Journal of Leukocyte Biology Myeloid cells, immune suppression, tumor immunology
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(Journal of Leukocyte Biology. 2002;71:238-246.)
© 2002 by Society for Leukocyte Biology

Bacterial metabolite interference with maturation of human monocyte-derived dendritic cells

Marcus D. Säemann*, Ornella Parolini*, Georg A. Böhmig{dagger}, Peter Kelemen*, Peter-Michael Krieger*, Josef Neumüller{ddagger}, Katharina Knarr*, Willibald Kammlander*, Walter H. Hörl{dagger}, Christos Diakos*, Karl Stuhlmeier§ and Gerhard J. Zlabinger*

* Institute of Immunology,
{dagger} Department of Internal Medicine III, and
{ddagger} Institute of Histology, University of Vienna, Austria; and
§ Ludwig Boltzmann Institute for Rheumatology, Vienna, Austria

Correspondence: Gerhard Zlabinger, Institute of Immunology, University of Vienna, Borschkegasse 8a, A-1090 Vienna, Austria. E-mail: Gerhard.Zlabinger{at}univie.ac.at


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Dendritic cells (DC), the most potent APC, are central to antimicrobial immunity. Because of evolutionary pressure, it is reasonable that pathogens have evolved strategies to also subvert this host-defense mechanism. In the present study, we describe a novel way of bacterial interference with DC maturation. The bacterial metabolite n-butyrate, which occurs physiologically in high concentrations in the gastrointestinal tract and has well-known anti-inflammatory effects, is able to prevent LPS-induced maturation of DC resulting in a reduced capability to stimulate T cells. In particular, n-butyrate prevents homotypic DC clustering, inhibits IL-12 while sparing IL-10 production, and at the molecular level, blocks NF-{kappa}B translocation. These results demonstrate efficient targeting of DC function by a bacterial metabolite, which might explain the particular type of immune responsiveness in the presence of this bacterial agent as exemplified in the gastrointestinal tract.

Key Words: bacteria • host defense • cytokine • cellular differentiation • immunomodulator


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Dendritic cells (DC) are highly specialized antigen-presenting cells (APC) that play a critical role in initiating primary T-cell responses [1 ]. Depending on their maturational state, they possess different functional properties. Resting or immature DC are poor stimulators of T-cell proliferation and have been shown to induce a state of tolerance in vitro and in vivo [2 3 4 ]. However, upon exposure to microorganisms and bacterial cell products, immature DC switch from an antigen-capturing to an antigen-presenting and T-cell-stimulating modes. Thus, the unique capacity of DC to respond to microbial signals and to subsequently activate naive T cells enables these cells to fundamentally determine the outcome of antimicrobial immunity [5 ].

Several lines of evidence indicate that bacteria and viruses have evolved strategies to evade immune surveillance and in particular to block DC function. Thus, infection of DC with herpes simplex or measles virus down-regulates cytokine production and the immunostimulatory capacity of these cells [6 7 8 ]. As an example of bacterial defense strategies, it was shown that the B subunits of cholera toxin strongly inhibit the T helper cell type 1 (Th1)-skewing factor interleukin (IL)-12, thus promoting T-cell polarization toward Th2 [9 , 10 ]. Nevertheless, it still remains largely enigmatic why distinct microbes are recognized as potentially dangerous or harmless as is the case for the intestinal microflora. In view of the central function of DC in regulating immune responsiveness, elucidation of the interaction of bacteria/bacterial products with this cell population is mandatory to better understand the mechanisms underlying the establishment of protective immunity or tolerance against certain bacteria [11 ].

We and others have shown that n-butyrate, a physiologically occurring short-chain fatty acid (SCFA) derived from breakdown of carbohydrates by the intestinal microflora, exerts profound anti-inflammatory effects in vitro [12 13 14 ]. It was demonstrated that this bacterial metabolite severely hampers interferon-{gamma} (IFN-{gamma}) production as a result of defective IL-12 production and IL-12-receptor ß1/2-chain expression [13 ]. Furthermore, mounting evidence suggests that apart from its physiological function as the essential energy source for colonocytes, n-butyrate exerts strong anti-inflammatory activity in several states of mucosal inflammation [15 16 17 18 ]. Finally, in vitro and in vivo experiments have demonstrated that n-butyrate is able to induce a state of T-cell anergy [19 20 21 ]. Although these data collectively demonstrate a clear immunomodulatory role for n-butyrate, a definitive mechanism explaining its beneficial anti-inflammatory efficacy is still lacking.

In this study, we investigated how DC respond to the bacterial DC stimulus lipopolysaccharide (LPS) when applied in the presence of n-butyrate, both of which occur at high concentrations in the mucosal immune system. We revealed a profound impact of this bacterial metabolite on phenotype and function at different stages of the DC life cycle. Importantly, early molecular events, such as the translocation of nuclear factor {kappa}B (NF-{kappa}B) to the nucleus, homotypic DC aggregation, as well as cytokine production, were inhibited as a result of the interaction of DC with this SCFA. Our demonstration that a soluble product of bacteria interferes with particular DC features describes a new aspect in the complex interaction of bacteria with DC, which may contribute to the peculiar host-bacterial relationship as established in the mammalian gastrointestinal system.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Media and reagents
RPMI 1640 (Gibco BRL, Grand Island, NY) supplemented with 2 mM L-glutamine, 100 µg/ml streptomycin, 100 U/ml penicillin, and 10% fetal calf serum (FCS; Hyclone, Logan, UT) was used as culture medium. The sodium salt of n-butyric acid and LPS (Escherichia coli 0111:B4) were purchased from Sigma Chemie GmbH Co. (Deisenhofen, Germany). IFN-{gamma} was from the Ernst Boehringer Institut für Arzneimittelforschung (Vienna, Austria). Recombinant human granulocyte-macrophage colony-stimulating factor (rh-GM-CSF) was obtained from Schering-Plough (Kenilworth, NJ), and rh-IL-4 was from Strathmann Biotech Gmbh (Hannover, Germany).

Cell separation
For cell isolation, heparinized blood was obtained from adult healthy volunteers. Peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation over Lymphoprep (Nycomed Pharma AS, Oslo, Norway). For isolation of monocytes, PBMC were first depleted of T cells by sheep erythrocyte-rosetting. T-cell-depleted PBMC were cultured for 2 h on plastic dishes, and subsequently, adherent cells were harvested gently using a cell scraper. As assessed by flow cytometry, monocyte preparations contained >85% CD14+ cells.

Resting T cells were isolated by magnetic selection. Briefly, freshly isolated PBMC were incubated with monoclonal antibodies (mAb) to CD14 (RMO52), CD11b (Bear1), CD20 (HRC20), and CD16 (3G8; all antibodies were purchased from Immunotech S.A., Marseille, France) at 4°C for 1 h. Each antibody was used at a final concentration of 1 µg/ml. After extensive washing, cells with surface-bound antibodies were removed using human anti-mouse immunoglobulin G (IgG)-coated magnetic beads (Dynal, Oslo, Norway). The resulting population contained >98% CD3+ cells.

DC maturation and differentiation
Highly enriched monocytes (>85% CD14+) were cultured in 24-well plates (Costar, Cambridge, MA) at a cell density of 5 x 105 cells/ml in RPMI 1640/10% FCS medium at 37°C in a humidified CO2-containing atmosphere. For induction of cell differentiation, the culture medium was supplemented for 7 days with 50 ng/ml rh-GM-CSF and 10 ng/ml rh-IL-4. To induce final maturation, LPS (100 ng/ml) was then added for 48 h with or without different concentrations of n-butyrate. To study the influence of n-butyrate on DC differentiation, monocytes were cultured for the first 7 days in the presence (nB-DC) or absence (Ctrl-DC) of n-butyrate.

Fluorescein-activated cell sorter (FACS) analysis
For evaluation of surface-marker expression, 50 µl cells (5x106/ml) were incubated with fluorescein isothiocyanate (FITC)- or phycoerythrin (PE)-conjugated mAb for 30 min at 4°C. For control, nonbonding isotype-matched FITC- and PE-conjugated mouse IgG were used. Cells were analyzed on a FACScalibur flow cytometer (Becton Dickinson, San Jose, CA). FITC-labeled anti-CD40 (IgG1, clone LOB7/6) and anti-HLA-ABC (IgG2a, clone W6/32) were from Immunotech S.A. FITC-conjugated anti-CD1a (IgG1, clone HI149), anti-CD14 (IgG2b, clone MOP9), anti-CD25 (IgG1a, clone 2A3), anti-HLA-DR (IgG2a, L243), and anti-CD83 (IgG1, clone HB15e) were from Becton Dickinson. FITC-labeled anti-CD32 (IgG2b, clone IV.3) was obtained from Medarex (Annandale, NJ). The following PE-labeled mAb (obtained from Becton Dickinson) were used: anti-CD80 (IgG1, L307.4), anti-CD86 (IgG2b, clone IT2.2), and antimannose receptor (IgG1, clone 19).

Morphological cell analysis
Immature DC were stimulated with LPS in the presence or absence of n-butyrate. After 4 h, the cells were analyzed by light microscopy on a Leitz Aristoplan microscope (Wetzlar, Germany). To perform scanning electron microscopy, cells were fixed onto 24 multiwell plates using 2 ml 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2, for 90 min at 4°C. After rinsing in cacodylate buffer, cells were postfixed with 1% OsO4 for 15 min. The samples were dried in a gradual series of ethanol, transferred to tertiary butanol (Merck, Darmstadt, Germany) for 60 min, and freeze-dried. The samples were examined in a Stereoscan S90 scanning electron microscope (Cambridge Instruments, Cambridge, UK).

Endocytosis and proliferation assay
To determine mannose receptor (MR)-mediated endocytosis, 1 x 106 cells/ml were incubated in medium with FITC-labeled dextran [molecular weight (Mr) 40,000; Sigma Chemie GmbH Co.] at a concentration of 1 mg/ml. After an incubation period of 60 min at 37°C or on ice as a control, cells were washed extensively with ice-cold phosphate-buffered saline (PBS) and analyzed on a FACScalibur. Fluid-phase endocytosis was measured via cellular uptake of lucifer yellow (LY; Sigma Chemie GmbH Co.) and was analyzed by flow cytometry.

After the differentiation or maturation period, the cells were washed extensively, irradiated (3000 rad, 137Cs source), and washed again. Then, the stimulator cells were added at increasing cell numbers to 1 x 105 allogeneic T cells in 96-well culture plates in RPMI 1640 medium supplemented with 10% FCS (total volume, 200 µl/well). After 4 days, cells were pulsed with 1 µCi [3H]-thymidine (ICN Pharmaceuticals, Irvine, CA). After another 18 h, the cells were harvested on glass-fiber filters (Packard, Topcount, Meriden, CT), and DNA-associated radioactivity was determined using a microplate scintillation counter (Packard). DNA synthesis was expressed as mean cpm of triplicate cultures.

Measurement of cytokine production
For evaluation of tumor necrosis factor (TNF)-{alpha}, 5 x 105 DC were stimulated with LPS (100 ng/ml) in 24-well plates (final volume, 1 ml). Cultures were performed in medium with or without n-butyrate. For assessment of IL-12p70 and IL-10 secretion, DC were precultured (24 h) with IFN-{gamma} (200 U/ml) in the presence or absence of n-butyrate before LPS (100 ng/ml) was added. Cell-free supernatants were harvested 48 h after addition of the bacterial stimulus. Cytokines were measured by sandwich enzyme-linked immunosorbent assay (ELISA) using matched-pair antibodies. Capture as well as detection antibodies to human IL-12p70 were obtained from R&D Systems (Minneapolis, MN). Antibodies to human TNF-{alpha} were from PharMingen (San Diego, CA). Standards consisted of human recombinant material from R&D Systems. Assays were set up in duplicates and were performed according to the recommendations of the manufacturers. The lower limit of detection was 20 pg/ml for all cytokines. For reverse transcriptase-polymerase chain reaction (RT-PCR), DC were stimulated with LPS plus IFN-{gamma} in the presence or absence of n-butyrate as described above. Total cellular RNA was isolated using the RNeasy Total RNA kit (Quiagen, Hilden, Germany), according to the manufacturer’s instruction. Approximately 200 ng total RNA was processed for reverse transcription. cDNA synthesis was performed with SuperScript II RT (Gibco BRL, Gaithersburg, MD) according to the manufacturer’s instructions. The cDNA reaction (5 µl) was used as template for IL-12p40 and IL-12p35 amplification; only 2 µl was used for actin amplification. The PCR reaction was performed in a total volume of 50 µl, 200 µM each dNTP, 25 pmol each primer, and 0.5 U Taq DNA polymerase (Perkin Elmer Cetus, Branchburg, NJ). DNA was denatured for 4 min at 94°C and amplified with different numbers of cycles at the following conditions: 94°C, 20 sec; 58°C, 30 sec; 72°C, 60 sec; final elongation step, 72° for 7 min; and stored at 4°C. Different numbers of cycles were tested to ensure linear-phase amplification of the cDNA. IL-12p40 and actin were amplified for 25 cycles, and IL-12p35 was amplified for 30 cycles. For IL-12p40 and IL-12p35, the primers used were as shown previously [13 ]: IL-12p40 sense, 5'-AGAGGCTCTTCTGACCCCCAG-3'; IL-12p40 antisense, 5'-CTCTTGCTCTTGCCCTGGACCTG-3'; IL-12p35 sense, 5'-TCAGCAACATGCTCCAGAAGGC-3'; IL-12p35 antisense, 5'-TGCATTCATGGTCTTGAACTCCACC-3'. For actin PCR amplification, the primers used were as shown previously [22 ]: sense primer, 5'-GCATCCCCCAAAGTTCACAAT-3', antisense primer, 5'-CGAAGGCTCATCATTCAAAAT-3'. In all experiments, H2O was included as a negative control in the RT procedure and in the PCR amplification (unpublished results). Amplified PCR products were resolved by gel electrophoresis on a 1.5% agarose gel and stained with ethidium bromide.

Electrophoretic mobility shift assay (EMSA)
Nuclear extracts from DC were prepared as described [23 ]. Oligonucleotides resembling the consensus binding site for NF-{kappa}B (5'-AGTTGAGGGGACTTTCCCAGGC-3') and activated protein-1 (AP-1; 5'-CGCTTGATGACTCAGCCGGAA-3') were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The double-stranded oligonucleotides used in all experiments were end-labeled using T4 polynucleotide kinase and [{gamma}-32P]-ATP. After labeling, 5 µg nuclear extract was incubated with 120,000 cpm labeled probe in the presence of 3 µg poly(dI-dC) at room temperature for 30 min. This mixture was separated on a 6% polyacrylamide gel in Tris/glycine/ethylenediaminetetraacetate (EDTA) buffer at pH 8.5. Control experiments were performed as described [24 ]. For specific competition, 5 pmol unlabeled NF-{kappa}B oligonucleotide was included, and for nonspecific competition, 5 pmol double-stranded AP-1 oligonucleotides was used.

Statistics
Comparisons were performed by Student’s t-test. A P value of <0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Phenotypical and functional impairment of DC maturation by n-butyrate
Because terminal DC maturation critically determines the outcome of antimicrobial immune responses, we evaluated the impact of n-butyrate on this stage of the DC life cycle. We first investigated the phenotypical changes in immature DC exposed to LPS under the influence of this bacterial metabolite. As shown in Figure 1 , addition of LPS to immature DC resulted in the neoexpression of the maturation markers CD83 and CD25, the {alpha}-subunit of the IL-2 receptor, and the up-regulation of major histocompatibility complex (MHC) class I and II and costimulatory molecules. However, concomitant treatment with n-butyrate yielded DC with a markedly altered phenotype (Figs. 1 and 2). Expression of CD25 and CD83 and of critical costimulatory molecules, i.e., CD40, CD80, and CD86, was reduced substantially. Furthermore, a clear suppression of the up-regulation of MHC class I and II antigens by n-butyrate was observed. When immature DC were first incubated with LPS for 48 h and subsequently exposed to n-butyrate for 2 days, the phenotype of the already activated DC remained stable (unpublished results).



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  		Figure 1. n-Butyrate prevents DC maturation. Monocytes were cultured for 7 days with GM-CSF (50 ng/ml) plus IL-4 (10 ng/ml). Subsequently, these immature DC (5x105/ml) were activated with LPS (100 ng/ml) with or without 1 mM n-butyrate for 48 h. Open profiles (fine line) in the upper panel represent a staining pattern with an isotype control, and open profiles (bold line) represent staining with mAb of the indicated specificity in immature DC before LPS activation. In the lower panel, surface expression results from cultures containing LPS (open profiles with bold line) or LPS plus 1 mM n-butyrate (solid profiles) are depicted. Open profiles (fine line) represent the staining pattern with an isotope control. Data are representative of three independent experiments.

 
A striking feature of activated DC is the occurrence of large cell clusters arising a few hours after addition of LPS (Fig. 2) . In comparison, little or no cell clustering occurs in cultures activated in the presence of n-butyrate (Fig. 2) . Moreover, the majority of cells in the n-butyrate-treated cultures imposed with widespread cytoplasmic projections. In addition to the reduced expression of DC maturation markers, we found a significant dose-dependent reduction of the allostimulatory capacity of immature DC treated with LPS and n-butyrate (Fig. 3 ). A series of experiments confirmed that n-butyrate does not impede the viability of DC; i.e., we found identical numbers of cells in cultures treated with or without n-butyrate for 48 h. More importantly, FACS analysis measuring propidium-iodide uptake confirmed the same number of viable cells in n-butyrate and nontreated cultures (unpublished results). Thus, the effects of this compound resulted from specific action rather than from unspecific effects, such as cell death.



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  		Figure 2. Effect of n-butyrate on maturation-associated clustering of DC. Immature DC were stimulated with LPS (100 ng/ml) in the absence (A, C) or presence(B, D) of 1 mM n-butyrate. After 4 h of cultivation, cells were analyzed by photomicrographs using light microscopy (A, B) or scanning electron microscopy(C, D). The insert in photomicrograph D shows a bulb-like ending of a cytoplasmic projection in n-butyrate-treated cells. Similar results were obtained in four different experiments.

 


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  		Figure 3. Reduced T-cell stimulatory capacity of DC matured in the presence of n-butyrate. Immature DC were stimulated with LPS (100 ng/ml) with or without n-butyrate at the indicated concentrations. After 48 h, the cells were washed extensively, irradiated, and cocultured with 1 x 105 allogeneic T cells at the indicated ratios. DNA synthesis was assessed at day 5. Shown are the means ± SE of six to nine experiments. *, P < 0.01.

 
Suppression of IL-12p70 and TNF-{alpha} production in DC by n-butyrate
Analyzing cytokine production during DC maturation, we found a dose-dependent inhibition of the production of IL-12p70 and TNF-{alpha} by n-butyrate (Fig. 4 A ). Inhibition was seen with doses as low as 0.25 mM (P<0.02). In contrast, this compound clearly differentially modulated IL-10 production, because significant amounts of this anti-inflammatory cytokine were still produced in the absence of IL-12 (Fig. 4A) . Semiquantitative RT-PCR revealed that the observed inhibition of IL-12 secretion is the result of decreased mRNA production. Stimulation of immature DC with LPS and IFN-{gamma} in the absence of n-butyrate resulted in increased mRNA for the p35 and p40 chain of the IL-12 heterodimer (Fig. 4B) , whereas treatment of DC with n-butyrate resulted in profound suppression of mRNA species.



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  		Figure 4. Suppression of IL-12 production by n-butyrate. Immature DC were stimulated with LPS (100 ng/ml) in the presence or absence of the indicated concentrations of n-butyrate. For induction of IL-10 and IL-12p70, cells were incubated with IFN-{gamma} (200 U/ml) and n-butyrate before LPS (100 ng/ml) was added. Cell-free supernatants were collected 24 h after addition of endotoxin and analyzed by ELISA (A). Mean % control responses ± SE calculated from four to seven independent experiments are shown. In unstimulated cultures, 47 ± 46 pg/ml TNF-{alpha} was detected. Levels of IL-12p70 and IL-10 were below the detection limit. Mean cytokine levels in stimulated cultures in the absence of n-butyrate were 2.01 ± 1.50 ng/ml (TNF-{alpha}), 368 ± 53 pg/ml (IL-10), and 1.67 ± 1.04 ng/ml (IL-12p70). *, P < 0.05 for given and all higher concentrations of n-butyrate. (B) DC were cultured as described above. After 18 h, cells were harvested, and total RNA was prepared. Results for RT-PCR are representative of three independent experiments.

 
n-Butyrate suppresses the LPS-induced activation of NF-{kappa}B
The transcription factor NF-{kappa}B is known to be implicated in the regulation of many genes encoding proinflammatory proteins as well as in the DC maturation process [25 , 26 ]. Our finding of impaired DC maturation in the presence of n-butyrate prompted us to examine the effect of this bacterial product on the constitutive expression and endotoxin-induced transactivation of this central transcription factor. As shown in Figure 5 using EMSA, we detected a clear increase in nuclear binding to the NF-{kappa}B consensus site upon LPS stimulation. The specificity of NF-{kappa}B binding was proven using excess, unlabeled NF-{kappa}B probe, which competed successfully for NF-{kappa}B binding, and a nonrelated competitor (AP-1 oligonucleotide) did not (unpublished results). Importantly, n-butyrate profoundly inhibited translocation of NF-{kappa}B to the nucleus in LPS-stimulated DC, suggesting that the inhibition of DC function by n-butyrate may be attributable to the failure to effectively activate NF-{kappa}B in DC.



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  		Figure 5. n-Butyrate suppresses the sequence-specific binding of NF-{kappa}B in DC. Immature DC were cultured for 2 h with or without n-butyrate (1 mM), and then LPS (100 ng/ml) or medium was added. After 60 min, total nucleoprotein was extracted. 32P-labeled oligonucleotides containing a NF-{kappa}B consensus sequence were incubated at room temperature with 5 µg nuclear extracts, followed by nondenaturating gel electrophoresis. Similar results were obtained in two independent experiments.

 
n-Butyrate interferes with differentiation of human monocyte-derived DC
Next, we explored the impact of n-butyrate on the process of DC differentiation, i.e., when this substance was added to freshly isolated monocytes together with GM-CSF plus IL-4 on day 0. After a 7-day culture period, profound differences between such differentiated cells (nB-DC) and control cells (Ctrl-DC) were detected regarding the expression of typical DC markers. Whereas >85% of Ctrl-DC expressed the Langerhans cell-associated marker CD1a, n-butyrate prevented the acquisition of this typical DC molecule (Fig. 6 A ). It is interesting that the characteristic disappearance of the monocyte marker CD14 was not hindered by n-butyrate (Fig. 6) . Expression of CD40 and CD80 was clearly suppressed in n-butyrate-treated cultures, whereas CD86 was even up-regulated as compared with Ctrl-DC, a further indicator of the functional integrity of n-butyrate-treated cells (Fig. 6A) . n-Butyrate did not essentially affect the expression of MHC molecules but inhibited the expression of the antigen-uptake molecules CD32 and MR (Fig. 6A and 6B) . Assessment of macropinocytosis, a special type of actin-dependent fluid-phase uptake, revealed impaired internalization of LY but a less pronounced inhibition of receptor-mediated endocytosis of dextran molecules (Fig. 6B) .



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  		Figure 6. n-Butyrate interferes with DC differentiation. Monocytes were cultured for 7 days with GM-CSF (50 ng/ml) plus IL-4 (10 ng/ml) in the absence (Ctrl-DC) or presence (nB-DC) of 1 mM n-butyrate (A). Open profiles represent a staining pattern with an isotype control, and solid profiles show staining with mAb of the indicated specificity. (B) Analysis of the DC antigen-uptake machinery of DC differentiated in the presence of n-butyrate. For assessment of CD32 or MR expression, cells were stained with the respective antibodies and analyzed by flow cytometry. For functional endocytosis assays, cells were pulsed with 1 mg/ml FITC-dextran (DEX) or 1 mg/ml LY for 60 min. Open profiles (fine line) show the background uptake on ice; open profiles with bold line (Ctrl-DC) and solid profiles (nB-DC) indicate antigen uptake of cells at 37°C. (C) Cells differentiated in the presence of n-butyrate do not acquire the phenotype of mature DC. LPS (100 ng/ml) was added for 48 h to Ctrl-DC or nB-DC. Open profiles (fine line) represent staining with an isotype control; open profiles with bold line (Ctrl-DC +us LPS) and solid profiles (nB-DC + LPS) show a staining pattern with mAb of the indicated specificity. Data are representative of at least four independent experiments.

 
To examine whether nB-DC could be differentiated further into fully mature DC, LPS was added to such cells differentiated by GM-CSF and IL-4 in the presence of n-butyrate. Importantly, the neoexpression of surface markers associated with mature DC was prevented completely (Fig. 6C) . Likewise, LPS-treated nB-DC displayed a profound defect in the up-regulation of molecules involved in antigen presentation and costimulation (Fig. 6C) . In n-butyrate-supplemented cultures, no increase in cell death was detectable as assessed by flow cytometry and cell number when compared with control cultures (unpublished results).

DC differentiated in the presence of n-butyrate exhibit impaired immunostimulatory capacity
Finally, we examined whether n-butyrate modulates APC function when present during the DC differentiation process (Fig. 7 ). Therefore, we tested the immunostimulatory capacity of nB-DC compared with Ctrl-DC in an allogeneic mixed-leukocyte reaction (MLR). As shown in Figure 7 (upper panel), the presence of n-butyrate during differentiation with GM-CSF and IL-4 resulted in a cellular population with markedly reduced stimulatory activity that was even inferior to freshly isolated monocytes. The functional immaturity of cells generated in the presence of n-butyrate was further corroborated by their inability to stimulate allogeneic T-cell proliferation even after LPS maturation. Whereas the addition of LPS to Ctrl-DC led to a further increase in the T-cell stimulatory capacity, nB-DC, in agreement with their insensitivity to maturation, retained their poor allostimulatory capability (Fig. 7 , lower panel).



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  		Figure 7. DC differentiated in the presence of n-butyrate have a reduced capacity to induce an allogeneic T-cell response. Freshly isolated monocytes, immature DC (Ctrl-DC), or cells differentiated in the presence of 1 mM n-butyrate (nB-DC) were washed extensively, irradiated (3000 rad), and subsequently cocultured with highly purified allogeneic T cells (1x105) at the indicated ratios (upper panel). To determine maturation sensitivity, Ctrl-DC or nB-DC (1 mM) was activated with 100 ng/ml LPS (lower panel). After 48 h, the cells were used as allogeneic stimulators as described above. DNA synthesis was assessed on day 5 and is expressed as mean cpm of a representative experiment. SD of triplicates was generally below 20%. Similar results were obtained in four other experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Selective pressure during coevolution results in a host-microbial relationship, which is characterized by the opposing goals of host-defense evasion on the part of the pathogen and elimination of the invading microorganisms on the part of the host. In this study, we provide phenotypical, morphological, and functional evidence that the bacterial metabolite n-butyrate profoundly suppresses the development of monocyte-derived DC in vitro. In particular, n-butyrate inhibited the development of mature DC, resulting in impaired DC function and defective cytokine production. Because DC are crucial to the initiation of effective immunity [1 ], such an interference may modify the outcome of the subsequent immune response substantially. In view of recent data showing that DC extend processes into the gut lumen and sample luminal contents including bacteria [27 ], our data imply that bacterial metabolites as major constituents of the human mucosal compartment might be directly involved in the modulation of resident DC function. Furthermore, the well-known beneficial effects of n-butyrate treatment in several inflammatory states [15 16 17 18 ] may occur at the level of professional APC.

Microbial stimuli such as LPS, gram-positive bacteria, or protozoa convert immature DC to a mature phenotype, characterized by increased expression of MHC antigens and costimulatory molecules, providing these cells with optimal T-cell stimulatory capacity [5 , 28 , 29 ]. In this study, we show that the bacterial metabolite n-butyrate substantially impairs the up-regulation of MHC antigens, costimulatory molecules, and DC-specific maturation markers, correlating with reduced antigen-specific T-cell proliferation. Inhibition of costimulatory molecule expression on DC by n-butyrate is also striking given their critical role in T-cell stimulation [30 31 32 ]. It is interesting that the early maturation-associated homotypic DC aggregation was abrogated completely by this bacterial substance, indicative of disturbed DC activation [33 ]. Moreover, the simultaneous absence of CD83 on n-butyrate-treated cells sensitively indicated a suppression of successful DC maturation, which was further corroborated by the impaired APC function of these cells. That prevention of DC maturation may indeed be of great biological and clinical relevance is illustrated by several studies suggesting that parasites, such as measles virus, herpes virus, and Plasmodium falciparum, use this strategy to subvert the host defense [6 7 8 , 34 ]. Importantly, n-butyrate differentially modulated the cytokine production of DC. Although production of the anti-inflammatory cytokine IL-10 was unaffected in activated DC, the major Th1 skewing factor IL-12 was substantially suppressed by this bacterial product, which may critically influence the development and polarization of a subsequent T-cell response. Because of the crucial role of IL-12 in the defense of parasitic infections, microbes might have developed strategies to suppress this critical defense cytokine. Indeed, several pathogens have been demonstrated to counteract IL-12 production by the host organism, which was suggested to lead to local immunosuppression [9 , 35 , 36 ]. Based on the demonstrated evidence that the bacterial product n-butyrate strongly influences DC maturation and IL-12 production, it, therefore, is tempting to speculate that the intestinal microflora in the gut uses this mechanism to escape immune surveillance too.

The intestinal lumen has been shown to contain n-butyrate concentrations from 0.1 mM to 16 mM in the small intestine and 40 mM in the colon depending on quality and quantity of daily food intake [37 ]. Because n-butyrate occurs in the portal blood at 0.04 mM [37 ], it is conceivable that the concentrations used in our study actually occur in the mucosa, where contact between butyrate and DC can be expected. In other studies dealing with apoptosis, differentiation, or cell-cycle progression n-butyrate has been used mostly in the range of 1–5 mM, whereas in the present analysis, effects were observed between 0.25 and 1 mM, indicating a high sensitivity of DC to the influence of n-butyrate. The influence of intestinal bacterial metabolites such as n-butyrate on DC morphology and function in vivo could be analyzed in an appropriate study that includes patients with high, normal, and low intestinal n-butyrate concentrations by testing colonic biopsies of patients with long-standing parenteral nutrition (low/no n-butyrate concentrations), patients that receive a high-fiber diet consisting of a fixed amount of amylase-resistant starch for a significant time period (high n-butyrate concentrations), as well as control subjects on a classical Western diet.

To reveal the mode of action of n-butyrate action, we analyzed the effect of this substance on the transcription factor NF-{kappa}B, which plays a decisive role for proper DC function. Thus, it has been shown that NF-{kappa}B translocation is essential for the ability of mature DC to present antigen to naive T cells [38 39 40 ]. Mice deficient in functional NF-{kappa}B have no mature DC and present with impaired cellular immunity [41 42 43 ]. Likewise, selective inhibition of NF-{kappa}B activity has also been shown to impair DC maturation [44 45 46 47 ]. To this end, we demonstrated that nuclear extracts from DC displayed a profoundly reduced binding to the NF-{kappa}B site when such cells were activated with LPS in the presence of n-butyrate. Impairment of nuclear translocation of this transcription factor by n-butyrate may account for most features of the altered DC phenotype [26 ] as well as for the sensitive inhibition of cytokine production by n-butyrate, because the human IL-12, but not the IL-10 promoter, contains crucial NF-{kappa}B binding sites within its promoter [48 , 49 ]. Our findings are in line with recent observations in intestinal epithelial cells demonstrating a suppression of cytokine-stimulated NF-{kappa}B transactivation because of stabilization of I{kappa} n-butyrate [50 51 52 ]. Endotoxin-stimulated PBMC also showed inhibited NF-{kappa}B activity in the presence of n-butyrate, which caused a decreased production of proinflammatory cytokines [14 ]. Finally, in animal models of colitis mucosal, NF-{kappa}B-DNA binding activity was reduced markedly when high intestinal n-butyrate concentrations were achieved by SCFA enemas or dietary means [14 , 16 ]. Although the detailed mechanism of suppressed NF-{kappa}B transactivation in n-butyrate-treated DC is currently unknown, it is tempting to speculate that this fatty acid may interfere directly with phosphorylation/dephosphorylation events of the LPS-triggered signaling program. Apart from the possibility that n-butyrate might directly induce the transcription of distinct I{kappa}B family members, this bacterial metabolite might also induce particular phosphatases to inhibit the I{kappa}B-inactivating kinase IKK, which is physiologically inactivated by putative okadaic acid-sensitive phosphatases [53 ]. Indeed, Cuisset et al. [55 ] have demonstrated recently that the transcriptional effects of n-butyrate are mediated directly by the induction of a distinct okadaic acid/calyculin A-sensitive phosphatase [54 , 55 ]. Clearly, the molecular mechanism of n-butyrate-mediated inhibition of NF-{kappa}B activity has to be analyzed in future studies.

Another key aspect of this study was the observed disruption of the cytokine-driven DC differentiation by n-butyrate. It is interesting that neoexpression of CD1a, the classical Langerhans cell-associated marker, was prevented in n-butyrate-treated cells, and simultaneously, CD14 was no longer expressed. Also, other markers of macrophages (CD16 and CD64) were not present in n-butyrate-treated DC, indicating that n-butyrate does not promote a classical differentiation program toward macrophages as has been demonstrated for corticosteroids, IL-10 and IL-6 [56 57 58 ]. More importantly, we revealed a functional alteration of these cells, because their T-cell-stimulatory capacity was also markedly reduced upon LPS activation. Recently, a similar phenotype with impaired CD1a up-regulation, unaffected loss of CD14 expression, and differential CD80/CD86 regulation has been shown for DC treated with IFN-{alpha} or nonsteroidal antiestrogens, which also exhibited an impairment to support T-cell proliferation [59 , 60 ]. These data suggest that the bacterial metabolite n-butyrate also acts at the first step of DC development by blocking the differentiation from monocytic precursors into mature DC and thus potentially impairs the normal turnover and function of DC.

In conclusion, DC are affected by n-butyrate at all major stages of their life cycle, ultimately leading to an impaired DC maturation. The emerging role of n-butyrate as a potent immunomodulator in vivo [18 , 52 ] is supported further by its effect on DC as described in this study. In addition to a direct inhibitory action in specific T-cell responses [19 , 20 ], modulation of the immune system may therefore also be expected through the effects of n-butyrate on DC, rendering these cells less supportive for inflammatory responses. Our study reveals that monocyte-derived DC are sensitive targets of a physiologically occurring substance derived from bacteria. These findings not only provide a novel interpretation of its potent anti-inflammatory properties but also suggest a possible in vivo immunomodulatory role of n-butyrate via interference with the function of DC.


   ACKNOWLEDGEMENTS
 
This work was supported in part by a grant (P 14874-PAT) from the Fonds zur Förderung der Wissenschaftlichen Forschung, Österreich. The authors thank Margarethe Merio and Birgit Kagerbauer for excellent technical assistance as well as M. Lehner and G. Staffler for critical reading of the manuscript.

Received May 26, 2001; revised July 10, 2001; accepted July 12, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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