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Keywords:

  • cytokine;
  • gene regulation;
  • innate immunity;
  • leucocyte;
  • signal transduction

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References

In the present study we have characterized T helper type 2 (Th2) [interleukin (IL)-10]/Th1 (IL-12) cytokine expression balance in human primary macrophages stimulated with multiple non-pathogenic Gram-positive bacteria used in the food industry and as probiotic substances. Bacteria representing Lactobacillus, Bifidobacterium, Lactococcus, Leuconostoc, Propionibacterium and Streptococcus species induced anti-inflammatory IL-10 production, although quantitative differences between the bacteria were observed. S. thermophilus was able to induce IL-12 production, while the production of IL-12 induced by other bacteria remained at a low level. The highest anti-inflammatory potential was seen with bifidobacteria, as evidenced by high IL-10/IL-12 induction ratios. All studied non-pathogenic bacteria were able to stimulate the expression of suppressor of cytokine signalling (SOCS) 3 that controls the expression of proinflammatory cytokine genes. Lactobacillus and Streptococcus species induced SOCS3 mRNA expression directly in the absence of protein synthesis and indirectly via bacteria-induced IL-10 production, as demonstrated by experiments with cycloheximide (CHX) and anti-IL-10 antibodies, respectively. The mitogen-activated protein kinase (MAPK) p38 signalling pathway played a key role in bacteria-induced SOCS3 gene expression. Enhanced IL-10 production and SOCS3 gene expression induced by live non-pathogenic Lactobacillus and Streptococcus is also likely to contribute to their immunoregulatory effects in vivo.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References

Macrophages are phagocytic cells that are present in almost all tissues of the body. Tissue macrophages originate from circulating monocytes, which after homing into peripheral tissues differentiate into tissue-specific mature macrophages [1]. The functions of macrophages are tightly regulated, as they control biological processes ranging from tissue development, bone remodelling and wound healing to inflammatory responses. Macrophages recognize, ingest and destroy infectious agents, initiate T cell responses by antigen presentation and act as effector cells for both humoral and cell-mediated immune responses. Interaction of macrophages with microbes or different antigens may lead to the production of cytokines, chemokines, anti-microbial peptides and various inflammatory mediators, which modulate host immune responses. The functions of macrophages may differ from one tissue to another depending on the local microenvironment and cytokine milieu [2].

Macrophages express a number of cell surface and intracellular receptors, called pattern recognition receptors (PRRs), that recognize conserved microbial structural components, genetic material and molecular patterns released by injured cells [3,4]. After ligand binding, PRRs activate intracellular signalling pathways including transcription factors nuclear factor kappa B (NF-κB), interferon regulatory factors (IRFs) and mitogen-activated protein kinases (MAPKs). Activation of these signalling pathways leads to the production of proinflammatory cytokines. Negative regulation of PRR-mediated responses is crucial in maintaining tissue homeostasis and for controlling host inflammatory responses. Suppressor of cytokine signalling (SOCS) proteins have been identified as important negative regulators of cytokine signalling [5]. SOCS proteins are inducible upon cytokine stimulation, and they have the ability to inhibit signalling through the Janus kinase signalling transducer and activator of transcription (JAK/STAT) pathway. The best-characterized SOCS family members include SOCS1, SOCS3 and cytokine-inducible SH-2 containing (CIS) protein. SOCS3 can interact with JAKs and gp130 [6], which leads to reduced activation of the respective signalling pathways.

Probiotic bacteria are defined as living microorganisms that have beneficial effects on host health. They can change the metabolic activity of the intestinal microbiota, modulate the immune system of the host and positively regulate intestinal homeostasis [7]. Bifidobacteria and lactobacilli are the most commonly used food supplements in human nutrition. Lactobacillus rhamnosus GG (LGG) has been studied extensively and found to reduce the severity and length of antibiotic-associated [8] and nosocomial diarrhoea in children [9]. LGG has also been shown to reduce the onset of atopy in early childhood [10,11]. The immunomodulatory effects of LGG are probably due to decreased production of proinflammatory cytokines, as evidenced by decreased production of interleukin (IL)-6 and tumour necrosis factor (TNF)-α in healthy individuals [12], and reduction in TNF-α production in allergic children [13]. A mixture of probiotic bacteria containing LGG, L. rhamnosus LC705, Propionibacterium freudenreichii ssp. shermanii PJS and Bifidobacterium breve Bb99 has been shown to alleviate the symptoms in irritable bowel disease [14].

The bacteria used in the present study are non-pathogenic Gram-positive bacteria. Although many probiotic bacterial strains are used as food supplements, with clinical evidence of their functionality, the mechanisms of action regarding their effects on the host immune responses have remained poorly characterized. The present study concentrates on analysing and comparing inflammatory versus anti-inflammatory cytokine expression patterns in human macrophages stimulated with different live non-pathogenic bacteria and pathogenic Streptococcus pyogenes. In addition, intracellular pathways involved in bacteria-induced anti-inflammatory responses are analysed.

Materials and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References

Bacterial strains

The bacterial strains used in the present study are described in Table 1. Two probiotic strains, L. rhamnosus GG (LGG; ATCC 53103) [9,13] and B. animalis ssp. lactis Bb12 (DSM15954) [18], and nine non-pathogenic bacteria, including L. rhamnosus LC705 (DSM 7061) [14,15], L. helveticus 161 [16], L. helveticus 1129 [17], B. longum 1/10, B. breve Bb99 (DSM 13692) [14,15], S. thermophilus THS [19,21], L. lactis ssp. cremoris ARH74 (DSM 18891) [20], Leuconostoc mesenteroides ssp. cremoris PIA2 (DSM 18892) [19] and P. freudenreichii ssp. shermanii PJS [14] were obtained from Valio Ltd R&D (Helsinki, Finland). A clinical isolate of S. pyogenes (GAS) serotype T1M1 (IH32030) from the collection of National Institute for Health and Welfare (Helsinki, Finland) functions as an example of a pathogenic bacterium. Bacteria were stored in skimmed milk at −70°C, and grown to the end of logarithmic growth phase as described previously [23] before they were used in stimulation experiments. All strains were passaged three times, except for the Bifidobacterium strains, which were passaged four times, before they were used in stimulation experiments. The number of bacteria was determined by counting in a Petroff–Hausser chamber.

Table 1.  Bacteria used in the study.
Bacterial species/subspeciesStrain abbreviationATCC/DSM numberReferencesUse in dairy products
  1. ATCC: American Type Culture Collection; n.a.: not applicable.

Lactobacillus    
 Lactobacillus rhamnosus GGLGGATCC 53103(Majamaa & Isolauri, 1997 [13], Szajewska et al., 2001 [9])Probiotic supplement
 Lactobacillus rhamnosus LC705LC705DSM 7061(Kajander et al., 2005 [14], Myllyluoma et al., 2005 [15])Cheese
 Lactobacillus helveticus161n.a.(Staaf et al., 2000 [16])Cheese
 Lactobacillus helveticus1129DSM 13137(Yang et al., 2000 [17])Cheese, fermented milk
Bifidobacterium    
 Bifidobacterium animalis subsp. lactisBb12DSM 15954(Schiffrin et al., 1995 [18])Probiotic supplement
 Bifidobacterium breveBb99DSM 13692(Kajander et al., 2005 [14], Myllyluoma et al., 2005 [15])n.a.
 Bifidobacterium longum1/10n.a.(Kekkonen et al., 2008 [19])n.a.
Lactococcus    
 Lactococcus lactis subsp. cremorisARH74DSM 18891(Yang et al., 1999 [20])Sour milk production
Leuconostoc    
 Leuconostoc mesenteroides subsp. cremorisPIA2DSM 18892(Kekkonen et al., 2008 [19])Sour milk production
Propionibacterium    
 Propionibacterium freudenreichii subsp. shermanii JSPJSDSM 7067(Kajander et al., 2005 [14])Cheese
Streptococcus    
 Streptococcus thermophilusTHSn.a.(Kekkonen et al., 2008 [19], Nordmark et al., 2005 [21])Yoghurt
 Streptococcus pyogenesGASn.a.(Miettinen et al., 2000 [22])n.a.

LGG, L. rhamnosus LC705, L. helveticus 161, L. helveticus 1129, and L. mesenteroides ssp. cremoris PIA2 strains were grown in de Man, Rogosa and Sharpe (MRS) medium (Lab M, Topley House, Lancashire, UK). Bifidobacterium strains were grown in MRS medium (Lab M) supplemented with 5 g/l l-cysteine (Merck, Darmstadt, Germany). S. thermophilus was grown in M17 agar (Lab M) supplemented with 20 g/l D (+) lactose monohydrate (J. T. Baker BV, Deventer, Holland) and M17 broth (Difco, Becton Dickinson, MD, USA) containing 20 g/l lactose (J. T. Baker BV). L. lactis was grown on calcium citrate agar (Valio Ltd) and M17 broth (Difco) containing 20 g/l lactose (J. T. Baker BV), as described previously [23]. P. freudenreichii ssp. shermanii PJS was grown anaerobically at +30°C in propioni-medium (Valio Ltd).

Monocyte purification and differentiation into macrophages

Human peripheral blood mononuclear cells (PBMCs) from leucocyte-rich buffy coats obtained from healthy blood donors (Finnish Red Cross Blood Transfusion Service, Helsinki, Finland) were isolated by density gradient centrifugation over a Ficoll-Pague gradient (Amersham Biotech, Uppsala, Sweden). Monocytes were purified further from PBMCs by adherence on 24-well plastic plates (Falcon, Becton Dickinson, Franklin Lakes, NJ, USA) at a concentration of 5 × 106 cells/well for 1 h at +37°C. To differentiate monocytes into macrophages, cells were grown in macrophage–serum-free substitution medium (Gibco Invitrogen, Grand Island, NY, USA) supplemented with 0·6 µg/ml penicillin, 60 µg/ml streptomycin and 10 ng/ml recombinant human granulocyte–macrophage colony-stimulating factor (GM-CSF) (Biosource, Camarillo, CA, USA), as described previously [22].

Stimulation experiments

All stimulation experiments were carried out with cells obtained from three to four blood donors and conducted in RPMI-1640 medium (Sigma St Louis, MO, USA). For stimulation experiments live bacteria were resuspended into RPMI-1640 medium. Macrophages were stimulated with a bacteria : host cell ratio of 2:1, 10:1 or 50:1.

Synthetic tripalmitoylated lipopeptide [Pam3Cys-Ser-(Lys)4 (Pam3CSK4)] (InvivoGen, San Diego, CA, USA) and Escherichia coli lipopolysaccharide (LPS) 0111 : B4 (Sigma, St Louis, MO, USA) were both used at a concentration of 100 ng/ml. After stimulation experiments, macrophages were collected and pooled for isolation of total cellular RNA, while the supernatants were collected separately and stored at −20°C until cytokine levels were determined by enzyme-linked immunosorbent assay (ELISA).

In cytokine priming experiments, macrophages were pretreated with IL-10 (10 ng/ml) (R&D Systems, Minneapolis, MN, USA) for 16 h prior to bacterial stimulation. Supernatants for cytokine measurements were collected 24 h after bacterial stimulation.

For cycloheximide (CHX; Sigma) and signalling inhibitor experiments macrophages were treated with CHX or inhibitors for 30 min before bacterial stimulation. CHX was used at the concentration of 10 µg/ml [22]. Signalling inhibitors PD98059 (used at 10 µmol/l) and LY294002 (50 µmol/l) were obtained from Calbiochem (San Diego, CA, USA) and SB202190 (10 µmol/l), SP600125 (10 µmol/l) and pyrrolidine dithiocarbamate (PDTC) (100 µmol/l) were purchased from Alexis Biochemicals (Lausen, Switzerland). Cells for RNA isolation were collected at 4 or 8 h after bacterial stimulation. Relative mRNA levels were determined by quantitative real-time reverse transcription–polymerase chain reaction (RT–PCR).

Cytokine-specific ELISAs

Cytokine levels (IL-12p70, IL-10) from cell culture supernatants were measured using FlowCytomix human Th1/Th2 11plex kit (Bender Medsystems, San Diego, CA, USA), as described previously [23]. TNF-α levels from cell culture supernatants were determined by a sandwich ELISA method using antibody pairs and standards obtained from BD PharMingen (San Diego, CA, USA), as described previously [24].

RNA isolation and quantitative RT–PCR

For RNA analysis, cells were collected, pooled and lysed with Trizol reagent (Invitrogen, Carlsbad, CA, USA), and total cellular RNA was isolated with the RNeasy Mini kit (Qiagen, Crawley, UK). Synthesis of cDNA was performed using Multiscribe RT (Applied Biosystems, Foster City, CA, USA) with oligo-d(T) primers (Applied Biosystems) [25]. To analyse mRNA expression by RT–PCR, cDNA amplification was performed using Master Mix Buffer with Assays-on-Demand gene expression assay primers and probes for SOCS3 (Hs02330328_s1) and beta-actin (Hs99999903_m1; both from Applied Biosystems). Relative mRNA levels were normalized against β-actin. Each cDNA sample was amplified in duplicate with MxPro 3005P (Stratagene, La Jolla, CA, USA) and relative amounts of mRNAs were calculated with the ΔΔ comparative threshold (Ct) method as instructed by the manufacturer.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References

The effects of different non-pathogenic bacteria besides LGG have not been characterized before in human primary macrophages, while their effects on human PBMCs and monocyte-derived dendritic cells (moDCs) have been described previously [19,23]. To compare the effects of non-pathogenic bacteria, we determined the optimal bacterial dose for macrophage stimulation. The amounts of bacteria for dose–response experiments were chosen based on previous studies where primary macrophages, dendritic cells, peripheral blood mononuclear cells or cell lines were stimulated with live bacteria [19,26–31]. Macrophage responses to non-pathogenic bacteria were compared to the ones induced by LGG and pathogenic S. pyogenes whose interactions with human macrophages have been well characterized previously [22,25,32]. Macrophage responses to bacteria were both dose- and strain-dependent (Fig. 1).

image

Figure 1. Gram-positive non-pathogenic and pathogenic bacteria induce interleukin (IL)-10 and IL-12 production in human monocyte-derived macrophages in a dose-dependent manner. Macrophages were stimulated with live bacteria at bacteria : host cell ratios of 2:1, 10:1 or 50:1. Cell culture supernatants were collected at 24 h after stimulation and cytokine levels were determined by FlowCytomix human T helper type 1 (Th1)/Th2 11plex kit. The columns represent the means and the error bars indicate standard deviations of four independent blood donors. The data are representative of two identical experiments.

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Different non-pathogenic Gram-positive bacteria have diverse anti-inflammatory potential

IL-10 is known to down-regulate the production of IL-12 [33]. Based on this Th2/Th1 cytokine balance, bacteria can be classified as anti-inflammatory Th2- or Th1-type inflammatory immune response-inducing strains [34,35]. Bifidobacterium strains were good inducers of anti-inflammatory IL-10, as also were LGG, L. helveticus 1129 and S. thermophilus (Fig. 1). L. lactis ARH74, L. mesenteroides PIA2 and L. helveticus 161 were weak inducers of IL-10. The amounts of IL-12 remained at a low level and S. thermophilus THS and S. pyogenes were the strongest IL-12 inducers (Fig. 1). The IL-10/IL-12 ratios (data presented for bacteria : host cell ratio of 10:1) were highest with Bifidobacterium strains (Fig. 2). LGG, L. rhamnosus LC705 and L. helveticus 1129 induced intermediate IL-10/IL-12 ratios, while L. helveticus 161 as well as Leuconostoc, Lactococcus, Propionibacterium and Streptococcus strains induced the lowest IL-10/IL-12 ratios.

image

Figure 2. Gram-positive bacteria induce anti-inflammatory cytokine response in human macrophages. The relative anti-inflammatory potential of each bacterium has been evaluated by calculating the IL-10/IL-12 ratios (n = 8) from experiments described in Fig. 1. Data from macrophages stimulated with 10:1 bacteria : host cell ratios have been used.

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SOCS3 mRNA is induced efficiently by non-pathogenic bacteria

It is not known what mechanisms participate in regulating the Th2/Th1 cytokine balance during bacterial stimulation. SOCS3 is known to inhibit cytokine production and signalling via a negative feedback loop [5] and could participate in also balancing Gram-positive bacteria-induced inflammatory responses. At present, no published reports on the effects of probiotic or non-pathogenic bacteria on SOCS3 gene expression in human macrophages are available. We analysed whether SOCS3 mRNA was inducible in macrophages after stimulation with non-pathogenic Gram-positive bacteria. All analysed bacteria induced SOCS3 mRNA expression at 8 h after stimulation (Fig. 3a). L. helveticus 161, B. breve Bb99 and S. thermophilus THS were the most efficient SOCS3 inducers. B. breve Bb99 and S. thermophilus THS were almost equally efficient SOCS3 mRNA inducers; S. thermophilus THS was chosen for further analysis, as it efficiently stimulated both IL-10 and IL-12p70 cytokine responses in macrophages (Fig. 1). LGG was chosen for further SOCS3 mRNA kinetics analyses, as it is a widely studied probiotic bacterial strain. In macrophages stimulated with LGG or S. thermophilus THS, SOCS3 expression was already induced at 2 h after stimulation (Fig. 3b). In LGG-stimulated macrophages the expression did not increase at later time-points, and remained high up to 24 h after stimulation. In S. thermophilus THS-stimulated cells SOCS3 mRNA levels continued to increase up to 24 h, and there was an approximately 150-fold induction at 24 h after S. thermophilus THS stimulation.

image

Figure 3. The expression of suppressor of cytokine signalling 3 (SOCS3) mRNA is induced in human macrophages stimulated with non-pathogenic bacteria. (a) Monocyte-derived macrophages were stimulated with 11 different non-pathogenic bacteria with bacteria : host cell ratio of 10:1 for 8 h. Macrophages were collected, pooled, total cellular RNA was isolated and SOCS3 mRNA levels were analysed by quantitative reverse transcription–polymerase chain reaction (qRT–PCR). Relative mRNA levels were normalized against β-actin and the relative level of SOCS3 mRNA was calculated with the ΔΔ comparative threshold (Ct) method. (b) Macrophages were stimulated with Lactobacillus rhamnosus GG (LGG) or Streptococcus thermophilus THS at a 10:1 bacteria : host cell ratio for times indicated in the figure. Note the differences in scales.

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LGG and S. thermophilus THS-induced SOCS3 gene expression is independent of ongoing protein synthesis

To analyse whether non-pathogenic Gram-positive bacteria-induced SOCS3 gene expression required ongoing protein synthesis, macrophages were stimulated with LGG or S. thermophilus THS in the presence of a protein synthesis inhibitor cycloheximide (CHX). In LGG and S. thermophilus THS-stimulated macrophages SOCS3 mRNA levels also increased in the presence of CHX (Fig. 4a). To verify the efficacy of CHX treatment we measured the production of TNF-α into the cell culture supernatant. The production of TNF-α was reduced by CHX treatment (Fig. 4b). We then analysed further the signalling pathways involved in LGG and S. thermophilus THS-induced direct protein synthesis-independent SOCS3 gene expression. Macrophages were treated with different pharmacological signalling inhibitors prior to stimulation with LGG or S. thermophilus THS (Fig. 5). In LGG-stimulated cells SOCS3 mRNA levels decreased to some extent after treatment with MAPK inhibitors PD98059 and SP600125, which inhibit extracellular-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), respectively. The strongest reduction in SOCS3 mRNA expression was seen with MAPK p38 inhibitor SB202190. PI3K and NF-κB signalling inhibitors LY294002 and PDTC had weak stimulatory effects on SOCS3 mRNA expression.

image

Figure 4. In macrophages bacteria-induced cytokine signalling 3 (SOCS3) mRNA expression does not require ongoing protein synthesis. (a) Macrophages were treated with protein synthesis inhibitor cycloheximide (CHX; 10 µg/ml) 30 min prior to stimulation with Lactobacillus rhamnosus GG (LGG), Streptococcus thermophilus THS or Streptococcus pyogenes (GAS). At 8 h after stimulation cells were collected, pooled and total cellular RNA was isolated. SOCS3 mRNA levels were analysed with quantitative reverse transcription–polymerase chain reaction (qRT–PCR). (b) Cell culture supernatants at 4 h after bacterial stimulation were analysed for tumour necrosis factor (TNF)-α by enzyme-linked immunosorbent assay (ELISA). The data are from one representative experiment out of three.

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image

Figure 5. The involvement of different signalling pathways in cytokine signalling 3 (SOCS3) gene expression in bacteria-stimulated macrophages. Macrophages were left untreated or treated with different pharmacological signalling inhibitors for 30 min prior to stimulation with live Lactobacillus rhamnosus GG (LGG), Streptococcus thermophilus THS or S. pyogenes (GAS) (bacteria : host cell ratio of 10:1). Cells were collected at 4 h after stimulation, total cellular RNA was isolated and SOCS3 mRNA levels were determined by quantitative reverse transcription–polymerase chain reaction (qRT–PCR). The used inhibitors were: extracellular-regulated kinase (ERK) inhibitor PD98059 (10 µmol/l), p38 inhibitor SB202190 (10 µmol/l), c-Jun N-terminal kinase (JNK) inhibitor SP600125 (10 µmol/l), PI3-kinase inhibitor LY294002 (50 µmol/l) and nuclear factor kappa B (NF-κ) inhibitor pyrrolidine dithiocarbamate (PDTC) (100 µmol/l). The data are a combination of three different experiments performed with macrophages obtained from total of 11 different blood donors. The columns represent the means and the error bars indicate standard deviations.

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S. thermophilus THS-induced IL-10 regulates SOCS3 mRNA expression

SOCS3 mRNA expression induced by LGG and S. thermophilus THS was independent of ongoing protein synthesis. However, the increase in SOCS3 mRNA expression up to 24 h after stimulation suggested that a positive feedback loop, e.g. via secreted cytokines, could exist. It has been shown previously that SOCS3 mRNA is up-regulated by IL-10 in human macrophages [6,36]. Because S. thermophilus THS was a potent inducer of both IL-10 production and SOCS3 gene expression, we wanted to analyse whether S. thermophilus THS-induced IL-10 has a role in SOCS3 gene expression. We neutralized IL-10 in S. thermophilus THS-stimulated macrophages, and then analysed S. thermophilus THS-induced SOCS3 mRNA expression. Treatment with anti-IL-10 antibodies led to reduced S. thermophilus THS-induced SOCS3 gene expression (Fig. 6a). To clarify further the regulatory role of IL-10 in bacteria-induced SOCS3 expression, we primed macrophages with IL-10 for 16 h followed by stimulation with live S. thermophilus THS or with purified bacterial components LPS and Pam3CSK4. In S. thermophilus THS-stimulated macrophages SOCS3 mRNA was expressed at higher levels than in LPS or Pam3CSK4-stimulated cells (Fig. 6b). Unlike in IL-10 primed LPS or Pam3CSK4-stimulated macrophages, S. thermophilus THS-induced SOCS3 mRNA expression was not increased further by IL-10 priming. IL-10 priming alone was able, to some extent, to induce SOCS3 gene expression (Fig. 6b).

image

Figure 6. Interleukin (IL)-10 regulates cytokine signalling 3 (SOCS3) gene expression in human macrophages during Gram-positive bacterial stimulation. (a) Effects of anti-IL-10 antibodies on Streptococcus thermophilus THS-induced SOCS3 gene expression. Macrophages were treated with anti-IL-10 antibodies 30 min prior to stimulation with S. thermophilus THS. Cells were collected at 8 h after stimulation followed by isolation of total cellular RNA and quantification of SOCS3 mRNA levels by quantitative reverse transcription–polymerase chain reaction (qRT–PCR). The data are from one representative experiment out of three. (b) The effect of IL-10 priming on SOCS3 gene expression in S. thermophilus THS, lipopolysaccharide (LPS) or Pam3Cys-Ser-(Lys)4 (Pam3CSK4)-stimulated cells. Macrophages were pre-treated with IL-10 (10 ng/ml) for 16 h, or left untreated prior to stimulation with live S. thermophilus THS (bacteria : host cell ratio 10:1), Toll-like receptor ligand (TLR)-4 ligand LPS (100 ng/ml) or TLR-2 ligand Pam3CSK4 (100 ng/ml). SOCS3 mRNA levels were analysed by qRT–PCR. The data are representative of two experiments.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References

In this study, we investigated the molecular mechanisms regulating interactions between non-pathogenic Gram-positive bacteria and human macrophages. Our results suggest that SOCS3 has a role in regulating macrophage responses after stimulation with non-pathogenic bacteria. We also show that the analysed non-pathogenic bacteria induce differential IL-10 and IL-12 cytokine levels in human primary macrophages.

IL-10 is an anti-inflammatory cytokine which can suppress the production of proinflammatory cytokines in macrophages [37]. The ability of LGG, L. rhamnosus LC705, B. breve Bb99 and P. freudenreichii PJS to induce IL-10 production in macrophages could be one factor contributing to their clinical anti-inflammatory effects seen as amelioration of the symptoms in inflammatory bowel disease [14]. In macrophages, LGG and S. thermophilus THS were potent inducers of IL-10, while in moDCs and PBMCs [19], IL-10 production remained at a low level.

In macrophages and PBMCs [19] IL-12 production was low, but was clearly induced in moDCs [23] in response to S. thermophilus THS stimulation. In moDCs, PBMCs and macrophages S. thermophilus THS was a strong IL-12 inducer [19,23]. LGG induced low IL-12 production in both PBMCs and moDCs [19,23].

One of the anti-inflammatory functions of IL-10 is the ability to down-regulate IL-12 production [33,38,39]. Therefore, calculating the IL-10/IL-12 ratio can be used as an indicator for anti-inflammatory potential of a given bacterium. The highest IL-10/IL-12 ratios were seen with bifidobacteria. These findings are in line with previous studies where some other strains of bifidobacteria were shown to have anti-inflammatory effects in vitro[40,41]. Previously, LGG has been shown to have anti-inflammatory properties in healthy adults [42]. Thus, by affecting the IL-10/IL-12 cytokine axis LGG could have a balancing effect on inflammatory reactions, also contributing to gut homeostasis. Our data are in line with previous findings that the ability of live non-pathogenic Gram-positive bacteria to regulate anti-inflammatory/inflammatory cytokine balance is dependent on the bacterial strain, and is not a universal feature for all non-pathogenic bacteria [19].

We show here that especially Bifidobacterium, S. thermophilus THS and Lactobacillus strains LGG and 1129 stimulated the expression of SOCS3. These bacterial strains also efficiently induced IL-10 production. SOCS3 is known to be regulated by both anti-inflammatory IL-10 and proinflammatory cytokines [6,43,44]. S. thermophilus-induced SOCS3 expression was partially dependent on IL-10 production, as neutralizing anti-IL-10 antibodies could inhibit S. thermophilus-induced SOCS3 gene expression. B. breve Bb99 was a strong inducer of SOCS3 gene expression and IL-10 cytokine production, but induced only low levels of IL-12p70 compared with S. thermophilus. Therefore, it would be of interest to also study the contribution of IL-10/IL-12 production balance on SOCS3 gene regulation for other bacterial species such as for bifidobacteria. Interestingly, IL-10 priming did not increase S. thermophilus THS induced SOCS3 expression further, while it had an additive or even partially synergistic effect on LPS and Pam3CSK4-induced SOCS3 expression. This shows that certain live whole bacteria are more potent inducers of SOCS3 expression in macrophages than purified TLR ligands.

Our data suggest that at early stages of bacteria-induced SOCS3 gene expression, when IL-10 expression is not yet induced, the p38 MAPK pathway is involved. Inhibitors for PI3K and NF-κB pathways had no reducing effect on SOCS3 expression in macrophages stimulated by the studied non-pathogenic bacteria. Because SOCS3 expression was not completely blocked by any of the inhibitors in LGG or S. thermophilus THS-stimulated cells, multiple simultaneously activated signalling pathways are likely to be involved in bacteria-induced SOCS3 gene expression. SOCS3 was also expressed in the absence of ongoing protein synthesis. This indicates that LGG and S. thermophilus THS stimulation directly enhances SOCS3 gene expression. Because CHX is known to prolong the half-life of cellular mRNAs, some enhanced expression of SOCS3 mRNA by CHX alone was seen, as expected. Our results thus indicate that during bacterial stimulation SOCS3 gene expression is inducible by two mechanisms: directly, via at least MAPK p38-mediated signalling pathway, and indirectly, via IL-10, which is produced by bacteria-stimulated macrophages. It has been shown previously in human macrophages that STAT3 is involved in IL-10-induced SOCS3 expression [45]. In mouse peritoneal macrophages, MAPK p38 and JNK pathways are required for both IL-10 and IL-12 production. In addition, the ERK pathway was shown to be critical for determining the IL-10/IL-12 production balance in mice [46]. The effects of signalling inhibitors on bacteria-induced SOCS3 mRNA expression were quite similar, suggesting that the signalling pathways regulating the responses induced by non-pathogenic LGG and S. thermophilus THS or pathogenic S. pyogenes are likely to be similar, if not identical.

The observed expression of anti-inflammatory IL-10 and SOCS3 in response to bacterial stimulation of macrophages may explain partially how probiotic or nonpathogenic bacteria such as LGG or S. thermophilus THS are able to modulate the immune system and generate health effects in humans. These data are of value when designing novel clinical trials and targeted treatments for different disease conditions.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References

This work was supported by The Medical Research Council of the Academy of Finland (Eranet Pathogenomics Programme, grant 130098), The Research Council for Biosciences and Environment of the Academy of Finland (Grants 119065 and 129954) and the Sigrid Juselius Foundation. The authors thank Mari Aaltonen for expert technical assistance.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References