Streptococcus thermophilus MN-ZLW-002 Can Inhibit Pre-adipocyte Differentiation through Macrophage Activation

Abstract

It is well documented that obesity and metabolic syndrome have a deep association with the intestinal immune system of the host animal. Recent studies indicate that some selected probiotics can modulate the immune responses of the host animal, thereby altering its lipid metabolism. However, the underlying mechanisms are still not fully understood. This study was conducted to investigate the possibility of probiotics to activate macrophages in the hosts, thus alter the differentiation of pre-adipocytes. In this study, Streptococcus thermophilus MN-ZLW-002 (MN-ZLW-002) was co-cultured with RAW264.7 macrophages, with Lactobacillus rhamnosus GG (LGG) as a control. The conditioned medium (CM) of the co-culture was collected and then added to 3T3-L1 pre-adipocytes. Viable and heat-killed (80 °C, 30 min) MN-ZLW-002 stimulated RAW264.7 cells to produce significant amounts of interleukin (IL)-6 and tumor necrosis factor (TNF)-α and induced intense phosphorylation of P38, p44/42 mitogen-activated protein kinase (MAPK) (extracellular signal-regulated kinase (ERK)) and nuclear factor κB (NF-κB). Cytokine production reduced dramatically when heat-killed MN-ZLW-002 was treated with Ribonuclease. Viable and heat-killed LGG induced less cytokine production and little signaling protein activation. Viable and heat-killed MN-ZLW-002-stimulated RAW264.7-CM notably suppressed pre-adipocytes differentiation. However, viable LGG-stimulated RAW264.7-CM had a weaker effect and heat-killed LGG-stimulated RAW264.7-CM had no effect. These findings suggest that viable and heat-killed (80 °C, 30 min) MN-ZLW-002 may alter its lipid metabolism by regulating its immune response, possibly via the release of cytokine, particularly TNF-α. The RNA of heat-killed MN-ZLW-002 may be a key component in its immune activation effect.

INTRODUCTION

Being overweight or obese is defined as having abnormal or excessive fat accumulation that presents a risk to health. Obesity is a major risk factor for type 2 diabetes, atherosclerotic cardiovascular disease, nonalcoholic fatty liver disease, and diverse cancers. According to the WHO, in 2016, more than 1.9 billion (39%) adults were overweight, of whom over 650 million (13%) were obese. Obesity in children and adolescents has increased tenfold in the past 40 years.¹⁾ Obesity has become a serious threat to public health worldwide.

It is generally believed that obesity is caused by an imbalance between energy intake and expenditure, resulting from an inadequate lifestyle, neuronal and hormonal mechanisms and genetic and epigenetic factors.²⁾ Recently, growing evidence has suggested that obesity is associated with chronic inflammation in metabolic tissues such as adipose tissue and the liver, and that the intestinal immune system may be an important contributor to metabolic disease. Researches have also shown that obesity and metabolic syndrome are associated with changes in the abundance and structure of gut microbiota, intestinal barrier function, and gut-residing immune cells.^3–7) In light of these reports, the intestinal immune system may become the new target in the future for the prevention and control of obesity and metabolic syndrome.

Probiotics are ‘live microorganisms, which when administered in adequate amounts, confer a health benefit on the host.’⁸⁾ Recent studies have shown that some specific probiotic strains (mainly belonging to Lactobacillus, Bifidobacterium and Pediococcus) have metabolic regulatory effects such as reducing the body weight, visceral fat and waist circumference, and have been reported to improve the indicators of lipid metabolism.^9–13) However, the underlying mechanisms of their actions have not been fully elucidated. A Japanese study shown that the supplementation of probiotics [Lactobacillus rhamnosus GG (LGG) and Lactobacillus gasseri TMC0356 (TMC0356)] with anti-allergic effects could regulate the blood lipid levels in humans. The same probiotics could also affect the differentiation of pre-adipocytes and alter the immune responses of macrophages in the adipose cells, even in the state of inactive.^14,15) Several studies have also shown that there may be a strong association between immune competent cells and adipose tissues.^16–18) In light of these findings, we hypothesize that probiotics, particularly those with immunoregulatory effects, may play a role in the management of obesity by regulating the host’s immune response, possibly via cytokine release. However, such effects may be strain-specific/dependent, and more scientific evidence should be obtained from well-designed strain-based studies.

In this study, we assessed the effects of in vitro exposure of Streptococcus thermophilus MN-ZLW-002 (MN-ZLW-002) on the activation of RAW264.7 macrophages as well as the effects of MN-ZLW-002-stimulated RAW264.7-conditioned medium (CM) on the growth and differentiation of 3T3-L1 pre-adipocytes. We also tested the potential effects of heat-killed MN-ZLW-002 and its cell components. Furthermore, intracellular pathways involved in bacteria-induced inflammatory responses were analyzed.

MATERIALS AND METHODS

Bacterial Strains and Heat-Killed Conditions

MN-ZLW-002 was first isolated from Yogurt Block, a traditional fermented dairy food that originates from the Gannan region of Gansu Province, China.¹⁹⁾ LGG was obtained from Chr. Hansen A/S (Hoersholm, Denmark). Bacteria were stored in 12% skimmed milk (Becton, Dickinson and Company, Sparks, MD, U.S.A.) at −80 °C and passaged three times before used. MN-ZLW-002 was grown in Modified Chalmers (MC) agar (Land Bridge Technology, Beijing, China) at 37 °C under aerobic conditions for 72 h. LGG was grown in de Man, Rogosa and Sharpe (MRS) agar (Land Bridge) at 37 °C under anaerobic conditions for 48 h. Cultures were collected and washed twice with sterilized 0.85% NaCl. The numbers of bacteria were determined by colony-forming unit (CFU)-absorbance standard curves of two bacterial strains that we made.

MN-ZLW-002 was suspended in sterilized 0.85% NaCl and heat-killed at 80, 100, 120, or 135 °C for 5, 10, or 30 min using water bath or an autoclave. After heat treatment, 100 µL each of the bacterial suspension was spread to the corresponding agar and cultured for 72 h to verify the viability of the bacteria. RAW264.7 cells (5 × 10⁵ cells/well) were then co-cultured with the heat-killed bacteria (1 × 10⁷ CFU/well) in a 24-well plate for 24 h. The supernatants were collected and assayed for the concentration of interleukin (IL)-6 and tumor necrosis factor (TNF)-α using enzyme-linked immunosorbent assay (ELISA). The heat-killed condition that had the least influence on the immunomodulatory effect of MN-ZLW-002 was selected (80 °C, 30 min; Fig. 1).

Fig. 1. Effects of MN-ZLW-002 under Different Heat-Killed Conditions on the Production of IL-6 and TNF-α by RAW264.7 Cells (n = 3)

RAW264.7 cells were co-cultured with MN-ZLW-002 under different heat-killed conditions for 24 h. Supernatants were harvested and assayed for (A) IL-6 and (B) TNF-α levels using ELISA. Results are expressed as means ± standard deviation (S.D.).

Culture of RAW264.7 Cells and Preparation of Bacteria-Stimulated CM

Murine macrophage cell line RAW264.7 was obtained from the American Type Culture Collection (ATC C, Manassas, VA, U.S.A.). RAW264.7 cells were cultured in RPMI-1640 medium containing 10% heat-inactivated fetal bovine serum (HI-FBS; Gibco, Carlsbad, CA, U.S.A.) and 2% antibiotics (100 U/mL penicillin and 100 µg/mL streptomycin; EMD Millipore Corporation, Billerica, MA, U.S.A.) at 37 °C in 5% CO₂. Cell number was assessed by Automated Cell Counter (ALIT Life Sciences, Shanghai, China). Cell viability test using Cell Counting Kit-8 (CCK8; Dojindo, Gaithersburg, MD, U.S.A.) was carried out to identify the best dosage for bacterial strains to treat RAW264.7 cells. Cells (5 × 10⁵ cells/well) were seeded in a 24-well plate for 2 h for adherence and then incubated with viable and heat-killed (80 °C, 30 min) MN-ZLW-002 and LGG (1 × 10⁷ CFU/well) respectively for 24 h, with peptidoglycan (PGN, 2 µg/well; Sigma Chemical Co., Ltd., St. Louis, MO, U.S.A.) as a positive control and RPMI-1640 medium as a negative control. Bacteria-stimulated RAW264.7-CM was collected (12000 rpm, 4 °C, 5 min) and stored at −80 °C until use in incubation with 3T3-L1 cells and ELISA analysis. For mRNA analysis, cells were pooled and collected as described below.

Culture and Differentiation of 3T3-L1 Pre-adipocytes

Mouse embryonic fibroblast adipose-like cell line 3T3-L1 (ATC C) was cultured in Dulbecco’s Modified Eagle Medium (DMEM; Gibco) containing 10% HI-FBS and 2% antibiotics (10% HI-FBS DMEM) in a 12-well plate at 37 °C in 5% CO₂. The differentiation procedure is shown in Fig. 4A. Briefly, two days after confluence (day 0), 3T3-L1 cells were induced to differentiate using 10% HI-FBS DMEM containing 0.5 mmol/L 3-Isobutyl-1-methylxanthine (IBMX; Sigma), 0.25 µmol/L dexamethasone (DEX; Sigma) and 10 µg/mL insulin (Sigma) for 72 h (days 0–3). To test their effects on differentiation, CMs (20%) from RAW264.7 cells were added from day 0 to day 3. On day 3, cells were washed twice with PBS and the medium was replaced with 10% fresh HI-FBS DMEM containing 10 µg/mL insulin for a further 72 h (days 3–6). On day 6, supernatants and cells were collected and frozen at −80 °C until used for ELISA and mRNA analysis. Cells in another 12-well plate cultured under the same conditions were fixed (4% paraformaldehyde; Solarbio Sciences & Technology Co., Ltd., Beijing, China) for 30 min, washed and then stained with oil red O solution (Solarbio) for a further 30 min. Thereafter, the stained cells were washed and were photographed by an inverted microscope (Olympus Corporation, Shinjuku, Tokyo, Japan) at 40× magnification.

Enzymatic Treatment

For Deoxyribonuclease I (DNase; Sigma) and Ribonuclease A (RNase; Sigma) treatment, heat-killed MN-ZLW-002 cells (1 × 10⁹ CFU/mL) were suspended in Tris–HCl buffer (10 mmol/L, pH 8.0; Solarbio Sciences & Technology Co., Ltd.) containing 2.5 mmol/L MgCl₂ (Jinshan Chemical Reagent Co., Ltd., Chengdu, China) and 0.5 mmol/L CaCl₂ (Jinshan), and were treated with DNase (400 µg/mL) or RNase (400 µg/mL) at 37 °C for 1 h. For DNase and RNase, cells were treated with both DNase (400 µg/mL) and RNase (400 µg/mL) under the same conditions. For lysozyme (Sigma) treatment, cells were suspended in Tris–HCl buffer and treated with lysozyme (10 mg/mL) at 37 °C for 30 min. After washing three times with phosphate buffer saline (PBS; Gibco; 13000 g, 4 °C, 5 min), cells were diluted ten times with Roswell Park Memorial Institute medium (RPMI-1640; Gibco) and co-cultured with RAW264.7 cells for 24 h. Supernatants were collected for ELISA.

ELISA

IL-6, IL-10, IL-12p70 and TNF-α levels of CM were analyzed using commercial ELISA kits (R&D Systems, Minneapolis, MN, U.S.A.). Adiponectin (ADP) level in supernatants of 3T3-L1 cells were also analyzed.

RNA Isolation and Quantitative RT-PCR

RAW264.7 cells and 3T3-L1 cells were collected and lysed with TRIzol reagent (Invitrogen, Carlsbad, CA, U.S.A.), and total cellular RNA was isolated using the Geneout TRIzol RNA Isolation Kit (Labgene Biotechnology Co., Ltd., Chengdu, China).

Synthesis of cDNA was performed at 25 °C for 5 min, followed by 46 °C for 20 min, and finally 94 °C for 1 min with an S1000 Thermal cycler (Bio-Rad Laboratories, Hercules, CA, U.S.A.). We determined mRNA expression of RAW264.7 cells (IL-6, IL-10, IL-12, TNF-α) and 3T3-L1 cells [peroxisome proliferator-activated receptor (PPAR)-γ, CCAAT/enhancer binding protein (C/EBP)-α] by real-time quantitative PCR (qPCR) using a CFX96 system (Bio-Rad). The qPCR cycling conditions were as follows: initial denaturation at 98 °C for 30 s, followed by 39 cycles of denaturation at 98 °C for 5 s, annealing and extension (IL-6: 55 °C; IL-10: 57.7 °C; IL-12: 59.5 °C; TNF-α: 59.5 °C; PPAR-γ: 56.5 °C; C/EBP-α: 61.4 °C and β-actin: 64.5 °C) for 5 s. Relative mRNA levels were normalized against β-actin.

Primer sequences were as follows (5′ to 3′): IL-6 (F: GTC ACA GAA GGA GTG GCT A; R: AGA GAA CAA CAT AAG TCA GAT ACC), IL-10 (F: GAC CAG CTG GAC AAC ATA CT; R: GAG GGT CTT CAG CTT CTC AC), IL-12 (F: CTC TGT CTG CAG AGA AGG TC; R: GCT GGT GCT GTA GTT CTC AT), TNF-α (F: CTC TTC AAG GGA CAA GGC TG; R: CGG ACT CCG CAA AGT CTA AG), PPAR-γ (F: TCA CAA GAG GTG ACC CAA TG; R: CCA TCC TTC ACA AGC ATG AA), C/EBP-α (F: GTG TGC ACG TCT ATG CTA AAC CA; R: GCC GTT AGT GAA GAG TCT CAG TTT G) and β-actin (F: GTG GGC CGC TCT AGG CAC CAA; R: CTC TTT GAT GTC ACG CAC GAT TTC). All primers were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China).

Western Blotting

Western blotting was conducted following the method described by Zhao et al. and Kaji et al. with some modification.^20,21) Briefly, RAW264.7 cells (5 × 10⁵ cells/well) were incubated with viable/heat-killed MN-ZLW-002 or LGG (1 × 10⁷ CFU/well) for 5 min, 1 h and 12 h in 12-well plates, with PGN as a positive control and RPMI-1640 medium as a negative control. Cells were washed twice with PBS and lysed with cell lysis buffer (20 mmol/L Tris pH 7.5, 150 mmol/L NaCl, 1% Triton X-100, sodium pyrophosphate, β-glycerophosphate, ethylenediaminetetraacetic acid (EDTA), Na₃VO₄ and leupeptin; Beyotime Biotechnology Co., Ltd., Shanghai, China) for the collection of total protein. The cell lysates were treated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample loading buffer (Beyotime), boiled for 5 min, separated by SDS-PAGE (120 V, 60 min), and then transferred onto a 0.22 µm polyvinylidene difluoride membrane (EMD Millipore). After blocking for 1 h with 5% BSA in PBS containing 0.1% Tween-20 (Jinshan), the membranes were blotted with anti-phosphorylated P38 (p-P38), anti-phosphorylated p44/42 mitogen-activated protein kinase (MAPK) (p-extracellular signal-regulated kinase (ERK)), anti-phosphorylated nuclear factor κB (p-NF-κB), anti-NF-κB (Cell Signaling Technology, Beverly, MA, U.S.A.) and anti-P38, anti-ERK (Santa Cruz Biotechnology, Santa Cruz, CA, U.S.A.) overnight at 4 °C. Secondary HRP-conjugated anti-rabbit immunoglobulin G (IgG) antibody or anti-mouse IgG antibody (Absin Bioscience Inc., Shanghai, China) was used to incubate for 1 h. Targeted proteins were visualized by ECL luminescence reagent (Absin) using the CFX96 system. Protein expression was quantified using Bio-Rad CFX Manager (version 1.6.541.1028; Bio-Rad).

Statistical Analysis

All statistical analyses were performed using the Statistical Package for Social Sciences version 21.0 (SPSS 21.0, SPSS Inc., Chicago, IL, U.S.A.). All data were expressed as mean ± standard deviation of the mean. Comparisons between groups were evaluated by one-way ANOVA followed by the Tukey test. A probability (p) value of <0.05 was assumed to be statistically significant.

RESULTS

Effects of MN-ZLW-002 on Cytokine mRNA Expression and Production of RAW264.7 Macrophages

Co-culture of RAW264.7 cells and viable/heat-killed MN-ZLW-002 revealed marked upregulation of IL-6, IL-12 and TNF-α mRNA expression compared with the control group (p < 0.05, all; Figs. 2A, E, G). While viable/heat-killed LGG could only induce TNF-α mRNA expression (p < 0.001, both; Fig. 2G). On the other hand, IL-6, IL-10 and TNF-α production of RAW264.7 cells were induced significantly by viable and heat-killed MN-ZLW-002 (p < 0.001, all; Figs. 2B, D, H). Viable LGG induced significant TNF-α production in RAW264.7 cells (p < 0.001; Fig. 2H) while heat-killed LGG did not induce any cytokine production. There was no toxic effect of bacterial strains on RAW264.7 cells according to the cell viability test using CCK8 (data not shown).

Fig. 2. mRNA Expression and Cytokine Production by RAW264.7 Cells after Exposure to Viable/Heat-Killed MN-ZLW-002 or LGG (n = 3)

RAW264.7 cells were co-cultured with viable/heat-killed MN-ZLW-002 or LGG with PGN as a positive control for 24 h. (A) IL-6, (C) IL-10, (E) IL-12 and (G) TNF-α mRNA expressions of RAW264.7 cells were examined using qPCR. Supernatants were harvested and assayed for (B) IL-6, (D) IL-10, (F) IL-12 and (H) TNF-α levels using ELISA. Results are expressed as means ± S.D. Means without a common letter differ significantly (p < 0.05). n.d, not detected.

Activation of P38/ERK/NF-κB Pathways in RAW264.7 by MN-ZLW-002

RAW264.7 cells were stimulated with viable/heat-killed MN-ZLW-002 or LGG, and the activation profiles of NF-κB, P38, and ERK were compared (Fig. 3). MN-ZLW-002 induced intense phosphorylation of P38, ERK, and NF-κB. Viable MN-ZLW-002 significantly increased p-P38/P38 level and p-ERK/ERK level by 335 and 195%, respectively, at 12 h after stimulating, compared to the control group (p < 0.05; p < 0.01). P38 and ERK activation induced by heat-killed MN-ZLW-002 peaked earlier than by the viable cells. Heat-killed MN-ZLW-002 significantly increased the phosphorylation level of P38 by 296% at 1 h (p < 0.05), and increased the p-ERK level by 292% at 5 min (p < 0.05). On the other hand, viable and heat-killed LGG induced little phosphorylation of all of the three proteins.

Fig. 3. Activation of P38/ERK/NF-κB Pathways in RAW264.7 Cells by MN-ZLW-002 and LGG (n = 3)

(A) RAW264.7 cells were treated with heat-killed/viable MN-ZLW-002 or LGG for 5 min, 1 h, and 12 h. Whole-cell lysates were collected and analyzed by Western blotting using specific antibiotics to p-P38, P38, p-ERK, ERK, p-NF-κB, and NF-κB. (B) Protein expressions were quantified. Results are expressed as means ± S.D. Compared with the control group, * p < 0.05; ** p < 0.01.

MN-ZLW-002-Stimulated RAW264.7-CM Inhibited the Differentiation of 3T3-L1 Pre-adipocytes

We transferred the CM from RAW264.7 cells to 3T3-L1 cells for 3 d while differentiating with IBMX, DEX and insulin (days 0–3; Fig. 4A). On the sixth day of differentiation, cells without CM treatment matured into round lipid-laden adipocytes. Compared to the control group, the MN-ZLW-002 (viable and heat-killed) and LGG (viable) groups revealed a reduction in adipocyte differentiation and lipid accumulation, as indicated by decreased oil red O staining. The heat-killed LGG group showed more oil red O staining (Fig. 4B).

Fig. 4. Effects of Bacteria-Stimulated RAW264.7-CM on Lipid Accumulation in Differentiating 3T3-L1 Cells

(A) 3T3-L1 cells were cultured with bacteria-stimulated RAW264.7-CM in days 0–3 during which time insulin, DEX, and IBMX were used to the induction of 3T3-L1 differentiation. On day 3, the medium was changed and insulin was added to induce differentiation for another 3 d. (B) On day 6, cells were stained with Oil Red O and imaged by a microscope under 400× magnification. (Color figure can be accessed in the online version.)

To evaluate the effect of CM-derived factors on adipogenesis, we isolated the RNA of differentiated 3T3-L1 cells (day 6) and tested the mRNA expression of adipogenesis-related transcription factors (Figs. 5A, B). MN-ZLW-002 (viable and heat-killed)-stimulated RAW264.7-CM significantly suppressed PPAR-γ and C/EBP-α mRNA expression (p < 0.05, all, compared with the control group). The viable LGG group, as opposed to the heat-killed LGG group, had the same effect (p < 0.05). We also collected the supernatants of differentiated 3T3-L1 cells (day 6) and tested the influence of CM on adipose-related cytokine production of 3T3-L1 cells (Fig. 5C). MN-ZLW-002 (viable and heat-killed)-stimulated RAW264.7-CM markedly reduced the production of ADP (p < 0.05, both, compared with the control group). The LGG (viable and heat-killed) groups showed no significant difference from the control group. There was no toxic effect of CM on 3T3-L1 cells according to the cell viability test using CCK8 (data not shown).

Fig. 5. Effects of Bacteria-Stimulated RAW264.7-CM on PPAR-γ, C/EBP-α Expression and on ADP Production in Mature Adipocytes (n = 3)

3T3-L1 cells were incubated with CM from RAW264.7 for 3 d, during which time insulin, DEX, and IBMX were used to induce differentiation of 3T3-L1 cells. In day 6, cells were collected for the detection of (A) PPAR-γ and (B) C/EBP-α mRNA expression using qPCR. Supernatants were harvested and assayed for (C) ADP level using ELISA. Results are expressed as means ± S.D. Means without a common letter differ significantly (p < 0.05).

Immune Activation of MN-ZLW-002 May Be Maximally Attributed to the RNA

To elucidate the components that contribute to the strong immune activation effect (particularly the ability to induce IL-6 and TNF-α) of heat-killed MN-ZLW-002, we treated bacteria with a different enzyme before co-culture with RAW264.7 cells. As shown in Fig. 6A, after treating with RNase and DNase + RNase, heat-killed MN-ZLW-002 lost the ability to activate RAW264.7 cells to produce IL-6 (p > 0.05, both, compared with the control group). DNase and lysozyme treatment to MN-ZLW-002 also reduced IL-6 production of RAW264.7 cells (p < 0.001, both, compared with the 002 group). In Fig. 6B, RNase and DNase + RNase treatment to MN-ZLW-002 decreased TNF-α production dramatically (p < 0.001, both, compared with the 002 group). To further clarify the differences between groups, we diluted the enzyme-treated MN-ZLW-002 ten times before co-cultured with RAW264.7 cells (Fig. 6C). A slightly greater reduction in TNF-α production was observed in the lysozyme- and DNase-treated group (p < 0.001, both, compared with the 002 group).

Fig. 6. Effects of Enzymatic Treatment on IL-6 and TNF-α Inducing Abilities of Heat-Killed MN-ZLW-002 (n = 3)

Heat-killed MN-ZLW-002 was treated with or without DNase, RNase, DNase + RNase or Lysozyme and then co-cultured with RAW264.7 cells for 24 h. Supernatants were harvested and assayed for (A) IL-6 and (B) TNF-α levels using ELISA. (C) Enzyme-treated MN-ZLW-002 was diluted ten times before co-culture with RAW264.7 for 24 h and TNF-α was assayed again. Results are expressed as means ± S.D. Means without a common letter differ significantly (p < 0.05).

DISCUSSION

Weight gain is due to an increase in the mass of adipose tissue, which generally results from an enhancement of lipid accumulation and an increase in the number of adipocytes through proliferation and differentiation.^22,23) A growing number of probiotics were tested for their ability to prevent or control excess weight or obesity.^2,9–12,24) However, in vivo, probiotics are not in direct contact with adipose tissue, which suggests that an indirect connection may take effect. Several studies have found that macrophage-CM markedly inhibits the differentiation of pre-adipocytes with in vitro models. Ingredients in the CM, particularly cytokines, may play a role.^15–18)

S. thermophilus has been widely used as a culture starter in the manufacturing of various fermented dairy products for a long time.²⁵⁾ Recently, accumulating scientific evidence suggest the possibility of using this bacterium as probiotic to contribute to human health.^26–28) However, studies focusing on the health benefits of S. thermophilus, particularly the heat-inactivated S. thermophilus, are still very limited, although the fermented milk containing heat-inactivated S. thermophilus has been marketed in many counties in the world, especially in developing countries such as China, where cold chains used to distribute the fermented milk are still not efficient enough.

MN-ZLW-002 strain in this study was originally isolated from a traditional fermented dairy food collected from the northwest of China, and the fermented milk prepared with this bacterium has been proven to regulate the innate immune responses and potentially prevent respiratory infections of mice.²⁹⁾ In this study, viable and heat-killed MN-ZLW-002 stimulated RAW264.7 cells to produce significant amounts of IL-6 and TNF-α, which are consistent with our previous studies with lactobacilli using similar methods.^15,30) These results indicated that viable and heat-killed MN-ZLW-002 might possess potent immune regulatory effect via macrophages as other lactobacilli well used as probiotics do, such as LGG and L. gasseri TMC0356¹⁵⁾; the activation of macrophages might be among the underlying mechanisms of MN-ZLW-002 to enhance the immunity of host animal observed in vivo.²⁹⁾

Macrophages are phagocytic cells derived from monocytes and they play an important part in the innate and adaptive immune response. By phagocytosing, presenting antigens and producing cytokines and mediators, macrophages can promote the proliferation and differentiation of immune cells and improve the body’s immune response.³¹⁾ The cell membrane and endosome of macrophages express various pattern-recognition receptors (PRPs). Toll-like receptors (TLRs) are one of the most important classes of PRPs and can sense various components of bacteria (for example, cell wall recognized by TLR2 and TLR4, genomic DNA recognized by TLR9, RNA recognized by TLR7). The recognition of bacterial components by TLRs leads to the recruitment of various adaptors such as MyD88 and TIR-domain-containing adapter-inducing interferon-β (TRIF). This triggers a signaling cascade of the signaling pathway, including the activation of mitogen-activated protein kinases such as p38, Janus kinases (JNKs), and ERK1/2. Ultimately, transcription factors such as NF-κB and interferon regulatory factors (IRFs) are activated and initiate the transcription of inflammatory cytokine genes and type I interferons.^22,32) In this study, viable and heat-killed MN-ZLW-002 induced intense phosphorylation of P38, ERK, and NF-κB and significant amounts of cytokine, whereas viable LGG induced less cytokine production and little signaling protein activation. These results suggest that MN-ZLW-002 and LGG can activate macrophages in a strain-specific manner, and MN-ZLW-002 might characteristically interact much more strongly with the signaling of p38, JNKs, and ERK1/2.

An interesting finding in this study is that heat-killed MN-ZLW-002 showed a strong immune activation effect, while heat-killed LGG did not. Both bacterial strains were confirmed as inactive by re-culture in the medium. What’s more, P38/ERK/NF-κB activation of RAW264.7 induced by heat-killed MN-ZLW-002 peaked earlier than by viable MN-ZLW-002 (Fig. 3B). We suspect that certain components of heat-killed MN-ZLW-002 may have taken effect. After treatment with RNase, both IL-6 and TNF-α produced by RAW264.7 were reduced dramatically, which suggests that the RNA of heat-killed MN-ZLW-002 is the main contributor to its immunoregulatory effect. The structure of RNA may be one of the key reasons that alter the strain-specific characteristics of LGG and MN-ZLW-002. These findings are similar to that of a study by Inoue et al., in which a drastic decrease in IL-12-inducing ability was observed when heat-killed E. faecalis strain EC-12 was treated with nuclease, particularly RNase.³³⁾ Kawashima et al. also found that the double-stranded RNA of intestinal commensal bacteria contributes to anti-inflammatory and protective immune responses.³⁴⁾ However, Miyazawa et al. found that heat-killed LGG can also activate macrophages.¹⁵⁾ The reason may be that different cell line (J774.1) and inactive conditions (90 °C, 5 min) were used. Our results in Fig. 1 also showed that heat-killed conditions can influence the immunoregulatory effect of bacteria. The effects of inactivated bacteria should be validated in different in vitro models or in vivo under different inactivated conditions.

In this study, the CM from viable/heat-killed MN-ZLW-002-stimulated macrophages and viable LGG-stimulated macrophages significantly inhibited PPAR-γ and C/EBP-α mRNA expression and ADP production in 3T3-L1 pre-adipocytes. PPAR-γ is a type-II nuclear receptor, which is an important regulator of adipocyte differentiation and maturation. PPAR-γ can regulate the storage of fatty acids and glucose metabolism, and the expression of PPAR-γ increases in the early stages of adipocyte differentiation.³⁵⁾ C/EBP-α is a nuclear transcription factor that plays an important role in the later stages of adipocyte differentiation, promoting the expression of PPAR-γ and maintaining the phenotype of differentiated cells.³⁶⁾ ADP is an important adipokine produced by mature adipocytes. The significant decrease in ADP production and the reduce of PPAR-γ and C/EBP-α mRNA expression found in the present study indicated that MN-ZLW-002 possesses an anti-adipogenesis effect, probably through macrophages activation, particularly the release of cytokines. These results have very good agreement with our previous study in which LGG and L. gasseri TMC0356 suppress the differentiation of pre-adipocytes in the same manner.¹⁵⁾

Furthermore, similar studies have shown that the pro-inflammatory cytokines released by macrophages, particularly TNF-α, may play a key role in such anti-adipogenesis effects of lactobacilli.^16–18) TNF-α has been reported to inhibit fatty acid intake by decreasing the activity of lipoprotein lipase—an important enzyme that regulates lipid synthesis and that can promote the uptake of fatty acids by adipocytes. TNF-α can also inhibit the gene expression of acetyl CoA carboxylase and fatty acid synthase, which control the production of lipids.^37–41) In our previous study, L. gasseri TMC0356-stimulated macrophages-CM were found to significantly inhibit the differentiation of pre-adipocytes; TNF-α, rather than IL-6, seemed to be the most important contributor on suppressing the mRNA expressions of PPAR-γ and C/EBP-α using anti-IL-6 antibody and anti-TNF-α antibody.¹⁵⁾ What’s more, TMC0356 showed apparent anti-obesity and anti-metabolic disorder effects in the later animal study.⁴²⁾ These findings suggest that TNF-α may be the key contributor in the anti-adipogenesis effect of MN-ZLW-002. However, it still needs to be verified by TNF-α antibody test.

Obesity-associated chronic inflammation has been found to mainly induced by infiltrating macrophages that aggregate around dead adipocytes in advanced obesity. Endogenous ligands from damaged or dead adipose are considered to be the main stimulators of this harmful chronic inflammation.⁴³⁾ However, lactic acid bacteria are similar to other exogenous ligands, which can enhance the host innate immunity. Such inflammation is considered as homeostatic and should be different from the obesity-associated chronic inflammation, which involves an acute phenotype. The enhancement of inflammatory immune responses with lactic acid bacteria, particularly with some selected probiotic strains, is considered to be a critical mechanism underlying the well-documented health-promoting effects of these probiotic strains on hosts, e.g., anti-allergic and anti-pathogenic.^44,45) Recent studies also found that the probiotics with anti-adipogenesis effects via the activation of macrophages can positively influence the lipid metabolism in animal and human body.^42,46) Therefore, the increase of TNF-α stimulated by MN-ZLW-002 might be useful in the management of obesity and related metabolic defects. Further studies will be conducted to evaluate these effects in animal and clinical trials.

In recent years, growing numbers of studies have proposed that some inactivated bacterial strains have similar health benefits to living microbes.^47–49) In a study by Pedret et al., the consumption of Bifidobacterium animalis ssp. lactis CECT 8145 improved anthropometric adiposity biomarkers in abdominally obese individuals, and heat-killed cells seemed to be more efficient than viable cells.⁹⁾ The mechanisms involved may be related to the regulation of the host immune system by inactivated bacteria.^50,51) On the other hand, inactivated probiotics are reported to be safer than live probiotics as the antibiotic-resistant genes are eliminated and the production of recombinant strains is prevented,⁵²⁾ which is of great significance to the development and application of probiotics.

This study is the first to demonstrate that S. thermophilus, a strain widely used in the dairy industry as yogurt culture, even in inactivated cells (80 °C, 30 min), has the potential to prevent obesity by stimulating immune cells and thus inhibiting pre-adipocytes differentiation. However, further in vivo studies are needed to confirm the results.

Conflict of Interest

The authors declare no conflict of interest.

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