS and blocked with a 1% bovine serum albumin solution. Next, experimental samples or the hyperimmune antiserum were appropriately diluted in 1% bovine serum albumin solution, added to the wells, and incubated at room temperature for 90 minutes. IgA antibodies in the samples and standard that bound to the immobilized RV antigen were detected by using an antimouse IgA secondary antibody conjugated with an enzyme horseradish peroxidase (Cappel, MP Biomedicals, Aurora, OH) that reacts with the chromogen O-phenylenediamine dihydrochloride (Sigma-Aldrich) to yield an absorbance of 490 nm.
For the determination of total IgA levels, the ELISA was carried out as described above, except that goat anti-mouse IgA (Cappel) as a primary antibody and recombinant IgA (Sigma-Aldrich) as a standard reagent were used instead of RV antigen and hyperimmune antiserum to RV, respectively.
The incidence of diarrhea was analyzed using the Fisher exact probability test. The difference in neutralizing activity and IgA levels among the 4 groups was analyzed using a 1-way analysis of variance with a post hoc Turkey-Kramer multiple comparison test. The difference between the 2 groups was evaluated by the Student t test. A value of P < 0.05 was considered significant. Estimates are expressed as mean ± standard deviation.
Incidence of RV-induced Diarrhea Among Mouse Pups
The incidence of diarrhea among mouse pups orally infected with RV 5 days after birth was determined (Table 1). Mouse pups born to dams immunized orally with RV alone (the C/RV group) had a significantly lower incidence of diarrhea (34.8%, P < 0.01) as compared with the 2 non–RV-immunized groups: the C/C group (89.5%) and the LG2055/C group (90.5%). Furthermore, a significantly lower incidence of diarrhea was observed in the LG2055/RV group, in which dams were both fed LG2055 and immunized orally with RV (10.0%), as compared with the C/RV group (P < 0.05).
RV-neutralizing Activity in the Extract From Breast Milk
The extract of the stomach content of mouse pups fed with breast milk was tested for its neutralizing activity against RV (Fig. 1A). The mean value of viral infectious units (10n TCID50/mL) was significantly lower (P < 0.05) in the pups that received breast milk from mouse dams that were both fed LG2055 and immunized orally with RV (the LG2055/RV group) as compared with the other groups (C/C, LG2055/C, and C/RV).
RV-specific IgA in the Extract From Breast Milk
The amount of RV-specific IgA in supernatant prepared from coagulated breast milk in the stomach of mouse pups was quantified by ELISA (Fig. 1B). The amount was significantly higher (P < 0.05) in the pups that received breast milk from mouse dams that were both fed LG2055 and immunized orally with RV (the LG2055/RV group) as compared with the other groups (C/C, LG2055/C, and C/RV).
RV-specific IgA Production in Peyer's Patch Cells of Mouse Dams
The amount of RV-specific IgA produced by Peyer's patch cells of mouse dams was determined (Fig. 2A). The amount of IgA produced was significantly greater (P < 0.05) in cells from mouse dams in the RV-immunized groups (the C/RV and LG2055/RV groups) as compared with the non–RV-immunized groups (the C/C and LG2055/C groups). There was, however, no significant difference between the C/RV and LG2055/RV groups.
RV-specific IgA Content in Feces of Mouse Dams
The amount of RV-specific IgA detected in the feces of mouse dams was determined (Fig. 2B). No RV-specific IgA was detected in the feces of mouse dams in the non–RV-immunized groups (the C/C and LG2055/C groups). Although RV-specific IgA was detected in the feces of the RV-immunized groups (the C/RV and LG2055/RV groups), there was no significant difference between the 2 groups.
Total IgA Production in Splenocytes From TLR-KO Mice
The involvement of TLR-2 and TLR-4 in the stimulation of IgA production was evaluated by comparing splenocytes prepared from TLR-2- and TLR-4-KO mice (Fig. 3). LG2055 bacterial cells did not stimulate the production of IgA in splenocytes from TLR-2-KO mice; rather, it decreased the production significantly. In contrast, LG2055 significantly enhanced the production of IgA in splenocytes from TLR-4-KO mice to the same extent as observed for wild-type mice.
The present study demonstrated a protective effect of breast-feeding on RV infection in a commonly used heterologous mouse model of infection due to the simian RV strain SA-11 (31–33). The RV strain SA-11, classified as group A and P-genotype, requires a sialic acid residue on the cell surface for its infectivity, and this sialic acid dependency is considered a factor that enables the virus to be examined in different species of animals and their cell cultures (34).
In the heterologous mouse model used here, maternal RV immunization was primarily necessary to induce RV-specific IgA in breast milk, and to prevent diarrhea in mouse pups. Mouse dams were first fed a diet with or without LG2055, and then immunized orally with RV. Although RV immunization itself had a preventive effect on the incidence of diarrhea among pups, the prevention was further augmented by maternal administration of LG2055 (the LG2055/RV group). Furthermore, only the LG2055/RV group showed a significant enhancement in both RV-neutralizing activity and RV-specific IgA production in breast milk as compared with the other experimental groups, indicating the adjuvant action of LG2055.
When considered in a human context, a natural infection or asymptomatic infection that occurs at some time will establish an antibody response to RVs, which could provide protection similar to that induced by artificial immunization or symptomatic infection (6). If the natural infection took place at the same time as the consumption of Lactobacillus, similar to our experimental conditions, then it is conceivable that an augmented protective response may be produced.
Heat-inactivated LG2055 cells were used in the present study; therefore, microbial components, rather than live cells in the intestine, are likely to be involved in the stimulation of IgA production. Such microbial components are recognized primarily by TLRs, which are expressed by antigen-presenting cells and are crucial elements for immune activation (35). Among the TLRs, components of Gram-positive bacteria including probiotics are sensed mainly through TLR-2, and those of Gram-negatives via TLR-4 (36). As expected, we observed that the LG2055 cells could not enhance IgA production when TLR-2 was knocked out, whereas it enhanced production in the absence of TLR-4 to the same extent as observed in wild-type mice. Moreover, no increase but rather a significant decrease in IgA production was observed in immune cells lacking TLR-2, indicating that stimulation of immune cells in the absence of TLR-2 may generate a negative signal to the IgA production pathways. Although this observation warrants further investigation, it is beyond the scope of the present study. Nevertheless, it is clear that TLR-2 is essential for the enhancement of IgA production.
The crucial involvement of TLR-2 in IgA production allows us to suggest a possible explanation for our observation that RV-specific IgA production was preferential in breast milk. First, we consider it of great interest that a significant increase in RV-specific IgA levels by maternal administration of LG2055, which was also accompanied by a significant increase in RV-neutralizing activity, was observed only in the breast milk of stomach origin; that is, in the stomach content of mouse pups. Such a significant increase in RV-specific IgA levels, by contrast, was not observed in the Peyer's patch cell cultures or feces of mouse dams. Second, this result is consistent with a previous study that showed significantly higher concentrations of RV-specific IgA particularly in breast milk (14). Therefore, we believe that the preferential secretion of IgA observed in breast milk of stomach origin is not an anomaly and may be due to the following mechanism. The breast milk–producing mammary gland expresses a mucosal epithelial chemokine CCL28, which accumulates IgA antibody-secreting cells (ASCs) via the cognate chemokine receptor CCR10 on the ASCs that secrete IgA into breast milk (37). The CCL28-CCR10 interaction is critical for efficient localization and accumulation of IgA-ASCs at the lactating mammary gland (38). Furthermore, the chemokine receptor CCR10 on ASCs has been found to be upregulated by TLR-2 stimulation with TLR-2-ligands (39). As a result, it is possible to envisage that TLR-2 stimulation by LG2055 may elevate CCR10 expression on IgA-ASCs, leading to a large accumulation of ASCs in the mammary gland, resulting in a significant increase in IgA secretion in breast milk. This idea may improve the utility of probiotics in the field of maternal nutrition; however, it remains to be corroborated further.
In summary, the administration of heat-treated LG2055 cells to mouse dams reduced the incidence of RV-induced diarrhea in their pups and enhanced the secretion of RV-specific IgA preferentially in breast milk of stomach origin. A possible involvement of TLR-2 stimulation by LG2055 can be proposed as a mechanism underlying this preferential secretion of IgA in breast milk.
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