Weifen Li, Key Laboratory of Molecular Animal Nutrition, Ministry of Education, Institute of Feed Science, College of Animal Science, Zhejiang University, Hangzhou China 310058. Tel: +86 571 88982108; fax: +86 571 88982729; e-mail: firstname.lastname@example.org
To investigate the immunomodulatory effects of Bacillus subtilis (B. subtilis) (natto) B4 spores on murine macrophage, RAW 264.7 cells were cultured alone or with B subtilis (natto) B4 spores at 37°C for 12 hrs, then both cells and culture supernatants were collected for analyses. Exposure of RAW 264.7 cells to B. subtilis (natto) B4 spores had no significant effects on macrophage viability and amounts of extracellular lactate dehydrogenase (LDH). However, it remarkably increased the activities of acid phosphatase (ACP), lactate dehydrogenase (LDH) and inducible nitric oxide synthase (iNOS) in cells and the amounts of nitric oxide (NO) and cytokines (tumor necrosis factor-alpha, interferon-gamma, interleukin [IL]-1 beta, IL-6, IL-12, IL-10 and macrophage inflammatory protein-2) in culture supernatants. These results demonstrate that B. subtilis (natto) B4 spores are harmless to murine macrophages and can stimulate their activation through up-regulation of ACP and LDH activities and enhance their immune function by increasing iNOS activity and stimulating NO and cytokine production. The above findings suggest that B. subtilis (natto) B4 spores have immunomodulatory effects on macrophages.
Bacillus subtilis (natto), also known as B. subtilis var. natto (1), is a Gram-positive spore-forming bacterium. It was originally used for the production of “natto”, a traditional Japanese staple made from fermented soybeans. Natto has multiple probiotic functions (2) and its bacterial component, B. subtilis (natto), can stimulate lymphocyte proliferation, modulate intestinal microflora and improve growth performance of chickens (3–6). Thus, this bacterium is thought to be a probiotic and its spores are currently widely used as a food supplement for humans and a feed additive for animals (7).
Macrophages play a key role in both innate and adaptive immune responses (8) and can recognize and eliminate pathogens through their phagocytic, cytotoxic and intracellular defense capabilities (9). Once activated, these cells may produce a variety of defense molecules, including oxygen-derived intermediates (10), COX-2 (11), NO, iNOS (also called NOS-2) (12), inflammatory cytokines (13,14) and antimicrobial peptides (15). Many studies have reported that some probiotics (e.g. bifidobacteria, lactobacillus) can enhance phagocytic activity and stimulate NO and cytokine production by macrophages (16–20).
The purpose of the present study was to explore the effects of B. subtilis (natto) B4 spores on murine macrophage viability and biological functions by using the RAW 264.7 cell co-culture model. We examined the viability, activation and immune functions of macrophages to clarify the immunomodulatory properties of B. subtilis (natto) B4 spores on macrophages.
MATERIALS AND METHODS
Bacterial strain and cell line
The B. subtilis (natto) stain B4 used in the present study was isolated from natto. The murine monocyte macrophage cell line RAW 264.7 was obtained from Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China.
Preparation of spores
Spores of B. subtilis (natto) B4 were prepared according to an exhaustion method using DSM as described elsewhere (21, 22). The spore suspension was treated with lysozyme and heat (68°C, 1 hr) to remove residual vegetative cells and stored at –20 C. Next, the spores were collected by centrifugation at 4000 g for 10 mins, resuspended and diluted to a concentration of 1 × 108 spores/mL with incomplete medium (RPMI-1640 medium [Gibco, Invitrogen, Auckland, New Zealand] supplemented with 10%[v/v] FBS [Gibco]) before being used.
Culture of macrophages
RAW 264.7 cells were cultured in complete medium (RPMI-1640 medium containing 10% FBS, 100 U/mL penicillin [Sigma-Aldrich, St Louis, MO, USA] and 100 μg/mL streptomycin [Sigma-Aldrich]) at 37 C in an incubator with 90% humidity and 5% CO2, refed every 24 hrs and passaged every 48 hrs.
Treatment of macrophage with Bacillus subtilis (natto) B4 spores
Cell suspension at a concentration of 1 × 106 cells/mL was seeded into six-well culture plates (1 mL per well) and grown in a CO2 incubator for 48 hrs. The culture medium was then removed and the monolayers washed twice with PBS (pH 7.2). 1 mL of incomplete medium and 1 mL of spore suspension were added to each well of the treatment group, whereas 2 mL of incomplete medium was used for the control group. The cells in both groups were cultured at 37 C in 5% CO2 for another 12 hrs.
Sampling and analyses
After a 12-hr co-culture, the culture supernatants were collected, centrifuged at 4000 g for 10 mins to remove B. subtilis (natto) spores and stored at –80 C until analyzed for extracellular LDH, NO and cytokines. The monolayers were washed three times with sterile 0.02 M PBS (pH 7.2), then treated by the following two methods: (i) detached with pre-warmed 0.05% trypsin-0.02% EDTA (Sigma-Aldrich) at 37 C for 2 mins, after which the cells were harvested for a viability test; and (ii) lysed in situ with 0.1% Triton X-100 (Sigma-Aldrich) for 5 mins at 37 C, after which the cell lysates were collected and frozen at −80°C until assayed for ACP, LDH and iNOS.
Safety evaluation of Bacillus subtilis (natto) B4 spores
The safety of B. subtilis (natto) B4 spores was determined by cytotoxicity evaluation, which included a cell viability test and LDH leakage assay. The viability of RAW 264.7 cells was measured by trypan blue dye exclusion test as described by Sarir et al. (23) and calculated according to the following formula: cell viability (%) = NV× 100/NT where NV is the number of viable cells and NT the total number of cells. The amount of LDH leakage from necrotic macrophages into the culture supernatant was determined by the method described by Weyermann et al. (24) using a Cytotoxicity Detection Kit (LDH) (Roche Diagnostics, Mannheim, Germany).
Analysis of macrophage functions
The biological functions of RAW 264.7 cells were analyzed by measuring cellular enzyme activities, NO production and cytokine secretion. Activation of macrophages was determined by ACP and LDH activities as described in previous studies (25, 26), whereas their immune function was evaluated by iNOS activity and concentrations of NO and cytokines (TNF-α, IFN-γ, IL-1β, IL-6, IL-12, IL-10 and MIP-2). All the above indexes were assayed by using commercial kits according to the manufacturers’ instructions. Mouse LDH, ACP, iNOS and NO kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China) and mouse TNF-α, IFN-γ, IL-1β, IL-6, IL-12, IL-10, MIP-2 ELISA kits from R & D Systems (Minneapolis, MN, USA).
Data analysis was carried out using software SPSS for Windows (version 16.0; SPSS, Chicago, IL, USA) and paired-samples t-tests were used to evaluate statistical differences between the treatment and control groups, the significance levels being set at P < 0.05 (significant) and P < 0.01(highly significant).
Cytotoxicity of Bacillus subtilis (natto) B4 spores
To evaluate the cytotoxic effect of B. subtilis (natto) B4 spores on murine macrophages, RAW 264.7 cells were cultured with B. subtilis (natto) B4 spores for 12 hrs and cell viability and LDH content in the culture supernatants measured by trypan blue dye exclusion and ELISA assay, respectively. The viability of treated macrophages (97.50 ± 1.30%) was a little higher than that of untreated ones (97.08 ± 1.24%) (P 0.05, Fig. 1a), whereas extracellular LDH activity in the treatment group (45.06 ± 4.11 U/mL) increased by 9.85% as compared with the control group (41.02 ± 4.54 U/mL)) (P 0.05, Fig. 1b). However, no statistically significant differences (P< 0.05) were found between the two groups for either cell viability or extracellular LDH activity, indicating that B. subtilis (natto) B4 spores had no apparent cytotoxic effect on murine macrophages.
Macrophage activation by Bacillus subtilis (natto) B4 spores
To determine the effect of B. subtilis (natto) B4 spores on murine macrophage activation, RAW 264.7 cells were incubated with B. subtilis (natto) B4 spores for 12 hrs and ACP and LDH activities in the cell lysates assessed by ELISA. ACP and LDH activities in macrophages cultured with B. subtilis (natto) B4 spores were 33.25 ± 2.11 U/mL and 423.45 ± 43.04 U/mL, respectively. Both these values were significantly greater than those in control cells (7.92 ± 0.78 U/mL and 327.11 ± 43.33 U/mL, respectively), the increases being 330.68% (P < 0.01) and 31.91% (P < 0.01), respectively (Fig. 2, a, b). The remarkable increases in ACP and LDH activities in the cells demonstrate clearly that murine macrophages are activated by B. subtilis (natto) B4 spores.
Enhancement of immune function of macrophages by Bacillus subtilis (natto) B4 spores
To analyze the effect of B. subtilis (natto) B4 spores on the immune function of murine macrophages, RAW 264.7 cells were treated with B. subtilis (natto) B4 spores for 12 hrs and NO and cytokine concentrations in the culture supernatants and iNOS activity in the cell lysates were assayed by ELISA.
Nitric oxide production in the control group was 31.42 ± 3.34 μM, significantly lower (by 24.80%) than that in the treatment group (41.78 ± 3.97 μM) (P < 0.01) (Fig. 3a). Treatment of the B. subtilis (natto) B4 spores resulted in a remarkable increase of 31.69% in iNOS activity in macrophages, which is an increase from 3.25 ± 0.23 U/mL (in control cells) to 4.28 ± 0.20 U/mL (in treated cells) (P < 0.01) (Fig. 3b).
Table 1 shows macrophage cytokine concentrations in the control and treatment groups. As expected, production of cytokines in macrophages cultured alone was weak, the amounts of IL-1β and IL-12 in the culture supernatants being below the detection limit. In the meantime, strong cytokine secretion was observed in macrophages stimulated with B. subtilis (natto) B4 spores. As a result of the remarkable cytokine induction by B. subtilis (natto) B4 spores, TNF-α, IFN-γ, IL-1β, IL-6, IL-12, IL-10 and MIP-2 concentrations in the treatment group all increased significantly by 761.64% (P < 0.01), 108.95% (P < 0.01), infinite amount (P < 0.01), 79.52% (P < 0.01), infinite amount (P < 0.01), 572.08% (P < 0.01) and 286.69% (P < 0.01), respectively, in comparison to those in the control group.
Table 1. Changes in cytokine production by RAW 264.7 cells (means ± SD, n= 6)
B. subtilis (natto) B4
ND, not detectable. **highly significant compared with control (P< 0.01).
19.29 ± 2.56
146.92 ± 5.78**
14.98 ± 1.74
31.30 ± 5.60**
6.67 ± 2.54**
22.17 ± 1.65
39.80 ± 7.26**
64.02 ± 5.5**
37.07 ± 10.41
249.14 ± 11.86**
28.09 ± 2.99
108.62 ± 17.50**
Because safety is a basic requirement for probiotics, this should be the first item assessed when evaluating a probiotic bacterium (27). Recently, several studies have focused on the safety of B. subtilis and found none of the strains investigated to be pathogenic (28–30). The cytotoxicity evaluation in this work indicates that B. subtilis (natto) B4 spores have no negative impact on the viability of RAW 264.7 macrophages. Taken together with the previous findings cited above, the B. subtilis (natto) strain may be considered safe.
An increase in cell membrane permeability always accompanies cell death (31) and this results in leakage of LDH (32). LDH is known to be a stable cytoplasmic enzyme that is present in all cells (24). Thus, extracellular LDH can be used as an indicator of cell death or cell damage (33), the amount decreasing as cell viability increases. However, it is interesting to note that a slight increase in LDH leakage accompanied the small rise in cell viability when we exposed RAW 264.7 cells to B. subtilis (natto) B4 spores. This phenomenon may have been due to the notable enhancement of cellular LDH activity observed in macrophages activated by B. subtilis (natto) B4 spores.
Macrophages possess many lysosomal enzymes (e.g. ACP, β-GLU) and cytoplasmic enzymes (e.g. LDH) which are closely involved in their phagocytic function of eliminating foreign particles (34). A number of studies have indicated that the activities of these enzymes are significantly increased after activation of macrophages (25, 34–36). Because ACP and LDH are marker enzymes of macrophages (25), their activities can serve as indexes of macrophage activation. In the current study, the B. subtilis (natto) B4 spores-treated group had remarkable increases in both ACP and LDH activities compared to the control group, showing that B. subtilis (natto) B4 spores do activate murine macrophages. Furthermore, B. subtilis (natto) B4 spores may enhance the phagocytic function of murine macrophages through positive modulation of lysosomal enzyme and cytoplasmic enzyme activities in the cells.
Macrophages are known to be essential for innate immunity and play an important part in host defense. Activation of macrophages by pathogens and pro-inflammatory cytokines (e.g. TNF-α, IFN-γ, IL-1β) leads to the production of various oxidant-generating enzymes, such as iNOS (12), which synthesizes NO from L-arginine (37, 38). NO, a paracrine-acting soluble gas, is recognized as a pleiotropic mediator of numerous physiological and pathological processes (17), including signal transduction (39, 40), redox regulation (41, 42), immunomodulation (43), anti-microbial defenses (44, 45), tumor cell killing (46), cytotoxicity and apoptosis (47). NO can also affect the functions of NK cells, neutrophils and mast cells (48–50). We observed that the amount of NO and activity of iNOS in macrophages cultured with B. subtilis (natto) B4 spores were both significantly higher than those in control macrophages, suggesting that B. subtilis (natto) B4 spores increase production of NO by increasing iNOS activity. B. subtilis (natto) B4 spores may enhance immune function of macrophages through induction and release of massive amounts of NO.
Cytokines are small multifunctional protein molecules that coordinate immune and inflammatory responses (51) and which can be divided into two main groups: pro-inflammatory (TNF-α, IFN-γ, IL-1, IL-2, IL-6, IL-8, IL-12) and anti-inflammatory (IL-4, IL-10, IL-13) (52). TNF, a key player in the cytokine network (53), promotes inflammatory responses through vascular endothelium and endothelial leukocytes, modulates host defenses against bacterial, viral and parasitic infections and mediates a variety of human diseases (e.g. rheumatoid arthritis, ankylosing spondylitis, psoriasis and various diseases of the central nervous system, cardiovascular system, respiratory system and kidneys) (54). IFN-γ, alternatively called type II IFN, an antiviral cytokine, increases MHC class I and class II protein expression, activates macrophages and induces NO production to kill pathogens and neoplastic cells, promotes inflammatory responses by enhancing TNF-α production and synergizing with TNF-α and plays a participatory or protective role in the development of various autoimmune responses (55). The IL-1 family is closely linked with innate immune responses. Its key member, IL-1β, functions as a T cell co-stimulator and B cell growth factor, affects lymphocyte function and is the primary mediator of a diverse group of inflammatory diseases (named autoinflammatory diseases) (56). IL-6 is critical for the resolution of innate immunity (e.g. it blocks neutrophil infiltration and promotes neutrophil apoptosis) and the development of acquired immunity (e.g. it recruits mononuclear cells, directs monocyte differentiation and regulates lymphocyte activities) (57). IL-8 (also named CXCL8) is a CXC chemokine characterized as a promoter of neutrophil chemotaxis and degranulation (58, 59), and may serve as a biomarker in various fields of medicine (60). Although the IL-8 gene has not been found in the mice genome, MIP-2 has been identified as a murine functional homolog of IL-8 (61). IL-12, a heterodimeric pro-inflammatory cytokine produced in response to pathogens (bacteria, fungi, intracellular parasites and viruses) induces production of IFN-γ, stimulates proliferation of NK and T cells, enhances the cytotoxicity of NK cells, favors differentiation of Th1 cells and bridges innate resistance and adaptive immunity (62). IL-10, a Type II cytokine, can inhibit DC differentiation and maturation, antigen presentation, MHC class II expression, and especially cytokine production (e.g. IL-1, IL-2, IL-4, IL-5, IL-6, IL-12, IFN-γ, TNF-α, G-CSF and GM-CSF, CC and CXC chemokines) (63, 64).
In this study, we measured the production of TNF-α, IFN-γ, IL-1β, IL-6, IL-12, IL-10 and MIP-2 by RAW 264.7 cells treated with B. subtilis (natto) B4 spores to assess macrophage immune and inflammatory responses. We found that B. subtilis (natto) B4 spores significantly induce production of the above pro-inflammatory cytokines (TNF-α, IFN-γ, IL-1β, IL-6, IL-12 and MIP-2), implying that B. subtilis (natto) B4 spores also enhance macrophage immune function by promoting immune and inflammatory responses.
The effect of B. subtilis (natto) B4 spores on pro-inflammatory cytokine production may be due to lipoteichoic acids. Opitz et al. demonstrated that highly purified lipoteichoic acids from B. subtilis induce translocation and activation of nuclear factor κB in RAW264.7 cells through Toll-like receptor-2 (65). In addition, activation of nuclear factor κB can further lead to transcriptional activation and subsequent release of pro-inflammatory cytokines such as IL-1, IL-6, and TNF-α (66). Furthermore, TNF-α and IL-1β are early cytokines in inflammatory responses and can induce other secondary cytokines such as IL-6, IL-8 and IL-10 (67). We found that TNF-α and IL-1β are the inflammatory cytokines with the greatest rate of increase (761.64% and infinite, respectively). Hence, we could infer that B. subtilis (natto) B4 first induces secretion of TNF-α and IL-1β by macrophages, which then results in the secondary release of other inflammatory cytokines (IFN-γ, IL-6, IL-12, IL-10 and MIP-2). It is important to note that the concentration of anti-inflammatory cytokine IL-10 was remarkably increased (P < 0.01) in parallel with the notable increases in pro-inflammatory cytokines (P < 0.01). However, it is believed that large amounts of IL-10 produced by macrophages in response to the high concentrations of pro-inflammatory cytokines exert an autoregulatory effect, as previously described for human monocytes (63, 68), and form part of a negative feedback loop that controls acute inflammatory responses (69).
Lactobacillus and Bifidobacterium are both most common probiotics. The effects of B. subtilis (natto) B4 spores on NO and cytokine production by murine macrophages determined in our study have similarities with those of Lactobacillus and Bifidobacterium found in previous studies. Morita et al. tested eleven strains of lactobacilli for their ability to induce the murine macrophage-like cell line J774.1 to secrete cytokines and found that lactobacilli activate macrophages to secrete both inflammatory (IL-6, IL-10, IL-12, and TNF-α) and anti-inflammatory (IL-10) cytokines (70). Meanwhile, Park et al. demonstrated that commercial Bifidobacterium strains stimulate NO, TNF-α and IL-6 production by RAW264.7 macrophage cells and that these effects are strain-dependent (19). Finally, B. subtilis (natto) B4 spores may have similar immunomodulatory potential to Lactobacillus and Bifidobacterium. Moreover the spores of B. subtilis are highly resistant to heat (71); therefore, most B. subtilis (natto) B4 spores can survive after feed processing. This outstanding advantage over other probiotics suggests a wide range of possible applications in the feed and food industry.
In conclusion, this study has elucidated the effects of B. subtilis (natto) B4 spores on viability and biological functions of murine macrophages. B. subtilis (natto) B4 spores are not only non-pathogenic to murine macrophages, but can also enhance macrophage phagocytic function through activation of macrophages and further promote the immune functions of macrophages by inducing NO and cytokine production. Thus, B. subtilis (natto) B4 spores have immunomodulatory effects on macrophages. Our findings may lay the basis for the development and application of B. subtilis (natto) B4 spores as immunomodulatory supplements.
This study was supported Zhejiang Provincial Science and Technology Foundation, China (No. 2006C12086) and the Specialized Research Fund for the Doctoral Program of Higher Education (No.20110101110101).
All authors certify that they have no financial arrangement with a company whose product figures prominently in the submitted manuscript or with a company making a competing product and no conflict of interest exists.