HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text (PDF) All Versions of this Article: 14/15/2380 00-0359fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by SÄEMANN, M. D. Articles by ZLABINGER, G. J. Search for Related Content PubMed PubMed Citation Articles by SÄEMANN, M. D. Articles by ZLABINGER, G. J.

Anti-inflammatory effects of sodium butyrate on human monocytes: potent inhibition of IL-12 and up-regulation of IL-10 production¹

Institute of Immunology and
^* Department of Internal Medicine III, University of Vienna, A-1090 Vienna, Austria

²Correspondence: Institute of Immunology, Borschkegasse 8a, A-1090 Vienna, Austria. E-mail: Gerhard.Zlabinger{at}univie.ac.at

SPECIFIC AIMS

Important clues to understand the anti-inflammatory action of the bacterial product butyrate in vivo might come from the analysis of the cytokine-modulating capabilities of this substance in immune cells. Here, we investigated whether butyrate influences the production of pro- and anti-inflammatory cytokines in leukocytes.

PRINCIPAL FINDINGS

1. Butyrate suppresses the production of interleukin-12 (IL-12) in peripheral blood monocytes
Staphylococcus aureus cells (SAC, 75 µg/ml) induced a massive production of the proinflammatory cytokines IL-12p40 and tumor necrosis factor (TNF-) in purified peripheral blood monocytes. Addition of butyrate to these cultures inhibited dose-dependently the production of both cytokines (Fig. 1A ). Moreover, butyrate effectively inhibited IL-12p70 heterodimer secretion, indicating that interferon (IFN- ) priming does not overcome the inhibitory effects of butyrate on IL-12 production. While inhibiting IL-12 and TNF- production, butyrate markedly increased SAC-induced release of the anti-inflammatory cytokine IL-10 resulting in a bell-shaped dose response curve (Fig. 1B ). In the absence of bacterial stimulation, butyrate did not induce cytokine secretion. Analysis of IL-12p40 and IL-12p35 mRNA by means of semiquantitative reverse transcription-polymerase chain reaction demonstrated that butyrate significantly inhibited both genes, indicating transcriptional suppression of both chains of the IL-12 heterodimer by butyrate. Determination of cell viability by means of FACS analysis 24 h after culture initiation did not reveal differences in butyrate-treated cultures vs. control cultures.

View larger version (16K): [in this window] [in a new window]   Figure 1. Butyrate inhibits monocyte-derived proinflammatory cytokines IL-12p40, IL-12p70, and TNF-, but enhances IL-10. For induction of IL-12p40, TNF- (A), and IL-10 (B), human monocytes (1x106 cells/well) were stimulated with SAC (75 µg/ml) in the presence or absence of butyrate at the concentrations indicated. For induction of IL-12p70 (A), cells were incubated with IFN- (200 U/ml) before SAC stimulation. Supernatants (24 h) were analyzed by ELISA. Mean % control responses ± SEM from 3 to 9 experiments are shown. *P < 0.05 for given and all higher concentrations of butyrate.

2. Butyrate does not inhibit IL-12 production via IL-10 or other endogenous inhibitors of IL-12
Impairment of IL-12 production by butyrate might result from increased IL-10 secretion. Although neutralization of IL-10 via anti-IL-10 mAb (10 µg/ml) in SAC-treated monocytes enhanced IL-12p70 secretion compared to untreated cultures, butyrate also inhibited IL-12 production in the presence of neutralizing IL-10 mAb. Similarly, pretreatment of monocytes with anti-transforming growth factor ß1 or indomethacin (to block the production of prostaglandins) did not reverse the inhibition of IL-12p70 by butyrate, indicating additional critical mechanism(s) involved in butyrate-mediated suppression of IL-12.

3. Butyrate modulates cytokine secretion by activated peripheral blood leukocytes
Since the functional characteristics of antigen-presenting cells determine the nature of the developing immune response, we evaluated the effects of butyrate on cytokine production in human peripheral blood mononuclear cell (PBMC) stimulated via T cell receptor ligation with CD3 mAb. While butyrate inhibited T cell proliferation, this substance also suppressed the production of the Th1-associated cytokines IL-2 and IFN-. The observed inhibition of IFN- secretion in butyrate-treated cultures was associated with a dose-dependent suppression of IL-12 release. Since impaired IL-12 production might be responsible for decreased IFN- secretion by butyrate, we added exogenous IL-12 (2 ng/ml) to restore deficient IFN- production. Indeed, recombinant IL-12 (rIL-12) led to complete restoration of IFN- secretion in cultures treated with butyrate at 0.25 mM. At higher concentrations of butyrate, however, the effect of rIL-12 was incomplete.

4. Butyrate inhibits cell surface expression of IL-12-receptor ß 1 (IL-12R ß 1) and IL-12R ß 2 chains on activated leukocytes
Then we examined the possibility that butyrate reduced IFN- secretion in anti-CD3-stimulated PBMC by inhibition of IL-12Rß1 and IL-12Rß2 expression. After a 72 h culture period, the anti-CD3-stimulated enhancement of IL-12Rß1 and IL-12Rß2 expression in PBMC was profoundly suppressed by addition of butyrate, indicating that down-regulated expression of IL-12R on activated leukocytes further contributes to impaired IFN- production in butyrate-treated cultures.

CONCLUSIONS

The four-carbon fatty acid butyrate plays a central role in the homeostasis of the gastrointestinal tract. Apart from its well-known function as an essential energy source for colonocytes, an anti-inflammatory role in certain states of mucosal inflammation has recently emerged. Here we demonstrate that butyrate strongly inhibits the production of proinflammatory cytokines IL-12 and TNF- by monocytes upon bacterial stimulation.

Monocytes/macrophages encountering bacteria or bacterial products release massive amounts of IL-12, which subsequently bind to its receptor (Fig. 2A ). This interaction guarantees optimal production of IFN-, which further primes monocytes/macrophages for effector functions, including the production of more IL-12. In regions of the gastrointestinal tract with abundant bacterial colonization, such as the terminal ileum and colon, the concentrations of butyrate and other short-chain fatty acids are the highest, reaching up to 30 mM depending on quality and quantity of daily food intake. Since butyrate occurs in the portal blood at 0.04 mM, it is conceivable that sufficiently high concentrations of butyrate occur in the mucosa to exert anti-inflammatory effects on leukocytes. In our proposed model of the anti-inflammatory role of butyrate in intestinal mucosal compartments (Fig. 2B ), both IL-12 production upon bacterial stimulation and IL-12R expression on activated T cells are suppressed. Butyrate further promotes a state of IL-12 deficiency, since the action of IFN- on monocytes/macrophages to activate the IL-12 gene promoter is prohibited. Simultaneously, the up-regulation of monocytes/macrophages-derived IL-10 by butyrate would further contribute to an anti-inflammatory state apart from the suppressed IL-12/IL-12R feedback loop.

View larger version (75K): [in this window] [in a new window]   Figure 2. Schematic diagram of the hypothesized action of butyrate on leukocytes. A) Monocytes/macrophages encounter bacteria and produce massive amounts of proinflammatory cytokines such as IL-12, which stimulates the production of the monocyte/macrophage activating cytokine IFN-. B) In the presence of butyrate, such as in the colonic mucosa, critical pathways leading to an inflammatory response are inhibited. MO, monocytes/macrophages; BUT, butyrate.

Mounting evidence suggests that some forms of mucosal inflammation, such as Cohn’s disease, are triggered by the nonpathogenic gut microbiota. In such patients as well as in many animal models of mucosal inflammation, IL-12 is critical for the initiation and maintenance of the disease; moreover, in experimental models, neutralization of IL-12 can prevent or even cure the disease process. Since a disturbed microbial/host relationship appears to be involved in the pathogenesis of these inflammatory diseases, such an anti-inflammatory bacterial factor might be of particular relevance in pathophysiological conditions of mucosal immunity. The crucial role of IL-10 in the gut is best illustrated in IL-10 knockout mice suffering from severe enterocolitis due to a Th1-dominated disease that manifests after significant colonization of the colon with nonpathogenic bacteria. Since butyrate selectively increases IL-10 production while strongly suppressing the production of IL-12, it is tempting to speculate that the induction of pro- and anti-inflammatory cytokines by the intestinal microbiota is differentially modulated by this bacterial metabolite to promote an anti-inflammatory state.

IL-12 is the most important factor in governing the differentiation and magnitude of a Th1 response and plays an important role in the defense of microbial infections. Therefore, it is conceivable that several pathogens have developed mechanisms to counteract IL-12 production in order to escape immune surveillance. Indeed, potent IL-12 suppression has been demonstrated for measles virus, rhinovirus, or cholera toxin, for example. We hypothesize that butyrate has a similar role also for commensal bacteria. Since the resident microflora grants nutritional advances for the host, an impediment of defense mechanisms against commensal bacteria is critical. Particular bacterial products may thus contribute to the delicate balance between the intestinal flora and the mucosal immune system.

Despite the evidence provided, the definite molecular mechanism for the anti-inflammatory effect of butyrate in monocytes remains unresolved. Since most cytokines are commonly controlled by the inducible nuclear factor NF-B, it could be hypothesized that interference with activation or mobilization of NF-B is responsible for the inhibitory effects of butyrate on cytokine secretion observed in this study. In contrast to IL-12, the promoter region of IL-10 contains crucial cAMP-responsive elements, but no NF-B binding sites. Therefore, it has to be tested whether butyrate also influences cAMP-dependent events.

We demonstrate here that the bacterial metabolite butyrate differentially modulates cytokine secretion in peripheral blood immune cells. These data demonstrate a novel anti-inflammatory property of butyrate that may have broad implications for the regulation of immune responses in vivo. Moreover, the molecular mechanisms of the described actions of butyrate could help to explore new therapeutic approaches in inflammatory conditions.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0359fje To cite this article, use (October 6, 2000) FASEB J. 10.1096/fj.00-0359fje

This article has been cited by other articles:

				S. Seifert and B. Watzl Inulin and Oligofructose: Review of Experimental Data on Immune Modulation J. Nutr., November 1, 2007; 137(11): 2563S - 2567S. [Abstract] [Full Text] [PDF]

				M. C. Noverr and G. B. Huffnagle Regulation of Candida albicans Morphogenesis by Fatty Acid Metabolites Infect. Immun., November 1, 2004; 72(11): 6206 - 6210. [Abstract] [Full Text] [PDF]

				P. Reddy, Y. Maeda, K. Hotary, C. Liu, L. L. Reznikov, C. A. Dinarello, and J. L. M. Ferrara Histone deacetylase inhibitor suberoylanilide hydroxamic acid reduces acute graft-versus-host disease and preserves graft-versus-leukemia effect PNAS, March 16, 2004; 101(11): 3921 - 3926. [Abstract] [Full Text] [PDF]

				C. Diakos, E. E. Prieschl, M. Saemann, V. Novotny, G. Bohmig, R. Csonga, T. Baumruker, and G. J. Zlabinger Novel Mode of Interference with Nuclear Factor of Activated T-cells Regulation in T-cells by the Bacterial Metabolite n-Butyrate J. Biol. Chem., June 28, 2002; 277(27): 24243 - 24251. [Abstract] [Full Text] [PDF]

				F. Leoni, A. Zaliani, G. Bertolini, G. Porro, P. Pagani, P. Pozzi, G. Dona, G. Fossati, S. Sozzani, T. Azam, et al. The antitumor histone deacetylase inhibitor suberoylanilide hydroxamic acid exhibits antiinflammatory properties via suppression of cytokines PNAS, February 20, 2002; (2002) 52702999. [Abstract] [Full Text] [PDF]

				M. D. Saemann, O. Parolini, G. A. Bohmig, P. Kelemen, P.-M. Krieger, J. Neumuller, K. Knarr, W. Kammlander, W. H. Horl, C. Diakos, et al. Bacterial metabolite interference with maturation of human monocyte-derived dendritic cells J. Leukoc. Biol., February 1, 2002; 71(2): 238 - 246. [Abstract] [Full Text] [PDF]

				F. Leoni, A. Zaliani, G. Bertolini, G. Porro, P. Pagani, P. Pozzi, G. Dona, G. Fossati, S. Sozzani, T. Azam, et al. The antitumor histone deacetylase inhibitor suberoylanilide hydroxamic acid exhibits antiinflammatory properties via suppression of cytokines PNAS, March 5, 2002; 99(5): 2995 - 3000. [Abstract] [Full Text] [PDF]

This Article Full Text (PDF) All Versions of this Article: 14/15/2380 00-0359fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by SÄEMANN, M. D. Articles by ZLABINGER, G. J. Search for Related Content PubMed PubMed Citation Articles by SÄEMANN, M. D. Articles by ZLABINGER, G. J.