Molecular Immunology 66 (2015) 107–116
Contents lists available at ScienceDirect
Molecular Immunology journal homepage: www.elsevier.com/locate/molimm
Effects of the non-commensal Methylococcus capsulatus Bath on mammalian immune cells Trine Eker Christoffersen a,∗ , Lene Therese Olsen Hult c , Henriette Solberg b , Anne Bakke b , Katarzyna Kuczkowska b , Eirin Huseby b , Morten Jacobsen b,c , Tor Lea b , Charlotte Ramstad Kleiveland b,c a
Faculty of Engineering, Ostfold University College, 1757 Halden, Norway Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, 1430 Aas, Norway c Ostfold Hospital Trust, 1603 Fredrikstad, Norway b
a r t i c l e
i n f o
Article history: Received 9 October 2014 Received in revised form 27 January 2015 Accepted 19 February 2015 Keywords: Methylococcus capsulatus Macrophage polarization Dendritic cell maturation Cytokines Immunomodulation
a b s t r a c t Dietary inclusions of a bacterial meal consisting mainly of the non-commensal, methanotrophic bacteria Methylococcus capsulatus Bath have been shown to ameliorate symptoms of intestinal inflammation in different animal models. In order to investigate the molecular mechanisms causing these effects, we have studied the influence of this strain on different immune cells central for the regulation of inflammatory responses. Effects were compared to those induced by the closely related strain M. capsulatus Texas and the well-described probiotic strain Escherichia coli Nissle 1917. M. capsulatus Bath induced macrophage polarization toward a pro-inflammatory phenotype, but not to the extent observed after exposure to E. coli Nissle 1917. Likewise, dose-dependent abilities to activate NF-B transcription in U937 cells were observed, with E. coli Nissle 1917 being most potent. High levels of CD141 on human primary monocyte-derived dendritic cells (moDCs) were only detected after exposure to E. coli Nissle 1917, which collectively indicate a superior capacity to induce Th1 cell responses for this strain. On the other hand, the M. capsulatus strains were more potent in increasing the expression of the maturation markers CD80, CD83 and CD86 than E. coli Nissle 1917. M. capsulatus Bath induced the highest levels of IL-6, IL-10 and IL-12 secretion from dendritic cells, suggesting that this strain generally the post potent inducer of cytokine secretion. These results show that M. capsulatus Bath exhibit immunogenic properties in mammalian in vitro systems which diverge from that of E. coli Nissle 1917. This may provide clues to how M. capsulatus Bath influence the adaptive immune system in vivo. However, further in vivo experiments are required for a complete understanding of how this strain ameliorates intestinal inflammation in animal models. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction Precise regulation of the intestinal barrier function is important for the maintenance of mucosal homeostasis and prevents the
Abbreviations: BP, BioProtein; DC, dendritic cells; DSS, dextran sulfate sodium; IECs, intestinal epithelial cells; IBD, inflammatory bowel diseases; iNOS, inducible nitric oxide synthase; LPS, lipopolysaccharide; moDCs, monocyte-derived DCs; MNC, mononuclear cells; PRR, pattern recognition receptors; TLR, toll-like receptor. ∗ Corresponding author. Tel.: +47 92293881. E-mail addresses: [email protected] (T.E. Christoffersen), [email protected] (L.T. Olsen Hult), [email protected] (H. Solberg), [email protected] (A. Bakke), [email protected] (K. Kuczkowska), [email protected] (E. Huseby), [email protected] (M. Jacobsen), [email protected] (T. Lea), [email protected] (C.R. Kleiveland). http://dx.doi.org/10.1016/j.molimm.2015.02.019 0161-5890/© 2015 Elsevier Ltd. All rights reserved.
onset of uncontrolled inflammation (Pastorelli et al., 2013). A finetuned cross-talk between intestinal epithelial cells (IECs), immune cells and the bacterial community enables discrimination between pathogenic and commensal bacteria in the gut lumen (Artis, 2008). Such discrimination plays a cardinal role in influencing the function of innate mononuclear cells and lymphocytes and the subsequent onset of an appropriate inflammatory response. Alterations in this inter-cell communication may disrupt intestinal homeostasis and provoke inflammation and injury leading to intestinal disorders such as ulcerative colitis (UC) and Crohn’s disease (CD), collectively known as inflammatory bowel diseases (IBDs) (Dignass et al., 2004; Clayburgh et al., 2004). A bacterial meal called BioProtein (BP® ), assumed to be a suitable protein source in fodder for the fish farming industry, has been reported to prevent soybean meal (SBM)-induced enteritis in Atlantic salmon in a dose dependent manner (Romarheim et al.,
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2011, 2013). Similar observations have been reported in mammals where intake of BP attenuated symptoms of dextran sulfate sodium (DSS)-induced UC in mice (Kleiveland et al., 2012a). Prophylactic treatment with BP reduced colitis-induced parameters such as reduced body weight, shortening of the colon and epithelial damage. Increased IEC-proliferation and enhanced mucin 2 gene transcription were also reported, suggesting that BP affects the mechanisms implicated in the maintenance of the intestinal barrier function. The main constituent of BP (88%) is the methanotrophic bacteria Methylococcus capsulatus Bath (Ward et al., 2004). Supplementary experiments have confirmed that the BP-induced effects observed in DSS-treated mice were due to M. capsulatus Bath and not any of the minor constituents in the preparation (Kleiveland et al., 2012a). This proposes M. capsulatus Bath as an interesting candidate for studying the molecular mechanisms involved in the development and treatment of IBD. Methanotrophs are Gram-negative bacteria that grow aerobically using methane as the sole carbon and energy source (Hakemian and Rosenzweig, 2007). The biochemical components and pathways enabling M. capsulatus Bath to oxidize methane have been widely studied (Hakemian and Rosenzweig, 2007; Lieberman and Rosenzweig, 2005; Myronova et al., 2006; Khmelenina et al., 2011), and the concomitant ability to assimilate carbon into useful biomass at the formaldehyde level has given this strain further attention. M. capsulatus Bath is not, to the best of our knowledge, a part of the mammalian microbiota. However, a human- or animal origin is commonly considered as a requirement for microorganisms to confer health benefits on mammalian hosts (Dunne et al., 2001). Non-commensals have not been subject to selection pressure in the mammalian gut during evolution and may retain unknown, and possibly beneficial, immunomodulatory properties. It is therefore of particular interest to understand the mechanisms by which M. capsulatus Bath ameliorate symptoms of intestinal inflammation in mammals. By studying the consequences of bacteria–host immune cell interactions we may not only increase our understanding of how a non-commensal can affect mammalian immunology, but also elucidate the molecular mechanisms involved in epithelial regulation. Dendritic cells (DCs) are professional antigen-presenting cells central to the regulation of both innate and adaptive immune responses at mucosal surfaces (Coombes and Powrie, 2008). Immature monocyte-derived DCs (moDCs) reside in peripheral tissues, such as the gut mucosa, and continuously sample the intestinal lumen and the microenvironment via pattern recognition receptors (PRRs), such as the Toll-like receptors (Rakoff-Nahoum et al., 2004). Activation of PRRs induces moDC maturation, and, importantly, modulates moDC maturation and differentiation in such a way that both tolerance toward commensals and generation of protective immune responses against pathogens are enabled (Coombes and Powrie, 2008). Thus, bacterial effects on immature moDCs are essential to understand the outcome of bacteria–host cell interactions in the intestinal mucosa (Christensen et al., 2002; Zoumpopoulou et al., 2009; Hart et al., 2004). Likewise, macrophages play a pivotal role in mucosal immune responses (Mowat and Bain, 2011; Hume, 2008), and may differentiate into pro- or anti-inflammatory phenotypes after antigen stimulation, indicated by an increase in inducible nitric oxide synthase- (iNOS) or arginase-1 gene expression, respectively (Lawrence and Natoli, 2011; Benoit et al., 2008). We have studied the consequences of exposing immature moDCs to M. capsulatus Bath in regards to the expression of co-stimulatory and maturation markers, and the subsequent secretion of cytokines. These outcomes, known to affect the adaptive immune system such as T-cell development and polarization, were
compared with those induced by other Gram-negative strains. Similarly, polarization of macrophages upon bacterial exposure was studied by quantifying iNOS and arginase-1 gene expression. Bacterial effects on NF-B transcription and moDC morphology are also described herein which increase the understanding how a non-commensal bacteria is able to affect the immune system in a mammalian host. 2. Materials and methods 2.1. Cells and culture conditions M. capsulatus Bath (NCIMB 11132, Aberdeen, UK) and M. capsulatus Texas (NCIMB 11851, Aberdeen, UK) were grown in batches of 5–20 ml nitrate minimal salt solution at 45 ◦ C, with shaking (200 rpm), in an atmosphere containing methane, CO2 and air (19:1:80 v/v), as previously described (Whittenbury et al., 1970). Escherichia coli Nissle 1917 (Villena et al., 2012) (Mutaflor, DSM 6601, serotype O6:K5:H1) was kindly provided by Ardeypharm GmbH, Herdecke, Germany and grown in Luria-Bertani Broth (Oxoid, UK) at 37 ◦ C without shaking. UV-inactivation of E. coli Nissle 1917 was performed for 60 min prior to experiments. Human blood mononuclear cells (MNC) were isolated from buffy coats of healthy volunteers obtained from Ostfold Hospital Trust, Fredrikstad, Norway, in accordance to institutional ethical guidelines. Both primary MNCs, the human monocytic leukemia cell line THP-1 (ATCC TIB-202, Rockville, USA) and the murine macrophage cell line RAW 267.4 (ATTC TIB-71, Rockville, USA) were maintained in RPMI 1640 medium with l-glutamine further supplemented with 100 M non-essential amino acids, 1 mM sodium pyruvate (all from PAA Laboratories, Austria), 10% heat-inactivated fetal calf serum (Gibco Life Technologies, UK) and 24 g/ml gentamicin (Lonza, Walkersville, ML). Tissue culture maintenance and experiments were carried out at 37 ◦ C in a 5% CO2 humidified air atmosphere. The human myeloblastic cell line U937, stably transfected with a construct containing the luciferase gene regulated by a promoter containing NF-B binding sites, was a kind gift from Rune Blomhoff, Faculty of Medicine, University of Oslo, Norway (Austenaa et al., 2009). Stably transfected U937 cells were cultured in supplemented RPMI 1640 medium. 2.2. Scanning electron microscopy (SEM) Samples were gently washed with phosphate buffered saline (PBS) (PAA Laboratories, Austria) and fixed using 5% glutaraldehyde in 0.1 M sodium cacodylate and 0.1 M sucrose (pH 7.4) at room temperature for 45 min. The fixative was then replaced with 0.1 M sodium cacodylate and 0.1 M sucrose (pH 7.4) for 30 min in room temperature. Samples were then washed, dehydrated in graded ethanol series and dried using a critical-point dryer (CDP 030, BALTEC GmBH, Germany). Dry samples were mounted on aluminum stubs using double-faced carbon tape (Agar Scientific, UK), and coated with approximately 500 A˚ platinum using a sputter coater (Polaron SC7640, Quorum Technologies, UK). Microscopic analyses were performed using an EVO-50 Zeiss microscope (Carl Zeiss AG, Germany). 2.3. Transmission electron microscopy (TEM) Samples were embedded in 0, 5% low-melting agarose to produce an agarose-plug. The plug was washed twice in distilled water prior to fixation in 2.5% glutaraldehyde/4% paraformaldehyde at room temperature for 45 min. Samples were washed twice in PBS and twice in 0.1 M saline cacodylate buffer and treated
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with 0.5% osmium tetroxide and 1.5% K3 Fe(CN)6 for 1.5 h. Samples were washed twice in saline cacodylate, dehydrated in graded ethanol series and gradually embedded in liquid LR White resin (SPI Supplies, UK). LR White resin polymerization was activated at 60 ◦ C overnight. Ultramicrotome sections (75 nm) were mounted on Formvar-treated cobber slot grids (Chemi-Teknik AS, Norway), and contrasted in uranyl acetate before visualized using a transmission electron microscopy (FEI, OR).
geNorm kit (PrimerDesign, Southhampton, United Kingdom) and the geNorm software (qbase) (Vandesompele et al., 2002). The following TaqMan® probes were used in this study: Mm00440502 m1 for Nos2, Mm00475988 m1 for Arg1 and Mm02528467 g1 for Rpl32.
2.4. Assaying NF-B activity in U937 cells
Peripheral blood mononuclear cells (MNCs) were isolated by density gradient centrifugation using Lymphoprep medium (specific gravity 1.077 g/ml; Fresenius Kabi, Norway). CD14 positive cells were selected using human CD14 MicroBeads (MACS Miltenyi Biotech, Germany) and seeded in 24-well plates. GM-CSF (50 ng/ml) and IL-4 (25 ng/ml) (both from ImmunoTools, Germany) were used to promote differentiation of CD14+ monocytes to immature moDCs. Cytokines were replenished on day 4 and experiments conducted on day 6. Immature moDCs were stimulated with 1 × 108 bacteria for 18 h. Equal bacterial load was ensured as described above. A cocktail containing 15 ng/ml TNF-␣ (ImmunoTools, Germany), 100 ng/ml LPS and 5 g/ml PGE2 (both from Sigma–Aldrich, St. Louis, MO) was used as a positive control for dendritic cell maturation (Landi et al., 2011). Antibody binding sites were blocked for 30 min in room temperature using 5% fetal calf serum prior to staining with fluorochrome-conjugated antibodies. CD80 was detected using PEconjugated mouse anti-CD80 antibodies (560925, BD Biosciences, NJ), CD83 was detected using PE-Cy5-conjugated mouse antihuman CD83 antibodies (130-094-181, MACS Miltenyi Biotech, Germany), CD 86 was detected using Alexa Fluor 700-conjugated mouse anti-human CD86 antibodies (130-094-876, MACS Miltenyi Biotech, Germany), CD141 was detected using PE-conjugated mouse anti-CD141 antibodies (344104, BioLegend, San Diego, CA) and HLA DR was detected using FITC-conjugated mouse anti-HLA DR antibodies (555558, BD Biosciences, NJ), all from (MACS Miltenyi Biotech, Germany). Samples were finally fixed in 4% paraformaldehyde and the fluorescence monitored using a MACSQuant flow cytometer. The data was analyzed using the MACSQuantify software (Miltenyi Biotech, Germany).
Stably transfected U937 cells (30,000) were seeded in 96-well plates and exposed to bacteria, LPS (0.1 g/well) or left untreated for 6 h. To ensure equal bacterial load to each sample, specific optical densities had been pre-determined for each strain by enumerating bacterial cells. None of the bacterial strains were able to multiply in the presence of 24 g/ml gentamicin. Luciferase substrate (100 l Bright-Glo Luciferase Assay System, Promega, Madison, WI) was added and luminescence measured using a TopCount NXTTM Luminometer (Packard BioScience Company, Meriden, CT). 2.5. Bacterial stimulation of the RAW 264.7 macrophage cell line RAW 264.7 cells (100.000) were seeded in 6-well tissue culture plates and cultured as described above for 48 h. Following 12 h starvation in serum-free medium, control samples for increased expression of iNOS and ariginase-1 were stimulated with either 100 ng/ml lipopolysaccaride (LPS) (Sigma–Aldrich, St. Louis, MO) or 40 ng/ml mouse IL-4 (Immunotools GmbH, Friesoythe, Germany) for 24 h, respectively. Bacteria were cultivated overnight, as described above, washed twice in PBS and re-suspended in supplemented RPMI. Ordinary samples were exposed to bacteria (1 × 108 bacteria/sample) for 3 h. Equal loading of different strains were ensured as described above. All strains were included in each experiment. The macrophages were finally washed, scraped of the plastic, harvested and stored at −80 ◦ C prior to qRT-PCR-analysis. 2.6. RNA isolation and cDNA synthesis Total RNA was extracted using the RNeasy Micro Kit (Qiagen, Germantown, MD) according to the manufacturers’ recommendations. Nucleic acid quantification was performed using a NanoDrop 2000 spectrophotometer (Thermo Fischer Scientific Inc., Waltham, MA). The RNA integrity number (RIN) was determined using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA) and ranged from 6.5–8. The SuperScript® VILOTM cDNA Synthesis Kit (Invitrogen, Carlsbad, CA) was used for cDNA synthesis using 2.3 g RNA per 50 l reaction, according to the manufacturer’s recommendations. Synthesis was performed using the following program: 25 ◦ C for 10 min, 42 ◦ C for 120 min and 85 ◦ C for 5 min with a final hold at 4 ◦ C (MJ Research Thermocycler, Watertown, MA).
2.8. Immunophenotyping of human-derived moDCs after bacterial exposure
2.9. Immunophenotyping of THP-1-derived mature moDCs after bacterial exposure THP-1 cells (750.000/well) were seeded in 24-well plates and allowed to differentiate into mature moDCs in the presence of 200 ng/ml IL-4, 100 ng/ml GM-CSF, 20 ng/ml TNF-␣ and 200 ng/ml ionomycin (Sigma–Aldrich, St. Louis, MO) for 72 h, as previously described (Berges et al., 2005). Samples were exposed to an equal loading of different bacteria (7.5 × 107 cells/well) or left untreated for 72 h. Expression of CD80, CD83 and CD86 were performed by flow cytometry, as described above.
2.7. Quantitative real-time PCR
2.10. Quantification of secreted cytokines
The TaqMan® Gene Expression Master Mix (Applied Biosystems, Carlsbad, CA) was employed, according to the manufacturer’s recommendations, using 20 ng cDNA-template calculated from the RNA input in the cDNA synthesis. All PCR reactions were set up in duplicates and analyzed using a Rotor Gene 6000 Real-Time PCR Machine (Qiagen, Germantown, MD). The following PCR program was used: 50 ◦ C for 2 min, 95 ◦ C for 5 min and then 40 cycles of 95 ◦ C for 15 s and 60 ◦ C for 1 min. All probes were from TaqMan® Gene Expression assay kits (Applied Biosystems, Carlsbad, CA). Rpl32 was identified as the most stable reference gene using the mouse
Human immature moDCs were co-inbucated with equal numbers of different bacteria as described above. Culture supernatant was collected after 24 h and stored at −80 ◦ C. Quantification of IL-10 and IL-12 were performed using human IL-10 (900-K21) and human IL-12 (900-K96) ELISA kits, as described by the manufacturer (PeproTech, Rockey Hill, NJ). Quantification of IL-6 was performed using the human IL-6 (31670069) ELISA kit, as described by the manufacturer (ImmunoTools GmbH, Friesoythe, Germany). Absorbance was measured at 405 nm using a Teacan Sunrise Basic plate reader (Teacan Group Ltd., Mannedorf, Switzerland).
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2.11. Statistics Data were analyzed using one-way ANOVA or t-tests. Tukey’s post hoc test was included when appropriate. Analyses were performed using the open-source statistical language and environment, R (www.r-project.org). Differences between means were considered significant if the P value was
Contents lists available at ScienceDirect
Molecular Immunology journal homepage: www.elsevier.com/locate/molimm
Effects of the non-commensal Methylococcus capsulatus Bath on mammalian immune cells Trine Eker Christoffersen a,∗ , Lene Therese Olsen Hult c , Henriette Solberg b , Anne Bakke b , Katarzyna Kuczkowska b , Eirin Huseby b , Morten Jacobsen b,c , Tor Lea b , Charlotte Ramstad Kleiveland b,c a
Faculty of Engineering, Ostfold University College, 1757 Halden, Norway Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, 1430 Aas, Norway c Ostfold Hospital Trust, 1603 Fredrikstad, Norway b
a r t i c l e
i n f o
Article history: Received 9 October 2014 Received in revised form 27 January 2015 Accepted 19 February 2015 Keywords: Methylococcus capsulatus Macrophage polarization Dendritic cell maturation Cytokines Immunomodulation
a b s t r a c t Dietary inclusions of a bacterial meal consisting mainly of the non-commensal, methanotrophic bacteria Methylococcus capsulatus Bath have been shown to ameliorate symptoms of intestinal inflammation in different animal models. In order to investigate the molecular mechanisms causing these effects, we have studied the influence of this strain on different immune cells central for the regulation of inflammatory responses. Effects were compared to those induced by the closely related strain M. capsulatus Texas and the well-described probiotic strain Escherichia coli Nissle 1917. M. capsulatus Bath induced macrophage polarization toward a pro-inflammatory phenotype, but not to the extent observed after exposure to E. coli Nissle 1917. Likewise, dose-dependent abilities to activate NF-B transcription in U937 cells were observed, with E. coli Nissle 1917 being most potent. High levels of CD141 on human primary monocyte-derived dendritic cells (moDCs) were only detected after exposure to E. coli Nissle 1917, which collectively indicate a superior capacity to induce Th1 cell responses for this strain. On the other hand, the M. capsulatus strains were more potent in increasing the expression of the maturation markers CD80, CD83 and CD86 than E. coli Nissle 1917. M. capsulatus Bath induced the highest levels of IL-6, IL-10 and IL-12 secretion from dendritic cells, suggesting that this strain generally the post potent inducer of cytokine secretion. These results show that M. capsulatus Bath exhibit immunogenic properties in mammalian in vitro systems which diverge from that of E. coli Nissle 1917. This may provide clues to how M. capsulatus Bath influence the adaptive immune system in vivo. However, further in vivo experiments are required for a complete understanding of how this strain ameliorates intestinal inflammation in animal models. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction Precise regulation of the intestinal barrier function is important for the maintenance of mucosal homeostasis and prevents the
Abbreviations: BP, BioProtein; DC, dendritic cells; DSS, dextran sulfate sodium; IECs, intestinal epithelial cells; IBD, inflammatory bowel diseases; iNOS, inducible nitric oxide synthase; LPS, lipopolysaccharide; moDCs, monocyte-derived DCs; MNC, mononuclear cells; PRR, pattern recognition receptors; TLR, toll-like receptor. ∗ Corresponding author. Tel.: +47 92293881. E-mail addresses: [email protected] (T.E. Christoffersen), [email protected] (L.T. Olsen Hult), [email protected] (H. Solberg), [email protected] (A. Bakke), [email protected] (K. Kuczkowska), [email protected] (E. Huseby), [email protected] (M. Jacobsen), [email protected] (T. Lea), [email protected] (C.R. Kleiveland). http://dx.doi.org/10.1016/j.molimm.2015.02.019 0161-5890/© 2015 Elsevier Ltd. All rights reserved.
onset of uncontrolled inflammation (Pastorelli et al., 2013). A finetuned cross-talk between intestinal epithelial cells (IECs), immune cells and the bacterial community enables discrimination between pathogenic and commensal bacteria in the gut lumen (Artis, 2008). Such discrimination plays a cardinal role in influencing the function of innate mononuclear cells and lymphocytes and the subsequent onset of an appropriate inflammatory response. Alterations in this inter-cell communication may disrupt intestinal homeostasis and provoke inflammation and injury leading to intestinal disorders such as ulcerative colitis (UC) and Crohn’s disease (CD), collectively known as inflammatory bowel diseases (IBDs) (Dignass et al., 2004; Clayburgh et al., 2004). A bacterial meal called BioProtein (BP® ), assumed to be a suitable protein source in fodder for the fish farming industry, has been reported to prevent soybean meal (SBM)-induced enteritis in Atlantic salmon in a dose dependent manner (Romarheim et al.,
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2011, 2013). Similar observations have been reported in mammals where intake of BP attenuated symptoms of dextran sulfate sodium (DSS)-induced UC in mice (Kleiveland et al., 2012a). Prophylactic treatment with BP reduced colitis-induced parameters such as reduced body weight, shortening of the colon and epithelial damage. Increased IEC-proliferation and enhanced mucin 2 gene transcription were also reported, suggesting that BP affects the mechanisms implicated in the maintenance of the intestinal barrier function. The main constituent of BP (88%) is the methanotrophic bacteria Methylococcus capsulatus Bath (Ward et al., 2004). Supplementary experiments have confirmed that the BP-induced effects observed in DSS-treated mice were due to M. capsulatus Bath and not any of the minor constituents in the preparation (Kleiveland et al., 2012a). This proposes M. capsulatus Bath as an interesting candidate for studying the molecular mechanisms involved in the development and treatment of IBD. Methanotrophs are Gram-negative bacteria that grow aerobically using methane as the sole carbon and energy source (Hakemian and Rosenzweig, 2007). The biochemical components and pathways enabling M. capsulatus Bath to oxidize methane have been widely studied (Hakemian and Rosenzweig, 2007; Lieberman and Rosenzweig, 2005; Myronova et al., 2006; Khmelenina et al., 2011), and the concomitant ability to assimilate carbon into useful biomass at the formaldehyde level has given this strain further attention. M. capsulatus Bath is not, to the best of our knowledge, a part of the mammalian microbiota. However, a human- or animal origin is commonly considered as a requirement for microorganisms to confer health benefits on mammalian hosts (Dunne et al., 2001). Non-commensals have not been subject to selection pressure in the mammalian gut during evolution and may retain unknown, and possibly beneficial, immunomodulatory properties. It is therefore of particular interest to understand the mechanisms by which M. capsulatus Bath ameliorate symptoms of intestinal inflammation in mammals. By studying the consequences of bacteria–host immune cell interactions we may not only increase our understanding of how a non-commensal can affect mammalian immunology, but also elucidate the molecular mechanisms involved in epithelial regulation. Dendritic cells (DCs) are professional antigen-presenting cells central to the regulation of both innate and adaptive immune responses at mucosal surfaces (Coombes and Powrie, 2008). Immature monocyte-derived DCs (moDCs) reside in peripheral tissues, such as the gut mucosa, and continuously sample the intestinal lumen and the microenvironment via pattern recognition receptors (PRRs), such as the Toll-like receptors (Rakoff-Nahoum et al., 2004). Activation of PRRs induces moDC maturation, and, importantly, modulates moDC maturation and differentiation in such a way that both tolerance toward commensals and generation of protective immune responses against pathogens are enabled (Coombes and Powrie, 2008). Thus, bacterial effects on immature moDCs are essential to understand the outcome of bacteria–host cell interactions in the intestinal mucosa (Christensen et al., 2002; Zoumpopoulou et al., 2009; Hart et al., 2004). Likewise, macrophages play a pivotal role in mucosal immune responses (Mowat and Bain, 2011; Hume, 2008), and may differentiate into pro- or anti-inflammatory phenotypes after antigen stimulation, indicated by an increase in inducible nitric oxide synthase- (iNOS) or arginase-1 gene expression, respectively (Lawrence and Natoli, 2011; Benoit et al., 2008). We have studied the consequences of exposing immature moDCs to M. capsulatus Bath in regards to the expression of co-stimulatory and maturation markers, and the subsequent secretion of cytokines. These outcomes, known to affect the adaptive immune system such as T-cell development and polarization, were
compared with those induced by other Gram-negative strains. Similarly, polarization of macrophages upon bacterial exposure was studied by quantifying iNOS and arginase-1 gene expression. Bacterial effects on NF-B transcription and moDC morphology are also described herein which increase the understanding how a non-commensal bacteria is able to affect the immune system in a mammalian host. 2. Materials and methods 2.1. Cells and culture conditions M. capsulatus Bath (NCIMB 11132, Aberdeen, UK) and M. capsulatus Texas (NCIMB 11851, Aberdeen, UK) were grown in batches of 5–20 ml nitrate minimal salt solution at 45 ◦ C, with shaking (200 rpm), in an atmosphere containing methane, CO2 and air (19:1:80 v/v), as previously described (Whittenbury et al., 1970). Escherichia coli Nissle 1917 (Villena et al., 2012) (Mutaflor, DSM 6601, serotype O6:K5:H1) was kindly provided by Ardeypharm GmbH, Herdecke, Germany and grown in Luria-Bertani Broth (Oxoid, UK) at 37 ◦ C without shaking. UV-inactivation of E. coli Nissle 1917 was performed for 60 min prior to experiments. Human blood mononuclear cells (MNC) were isolated from buffy coats of healthy volunteers obtained from Ostfold Hospital Trust, Fredrikstad, Norway, in accordance to institutional ethical guidelines. Both primary MNCs, the human monocytic leukemia cell line THP-1 (ATCC TIB-202, Rockville, USA) and the murine macrophage cell line RAW 267.4 (ATTC TIB-71, Rockville, USA) were maintained in RPMI 1640 medium with l-glutamine further supplemented with 100 M non-essential amino acids, 1 mM sodium pyruvate (all from PAA Laboratories, Austria), 10% heat-inactivated fetal calf serum (Gibco Life Technologies, UK) and 24 g/ml gentamicin (Lonza, Walkersville, ML). Tissue culture maintenance and experiments were carried out at 37 ◦ C in a 5% CO2 humidified air atmosphere. The human myeloblastic cell line U937, stably transfected with a construct containing the luciferase gene regulated by a promoter containing NF-B binding sites, was a kind gift from Rune Blomhoff, Faculty of Medicine, University of Oslo, Norway (Austenaa et al., 2009). Stably transfected U937 cells were cultured in supplemented RPMI 1640 medium. 2.2. Scanning electron microscopy (SEM) Samples were gently washed with phosphate buffered saline (PBS) (PAA Laboratories, Austria) and fixed using 5% glutaraldehyde in 0.1 M sodium cacodylate and 0.1 M sucrose (pH 7.4) at room temperature for 45 min. The fixative was then replaced with 0.1 M sodium cacodylate and 0.1 M sucrose (pH 7.4) for 30 min in room temperature. Samples were then washed, dehydrated in graded ethanol series and dried using a critical-point dryer (CDP 030, BALTEC GmBH, Germany). Dry samples were mounted on aluminum stubs using double-faced carbon tape (Agar Scientific, UK), and coated with approximately 500 A˚ platinum using a sputter coater (Polaron SC7640, Quorum Technologies, UK). Microscopic analyses were performed using an EVO-50 Zeiss microscope (Carl Zeiss AG, Germany). 2.3. Transmission electron microscopy (TEM) Samples were embedded in 0, 5% low-melting agarose to produce an agarose-plug. The plug was washed twice in distilled water prior to fixation in 2.5% glutaraldehyde/4% paraformaldehyde at room temperature for 45 min. Samples were washed twice in PBS and twice in 0.1 M saline cacodylate buffer and treated
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with 0.5% osmium tetroxide and 1.5% K3 Fe(CN)6 for 1.5 h. Samples were washed twice in saline cacodylate, dehydrated in graded ethanol series and gradually embedded in liquid LR White resin (SPI Supplies, UK). LR White resin polymerization was activated at 60 ◦ C overnight. Ultramicrotome sections (75 nm) were mounted on Formvar-treated cobber slot grids (Chemi-Teknik AS, Norway), and contrasted in uranyl acetate before visualized using a transmission electron microscopy (FEI, OR).
geNorm kit (PrimerDesign, Southhampton, United Kingdom) and the geNorm software (qbase) (Vandesompele et al., 2002). The following TaqMan® probes were used in this study: Mm00440502 m1 for Nos2, Mm00475988 m1 for Arg1 and Mm02528467 g1 for Rpl32.
2.4. Assaying NF-B activity in U937 cells
Peripheral blood mononuclear cells (MNCs) were isolated by density gradient centrifugation using Lymphoprep medium (specific gravity 1.077 g/ml; Fresenius Kabi, Norway). CD14 positive cells were selected using human CD14 MicroBeads (MACS Miltenyi Biotech, Germany) and seeded in 24-well plates. GM-CSF (50 ng/ml) and IL-4 (25 ng/ml) (both from ImmunoTools, Germany) were used to promote differentiation of CD14+ monocytes to immature moDCs. Cytokines were replenished on day 4 and experiments conducted on day 6. Immature moDCs were stimulated with 1 × 108 bacteria for 18 h. Equal bacterial load was ensured as described above. A cocktail containing 15 ng/ml TNF-␣ (ImmunoTools, Germany), 100 ng/ml LPS and 5 g/ml PGE2 (both from Sigma–Aldrich, St. Louis, MO) was used as a positive control for dendritic cell maturation (Landi et al., 2011). Antibody binding sites were blocked for 30 min in room temperature using 5% fetal calf serum prior to staining with fluorochrome-conjugated antibodies. CD80 was detected using PEconjugated mouse anti-CD80 antibodies (560925, BD Biosciences, NJ), CD83 was detected using PE-Cy5-conjugated mouse antihuman CD83 antibodies (130-094-181, MACS Miltenyi Biotech, Germany), CD 86 was detected using Alexa Fluor 700-conjugated mouse anti-human CD86 antibodies (130-094-876, MACS Miltenyi Biotech, Germany), CD141 was detected using PE-conjugated mouse anti-CD141 antibodies (344104, BioLegend, San Diego, CA) and HLA DR was detected using FITC-conjugated mouse anti-HLA DR antibodies (555558, BD Biosciences, NJ), all from (MACS Miltenyi Biotech, Germany). Samples were finally fixed in 4% paraformaldehyde and the fluorescence monitored using a MACSQuant flow cytometer. The data was analyzed using the MACSQuantify software (Miltenyi Biotech, Germany).
Stably transfected U937 cells (30,000) were seeded in 96-well plates and exposed to bacteria, LPS (0.1 g/well) or left untreated for 6 h. To ensure equal bacterial load to each sample, specific optical densities had been pre-determined for each strain by enumerating bacterial cells. None of the bacterial strains were able to multiply in the presence of 24 g/ml gentamicin. Luciferase substrate (100 l Bright-Glo Luciferase Assay System, Promega, Madison, WI) was added and luminescence measured using a TopCount NXTTM Luminometer (Packard BioScience Company, Meriden, CT). 2.5. Bacterial stimulation of the RAW 264.7 macrophage cell line RAW 264.7 cells (100.000) were seeded in 6-well tissue culture plates and cultured as described above for 48 h. Following 12 h starvation in serum-free medium, control samples for increased expression of iNOS and ariginase-1 were stimulated with either 100 ng/ml lipopolysaccaride (LPS) (Sigma–Aldrich, St. Louis, MO) or 40 ng/ml mouse IL-4 (Immunotools GmbH, Friesoythe, Germany) for 24 h, respectively. Bacteria were cultivated overnight, as described above, washed twice in PBS and re-suspended in supplemented RPMI. Ordinary samples were exposed to bacteria (1 × 108 bacteria/sample) for 3 h. Equal loading of different strains were ensured as described above. All strains were included in each experiment. The macrophages were finally washed, scraped of the plastic, harvested and stored at −80 ◦ C prior to qRT-PCR-analysis. 2.6. RNA isolation and cDNA synthesis Total RNA was extracted using the RNeasy Micro Kit (Qiagen, Germantown, MD) according to the manufacturers’ recommendations. Nucleic acid quantification was performed using a NanoDrop 2000 spectrophotometer (Thermo Fischer Scientific Inc., Waltham, MA). The RNA integrity number (RIN) was determined using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA) and ranged from 6.5–8. The SuperScript® VILOTM cDNA Synthesis Kit (Invitrogen, Carlsbad, CA) was used for cDNA synthesis using 2.3 g RNA per 50 l reaction, according to the manufacturer’s recommendations. Synthesis was performed using the following program: 25 ◦ C for 10 min, 42 ◦ C for 120 min and 85 ◦ C for 5 min with a final hold at 4 ◦ C (MJ Research Thermocycler, Watertown, MA).
2.8. Immunophenotyping of human-derived moDCs after bacterial exposure
2.9. Immunophenotyping of THP-1-derived mature moDCs after bacterial exposure THP-1 cells (750.000/well) were seeded in 24-well plates and allowed to differentiate into mature moDCs in the presence of 200 ng/ml IL-4, 100 ng/ml GM-CSF, 20 ng/ml TNF-␣ and 200 ng/ml ionomycin (Sigma–Aldrich, St. Louis, MO) for 72 h, as previously described (Berges et al., 2005). Samples were exposed to an equal loading of different bacteria (7.5 × 107 cells/well) or left untreated for 72 h. Expression of CD80, CD83 and CD86 were performed by flow cytometry, as described above.
2.7. Quantitative real-time PCR
2.10. Quantification of secreted cytokines
The TaqMan® Gene Expression Master Mix (Applied Biosystems, Carlsbad, CA) was employed, according to the manufacturer’s recommendations, using 20 ng cDNA-template calculated from the RNA input in the cDNA synthesis. All PCR reactions were set up in duplicates and analyzed using a Rotor Gene 6000 Real-Time PCR Machine (Qiagen, Germantown, MD). The following PCR program was used: 50 ◦ C for 2 min, 95 ◦ C for 5 min and then 40 cycles of 95 ◦ C for 15 s and 60 ◦ C for 1 min. All probes were from TaqMan® Gene Expression assay kits (Applied Biosystems, Carlsbad, CA). Rpl32 was identified as the most stable reference gene using the mouse
Human immature moDCs were co-inbucated with equal numbers of different bacteria as described above. Culture supernatant was collected after 24 h and stored at −80 ◦ C. Quantification of IL-10 and IL-12 were performed using human IL-10 (900-K21) and human IL-12 (900-K96) ELISA kits, as described by the manufacturer (PeproTech, Rockey Hill, NJ). Quantification of IL-6 was performed using the human IL-6 (31670069) ELISA kit, as described by the manufacturer (ImmunoTools GmbH, Friesoythe, Germany). Absorbance was measured at 405 nm using a Teacan Sunrise Basic plate reader (Teacan Group Ltd., Mannedorf, Switzerland).
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2.11. Statistics Data were analyzed using one-way ANOVA or t-tests. Tukey’s post hoc test was included when appropriate. Analyses were performed using the open-source statistical language and environment, R (www.r-project.org). Differences between means were considered significant if the P value was
