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The murine macrophage-like cell line J774.1 was treated with heat-killed cells of Lactobacillus GG (LGG) and L. gasseri TMC0356 (TMC 0356). Interleukin (IL)-6, IL-12, and tumor necrosis factor-α were profiled from the J774.1 cells using enzyme-linked immunosorbent assay methods. The conditioned medium from cultured J774.1 cells was transferred to the preadipocyte cell line 3T3-L1 (which is a mouse embryonic fibroblast-adipose-like cell line). Growth and differentiation of 3T3-L1 cells were monitored by analyzing lipid accumulation and expression of peroxisome proliferator-activated receptor (PPAR)-γ mRNA. The medium conditioned by 3T3-L1 cells was added to J774.1 cells and the cytokines in the supernatant analyzed. Compared with that of cells exposed to a PBS-conditioned medium, lipid accumulation in 3T3-L1 cells was significantly suppressed in a dose-dependent manner by each medium that had been conditioned with LGG and TMC0356. PPAR-γ mRNA expression in 3T3-L1 cells was also significantly downregulated (P < 0.01, P < 0.05, respectively). The conditioned medium of 3T3-L1 adipose phenotype significantly stimulated production of IL-6 and IL-12 in J774.1 cells treated with LGG and TMC0356. These results suggest that lactobacilli may suppress differentiation of preadipocytes through macrophage activation and alter the immune responses of macrophages to adipose cells.
Obesity is defined as “body fat accumulated abnormally or excessively.” In general, a person with a body mass index ≥ 30 is considered obese. In the past two decades, changes in lifestyles (including eating behavior) have led to a rapid increase in the prevalence of obesity in “developed” and “developing” countries. Furthermore, the increasing prevalence of obesity among young people and children has been recently documented (1, 2).
Obese persons have a high risk of developing cardiovascular diseases, type-2 diabetes mellitus, hypertension and various types of cancer. Obesity also leads to immune dysfunction and increased susceptibility to bacterial and viral infections (3). For example, obese people reportedly had an increased risk of influenza-associated hospitalization and deaths during the 2009 influenza pandemic (4, 5, 6). Therefore, obesity is one of the most important global public health concerns.
Excessive growth of adipose tissue is the most important pathogenic aspect of obesity. In general, such excessive growth is considered to result from enlargement of existing adipocytes (hypertrophy) and formation of new adipocytes (hyperplasia) through differentiation of stromal preadipocytes (adipogenesis) (7). Adipocytes are important for maintenance of an adequate balance of energy, for example by storing calories in the form of lipids, mobilizing energy sources in response to hormonal stimulation and modulation of body functions by signal secretion. Recent studies have shown that adipose tissue may have greater involvement in animals’ physiology and metabolism than considered previously and that most obesity-associated pathogenic events may be attributable to adipocyte activity. Therefore, regulation of the overgrowth of adipose tissue through modification of hypertrophy and hyperplasia may be a practical strategy for the management of obesity.
It has been estimated that at least 400 microbial species, including lactobacilli, reside in the human gut. Complex microbial communication organizes them efficiently, thereby forming the human intestinal microbiota (8). Overall, the intestinal microbiota is strongly involved in nutrient absorption, vitamin production, metabolism, and host immunity (8). Emerging scientific evidence suggests that obesity may be closely associated with an abnormal intestinal microbiota (9). Recently, oral administration of lactobacilli (especially specific strains) was shown to protect host animals from obesity (10, 11, 12, 13). Conversely, lactobacilli (especially selected strains) reportedly activate/cause proliferation of macrophages and other immune cells, thus altering the immunity of the host animal (8, 14). Recent studies also suggest that the association/interaction between immune competent cells and adipose tissue of host animals may be much deeper and more complicated than previously believed (7, 15, 16, 17, 18).
We investigated the possibility that lactobacilli modify the growth of adipose tissue through host animals’ immune-competent cells. We treated the murine macrophage-like cell line J774.1 with two probiotic strains, LGG and TMC0356. We tested conditioned media from lactobacilli-stimulated macrophages to determine the inhibitory effects of these macrophages on the growth and differentiation of 3T3-L1 preadipocytes. Furthermore, to ascertain how potent an influence adipose tissue has on the immunity of host animals, we investigated conditioned media from 3T3-L1 to determine the effects of 3T3-L1 on the lactobacilli-derived immune responses of J774.1 cells.
We isolated TMC0356 from the human intestine and deposited it in the Takanashi Microbiological Collection (Yokohama, Japan). LGG was kindly supplied by Valio (Helsinki, Finland).
We used FG medium to culture TMC0356 (14). We replaced proteose peptone number 3 and the beef extract used in MRS medium with yeast peptone (Bio Springer, Maisons-Alfort, France) in FG medium. Furthermore, we eliminated the manganese sulfate, which is in MRS medium, from the FG medium. We cultured LGG with MRS medium (Becton Dickinson, Sparks, MD, USA)
After continually culturing them twice, we grew the lactobacilli in each medium at 37°C for 24 hrs. We collected TMC0356 by centrifugation at 7000 g, washed twice with sterilized 0.85% NaCl, and lyophilized. We incubated, collected, and washed TMC0356 and LGG before resuspension in sterilized 0.85% NaCl. We then heat killed them at 90°C in a water bath for 5 min and subjected them to lyophilization. We stored the bacterial preparations at −80°C until use.
We purchased J774.1 cells from Riken Gene Bank (Tsukuba, Japan) and maintained them in RPMI 1640 medium (Sigma–Aldrich, St. Louis, MO, USA) containing 10% heat-inactivated FBS (Gibco, Rockville, MD, USA) at 37°C, in an atmosphere of air and 5% CO2. To test the effects of lactobacilli, we co-cultured J774.1 cells and lactobacilli for 24 hrs. We collected the supernatants of the J774.1 cell cultures by centrifugation at 956 g for 10 min and stored them at −80°C until use. To test the effects of 3T3-L1 cells, we added the supernatants from the 3T3-L1 cell culture to the medium in which we had co-cultured the J774.1 cells and TMC0356.
We obtained 3T3-L1 cells from the Health Science Research Resources Bank (Osaka, Japan). We cultured them in DMEM supplemented with 10% heat-inactivated FBS (Gibco) at 37°C in an atmosphere of air and 5% CO2. Two days after confluence, we induced the 3T3-L1 cells to differentiate using DMEM containing 0.25 μM DEX, 0.5 mM IBMX and 10 μg/mL insulin. After treatment for 72 hrs (days 0–2), we replaced the medium with fresh medium containing 10 μg mL−1 insulin for a further 72 hrs (days 3–5). We then replaced the medium with fresh medium containing 10% FBS, every 2–3 days for 14 days (day 20). On day 20, we froze the supernatants of the 3T3-L1 cells at −80°C until use in the experimental incubation with J774.1 cells. To investigate their effects on differentiation, we added the supernatants from the J774.1 cell cultures to the culture medium for the first 2 days, whereas we collected the supernatants from the 3T3-L1 cells on day 10 and froze them at −80°C until use in the adipogenesis experiments.
In addition, we added LGG- or TMC0356-stimulated J774.1 conditioned medium with or without mouse IL-6 antibody and mouse TNF-α antibody (R & D Systems, Minneapolis, MN, USA) at 10 μg/mL to the culture medium of 3T3-L1 cells (30%) for 2 days. DEX, IBMX and insulin were used to stimulate the differentiation of 3T3-LI cells.
Ten days after we had induced the 3T3-L1 cells to differentiate, we stained them using an Adipogenesis Assay kit (Cayman Chemical Company, Ann Arbor, MI, USA), according to the manufacturer's instructions.
We isolated total RNA from 3T3-L1 using a NucleoSpin RNA kit (Macherey Nagel, Dueren, Germany) and performed reverse transcription using a PrimeScript RT Reagent kit (Takara Bio, Shiga, Japan). We determined the degree of mRNA PPAR-γ expression by qRT-PCR using SYBR Premix Ex Taq (Takara Bio) and detected fluorescent signals using a Thermal Cycler Dice Real Time System TP800 (Takara Bio). We designed primers using a Perfect Real Time Support System (Takara Bio). We measured amounts of β-actin mRNA for all samples to normalize gene expression and determined supernatant concentrations using a commercial ELISA kit (Thermo Scientific, Waltham, MA, USA).
Results are expressed as mean ± SD. We performed statistical analyses with the Tukey Kramer test. We assessed differences in IL-12 production from J774.1 cells cultured with conditioned medium in the presence of TMC0356 and LPS with Student's t-test. We considered P < 0.05 significant.
We cultured the J774.1 cell line in the presence of LGG and TMC0356. After 24 hrs, we collected the supernatants from the J774.1 cell cultures and analyzed the concentrations of IL-6, IL-12, and TNF-α by ELISA (Fig. 1). LGG and TMC0356 significantly stimulated production of IL-6, IL-12, and TNF-α. Furthermore, TMC0356 showed a significantly stronger capacity to elicit production of these cytokines than did LGG (Fig. 1).
Figure 1. Cytokine production by J774.1 cells after exposure to lactobacilli. The murine macrophage cells line J774.1 was cultured in the presence of heat-inactivated lactobacilli with PBS as a negative control for 24 hrs. The concentrations of cytokines in the supernatant were analyzed with commercial cytokine ELISA kits. Results are the average of three independent experiments and SD. *, P < 0.05; **, P < 0.01.
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We transferred the supernatants collected from lactobacilli-stimulated J774.1 cells to 3T3-L1 cells for 2 days while we initiated differentiation with DEX, IBMX, and insulin. We then measured amounts of lipids in 3T3-L1 cells (Fig. 2). LGG- and TMC0356-stimulated J774.1-conditioned medium significantly inhibited lipid accumulation in 3T3-L1 cells (P < 0.01; P < 0.01, respectively) in a dose-dependent manner (Figs 2, 3).
Figure 2. Lipid accumulation in 3T3-L1 cells J774.1 cells were treated with LGG or TMC0356 or without bacteria (negative control) for 24 hrs. Their cultured supernatants were collected as test samples (TMC0356 and LGG) and added to the culture medium of 3T3-L1 at doses of 0% (control), 5%, 20%, and 30%, respectively, for 2 days during which time DEX, IBMX, and insulin were used to induce differentiation of 3T3-L1 cells. Lipid accumulation in 3T3-L1 cells was analyzed using Oil Red O staining 10 days after 3T3–1 cells had been induced to differentiate. Values are expressed as mean ± SD. **, P < 0.01. M, medium.
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Figure 3. Microscopic assessment of 3T3-L1 cells. The supernatants of J774.1 cell cultures were added to the culture medium of 3T3-L1 at a dose of 5% for 2 days during which time DEX, IBMX, and insulin were used to induce differentiation of 3T3-L1 cells. Lipids in 3T3-L1 cells were analyzed by Oil Red O staining 10 days after 3T3–1 cells had been induced to differentiate.
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We also added the conditioned medium from lactobacilli-stimulated J774.1 cells to the differentiated 3T3-L1 cells from 20 days for 2 days at doses of 5%, 20%, and 30%. We assessed lipid accumulation in 3T3-L1 cells using Oil Red O staining at 22 days. However, we did not observe significant changes in the lipid accumulated in the differentiated 3T3-L1 cells (Fig. 4).
Figure 4. Lipid accumulation in 3T3-L1 cells LGG- or TMC0356-stimulated J774.1 conditioned medium as well as J774.1-conditioned medium were added to differentiated 3T3-L1 cells from 20 days for 2 days at doses of 5%, 20%, and 30%. Lipid accumulation in 3T3-L1 cells was analyzed using Oil Red O staining at 22 days. M, medium.
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TMC0356- and LGG-stimulated J774.1-conditioned medium also significantly suppressed PPAR-γ mRNA expression (Fig. 5) (P < 0.01, P < 0.05, respectively).
Figure 5. mRNA expression of PPAR-γ in 3T3-LI cells. The supernatants of J774.1 cell cultures were added to the culture medium of 3T3-L1 cells at a dose of 30% for the first 2 days of differentiation. mRNA expression of PPAR-γ in 3T3-LI cells was analyzed by RT-PCR 10 days after 3T3–1 cells had been induced to differentiate. Values are mean ± SD. *, P < 0.05; **, P < 0.01.
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We tested the supernatants of differentiated 3T3-L1 to investigate the possibility that these cells regulate cytokine production in LGG- and TMC0356-stimulated J774.1 cells (Fig. 6). When we added 5% 3T3-L1-conditioned medium to J774.1 cultures, we observed no significant change in IL-12 production by J774.1 cells compared with cells not treated with 3T3-L1-conditioned medium. When we increased the 3T3-L1-conditioned medium concentration to 25% and 50%, IL-12 production in J774.1 cells was significantly increased compared with J774.1 cells not treated with 3T3-L1-conditioned medium (P < 0.05). Addition of 3T3-L1-conditioned medium IL-6 also significantly increased production in TMC0356-stimulated J774.1 cells (P < 0.05). 3T3-L1-conditioned medium did not significantly affect TNF-α production in J774.1 cells exposed to TMC0356 and LGG.
Figure 6. Cytokine production by J774.1 cells after exposure to lactobacilli with the conditioned medium of 3T3-L1 cells. Concentrations of IL-12, IL-16, and TNF-α in the supernatant of J774.1 cells cultured with the conditioned medium of 3T3 L1 cells in the presence of TMC0356 and LGG after 24 hrs were analyzed using commercial ELISA kits. Values are expressed as mean ± SD. *, P < 0.05; **, P < 0.01.
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Mouse TNF-α antibody significantly neutralized the inhibitory effects of LGG- or TMC0356-conditioned medium against differentiation of 3T3-L1 cells (Fig. 7). However, mouse IL-6 antibody did not greatly alter the inhibitory effects of LGG- or TMC0356-conditioned medium with respect to differentiation of 3T3-L1 cells.
Figure 7. Lipid accumulation in 3T3-L1 cells J774.1 cells were treated with LGG or TMC0356 or without bacteria (negative control) for 24 hrs. Culture supernatants were then collected as test samples (TMC0356 and LGG) and added to the culture medium of 3T3-L1 with or without anti-IL-6 and anti-TNF-α antibody at 10 μg/mL at a dose of 30% for 2 days during which time DEX, IBMX, and insulin were used to induce differentiation of 3T3-L1 cells. Lipid accumulation in 3T3-L1 cells was analyzed using Oil Red O staining 10 days after 3T3–1 cells had been induced to differentiate. Values are expressed as mean ± SD. **, P < 0.01.
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Macrophages are tissue-based phagocytic cells derived from monocytes. They play an important part in innate and adaptive immune responses (15). Macrophages are activated by microbial metabolic products (such as endotoxins), molecules such as the CD40 ligand and T-cell cytokines (such as interferon-γ). Activated macrophages can secrete pro- and anti-inflammatory cytokines, which are the main constituents of the cytokine-regulating local inflammatory response.
Recently, researchers have found that macrophage-conditioned medium significantly inhibits differentiation of preadipocytes into adipocytes in several human and murine preadipocyte and macrophage experimental models (7, 16, 17, 18). Such potent anti-adipogenic effects may be because secreted macrophage products inhibit differentiation-induced Rb phosphorylation and activate cyclin-dependent kinase 2 in preadipocytes (19).
Lactobacillus GG is used widely in the dairy industry as a probiotic bacterium with well-documented health-promoting benefits (20). TMC0356 is a new probiotic strain that was isolated originally from the intestines of healthy human adults, and which has an apparent strain-dependent immunoregulatory effect (21, 22, 23). LGG and TMC0356 reportedly significantly improve the allergic symptoms of patients with Japanese cedar pollinosis as well as those of ovalbumin-immunized animals (24–26). They also have protective effects against infection by the influenza virus (27, 28; 29, 30). These anti-allergic and anti-pathogenic effects have resulted (at least in part) from strain-dependent regulatory effects on the cell-mediated immunity of host animals, these effects being characterized by increased production of IFN-γ and upregulated Th1-type immunity (31, 32). LGG and TMC0356 possess strain-dependent characteristic immunomodulatory effects on host animals; however, both bacteria can activate macrophages to secrete pro-inflammatory (IL-12) and anti-inflammatory (IL-10) cytokines in a strain-dependent manner (23). Recently, LGG was found to promote loss of body weight and fat in aP2-agouti transgenic mice after energy restrictions (personal communication, Dr. Zemel, Nutrition Institute, University of Tennessee, Knoxville, TN, USA). TMC0356 protects rats from obesity caused by a high-fat diet, this protection being associated with increased inflammatory responses of host animals (33). LGG can improve glucose tolerance and suppress post-prandial hyperglycemia (34). These findings suggest that lactobacilli (especially some strains with immunoregulatory effects) may regulate the postulated association between adipose tissue and immune tissue.
In the present study, LGG and TMC0356 activated macrophages in a strain-specific manner to produce the pro-inflammatory cytokines IL-6, IL-12, and TNF-α, as observed in previous studies (14, 23). The conditioned media from LGG- and TMC0356-stimulated macrophages significantly inhibited lipid accumulation in 3T3-L1 cells, these effects being dose-dependent. Furthermore, PPAR-γ expression of 3T3-L1 preadipocytes was also significantly downregulated by LGG and TMC0356. PPAR-γ is known as the glitazone receptor (also known as nuclear receptor subfamily 1, group C, member 3), which is a type-II nuclear receptor encoded by the PPAR-γ gene in humans. PPAR-γ can regulate the storage of fatty acids and glucose metabolism and is a regulator of adipocyte differentiation. The significant decrease in lipid accumulation and reduced PPAR-γ expression found in the present study indicate that there was significant inhibition of differentiation of 3T3-L1 preadipocytes. Therefore, these results suggest that LGG and TMC0356 influences differentiation of preadipocytes (adipogenesis).
However, the conditioned medium from the lactobacilli-stimulated macrophages had no effect on lipid accumulation in differentiated 3T3-L1 cells or their expression of PPAR-γ. These results suggest that the tested lactobacilli did not affect the activation of existing adipocytes (hypertrophy) through enhancement of macrophage-derived inflammatory responses. Furthermore, unlike other functional food ingredients that are directed against obesity (26), the tested lactobacilli strains did not directly affect differentiation or other activities of 3T3-L1 (data not shown).
Several studies have demonstrated that inflammatory cytokines such as TNF-α and IL-6 may negatively influence the differentiation and growth of preadipocytes by inducing insulin resistance and apoptosis (19, 35). In the present study, TNF-α antibody significantly neutralized the inhibitory effects of LGG- or TMC0356-conditioned J774.1 medium against differentiation of 3T3-L1 cells. However, IL-6 antibody did not significantly alter the inhibitory effects of LGG- or TMC0356-conditioned medium with respect to differentiation of 3T3-L1 cells. In addition, LGG and TMC0356 induced production of TNF-α, IL-6, and IL-12 from murine macrophages and J774.1 cells. Therefore, the inflammatory factor TNF-α present in the conditioned medium of LGG and TMC0356 stimulated J774.1 cells may be a key contributor to the observed anti-adipogenic effects. However, we observed similar inhibitory effects in the conditioned medium of LGG- and TMC0356-stimulated macrophages and J774.1 cells (although TMC0356 induced significantly more TNF-α production from J774.1 cells). These results suggest that, in addition to inflammatory cytokines such as TNF-α, other functional molecules produced by macrophages upon stimulation by certain lactobacilli in a strain-dependent manner may influence the differentiation and growth of preadipocytes.
In the present study, we added the conditioned medium of the 3T3-L1 adipose phenotype to J774.1 cell cultures to investigate the influence of adipose tissue on the immune responses of macrophages. Interestingly, the conditioned medium of the 3T3-L1 adipose phenotype significantly stimulated production of IL-6 and IL-12 (but not of TNF-α) by J774.1 cells in the presence of heat-killed LGG and TMC0356. These results suggest that lactobacilli enhance the inflammatory responses of host immune cells against signals from adipose tissue. Recently, researchers have reported that obesity leads to immune dysfunction and increased susceptibility to bacterial and viral infection. For example, obese individuals appeared to be at increased risk of influenza-associated hospitalizations and deaths in the 2009 influenza pandemic (4). Mice with diet-induced obesity infected with influenza virus reportedly have higher mortality rates than do infected lean controls (3). In addition, the obese mice not only had reduced expression of the anti-viral cytokines IFN-α and IFN-β, but also demonstrated a notable delay in expression of the pro-inflammatory cytokines IL-6 and TNF-α in their lungs. In our previous study, mRNA expression of IFN-γ, IL-2Rb and perforin 1 significantly decreased in the lungs of mice with diet-induced obesity (36). Oral administration of heat-killed TMC0356 suppressed the decline in mRNA expression of IFN-γ, IL-2Rb, and perforin 1 in their lungs and increased their degree of expression up to those or higher of the control group (28). The results of the present study support the observation that oral administration of heat-killed TMC0356 significantly enhances pulmonary mRNA expression of cytokines in mice with diet-induced obesity.
Inflammation is part of the complex biological response of vascular tissues to harmful stimuli such as pathogens, damaged cells, or irritants. It is a protective response by the organism to remove the injurious stimuli and initiate the healing process. Inflammation is not a synonym for infection, even where inflammation is caused by infection. Current opinion in immunology is that inflammation is a stereotyped response that is not only an important mechanism of innate immunity, but can also affect adaptive immunity. Lactobacillus, Bifidobacterium, and other lactic acid-associated bacteria have been selected as candidate probiotics because of their potent stimulation of production of pro-inflammatory cytokines such as IL-12 and TNF-α (37, 38). Several of these bacteria also have anti-obesity effects in animal and human studies. The current study suggests that lactobacilli can modify the crosstalk between immune-component cells and adipose tissue by enhancement of macrophage-derived inflammatory responses. However, further studies are required to clarify whether alterations in the interactions between immune and adipose tissue induced by lactobacilli contribute to the anti-obesity effects of these bacteria.
Chronic subacute inflammation, as indicated by changes in inflammatory cells and the biochemical markers of inflammation, is reportedly present in overweight people (39). Obesity-associated inflammation derives mainly from macrophages resident within adipose tissue and is generally considered to be a harmful immune event because of the rapidly expanding body of the animal. Clinical data support a potential role for inflammation in the pathogenesis of insulin resistance and type-2 diabetes mellitus (40). Therefore, reduction or suppression of obesity-associated chronic inflammation is a practical strategy for reducing the harmful effects of obesity; several anti-inflammatory agents for treatment of obesity have been proposed.
Obesity-associated chronic inflammation is induced mainly by infiltration and aggregation of macrophages to constitute granulysins surrounding dead adipocytes (39). Endogenous ligands (“danger signals”) from damaged/dead adipose cells such as saturated fatty acids and damage-associated molecular pattern molecules are probably the main stimulators of such harmful chronic inflammation. However, lactobacilli are identical to other exogenous ligands that can enhance the innate immunity of the host animal. This inflammation should be different to obesity-associated chronic inflammation, which has an acute phenotype and is considered to be “homeostatic” inflammation. Enhancement of inflammatory immune responses with lactobacilli such as LGG and TMC0356 is considered a critical mechanism underlying their well-documented health-promoting effects (e.g., anti-allergic and anti-pathogenic) in host animals (23, 24, 25, 26, 29, 30, 31). The results of the present study suggest that lactobacilli-stimulated acute inflammation may express one more function in host animals by suppressing adipogenic differentiation and thus altering adipocyte hypertrophy.