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Research article
Micronized and Heat-Treated Lactobacillus plantarum LM1004 Stimulates Host Immune Responses Via the TLR-2/MAPK/NF-κB Signalling Pathway In Vitro and In Vivo
1Department of Biotechnology, The Catholic University of Korea, Bucheon 14662, Republic of Korea
2LACTOMASON, Jinju 52840, Republic of Korea
3Department of Biochemistry and Molecular Biology, School of Medicine, Kyung Hee University, Seoul 02447, Republic of Korea
J. Microbiol. Biotechnol. 2019; 29(5): 704-712
Published May 28, 2019 https://doi.org/10.4014/jmb.1812.12059
Copyright © The Korean Society for Microbiology and Biotechnology.
Abstract
Keywords
Introduction
Probiotics are generally defined as live microorganisms, such as
Previously, a strain of
Materials and Methods
Materials
Fetal bovine serum (FBS), penicillin-streptomycin, and trypsin– EDTA were purchased from Lonza (USA). 3-(4,5-dimethylthiazol-yl)-diphenyl tetrazolium bromide (MTT) was provided by Duchefa Biochemie (Netherlands). Dulbecco’s Modified Eagle’s Medium (DMEM) and RPMI-1640 medium (RPMI) were purchased from WelGENE (Korea). The antibodies against total ERK (t-ERK), phospho-ERK (p-ERK), NF-κB p65, β-actin, goat anti-mouse IgG-HRP and goat anti-rabbit IgG-HRP were obtained from Cell Signaling Technology (USA).
Preparation of MHT-LM1004
Micronized and heat-treated
Cell Culture
RAW 264.7 macrophage cells were purchased from the Korea Cell Line Bank (Korea) and grown in DMEM supplemented with 10% (v/v) FBS, 2 mM L-glutamine and 100 U/ml penicillin-streptomycin. The cytotoxicity of MHT-LM1004 was assessed by measuring cell viability using the MTT assay [15]. To examine the role of TLR-2 in cytokine production, RAW 264.7 cells were pre-incubated with medium containing the TLR-2-specific antibody (10 μg/ml) or IgG1 antibody (isotype control antibody, 10 μg/ml) (Invivogen, USA) for 1 h. Cells were then treated with MHT-LM1004 (107 cells/ml) for 4 h.
Assay for TNF-α and IL-6 Secretion
The cells were cultured in DMEM for 24 h with various concentrations of MHT-LM1004 (102 ~ 107 cells/ml) and the supernatants were used for the quantification of TNF-α and IL-6 secretion using an ELISA kit (R&D Systems, United Kingdom) according to the manufacturer’s protocol.
Measurement of NO Production
The RAW 264.7 cells were cultured for 24 h in DMEM supplemented with 10% FBS and 1% PEST. The cells were then treated with different concentrations of MHT-LM1004 (103 ~ 107 cells/ml) for 24 h. NO levels in the culture supernatants were determined using Griess reagent (Sigma, USA). Briefly, samples of the culture supernatants were mixed with an equal volume of Griess reagent [0.1% naphthyl ethylenediamine (w/v) and 1% sulfanilamide (w/v) in 5% phosphoric acid (v/v)], and the absorbance at 540 nm was measured. Sodium nitrite (NaNO2, Sigma) was used as a reference.
Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) and Western Blot Analysis
To evaluate the mRNA expression levels of iNOS, TLR-2 and TLR-4, the total RNA from MHT-LM1004-treated RAW 264.7 cells was prepared using a Total RNA Extraction Kit (Intron Biotechnology, Korea). RT-PCR was performed using the One-Step RT-PCR PreMix Kit (Intron Biotechnology) with appropriate sense and antisense. The products were separated on a 1.5% agarose gel, stained with ethidium bromide and then viewed under UV transillumination. Western blot was performed as described previously [16]. The mRNA and protein were quantified using ImageJ software from the NIH (USA).
Immunocytochemistry
Cells were grown on an 8-chamber slide, fixed with 4% formaldehyde in PBS, and then permeabilized and blocked for 1 h with blocking buffer (5% normal goat serum and 0.3% Triton X-100 in PBS). For NF-κB visualizations, cells were incubated overnight with primary antibody and diluted 1:100 in antibody dilution buffer (1% BSA and 0.3% Triton X-100 in PBS). Cells were then incubated for 2 h with fluorochrome-conjugated secondary antibody, diluted 1:1,000, and then washed three times with PBS. The cover glass was applied to the slide with Prolong Gold Antifade Reagent with DAPI (all from Cell Signaling Technology). The stained cells were monitored using a Zeiss LSM800 confocal laser scanning microscope (Carl Zeiss, Germany, X630).
Animals
The animal protocol (Approval number: 2016-021) was approved by the Institutional Animal Care and Use Committee of The Catholic University of Korea. The 6-week-old female BALB/c mice were purchased from Orientbio (Korea) and randomly divided into three equal groups (
Isolation of Splenocytes
To isolate splenocytes, the tissues were disaggregated via passage through a 70-μm nylon mesh (Becton-Dickinson, USA) in RPMI. The cells were isolated via centrifugation at 470
Measurement of Splenocyte Proliferation Rate and Serum Cytokine
The splenocytes were suspended in RPMI supplemented with 10% (v/v) FBS and dispensed into 12-well plates (5 × 106 cells/ml). Cells were then incubated in RPMI containing mitogens – concanavalin A (ConA, 5 μg/ml, Sigma-Aldrich) as a T-cell mitogen or
Statistical Analysis
Measurements were performed in triplicate, and data are expressed as the means ± SD. The statistical analyses were performed using the unpaired Student’s
Results
Preparation of MHT-LM1004
Micronized and heat-treated
-
Fig. 1.
Effects of MHT-LM1004 on the viability of RAW 264.7 cells. (A ) Scanning electron micrograph (left) and particle size distribution (right) of MHT-LM1004. (B ) Cell viability was assessed using the MTT assay after treatment with LM1004 for 24 h. The data are expressed as the percentage normalized to the untreated control (Con); Data = the means ± SD,n = 3.
Effect of MHT-LM1004 on the Viability of RAW 264.7 Cells
To determine whether MHT-LM1004 is detrimental to RAW 264.7 cells, the effects of MHT-LM1004 on the viability were tested using the MTT assay after treatment with increasing concentrations of MHT-LM1004 for 24 h. MHT-LM1004 did not affect the viability of RAW 264.7 cells up to the concentrations of 108 cells/ml (Fig. 1B), showing that MHT-LM1004 has no cytotoxicity on RAW 264.7 cells. Thus, concentrations of less than 108 cells/ml of the MHT-LM1004 were used for subsequent experiments to evaluate its immunostimulatory activities.
Effects of MHT-LM1004 on Cytokine Production
Activated macrophages release pro-inflammatory cytokines such as TNF-α and IL-6, which are potent immunomodulators [16, 18]. As shown in Fig. 2, MHT-LM1004 significantly induced TNF-α (Fig. 2A) and IL-6 (Fig. 2B) secretion in a dose-dependent manner. MHT-LM1004 treatment resulted in approximately 24.9-fold and 19.3-fold higher levels of TNF-α and IL-6, respectively, at 107 cells/ml compared to the untreated control (Con), suggesting that MHT-LM1004 is a strong stimulator of TNF-α and IL-6 secretion.
-
Fig. 2.
Effects of MHT-LM1004 on TNF-α and IL-6 secretion. Cells were incubated with the indicated doses of MHT-LM1004 for 24 h. The levels of (A ) TNF-α and (B ) IL-6 in the culture supernatants were determined using ELISA. Data = mean ± SD,n = 3; *,p < 0.05; **,p < 0.01; ***,p < 0.001, Student’st -test compared to the untreated control (Con).
Effects of MHT-LM1004 on iNOS Expression and NO Production
NO production plays a crucial role in the immune response [19]. When the RAW 264.7 cells were incubated with MHT-LM1004 for 24 h, the iNOS expression was significantly enhanced (Fig. 3A), with approximately 17.7-fold higher expression at 10 cells/ml MHT-LM1004, compared to the untreated cells (Con) (Fig. 3B). This result was further confirmed by measuring the level of NO production. When the cells were treated with varying concentrations of MHT-LM1004 (10 ~ 10 cells/ml) for 24 h, the levels of NO were significantly enhanced in a dose-dependent manner (Fig. 3C). These results suggested that NO is a mediator of MHT-LM1004-stimulated immune signalling pathways.
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Fig. 3.
Effects of MHT-LM1004 on iNOS mRNA expression. (A ) The mRNA level of iNOS was determined using RT-PCR. (B ) The amount of iNOS mRNA is expressed as the ratio of the densitometric measurement of mRNA in MHT-LM1004-treated cells to the corresponding internal standard (β-Actin). (C ) Nitric oxide levels in the culture media of MHT-LM1004-stimulated cells were measured using the Griess reagent. Data = mean ± SD,n = 3; **,p < 0.01; ***,p < 0.001, Student’st -test compared to the untreated control (Con).
MHT-LM1004 Induces Cytokine Production Dependent on TLR-2 Gene Expression
Toll-like receptors (TLRs) are pattern recognition receptors that have important functions in the initiation of mammalian immune responses [20]. Thus, to understand how the MHT-LM1004 affects the innate immune response, responsible receptors on the surfaces of macrophages were monitored. The RAW 264.7 cells were treated with MHT-LM1004 for 24 h, and then the mRNA expression levels of the candidate membrane receptors including TLR-2 and TLR-4 were determined using RT-PCR. As shown in Fig. 4A, MHT-LM1004 treatment resulted in a remarkable increase of TLR-2 mRNA expression levels, whereas TLR-4 expression levels did not change. This result was further confirmed using anti-TLR-2 antibody. The treatment of macrophages with the TLR-2 antibody significantly reduced MHT-LM1004-induced up-regulation of TNF-α secretion (Fig. 4B), clearly demonstrating that the target receptor of MHT-LM1004 was TLR-2 on the RAW 264.7 cells.
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Fig. 4.
MHT-LM1004 induces TNF-α production through TLR-2 gene expression in RAW 264.7 cells. (A ) RAW 264.7 cells were incubated with the indicated doses of MHT-LM1004 for 24 h. The mRNA expression levels of TLR-2 and TLR-4 were measured using RT-PCR. (B ) Cells were pre-treated with isotype antibody (10 μg/ml) or anti-TLR-2 antibody (10 μg/ml) for 1 h and then stimulated with MHT-LM1004 (107 cells/ml) for 4 h. TNF-α in the culture supernatants was determined using ELISA. Data = mean ± SD,n = 3; **,p < 0.01, Student’st -test compared to the control without MHT-LM1004 or antibody-only treated cells; ##,p < 0.01, Student’st -test compared to the MHT-LM1004 plus isotype antibody treated cells.
MHT-LM1004 Stimulates the Immune System Via the MAPKs/NF-κB Pathway
Interestingly, the TLR-2 activation recruits the myeloid differentiation primary response gene 88 (MyD88) adapter protein and activates MAPK signalling pathways including extracellular-signal-regulated kinase (ERK) for regulating the immune response in macrophages [16, 20, 21]. The western blot analysis showed that while the total protein LM1004 activates the TLR-2/MAPK/NF-κB pathway in RAW 264.7 cells for immune modulation.
Effects of MHT-LM1004 on the Splenocyte Proliferation Rate and Cytokine Production In Vivo
To prove if MHT-LM1004 is equally effective in vivo, the mice were fed with MHT-LM1004 (4 × 109 or 4 × 1011 cells/ml) levels of ERK (t-ERK) remained unchanged, the treatment of cells with MHT-LM1004 for 24 h significantly increased the phosphorylation levels of ERK (Fig. 5A), suggesting that MHT-LM1004 activates the TLR-2/MAPKs (ERK) signalling pathway to enhance the immune response in RAW 264.7 cells. On the other hand, NF-κB is known as a key transcription factor, which regulates immune response genes. The expression of iNOS is directly coupled to the up-regulation of NF-κB [22]. The immunocytochemistry result showed that MHT-LM1004 treatment increased the translocation of NF-κB from the cytosol to the nucleus compared to the untreated control (Con) (Fig. 5B). Taken collectively, these data clearly demonstrated that MHT-for 28 days, and the splenocytes were isolated from the spleens of the mice and then treated with mitogens (ConA or LPS). As shown in Figs. 6A and 6B, ConA and LPS treatment increased the proliferation of splenocytes isolated from the mice treated with 4 × 1011 cells/ml MHT-LM1004 up to 2.3-fold and 1.6-fold, respectively, (
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Fig. 5.
MHT-LM1004 activates the MAPKs/NF-κB pathway in RAW 264.7 cells. (A ) Whole cell lysates were collected after treatment with MHT-LM1004 for 24 h. t-ERK was used as a loading control for the western blot analysis of p-ERK. (B ) The cells were exposed to MHT-LM1004 (107 cells/ml) for 24h, stained with DAPI solution and anti-NF-κB p65 (Rhodamine) antibody, and then visualized under a Zeiss LSM800 confocal laser scanning microscope (Carl Zeiss, Germany, X630).
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Fig. 6.
Effects of MHT-LM1004 on the splenocyte proliferation rate and serum cytokine production in vivo. (A and B) Splenocytes (5 × 106 cells per well) were individually isolated from the BALB/c mice orally administered with MHT-LM1004 for 28 days and cultured with mitogens (ConA or LPS) for 48 h (n = 6 in each group). The proliferation of splenocytes was determined using the MTT assay. The data are expressed as the percentage normalized to the untreated control cells (Con). (C and D) Blood was withdrawn via cardiac puncture from the mice orally administered MHT-LM1004 for 28 days, and serum TNF-α and IL-6 was measured using the ELISA kit (n = 5 in each group). All values are expressed as the mean ± SE. Values with different superscripts are significantly different among the groups according to ANOVA Duncan’s multiple range test atp < 0.05.
Discussion
Generally, it is well known that heat-killed probiotics, such as
Here, we focused on the advantages of heat-treated probiotics, such as a longer shelf-life and enhanced immunomodulatory activities. When the live
Previously, several studies suggested that probiotics modulate the cytokine profile in the cells through TLR stimulation (TLR-2 and TLR-4) [24, 25]. In this context, the studies on TLRs in immune recognition of immunomodulators have helped us to understand how probiotics function as an immunostimulator or immunosuppressor in immune cells. TLRs initiate and regulate the innate immune response through the NF-κB signalling pathway [26]. Thus, the TLRs are the key sensors of the probiotics for controlling many cellular activities in the innate immune response. One of our striking findings was that MHT-LM1004 remarkably activated macrophages by enhancing the TLR-2 expression levels, but not TLR-4, suggesting that the immunostimulating activity of MHT-LM1004 was associated with the TLR-2 sensor. In accordance with our results, some reports demonstrated the role of TLR-2 in the sensing of
Macrophage activation is an important strategy in the host defense response and immune enhancement. When macrophages are activated, a series of signalling molecules selectively turn on immunostimulatory genes including cytokines and iNOS through NF-κB [16]. Among these immune signalling pathways, the MAPK/NF-κB pathway is crucial for orchestrating early innate immune responses, which are essential for the host defense against a wide range of pathogens [16, 30]. Consistently, our data indicated that upon treatment with MHT-LM1004, MAPK (ERK) phosphorylation was enhanced and NF-κB was translocated from the cytoplasm to the nucleus. These results revealed that MAPK/NF-κB signalling is involved in MHT-LM1004-induced cytokine release and iNOS expression in RAW 264.7 macrophages.
Taken together, our study demonstrated that micronized and heat-treated
Acknowledgments
This work was supported by the Ministry of Trade, Industry, and Energy (MOTIE), Korea, under the “Regional Specialized Industry Development Program (Project No. R0005616)” supervised by the Korea Institute for Advancement of Technology (KIAT).
Conflict of Interest
The authors have no financial conflicts of interest to declare.
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Related articles in JMB

Article
Research article
J. Microbiol. Biotechnol. 2019; 29(5): 704-712
Published online May 28, 2019 https://doi.org/10.4014/jmb.1812.12059
Copyright © The Korean Society for Microbiology and Biotechnology.
Micronized and Heat-Treated Lactobacillus plantarum LM1004 Stimulates Host Immune Responses Via the TLR-2/MAPK/NF-κB Signalling Pathway In Vitro and In Vivo
Jisun Lee 1, Ilseon Jung 2, Ji Won Choi 1, Chang Won Lee 1, Sarang Cho 1, Tae Gyu Choi 3, Minn Sohn 2 and Yong Il Park 1*
1Department of Biotechnology, The Catholic University of Korea, Bucheon 14662, Republic of Korea
2LACTOMASON, Jinju 52840, Republic of Korea
3Department of Biochemistry and Molecular Biology, School of Medicine, Kyung Hee University, Seoul 02447, Republic of Korea
Correspondence to:Yong Il Park
yongil382@catholic.ac.kr
Abstract
Although nanometric dead Lactobacillus plantarum has emerged as a potentially important modulator of immune responses, its underlying mechanism of action has not been fully understood. This study aimed to identify the detailed biochemical mechanism of immune modulation by micronized and heat-treated L. plantarum LM1004 (MHT-LM1004, <1 μm in size). MHT-LM1004 was prepared from L. plantarum LM1004 via culture in a specifically designed membrane bioreactor and heat treatment. MHT-LM1004 was shown to effectively induce the secretion of TNF-α and IL-6 and the mRNA expression of inducible nitric oxide synthase (iNOS). MHT-LM1004 enhanced the expression of TLR-2, phosphorylation of MAPKs (ERK), and nuclear translocation of NF-κB in a dose-dependent manner. Oral administration of MHT-LM1004 (4 × 109 or 4× 1011 cells/kg mouse body weight) increased the splenocyte proliferation and serum cytokine levels. These results suggested that MHT-LM1004 effectively enhances early innate immunity by activating macrophages via the TLR-2/MAPK/NF-κB signalling pathway and that this pathway is one of the major routes in immune modulation by the Lactobacillus species.
Keywords: Nanometric heat-killed Lactobacillus plantarum, immunostimulating activity, action mechanism, TLR-2, MAPK-NF-&kappa,B signalling pathway
Introduction
Probiotics are generally defined as live microorganisms, such as
Previously, a strain of
Materials and Methods
Materials
Fetal bovine serum (FBS), penicillin-streptomycin, and trypsin– EDTA were purchased from Lonza (USA). 3-(4,5-dimethylthiazol-yl)-diphenyl tetrazolium bromide (MTT) was provided by Duchefa Biochemie (Netherlands). Dulbecco’s Modified Eagle’s Medium (DMEM) and RPMI-1640 medium (RPMI) were purchased from WelGENE (Korea). The antibodies against total ERK (t-ERK), phospho-ERK (p-ERK), NF-κB p65, β-actin, goat anti-mouse IgG-HRP and goat anti-rabbit IgG-HRP were obtained from Cell Signaling Technology (USA).
Preparation of MHT-LM1004
Micronized and heat-treated
Cell Culture
RAW 264.7 macrophage cells were purchased from the Korea Cell Line Bank (Korea) and grown in DMEM supplemented with 10% (v/v) FBS, 2 mM L-glutamine and 100 U/ml penicillin-streptomycin. The cytotoxicity of MHT-LM1004 was assessed by measuring cell viability using the MTT assay [15]. To examine the role of TLR-2 in cytokine production, RAW 264.7 cells were pre-incubated with medium containing the TLR-2-specific antibody (10 μg/ml) or IgG1 antibody (isotype control antibody, 10 μg/ml) (Invivogen, USA) for 1 h. Cells were then treated with MHT-LM1004 (107 cells/ml) for 4 h.
Assay for TNF-α and IL-6 Secretion
The cells were cultured in DMEM for 24 h with various concentrations of MHT-LM1004 (102 ~ 107 cells/ml) and the supernatants were used for the quantification of TNF-α and IL-6 secretion using an ELISA kit (R&D Systems, United Kingdom) according to the manufacturer’s protocol.
Measurement of NO Production
The RAW 264.7 cells were cultured for 24 h in DMEM supplemented with 10% FBS and 1% PEST. The cells were then treated with different concentrations of MHT-LM1004 (103 ~ 107 cells/ml) for 24 h. NO levels in the culture supernatants were determined using Griess reagent (Sigma, USA). Briefly, samples of the culture supernatants were mixed with an equal volume of Griess reagent [0.1% naphthyl ethylenediamine (w/v) and 1% sulfanilamide (w/v) in 5% phosphoric acid (v/v)], and the absorbance at 540 nm was measured. Sodium nitrite (NaNO2, Sigma) was used as a reference.
Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) and Western Blot Analysis
To evaluate the mRNA expression levels of iNOS, TLR-2 and TLR-4, the total RNA from MHT-LM1004-treated RAW 264.7 cells was prepared using a Total RNA Extraction Kit (Intron Biotechnology, Korea). RT-PCR was performed using the One-Step RT-PCR PreMix Kit (Intron Biotechnology) with appropriate sense and antisense. The products were separated on a 1.5% agarose gel, stained with ethidium bromide and then viewed under UV transillumination. Western blot was performed as described previously [16]. The mRNA and protein were quantified using ImageJ software from the NIH (USA).
Immunocytochemistry
Cells were grown on an 8-chamber slide, fixed with 4% formaldehyde in PBS, and then permeabilized and blocked for 1 h with blocking buffer (5% normal goat serum and 0.3% Triton X-100 in PBS). For NF-κB visualizations, cells were incubated overnight with primary antibody and diluted 1:100 in antibody dilution buffer (1% BSA and 0.3% Triton X-100 in PBS). Cells were then incubated for 2 h with fluorochrome-conjugated secondary antibody, diluted 1:1,000, and then washed three times with PBS. The cover glass was applied to the slide with Prolong Gold Antifade Reagent with DAPI (all from Cell Signaling Technology). The stained cells were monitored using a Zeiss LSM800 confocal laser scanning microscope (Carl Zeiss, Germany, X630).
Animals
The animal protocol (Approval number: 2016-021) was approved by the Institutional Animal Care and Use Committee of The Catholic University of Korea. The 6-week-old female BALB/c mice were purchased from Orientbio (Korea) and randomly divided into three equal groups (
Isolation of Splenocytes
To isolate splenocytes, the tissues were disaggregated via passage through a 70-μm nylon mesh (Becton-Dickinson, USA) in RPMI. The cells were isolated via centrifugation at 470
Measurement of Splenocyte Proliferation Rate and Serum Cytokine
The splenocytes were suspended in RPMI supplemented with 10% (v/v) FBS and dispensed into 12-well plates (5 × 106 cells/ml). Cells were then incubated in RPMI containing mitogens – concanavalin A (ConA, 5 μg/ml, Sigma-Aldrich) as a T-cell mitogen or
Statistical Analysis
Measurements were performed in triplicate, and data are expressed as the means ± SD. The statistical analyses were performed using the unpaired Student’s
Results
Preparation of MHT-LM1004
Micronized and heat-treated
-
Figure 1.
Effects of MHT-LM1004 on the viability of RAW 264.7 cells. (A ) Scanning electron micrograph (left) and particle size distribution (right) of MHT-LM1004. (B ) Cell viability was assessed using the MTT assay after treatment with LM1004 for 24 h. The data are expressed as the percentage normalized to the untreated control (Con); Data = the means ± SD,n = 3.
Effect of MHT-LM1004 on the Viability of RAW 264.7 Cells
To determine whether MHT-LM1004 is detrimental to RAW 264.7 cells, the effects of MHT-LM1004 on the viability were tested using the MTT assay after treatment with increasing concentrations of MHT-LM1004 for 24 h. MHT-LM1004 did not affect the viability of RAW 264.7 cells up to the concentrations of 108 cells/ml (Fig. 1B), showing that MHT-LM1004 has no cytotoxicity on RAW 264.7 cells. Thus, concentrations of less than 108 cells/ml of the MHT-LM1004 were used for subsequent experiments to evaluate its immunostimulatory activities.
Effects of MHT-LM1004 on Cytokine Production
Activated macrophages release pro-inflammatory cytokines such as TNF-α and IL-6, which are potent immunomodulators [16, 18]. As shown in Fig. 2, MHT-LM1004 significantly induced TNF-α (Fig. 2A) and IL-6 (Fig. 2B) secretion in a dose-dependent manner. MHT-LM1004 treatment resulted in approximately 24.9-fold and 19.3-fold higher levels of TNF-α and IL-6, respectively, at 107 cells/ml compared to the untreated control (Con), suggesting that MHT-LM1004 is a strong stimulator of TNF-α and IL-6 secretion.
-
Figure 2.
Effects of MHT-LM1004 on TNF-α and IL-6 secretion. Cells were incubated with the indicated doses of MHT-LM1004 for 24 h. The levels of (A ) TNF-α and (B ) IL-6 in the culture supernatants were determined using ELISA. Data = mean ± SD,n = 3; *,p < 0.05; **,p < 0.01; ***,p < 0.001, Student’st -test compared to the untreated control (Con).
Effects of MHT-LM1004 on iNOS Expression and NO Production
NO production plays a crucial role in the immune response [19]. When the RAW 264.7 cells were incubated with MHT-LM1004 for 24 h, the iNOS expression was significantly enhanced (Fig. 3A), with approximately 17.7-fold higher expression at 10 cells/ml MHT-LM1004, compared to the untreated cells (Con) (Fig. 3B). This result was further confirmed by measuring the level of NO production. When the cells were treated with varying concentrations of MHT-LM1004 (10 ~ 10 cells/ml) for 24 h, the levels of NO were significantly enhanced in a dose-dependent manner (Fig. 3C). These results suggested that NO is a mediator of MHT-LM1004-stimulated immune signalling pathways.
-
Figure 3.
Effects of MHT-LM1004 on iNOS mRNA expression. (A ) The mRNA level of iNOS was determined using RT-PCR. (B ) The amount of iNOS mRNA is expressed as the ratio of the densitometric measurement of mRNA in MHT-LM1004-treated cells to the corresponding internal standard (β-Actin). (C ) Nitric oxide levels in the culture media of MHT-LM1004-stimulated cells were measured using the Griess reagent. Data = mean ± SD,n = 3; **,p < 0.01; ***,p < 0.001, Student’st -test compared to the untreated control (Con).
MHT-LM1004 Induces Cytokine Production Dependent on TLR-2 Gene Expression
Toll-like receptors (TLRs) are pattern recognition receptors that have important functions in the initiation of mammalian immune responses [20]. Thus, to understand how the MHT-LM1004 affects the innate immune response, responsible receptors on the surfaces of macrophages were monitored. The RAW 264.7 cells were treated with MHT-LM1004 for 24 h, and then the mRNA expression levels of the candidate membrane receptors including TLR-2 and TLR-4 were determined using RT-PCR. As shown in Fig. 4A, MHT-LM1004 treatment resulted in a remarkable increase of TLR-2 mRNA expression levels, whereas TLR-4 expression levels did not change. This result was further confirmed using anti-TLR-2 antibody. The treatment of macrophages with the TLR-2 antibody significantly reduced MHT-LM1004-induced up-regulation of TNF-α secretion (Fig. 4B), clearly demonstrating that the target receptor of MHT-LM1004 was TLR-2 on the RAW 264.7 cells.
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Figure 4.
MHT-LM1004 induces TNF-α production through TLR-2 gene expression in RAW 264.7 cells. (A ) RAW 264.7 cells were incubated with the indicated doses of MHT-LM1004 for 24 h. The mRNA expression levels of TLR-2 and TLR-4 were measured using RT-PCR. (B ) Cells were pre-treated with isotype antibody (10 μg/ml) or anti-TLR-2 antibody (10 μg/ml) for 1 h and then stimulated with MHT-LM1004 (107 cells/ml) for 4 h. TNF-α in the culture supernatants was determined using ELISA. Data = mean ± SD,n = 3; **,p < 0.01, Student’st -test compared to the control without MHT-LM1004 or antibody-only treated cells; ##,p < 0.01, Student’st -test compared to the MHT-LM1004 plus isotype antibody treated cells.
MHT-LM1004 Stimulates the Immune System Via the MAPKs/NF-κB Pathway
Interestingly, the TLR-2 activation recruits the myeloid differentiation primary response gene 88 (MyD88) adapter protein and activates MAPK signalling pathways including extracellular-signal-regulated kinase (ERK) for regulating the immune response in macrophages [16, 20, 21]. The western blot analysis showed that while the total protein LM1004 activates the TLR-2/MAPK/NF-κB pathway in RAW 264.7 cells for immune modulation.
Effects of MHT-LM1004 on the Splenocyte Proliferation Rate and Cytokine Production In Vivo
To prove if MHT-LM1004 is equally effective in vivo, the mice were fed with MHT-LM1004 (4 × 109 or 4 × 1011 cells/ml) levels of ERK (t-ERK) remained unchanged, the treatment of cells with MHT-LM1004 for 24 h significantly increased the phosphorylation levels of ERK (Fig. 5A), suggesting that MHT-LM1004 activates the TLR-2/MAPKs (ERK) signalling pathway to enhance the immune response in RAW 264.7 cells. On the other hand, NF-κB is known as a key transcription factor, which regulates immune response genes. The expression of iNOS is directly coupled to the up-regulation of NF-κB [22]. The immunocytochemistry result showed that MHT-LM1004 treatment increased the translocation of NF-κB from the cytosol to the nucleus compared to the untreated control (Con) (Fig. 5B). Taken collectively, these data clearly demonstrated that MHT-for 28 days, and the splenocytes were isolated from the spleens of the mice and then treated with mitogens (ConA or LPS). As shown in Figs. 6A and 6B, ConA and LPS treatment increased the proliferation of splenocytes isolated from the mice treated with 4 × 1011 cells/ml MHT-LM1004 up to 2.3-fold and 1.6-fold, respectively, (
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Figure 5.
MHT-LM1004 activates the MAPKs/NF-κB pathway in RAW 264.7 cells. (A ) Whole cell lysates were collected after treatment with MHT-LM1004 for 24 h. t-ERK was used as a loading control for the western blot analysis of p-ERK. (B ) The cells were exposed to MHT-LM1004 (107 cells/ml) for 24h, stained with DAPI solution and anti-NF-κB p65 (Rhodamine) antibody, and then visualized under a Zeiss LSM800 confocal laser scanning microscope (Carl Zeiss, Germany, X630).
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Figure 6.
Effects of MHT-LM1004 on the splenocyte proliferation rate and serum cytokine production in vivo. (A and B) Splenocytes (5 × 106 cells per well) were individually isolated from the BALB/c mice orally administered with MHT-LM1004 for 28 days and cultured with mitogens (ConA or LPS) for 48 h (n = 6 in each group). The proliferation of splenocytes was determined using the MTT assay. The data are expressed as the percentage normalized to the untreated control cells (Con). (C and D) Blood was withdrawn via cardiac puncture from the mice orally administered MHT-LM1004 for 28 days, and serum TNF-α and IL-6 was measured using the ELISA kit (n = 5 in each group). All values are expressed as the mean ± SE. Values with different superscripts are significantly different among the groups according to ANOVA Duncan’s multiple range test atp < 0.05.
Discussion
Generally, it is well known that heat-killed probiotics, such as
Here, we focused on the advantages of heat-treated probiotics, such as a longer shelf-life and enhanced immunomodulatory activities. When the live
Previously, several studies suggested that probiotics modulate the cytokine profile in the cells through TLR stimulation (TLR-2 and TLR-4) [24, 25]. In this context, the studies on TLRs in immune recognition of immunomodulators have helped us to understand how probiotics function as an immunostimulator or immunosuppressor in immune cells. TLRs initiate and regulate the innate immune response through the NF-κB signalling pathway [26]. Thus, the TLRs are the key sensors of the probiotics for controlling many cellular activities in the innate immune response. One of our striking findings was that MHT-LM1004 remarkably activated macrophages by enhancing the TLR-2 expression levels, but not TLR-4, suggesting that the immunostimulating activity of MHT-LM1004 was associated with the TLR-2 sensor. In accordance with our results, some reports demonstrated the role of TLR-2 in the sensing of
Macrophage activation is an important strategy in the host defense response and immune enhancement. When macrophages are activated, a series of signalling molecules selectively turn on immunostimulatory genes including cytokines and iNOS through NF-κB [16]. Among these immune signalling pathways, the MAPK/NF-κB pathway is crucial for orchestrating early innate immune responses, which are essential for the host defense against a wide range of pathogens [16, 30]. Consistently, our data indicated that upon treatment with MHT-LM1004, MAPK (ERK) phosphorylation was enhanced and NF-κB was translocated from the cytoplasm to the nucleus. These results revealed that MAPK/NF-κB signalling is involved in MHT-LM1004-induced cytokine release and iNOS expression in RAW 264.7 macrophages.
Taken together, our study demonstrated that micronized and heat-treated
Acknowledgments
This work was supported by the Ministry of Trade, Industry, and Energy (MOTIE), Korea, under the “Regional Specialized Industry Development Program (Project No. R0005616)” supervised by the Korea Institute for Advancement of Technology (KIAT).
Conflict of Interest
The authors have no financial conflicts of interest to declare.
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