Dietary supplementation of milk fermented with probiotic Lactobacillus fermentum enhances systemic immune response and antioxidant capacity in aging mice.

N U TR IT ION RE S E ARCH XX ( 2 0 14 ) X XX– X XX

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Dietary supplementation of milk fermented with probiotic Lactobacillus fermentum enhances systemic immune response and antioxidant capacity in aging mice Rohit Sharma, Rajeev Kapila⁎, Meena Kapasiya, Vamshi Saliganti, Gulshan Dass, Suman Kapila Animal Biochemistry Division, National Dairy Research Institute, Karnal, India, 132001

ARTI CLE I NFO

A BS TRACT

Article history:

Although probiotics are known to enhance the host immune response, their roles in

Received 25 January 2014

modulating immunosenescence, resisting infection, and improving redox homeostasis during

Revised 12 September 2014

aging remain unclear. Therefore, the present study was devised in aging mice to assess the

Accepted 19 September 2014

antiimmunosenescence potential from the consumption of milk that is fermented with probiotic Lactobacillus fermentum MTCC 5898 (LF). We hypothesized that probiotic supplementation would

Keywords:

boost immunity, improve antioxidant capacity, and resist severity of pathogenic infection in

Immunosenescence

aging mice. To test this hypothesis, during a trial period of 2 months, 16-month-old male Swiss

Lactobacillus

mice were kept on 3 experimental diets: basal diet (BD), BD supplemented with skim milk,

Infection

and BD supplemented with probiotic LF-fermented milk. A concurrent analysis of several

Antioxidant

immunosenescence markers that include neutrophil functions, interleukins profile,

Mice

inflammation and antibody responses in the intestine as well as analysis of antioxidant

Inflammaging

enzymes in the liver and red blood cells was performed. Neutrophil respiratory burst enzymes and phagocytosis increased significantly in probiotic LF-fed groups, whereas no exacerbation in plasma levels of monocyte chemotactic protein 1 and tumor necrosis factor α was observed. Splenocytes registered increased interferon-γ but decreased interleukin 4 and interleukin 10 production, whereas humoral antibodies registered decreases in immunoglobulin G1 (IgG1)/IgG2a ratio and IgE levels in the probiotic-fed groups. Antioxidant enzymes (superoxide dismutase, catalase, and glutathione peroxidase) in LF-fed groups showed increased activities, which were more pronounced in the liver than in red blood cell. An Escherichia coli–based infection model in aging mice was also designed to validate the protective attributes of LF. Administration of probiotic LF significantly reduced E coli population in organs (intestine, liver, spleen, and peritoneal fluid), as compared with control groups, by enhancing E coli–specific antibodies and inflammatory proteins. Based on these results, it appears that LF supplementation alleviated immunosenescence, enhanced antioxidant enzyme activities, and resisted E coli infection in aging mice; thereby, signifying its potential in augmenting healthy aging. © 2014 Elsevier Inc. All rights reserved.

Abbreviations: ANOVA, analysis of variance; BD, basal diet; CAT, catalase; DCs, dendritic cells; E coli, Escherichia coli; ELISA, enzyme-linked immunosorbent assay; GPx, glutathione peroxidase; IgG1, immunoglobulin G1; IL-4, interleukin 4; LF, Lactobacillus fermentum MTCC 5898; MCP-1, monocyte chemotactic protein 1; MPO, myeloperoxidase; MRS, agar de Man Rogosa and Sharpe agar; NDRI, National Dairy Research Institute; PBS, phosphate-buffered saline; RBC, red blood cell; SM, skim milk; SOD, superoxide dismutase; TNF-α, tumor necrosis factor α. ⁎ Corresponding author. Tel.: +91 184 2259128. E-mail address: [email protected] (R. Kapila). http://dx.doi.org/10.1016/j.nutres.2014.09.006 0271-5317/© 2014 Elsevier Inc. All rights reserved.

Please cite this article as: Sharma R, et al, Dietary supplementation of milk fermented with probiotic Lactobacillus fermentum enhances systemic immune response and antioxidant..., Nutr Res (2014), http://dx.doi.org/10.1016/j.nutres.2014.09.006

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N UTR IT ION RE S EA RCH XX ( 2 01 4 ) X XX– X XX

1.

Introduction

Immunosenescence refers to the inevitable, multifaceted decline in the functions of the immune system during progressive aging. In a multitude of ways, the process affects all cells of the immune system, including neutrophils, macrophages, dendritic cells (DCs), and lymphocytes and renders the elderly more prone to infectious diseases and inflammatory disorders [1,2]. A disruption in cellular redox homeostasis is another critical manifestation of aging, which culminates in oxidative damage to cells and tissues. Together, immunosenescence, chronic infections, and oxidative stress constitute a grave threat for the rationale of healthy aging and pose a challenge to public health systems throughout the world. Thus, a particular global emphasis is focused on developing food- and nutrition-based strategies to rejuvenate the aging immune system as an attempt to ultimately boost immunity, prevent infections, and augment healthy aging. Therefore, it is not surprising that various interventions, such as vaccinations, nutritional supplements, and antiinflammatory treatments have been suggested to counter immunosenescence and age-inflicted disorders [3-5]. Dietary consumption of probiotics has long been considered beneficial for human health, primarily due to their ability to boost the host immune system [6]. However, most of these observations are the result of studies focusing on young adult populations, whereas the effects of probiotic supplementation in modulating immunosenescence and improving antioxidant capacity in the elderly are only partly understood. In addition, probiotic effects appear to vary with the immunophysiologic state of the host [7]. This implies that a distinctive analysis of probiotic immunomodulatory attributes on the aging immune system is more imperative than extrapolating results from studies in younger subjects. Considering these aspects, the present investigation was designed to provide a holistic view of the antiimmunosenescence effects of probiotic Lactobacillus fermentum MTCC 5898 (LF) in aging mice. We hypothesized that probiotic LF administration alleviates the deleterious aspects of immunosenescence, improves antioxidant capacity, and resists invading pathogen in aging animals. The hypothesis was tested by evaluating various cellular and humoral immunologic parameters as well as analyzing of antioxidant enzyme activities in liver and red blood cell (RBC) of experimental animals. Furthermore, an Escherichia coli–based infection model was used to validate the immunomodulatory attributes of this specific probiotic strain, thereby testing its potency in a real-time pathogen challenge. Probiotic LF was chosen in the present investigation because it had shown promising immunomodulatory potential in our preliminary analysis of macrophage functions, interleukins production in splenocytes, humoral response in serum/intestine, etc (data unpublished). Thus, the overall aim of this study was to assess the beneficial characteristics of probiotic LF in augmenting healthy aging.

2.

Methods and materials

2.1.

Microorganisms

The LF used in the present study was isolated from the feces of a 10-month-old infant, after obtaining informed written consent

from the parents. The lactobacilli colonies were isolated from fecal sample using agar de Man, Rogosa and Sharpe agar (MRS) agar plates. The bacterial culture was characterized for standard probiotic attributes, such as acid resistance, bile tolerance, and cell-surface hydrophobicity as per methods previously described [8]. Scanning electron microscopy was performed to visualize the adherence of LF to Caco-2 (National Center for Cell Science, Pune, India) cells [9]. The probiotic bacterium was identified using a commercially available API (Biomeriux, Durham, NC, USA) microorganism identification kit with apiweb (version API 50 CHL V5.1) analysis software. Subsequently, the culture was subjected to 16S ribosomal RNA analysis as previously described [10], along with DNA sequencing to taxonomically confirm bacterial species. For the in vivo experiments, the bacterial culture was stored at −80°C in MRS broth, which was supplemented with 20% (vol/vol) glycerol and activated before use by subculturing twice in MRS broth for 18 hours at 37°C. Fermented milk was prepared by inoculating aliquots of sterile skim milk (SM) with bacterial strain, followed by incubation for 18 hours at 37°C. The number of bacteria in the fermented milk was determined by plate counting on MRS agar plates after aerobic incubation at 37°C for 24 to 48 hours. A milk-based delivery system of probiotics was favored, as it is the most common way of probiotic consumption in the general population. E coli (ATCC 14948) was procured from the National Collection of Dairy Cultures, National Dairy Research Institute (NDRI), Karnal, India. The culture was grown in brain-heart infusion broth (Himedia Laboratories, Mumbai, India) at 37°C for 24 hours and then washed and resuspended in sterile phosphate-buffered saline (PBS), after adjusting the cell concentration to 108 CFU/mL using agar plate counting.

2.2.

Animal care and experimental design

To fully comprehend the extent of the effects of probiotics on aging physiology, earlier, we reported an age-associated profile of immunologic changes in Swiss albino mice [11]. In the present study, we devised 2 separate experiments with 16-month-old male Swiss albino mice. The first experiment was designed to evaluate the influence of LF consumption on immunologic and redox homeostasis parameters in aging mice. A second pathogen challenge experiment was conducted to determine the protective attributes of LF-fermented milk consumption in aging animals. To eliminate any effect from feeding or infection history on this aging study, the male Swiss albino mice used were procured from the small animal house of NDRI, Karnal, at the age of 2 weeks and raised on a basal diet (BD) until the age of 16 months [12]. The BD (Table) was prepared as reported earlier by Kaushal and Kansal [13]. This diet met the daily nutritional requirements for adequate maintenance and growth of aging mice. All animal experiments were conducted with the approval of the institutional animal ethics committee (approval letter no. NDRI 382/01CPCSEA; dated, 29.06.2011). For the first experiment, animals (weighing 38-40 g) were kept in 2 sets of 3 diets containing 6 animals each: BD, BD supplemented with SM, and BD supplemented with fermented milk prepared from LF. The study duration was divided into 2 intervals; one set of animals was fed for 1 month, whereas the other set continued for 2 months on their respective diet regimen. Animals kept in the LF group were trained to consume



Table – Ingredient composition of the BD fed to mice Ingredient

Grams/kilogram

Starch Casein Soybean oil Cellulose Mineral mixture a Vitamin mixture a Choline chloride Methionine

630 200 55 50 50 10 2 2

a

As per Association of Analytical Communities (2005).

a thin slurry of BD from glass plates, for at least 1 month before the onset of the actual experiments. During the feeding trials, milk fermented with LF (18.0 mL at 3.0 mL/animal) was lyophilized (approximately equal to 1/2 volume and finally containing 2.0 × 109 CFU/mL) and supplied to the LF animals in glass plates. In the control SM group, SM (18.0 mL at 3.0 mL/ animal) was mixed with the BD during preparation (30 g at 5 g/ animal per day) to a consistency of a dough, instead of water, which was used in the BD group. Animals in the 2 control groups (BD and SM) were fed 3 times a day, at regular intervals of 3 to 4 hours by dividing their total required diet into 3 equal parts. However, mice in the experimental group (LF) were initially given a dose of probiotic bacteria (3 × 109 CFU/animal per day) in 9.0 mL lyophilized fermented milk during the morning hours (9.30 AM Indian Standard Time). Later, after its consumption, the animals resumed the BD that was distributed in 3 equal parts as in control groups. During the night, all groups of animals were provided water ad libitum, but it was removed at least 3 to 4 hours (approximately equal to 6.00 AM Indian Standard Time) before feeding their respective diets (BD, SM, and LF) in the morning. The food bowls were also removed from the cages each night. This ensured that animals felt an adequate urge for food and water in the morning, when the experimental diets were supplied. To negate any handling stress to animals during the longer feeding trials, the oral intubation/cannula feeding method was not used, and animals were instead trained to obtain food/fermented milk from feeding plates/bowls. The selected dose of probiotic bacteria (3 × 109 CFU/animal per day) in fermented milk was based on our previous in vivo studies of probiotic immunomodulatory attributes [14,15]. Basically, it was extrapolated from recommended human doses (1-3 × 1010-1011 CFU/subject per day) using the dose translation formula (human: mouse), which is based on the ratio of body weight (kilograms) to body surface area (square meter) [16]. This is mentioned in practice guidelines for probiotics and prebiotics by the World Gastroenterology Organization [17] for a healthy or therapeutic outcome in clinical trials. At the end of their respective study periods, animals were euthanized by diethyl ether overdose; and then blood, peritoneal fluid, spleen, intestine, and liver of each animal were collected to assess various immunologic and antioxidant parameters. For the second experiment (pathogen challenge), animals were kept on 2 diet groups with 8 animals each: BD supplemented with SM (control) and BD supplemented with LFfermented milk. The same quantity and daily feeding procedure, as discussed above, were followed. Under this experiment, animals were given their respective diets for 30 days. On the

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31st day, animals were then administered a single dose (100 μL) of E coli containing 108 CFU, using a cannula. The same feeding regimen was continued for 7 more days postinfection. Mice were euthanized on the eighth day by diethyl ether overdose. Spleen, liver, intestine, and peritoneal fluid of animals were then collected for analysis of pathogen colonization; whereas, blood plasma was used to assess inflammatory proteins.

2.3.

Neutrophil isolation and functional analysis

Neutrophils were isolated from whole blood by density gradient centrifugation using Histopaque (Sigma, St. Louis, MO, USA) solutions 1077 and 1119. Briefly, 3 mL of Histopaque 1077 was carefully layered on top of 3 mL of Histopaque 1119, and 1.5 mL of collected blood was layered on top of the gradient, which was followed by centrifugation at 800g for 30 minutes at room temperature. The neutrophil layer in the gradient was carefully removed and subjected to lysis of any remnants of RBCs as previously described [18]. The resulting cell suspension contained more than 90% of neutrophils, with the overall viability greater than 95% as determined by the trypan blue exclusion method. Peripheral blood neutrophils were analyzed to evaluate the effects of LF consumption, in terms of respiratory burst potential and phagocytic activity. Neutrophil respiratory burst potential was assessed by analyzing cytochrome c reductase and myeloperoxidase (MPO) activities. Neutrophil cytochrome c reductase activity was evaluated using a cytochrome c reductase (nicotinamide adenine dinucleotide phosphate [NADPH]) Assay Kit (CY0100; Sigma) as per the manufacturer's protocol. Briefly, neutrophil cell suspensions (1 × 106 neutrophils/mL) were sonicated in the enzyme dilution buffer (300 mmolar, pH 7.8) containing 0.05% Triton X-100. The suspension was centrifuged at 12000g for 10 minutes, and the supernatant was analyzed for cytochrome c reductase activity. One unit of cytochrome c reductase activity was defined as a reduction of 1.0 nmol of oxidized cytochrome c in the presence of 100 μmol of NADPH per minute at pH 7.8 at 25°C. Myeloperoxidase activity was measured according to Bradley et al [19], with some modifications. Briefly, neutrophil suspensions were homogenized in 9 volumes of ice cold potassium phosphate buffer (50 mmol, pH 6.0) containing 0.5% cetyl trimethyl ammonium bromide, followed by sonication (10 seconds), and 3 cycles of freeze thawing. The suspension was centrifuged at 12000g for 15 minutes, and the supernatant was analyzed for MPO activity by mixing assay buffer (50 mmol potassium phosphate buffer, pH 6.0) containing 0.5 mmol o-dianisidine dihydrochloride and 0.0005% hydrogen peroxide (H2O2) as substrates. The breakdown of H2O2 is directly proportional to oxidation of o-dianisidine dihydrochloride, which was measured at 460 nm (UV-visible double beam spectrophotometer, UVD-3500; Labomed Inc., Los Angeles, CA, USA). The concentration of oxidized o-dianisidine dihydrochloride was calculated from its molar extinction coefficient (1.13 × 104/ cmM). One unit of enzyme activity was defined as that oxidizing 1.0 nmol of o-dianisidine per minute at 25°C. Neutrophil suspensions (1 × 10 6 cells/mL) were further assessed for phagocytic activity using yeast cells, according to the method of Hay and Westwood [20]. Phagocytosis was observed at ×1000 magnification, under oil immersion


4


(Olympus Optical Co., Tokyo, Japan). The following observations were recorded: Percentage of phagocytosis = number of neutrophils with yeast cells internalized per 100 neutrophils. Phagocytic index = (% phagocytic neutrophils containing ≥1 yeast) × (mean number of yeasts/phagocytic neutrophils containing yeasts).

2.4.

Cytokine response

Splenocytes were isolated and cultured from the spleen tissue as previously described, using concanavalin A (5 μg/mL; Sigma) as a stimulant [21]. Supernatants of cultured splenocytes were used to estimate levels of interleukins (interferon-γ (IFN-γ), interleukin 4 [IL-4], and IL-10) by commercially available quantitative sandwich enzyme-linked immunosorbent assay (ELISA) kits (eBiosciences, San Diego, CA, USA), according to the manufacturer's protocol. Briefly, NUNC (Waltham, MA, USA) Maxisorp 96 well plates were coated with 100 μL of ×1 capture antibody (goat antimouse, IFN-γ/IL-4/IL-10) and incubated overnight at 4°C. The samples were diluted 2 times before adding to the experimental wells and followed by the addition of detection antibody and 100 μL of avidin horseradish peroxidase. Plates were allowed to develop with the tetramethyl diamine benzidine substrate (3,3,5,5-tetramethyl diamine benzidine containing 0.03% H2O2), and the reaction was finally stopped with 50 μL of 2 mol sulfuric acid. Plates were read at 450 nm. Results are expressed as per milligram of total protein. Folin-phenol reagent method was used to estimate total protein content in culture supernatants as previously described [22]. The basal level of circulatory inflammation in plasma was also analyzed by estimation of monocyte chemotactic protein 1 (MCP-1) and tumor necrosis factor α (TNF-α) levels, in undiluted samples using the commercially available quantitative sandwich ELISA kits (eBiosciences) as described above.

2.5.

Humoral immune response in the intestine

Intestinal fluid was collected as per the procedure described by Lim et al [23]. Briefly, intestine from the gastroduodenal to ileocecal junctions was carefully removed, and the contents were washed with 5 mL PBS (pH 7.2) and followed by centrifugation at 2000g for 30 minutes. The resultant supernatant, that is, intestinal fluid, was recovered and stored at −80°C until used for estimation of total immunoglobulin A (IgA), total IgE, IgG1, and IgG2a antibodies. These antibodies were detected in a sandwich ELISA format as described in the previous section and according to the manufacturer's protocol (Komabiotech, Seoul, Korea; and eBiosciences). Results are expressed as per milligram of total protein.

2.6.

Immunofluorescence assay for IgA + cells

Two centimeters of tissue from the small intestine of mice were used for the preparation of the histologic slides, by the method of Kiernan [24]. Sections (3 μm) of tissues were cut with a Senior Rotary Microtome (RMT-30; Radical, Ambala, India), and the slides were prepared for direct immunofluorescence assay for IgA+ cells. After deparaffinization using xylene and rehydration

in a decreasing gradient of ethanol, sections were blocked with 2% bovine serum albumin for 1 hour. The slides were washed 2 to 3 times with PBS, followed by incubation with 1:100 dilution of α-chain monospecific antibody that was conjugated with fluorescein isothiocyanate (Cayman Chemical, Ann Arbor, MI, USA) for 1 hour. They were then observed with a fluorescent light microscope (CKX41; Olympus). The number of fluorescent cells was counted in a minimum of 30 fields at ×200 magnification. The results were expressed as the number of positive fluorescent cells per 5 fields of vision.

2.7.

Antioxidant enzyme activity

To assess antioxidant capacity, catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx) activities were determined in liver and RBC lysate. Catalase activity was measured spectrophotometrically by analyzing the rate of H2O2 decomposition at 240 nm [25]. One unit of CAT corresponds to degradation of 1 μmol of H2O2 per minute. Superoxide dismutase was measured according to the method of Marklund and Marklund [26], and the unit activity was defined as the amount of enzyme that causes 50% inhibition of pyrogallol autooxidation under experimental conditions. Glutathione peroxidase was assayed by measuring the rate of oxidation of NADPH, using cumene hydroperoxide as a substrate [27]. The enzyme activity was calculated using an extinction coefficient of 6.22 × 103 M−1 cm−1, and 1 U was defined as 1 mmol of NADPH oxidized per minute. Results are expressed as units/milligram of total protein for liver enzymes and units/milligram of total hemoglobin level for enzymes in the RBC lysate.

2.8.

Quantitative estimation of E coli translocation

The translocation and colonization of E coli were determined in peritoneal fluid and various organs of the animals. Intestine, liver, and spleen of each animal were individually homogenized in 0.1% peptone water. The tissue homogenate and peritoneal fluid were then serially diluted in peptone water and plated on eosin methylene blue agar (Himedia Laboratories). E coli bacterial colonies with characteristic green metallic sheen were identified and enumerated after 48 hours incubation at 37°C.

2.9. Estimation of E coli–specific antibodies in intestinal fluid A section of the small intestine was used to collect intestinal fluid for estimation of pathogen-specific antibodies. E coli– specific IgA (Komabiotech) and IgG1 (eBiosciences) antibodies were estimated in intestinal fluid by ELISA, according to manufacturer's protocol and as described by Engwall and Perlmann [28]. Briefly, wells of microtiter plates (NUNC) were coated with E coli suspension (100 μL) at the rate of 108 CFU/ mL in 0.06 mol carbonate buffer (pH 9.6). Control wells were coated with 100 μL of carbonate buffer alone. Plates were incubated for 18 to 20 hours at 37°C for drying. Subsequently, 200 μL of 70% methanol were added and left for 20 minutes to fix antigen on the plate surface and then dried again. Free binding sites were blocked by adding 200 μL of blocking solution (2% bovine serum albumin in PBS–Tween-20) and



then incubated at room temperature for 2 hours with occasional shaking. Blocking solution was eliminated, and the wells were washed 3 times with 0.05% PBS–Tween-20. Samples and detection antibody (IgG1) were added, and the plates were incubated for 3 hours at 37°C, with occasional shaking. For IgA, samples were incubated for 1 hour, followed by the addition of 100 μL of goat antimouse peroxidase conjugate antibody (IgA) and incubation at 37°C for 1 hour. Substrate solution (0.1 mL) (0.04% o-phenylene-diamine hydrochloride, 0.012% H2O2 in phosphate citrate buffer; pH 5.0) was added to each well, and the reaction was carried out at room temperature for 30 minutes in the dark. The reaction was stopped with 0.1 mL of sulfuric acid (2N), and absorbance was measured at 450 nm using an ELISA plate reader. The antibody concentration in each sample was expressed as absorbance/100 μL.

2.10.

Inflammatory proteins

The levels of circulatory inflammatory proteins (IFN-γ/MCP-1/ TNF-α) were assessed in the plasma of the experimental animals via quantitative sandwich ELISA as described in section 2.4.

2.11.

Statistical analyses

Data were analyzed by using GraphPad Prism (La Jolla, CA, USA) (version 5.01) software. Resource equation method was used to ascertain the sample size as previously described [29]. Results are expressed as means ± SEM. Differences between the means were tested for statistical significance using a 2-way analysis of variance (ANOVA) and followed by Bonferroni post hoc test. The significance level was set at 5% (P < .05) for all calculations.

3.

Results

3.1.

Probiotic attributes of LF

The probiotic attributes of LF are presented in Fig. 1. Even in a strongly acidic environment (pH 2), LF showed a high survival rate (Fig. 1A). Except for pH 2 after a 2-hour incubation, no significant difference in the survivability of LF with time in different acidic conditions could be observed. Lactobacillus fermentum MTCC 5898 also showed strong bile tolerance, even after prolonged incubation (6 hours) under increasing bile concentrations (Fig. 1B). Lactobacillus fermentum MTCC 5898 exhibited excellent percent cell-surface hydrophobicity with xylene, followed by octane and n hexadecane (Fig. 1C). Cellsurface hydrophobicity is used as an indicator of the ability of probiotics to adhere to the intestinal epithelial cells. A representative scanning electron micrograph of LF attached to Caco-2 cells is also shown in Fig. 1D.

3.2.

Neutrophil functions

The impact of LF-fermented milk on neutrophil functions is depicted in Fig. 2. A significant (P < .05) increase in activities of both cytochrome c reductase and MPO was observed, as compared with the control groups BD and SM (Fig. 2A and B). Percent phagocytosis and the phagocytic index of neutrophils also

5

significantly (P < .05) increased in the LF-fed groups, in comparison with the BD group. This appeared to be maintained over the 2month feeding period (Fig. 2C and D). Representative photographs of neutrophils phagocytizing yeast cells are depicted in Fig. 2E.

3.3.

Inflammaging markers

The effect of probiotic administration on circulatory inflammatory markers in plasma is depicted in Fig. 3. No significant difference in levels of MCP-1 in any experimental group was observed after 1 month of feeding. However, a significant (P < .05) decrease in MCP1 was recorded in the LF-fed group, as compared with the control groups after 2 months of feeding (Fig. 3A). A significant (P < .05) increase in TNF-α levels in the BD-fed group was observed during 1 and 2 months of the study duration (Fig. 3B). However, the LF-fed group maintained its TNF-α levels and, thus, registered a significant (P < .05) decline as compared with the BD-fed group after 2 months.

3.4.

Interleukins profile

A remarkable increase (P < .001) in IFN-γ production was recorded in the LF-fed groups, as compared with control (BD and SM) groups (Fig. 4A). However, the levels of IL-4 and IL-10 recorded a considerable (P < .01) decrease in the probiotic-fed group, as compared with the BD group after the 2-month feeding period (Fig. 4B and C).

3.5.

Humoral immune response

After 2 months of feeding, IgG1 levels registered a significant increase (P < .05) in both BD and SM control groups, whereas the LF-fed group maintained its original levels (Fig. 5A). No significant variations were observed in IgG2a levels while comparing the LF group with the control groups (Fig. 5B). However, the ratio of IgG1/IgG2a observed a significant (P < .05) decrease in the LF-fed groups after 2 months of feeding, as compared with the BD group (Fig. 5C). In addition, a significant (P < .05) increase in the ratio of IgG1/IgG2a in both control groups throughout the entire feeding trial was observed. A similar trend was observed in the levels of the IgE; whereas, the BD group recorded a significant increase among both months of feeding, and the LF-fed group registered a significant (P < .05) decrease after 2 months (Fig. 5D). In contrast, no significant variations in IgA levels were observed among different experimental groups over the entire study duration (Fig. 5E).

3.6.

IgA + cells in intestine

Changes in numbers of IgA+ cells in various groups are illustrated in Fig. 6A. No statistically significant differences in IgA+ cells were observed among any of the experimental groups over the 2-month study duration. Fig. 6B depicts a representative photograph of fluorescent IgA+ cells.

3.7.

Antioxidant capacity

Catalase and GPx activities in the liver recorded a significant (P < .05) increase in LF-fed groups, as compared with BD groups throughout the feeding duration. Superoxide dismutase activity


6


A

B

8

log cfu/ml

6

4

8

4

2

2

0

0 2.0

2.5

3.5

Control

6.5

0.5

1.0

1.5

2.0

Bile salt conc. (%)

pH

C

D 1h 2h

100

% Hydrophobicity

0h 2h 4h 6h

6

log cfu/ml

0h 1h 2h

80 60 40 20 0 Xylene

Octane

n-Hexadecane

Fig. 1 – Probiotic attributes of LF. Acid resistance (A), bile salt tolerance (B), and cell surface hydrophobicity (C). Scanning electron micrograph showing adherence of LF to Caco-2 cells (D). The data were tested using a 2-way ANOVA followed by Bonferroni post hoc test. All experiments were performed in triplicate, and values presented are means ± SEM. ⁎P < .05 represents values, which are significantly different from 0 hour in a given group.

registered a significant increase only after 2 months of probiotic feeding (Fig. 7B, D, and F). However, the differences in enzyme activities in RBC were less distinctive, and only GPx activity recorded a significant increase when compared with control groups during the feeding period (Fig. 7A, C, and E).

3.8.

E coli infection symptoms and pathogen colonization

By the third day of the postpathogen challenge, apparent changes in behavior, which included dormant and isolated behavior, massive diarrhea, continuous erratic heartbeat, and loss of appetite, were evident in the SM-fed mice. Conversely, LF-fed animals were more active and respondent, and they maintained their appetite. No mortalities were observed among any of the animal groups during the feeding trial. E coli infection heavily infiltrated body organs and peritoneal fluid of mice. However, a significant (P < .01) decrease in pathogen colonization in the intestine, liver, spleen, and peritoneal fluid was observed in the LF-fed groups, as compared with the SM-fed group (Fig. 8A). The colonization in various organs and peritoneal fluid was 1 to 2 log units less in the LF-fed groups when compared with the SM group.

3.9.

E coli–specific antibodies

A remarkable (P < .001) 1.34-fold increase in E coli–specific IgA antibodies and a 1.15-fold increase (P < .01) in E coli–specific

IgG1 were observed in the LF-fed group, as compared with the SM group (Fig. 8B).

3.10.

Inflammatory proteins

A significant presence of inflammatory markers was noted in plasma of LF-fed animals (Fig. 8C). Compared with control SM group, IFN-γ registered an increase of 4.19-fold (P < .01), TNF-α recorded an increase of 1.61-fold (P < .01), and MCP-1 registered an increase of 1.38-fold (P < .05) in the LF-fed group.

4.

Discussion

The primary focus of the present investigation was to assess the antiimmunosenescence attributes of probiotic LF while maintaining general health during the aging process. Our results support our hypothesis indicating that probiotic LF supplementation alleviates immunosenescence, resists infections, and improves antioxidant capacity, thereby augmenting healthy aging. Several previous studies reported various age-associated discrepancies in neutrophil functions, including impaired respiratory burst and phagocytosis [30-32]. Neutrophils constitute the first line of immune defense and are among the principal cells to respond against invading pathogens. In the first experiment of the present study, supplementation of LF


7


B

200

B BD S SM L LF

Units/106 cells

b b

a

150

a

a

a

100

50

50 40

Units/106 cells

A

a a

30

BD D M SM LF F

a

20 10

0

0 1 Month

2 Months

1 Month

2 Months

D

50

BD D SM M LF F

b

40

b a

30

a

a

a

20 10

50

Phagocytic Index

C % Phagocytosis

b

b a

0

b b

40

a

a

B BD SM S LF L

a

a

30 20 10 0

1 Month

2 Months

1 Month

2 Months

E

Fig. 2 – Modulation of peripheral blood neutrophils respiratory burst enzyme activities (A and B) and phagocytosis (C and D) on consumption of LF-fermented milk after 1 and 2 months of feeding in aging mice. Cytochrome c reductase activity (A), MPO activity (B), percent phagocytosis (C), and phagocytic index (D). E, Representative microphotographs showing ex vivo internalized yeast cells by neutrophils at ×1000 magnification. The data were tested using a 2-way ANOVA followed by Bonferroni post hoc test. Values are means ± SEM of 6 animals per group. Means that do not share a common letter indicate statistical difference at P < .05.

B

A

20

200

pg/ml

a

a

a

a 100

b

10

a

b

a

5

50

BD SM LF

a

15

pg/ml

a

150

BD SM LF

b

b

0

0

1 Month

2 Months

1 Month

2 Months

Fig. 3 – Effect of probiotic LF-fermented milk administration on circulatory inflammation status in aging animals after 1 and 2 months of feeding period as indicated by levels of MCP-1 (A) and TNF-α (B). The data were tested using a 2-way ANOVA followed by Bonferroni post hoc test. Values are means ± SEM of 6 animals per group. Means that do not share a common letter indicate statistical difference at P < .05. ⁎P < .05 for treatments fed for 1 vs 2 months. Please cite this article as: Sharma R, et al, Dietary supplementation of milk fermented with probiotic Lactobacillus fermentum enhances systemic immune response and antioxidant..., Nutr Res (2014), http://dx.doi.org/10.1016/j.nutres.2014.09.006

8


A

B BD SM LF

pg/mg protein

b 80 60 40 20

b a

a

a

a

pg/mg protein

30

100

a

BD SM LF

a

a

a

20

b b

10

0

0 1 Month

2 Months

1 Month

C

a

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Fig. 4 – Effect of probiotic LF-fermented milk consumption on interleukins production in splenocytes of aging animals after 1 and 2 months of feeding. IFN-γ (A), IL-4 (B), and IL-10 (C). Splenocytes were isolated followed by incubation with concanavalin A to stimulate T cells, and culture supernatants were analyzed for interleukins. The data were tested using a 2-way ANOVA followed by Bonferroni post hoc test. Values are means ± SEM of 6 animals per group. Means that do not share a common letter indicate statistical difference at P < .05. ⁎⁎P < .01 for treatments fed for 1 vs 2 months.

in aging mice resulted in increased enzyme activities of neutrophil cytochrome c reductase and MPO. Furthermore, a corresponding increase in phagocytosis was also observed in the LF-fed groups. A decrease in MPO activity has been directly implicated in the impaired phagocytic response of neutrophils [33]. Thus, it seems plausible that the enhanced activities of MPO and cytochrome c reductase on LF consumption could be responsible for the increased phagocytic potential of neutrophils. Our results showing enhanced neutrophil functions in aging mice by probiotic supplementation are also related to previous human-based studies. A report by Maneerata et al [34] showed increased phagocytic activities of monocytes and granulocytes on supplementation with Bifidobacterium lactis Bi-07 in healthy elderly individuals. Similarly, Stadlbauer et al [35] reported enhanced phagocytosis in neutrophils after dietary supplementation with Lactobacillus casei Shirota in patients with alcoholic cirrhosis. Earlier reports have suggested that aging in mice is accompanied by an imbalance in type 1 T helper cell (Th1)/type 2 T helper cell (Th2) cytokine production. This discrepancy in T-cell differentiation is often held accountable for vulnerability to infections and other adverse immunologic outcomes during aging. However, the nature of this imbalance is often ambiguous and contradictory. Although some studies suggest increased Th1 response with aging, others implicate increased Th2 response [36-39]. We argued that without explicit knowledge of the nature of this dysregulation and the immunologic state of the subjects used, it is difficult to comprehend and contemplate

the precise effects of probiotics. Considering this, we reported earlier that the Swiss albino mouse strain used in the present study shows a decrease in Th1 response with an aggravation of Th2 response during progressive aging [11]. Probiotics have been shown to restore Th1/Th2–related imbalances in clinical studies and murine allergic models [40,41]. In the first experimental design of the present investigation, we reported that the age-associated skewed pattern toward Th2 response can also be countered by probiotic lactobacilli supplementation. A robust increase in IFN-γ levels and a concurrent decrease in IL-4 and IL-10 levels in the LF-fed group suggested a strong inclination toward cell-mediated immune response (Th1). This scenario proposes an effective reversal of the immune response from prevalent Th2 response in aging control groups (BD and SM) to a predominant Th1 response in lactobacilli-supplemented groups. The mechanisms governing the observed effects were not addressed in the present study but may be related to DCs, which play a critical role in the polarization of Th0 cells to Th1 or Th2 subsets. It has been shown that probiotic lactobacilli can modulate the phenotype and function of DC, thus resulting in deviation toward Th1 response [42-44]. The increased Th1 response, IFN-γ in particular, is also shown to enhance various neutrophil functions [45]. Therefore, this could be attributed to the increase in neutrophil activities that were observed in the LF-supplemented groups. The analysis of circulatory proinflammatory proteins in the different experiments was intriguing. During the


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Fig. 5 – Effect of probiotic LF-fermented milk administration on immunoglobulin production in intestine of aging mice after 1 and 2 months of feeding as observed in IgG1 (A), IgG2a (B), IgG1/IgG2a ratio (C), IgE (D), and IgA (E). The data were tested using a 2-way ANOVA followed by Bonferroni post hoc test. Values are means ± SEM of 6 animals per group. Means that do not share a common letter indicate statistical difference at P < .05. ⁎⁎⁎P < .001, ⁎⁎P < .01, and ⁎P < .05 for treatments fed for 1 vs 2 months.

assessment of the inflammatory status in the first experiment, TNF-α levels in the control BD-fed animals registered a significant increase (71.9%) with age as evidenced from both the 1- and 2-month durations of the trial period. This suggests the presence of a chronic low-grade inflammation state in aging mice (inflammaging), which predisposes the elderly to inflammatory disorders [46-48,11]. In the present study, LF supplementation apparently suppressed concentrations of both TNF-α and MCP-1, which are potent markers of inflammation. Thus, it appears that aging animals in the control group continued to show signs of inflammaging, whereas probiotic LF-fed animals were resistant to aggravation of circulatory inflammation. A recent study in healthy elderly adults by Moro-Garcí et al. [49] showed that supplementation of probiotic Lactobacillus delbrueckii subspecies Bulgaricus 8481 reduces proinflammatory cytokine IL-8 levels with general improvement in parameters defining the immune

risk profile. However, to the best of our knowledge, there is no specific analysis of the antiinflammaging attributes of probiotic supplementation in elderly humans. The apparent inflammaging modulating properties of probiotic LF in the present study suggest its potential in maintaining inflammatory homeostasis in elderly. Nonetheless, there are reports in adult human subjects suggesting that various strains of probiotics can attenuate levels of proinflammatory proteins, such as IL-1β, TNF-α, and IL-6, beyond the gastrointestinal mucosa and in the periphery [50]. It is also shown that healthy adults consuming a milk-based drink containing Lactobacillus rhamnosus GG, Bifidobacterium animalis species lactis Bb12, or Propionibacterium freudenreichii subspecies lactis/shermanii led to a significant decrease in the levels of serum C-reactive protein and proinflammatory cytokines in peripheral blood mononuclear cells; thus, suggesting strain-dependent antiinflammatory effects of probiotics [51]. The precise mechanism pertaining to


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Fig. 6 – Fluorescent microscopy (×200) of intestinal villi with fluorescein isothiocyanate–labeled rabbit antimouse IgA for IgA+ cells. A, No changes in numbers of IgA+ cells/5 fields could be observed in different experimental groups. B, Representative image of fluorescent IgA+ cells. The data were tested using a 2-way ANOVA followed by Bonferroni post hoc test. Values are means ± SEM of 6 animals per group. Means that do not share a common letter indicate statistical difference at P < .05.

these observations is not fully understood. However, a recent hypothesis linked increased production of polyamines as probable mediators of antiinflammatory effects of probiotics during aging, but this requires further validation [52]. During E coli infection, all measured inflammatory proteins recorded a remarkable increase in the LF-fed group, as compared with control group. This apparent contradiction in inflammatory networks could be attributed to the fact that infectious agents invade the intestinal epithelium and stimulate an intense acute inflammatory response. We reason that the changed dynamics of the immune system under the threat of an infection, coupled with the Th1-stimulating ability of LF, prevailed and resulted in increased inflammatory proteins (IFN-γ) in the pathogen challenge experiment. This increase in Th1 response could have helped in resisting the invading pathogen by further activating innate immune cells through the observed increase in other inflammatory proteins (TNF-α and MCP-1). These observations are indicative of differential probiotic-host cross talk and effector immune response, in relation to inflammaging and infection. Evaluation of the humoral response in intestinal fluid revealed significant effects from probiotic supplementation in modulating immunoglobulin class switching. On one hand, a significant increase in IgG1/IgG2a ratio and IgE levels was observed in both control groups (BD and SM) between 1 and 2 months of feeding. On the other hand, LF-fed groups recorded a significant decrease in both IgG1/IgG2a ratio and IgE levels after 2 months of feeding. This suggests the robust effects of probiotic LF in countering prevalent immunosenescence in aging animals. Interleukins are responsible for stimulating the class-switching process in B cells. In particular, IL-4 is considered the main driving force favoring IgG1 and IgE production, whereas IFN-γ is responsible for IgG2a synthesis in mice. Thus, the altered interleukins profile in the LF-fed animals (increased IFN-γ and decreased IL-4) could be responsible for apparent changes in downstream immunoglobulin production. In contrast, secretory IgA levels and numbers of IgA + cells in the intestine remained unchanged in the various experimental groups over the entire feeding duration.

Infectious agents, that is, E coli, can bypass gastric defenses, penetrate into intestinal mucosa, and multiply within macrophages of the reticuloendothelial system to disseminate via systemic circulation, thus reaching different organs such as liver and spleen. Lactobacillus fermentum MTCC 5898 consumption resisted invading pathogen in all organs and peritoneal fluid, suggesting that this probiotic has the potential to inhibit systemic infection. The exact mechanism of LF resistance of E coli is speculative, but it is possible that prefeeding (30 days) LF to animals could have enriched the gut microbiota and created a protective layer over the intestinal epithelial cells, therefore resulting in competition for adherence space for the invading pathogen and providing preliminary protection. This resistance for colonizing space is a known mechanism of probiotic action against pathogens and is supported by probiotic attributes of LF, suggesting excellent survivability and adhesion potential in the gut. The apparent Th1-stimulating potential of LF likely took over to mount an inflammatory response, resulting in observed clearance of E coli in LF-fed animals. This is also evident from the remarkable increase in E coli–specific IgA and IgG1 antibodies and inflammatory proteins in the LF group. Lactobacillus fermentum MTCC 5898 consumption in the present study had profound effects on several immune parameters in aging mice, which was also evident during an infection challenge. To further substantiate these findings, we hypothesized that this beneficial immunologic state might also be reflected in the oxidative status of the aging animals. Free radical theory of aging proposes that an increase in ageinflicted oxidative stress coupled with decreased activities of various antioxidant enzymes results in tissue damage and inflammatory aggravation. Lactobacillus fermentum MTCC 5898 administration in the present study enhanced the activities of SOD, CAT, and GPx in the liver; whereas, an increase in GPx activity was also observed in RBC. Together, this signifies an improved free radical clearance system. These findings can also be directly related to humans because weakened cellular antioxidant potential is a characteristic feature of human aging [53]. It has been reported that supplementation of


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Fig. 7 – Effect of probiotic LF-fermented milk consumption in aging mice on antioxidant SOD activity in RBCs (A) and liver (B), CAT activity in RBCs (C) and liver (D), GPx activity in RBCs (E), and Liver (F). Liver enzymes showed distinct changes as compared with enzymes in RBCs. The data were tested using a 2-way ANOVA followed by Bonferroni post hoc test. Values are means ± SEM of 6 animals per group. Means that do not share a common letter indicate statistical difference at P < .05. ⁎P < .05 for treatments fed for 1 vs 2 months.

probiotic L rhamnosus IMC 501 and Lactobacillus paracasei IMC 502 increased plasma antioxidant levels in adult athletes [54]. Previous studies also show that oral administration of lactobacilli and fermented milk whey promote glutathione biosynthesis in tissues, which could explain the increased GPx activity in LF-fed groups [55,56]. The liver is the major site of glutathione synthesis and transport and, thus, could explain the apparent effects of LF consumption on GPx activity in liver and RBC. Similarly, probiotic consumption is also shown to enhance activities of SOD and CAT in various clinical and experimental studies [57-59]. There are several limitations with the current study that includes the identification of a mechanism. Firstly, there is a need for more direct and deeper analyses of the factors involved in the observed immunomodulatory effects of probiotic LF during aging. Our results provide indirect evidence of the underlying mechanisms relating to the effects of probiotic administration in countering immunosenescence

and resisting the severity of infection. Future studies are required to examine the direct effects of LF on intestinal epithelial tight junction structure and function in the coordination of appropriate immune responses, ranging from tolerance to antipathogen immunity. Secondly, the mice used in the present study showed a weakened Th1 response with age [11], which appeared to be enhanced on account of probiotic administration. However, there is no consensus on the nature of immune deregulation (Th1/Th2) with aging in humans, and thus, it is critical to assess the immunomodulatory behavior of probiotic LF in Th1-polarized circumstances. Thirdly, the influence of LF administration on the composition of intestinal microbiota was not addressed in the present study, and this could provide more insights into the observed effects of probiotics. Lastly, there is no consensus regarding the most optimal dose, duration of the treatment, and interspecies extrapolation of probiotic effects in various studies [60].


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Fig. 8 – Influence of consumption of LF-fermented milk on pathogenic E coli infection in aging mice. A, Lactobacillus fermentum MTCC 5898–fed animals showed significant clearance of E coli from body organs and peritoneal fluid. B, E coli–specific antibodies in intestine increased remarkably in LF-fed group. C, An increase in inflammatory proteins was also observed in plasma of LF group animals. The data were tested using a 2-way ANOVA followed by Bonferroni post hoc test. Values are means ± SEM of 6 animals per group; ⁎Represents significant difference as compared with SM group; ⁎⁎⁎P < .001, ⁎⁎P < .01, and ⁎P < .05.

It is also necessary to assess the dose and efficacy of probiotic LF in human subjects before considering any therapeutic interventions based on this study. The dose of 3 × 109 CFU/mL per day of LF in fermented milk in our investigation is extrapolated from human studies as mentioned in a report by the World Gastroenterology Organization [17]. However, one particular dose level cannot be assumed to be effective for all strains. For instance, the efficacy of Bifidobacterium infantis 35264 has been documented at 108 CFU/d [61], whereas the recommended dose of VSL no. 3 (VSL Pharmaceuticals, Gaithersburg, MD, USA) is 1.8 × 1012 CFU/d [62]. As of this study, little is known about the minimal dosage and frequency of probiotics that are required for any significant effect. Earlier, we showed that 1 billion CFU/d of probiotic bacteria is sufficient to induce immunomodulatory response in mice [14,15]. However, clinical trials regarding the relationship between the dose of probiotics and the duration of the treatment need to be addressed in future studies. In summary, the present investigation focused on providing a holistic scenario of the immunomodulatory attributes of the probiotic LF in an aging immune system. Our previous analysis of age-associated changes in a murine immune system greatly substantiated our interpretation and contemplation of the probiotic effects in the present study. Several of the immune parameters investigated indicated signs of prevalent immunosenescence and polarized Th2 response in control groups (BD and SM), which were effectively

ameliorated by the restoration of Th1/Th2 homeostasis on LF supplementation. Although probiotics modulated immune functions, no indiscriminate aggravation of inflammatory status was apparent until animals were subjected to a real-time pathogenic challenge. However, the present study did not address the precise mechanisms governing these probiotic effects; and further investigations aimed at understanding the modulation of inflammaging, in conjunction with pathogenic infection, are required to fully comprehend the probiotic-host interactions during aging.

Acknowledgment The financial assistance provided by the Department of Biotechnology and the Government of India are appreciated. The laboratory facilities provided by NDRI, Karnal, are also acknowledged.

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Dietary Supplementation of Phoenix dactylifera Seeds Enhances Performance, Immune Response, and Antioxidant Status in Broilers.

Th2 immune homeostasis, antioxidative status and resistance to pathogenic E. coli on consumption of probiotic Lactobacillus rhamnosus fermented milk in aging mice.

Technological properties and probiotic potential of Lactobacillus fermentum strains isolated from West African fermented millet dough.

Effects of dietary supplementation with fermented ginkgo leaves on antioxidant capacity, intestinal morphology and microbial ecology in broiler chicks.

Dietary supplementation with Lactobacilli improves emergency granulopoiesis in protein-malnourished mice and enhances respiratory innate immune response.

Whole Genome Sequence of the Probiotic Strain Lactobacillus paracasei N1115, Isolated from Traditional Chinese Fermented Milk.

Optimisation of oat milk formulation to obtain fermented derivatives by using probiotic Lactobacillus reuteri microorganisms.

Anti-colon cancer and antioxidant activities of bovine skim milk fermented by selected Lactobacillus helveticus strains.

Complete Genome Sequence of Lactobacillus fermentum MTCC 25067 (Formerly TDS030603), a Viscous Exopolysaccharide-Producing Strain Isolated from Indian Fermented Milk.

Dietary supplementation with white button mushrooms augments the protective immune response to Salmonella vaccine in mice.

Antioxidant supplementation enhances bacterial peritonitis in mice by inhibiting phagocytosis.

Training Enhances Immune Cells Mitochondrial Biosynthesis, Fission, Fusion, and Their Antioxidant Capabilities Synergistically with Dietary Docosahexaenoic Supplementation.

Effects of probiotic supplementation on systemic and intestinal oxidant-antioxidant events in splenectomized rats.

Dietary milk fat globule membrane improves endurance capacity in mice.

The Influence of Probiotic Lactobacillus casei in Combination with Prebiotic Inulin on the Antioxidant Capacity of Human Plasma.

Dietary supplementation with lactose or artificial sweetener enhances swine gut Lactobacillus population abundance.

Supplementation with two probiotic strains, Lactobacillus curvatus HY7601 and Lactobacillus plantarum KY1032, reduces fasting triglycerides and enhances apolipoprotein A-V levels in non-diabetic subjects with hypertriglyceridemia.

Inhibition of Growth and Metastasis of Breast Cancer in Mice by Milk Fermented With Lactobacillus casei CRL 431.

Glucan supplementation enhances the immune response against an influenza challenge in mice.

c Mice.

Functional Profile Evaluation of Lactobacillus fermentum TCUESC01: A New Potential Probiotic Strain Isolated during Cocoa Fermentation.

Inoculated fermentation of green olives with potential probiotic Lactobacillus pentosus and Lactobacillus plantarum starter cultures isolated from industrially fermented olives.

Effect of orally administered soy milk fermented with Lactobacillus plantarum LAB12 and physical exercise on murine immune responses.

Draft Genome Sequence of a Probiotic Strain, Lactobacillus fermentum UCO-979C.