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Butyrate Switches the Pattern of Chemokine Secretion by Intestinal Epithelial Cells through Histone Acetylation
Molecular Medicine volume 5, pages 631–640 (1999)
Butyrate, a fermentation product of intestinal bacteria, modifies chromatin structure through histone acetylation, thereby altering gene transcription. EL-8 and MCP-1 are chemokines, expressed by intestinal epithelial cells, which attract neutrophils and monocytes, respectively. We hypothesized that butyrate may alter IL-8 and MCP-1 expression by intestinal epithelial cells through histone acetylation.
Materials and Methods
IL-8 and MCP-1 expression was measured by ELISA and RNA transfer blots. Acetylated histones were separated on acetic acid-urea-triton gels. Butyrate was compared to Trichostatin-A, a specific inhibitor of histone deacetylase and to other short chain fatty acids.
Caco-2 cells constitutively secreted MCP-1 but not IL-8. Butyrate reversibly decreased MCP-1 secretion. In contrast, butyrate increased IL-8 production. The effects of butyrate and Trichostatin-A were greater when cells were stimulated with EL-1β. Butyrate and Trichostatin-A both increased histone acetylation. Trichostatin-A and other short chain fatty acids altered chemokine secretion according to their effect on histone acetylation.
Butyrate reversibly switches chemokine secretion by epithelial cells through histone acetylation. We speculate that butyrate carries information from resident bacteria to epithelial cells. Epithelial cells transduce this signal through histone acetylation, modulating the secretion of chemokines.
The small intestinal epithelium regulates the absorption of nutrients and also serves as a barrier to the external environment (1). Epithelial cells may also participate actively in host defense mechanisms (2). They express class II major histocompatibility complex (MHC) molecules and they secrete chemokines in response to invasive pathogens (3,4). The location of the intestinal epithelium between the luminal environment containing bacterial products and the immuno-cytes of the mucosal lamina propria puts it in a strategic position to respond quickly to changes in the external environment to maintain host equilibrium.
Luminal short-chain fatty acids, such as acetate, propionate, and butyrate, are the principal products of bacterial fermentation of ingested carbohydrates (5) in the gastrointestinal tract. The concentrations of short-chain fatty acids largely depend on dietary intake and on the types of bacteria present (6). Short-chain fatty acids are not present in the intestinal lumen before birth, but reach adult levels by the age of 1 year (7). Among the short-chain fatty adds, butyrate is the principal source of energy for colonocytes. Butyrate also has a stimulatory effect on epithelial cell proliferation in vivo (8), which contributes to epithelial cell restitution and integrity. Butyrate concentrations are higher in the large intestine (5–24 mM) (5,9) than in the small intestine. However, concentrations of butyrate in the intestine increase significantly with bacterial overgrowth, as seen in short bowel syndrome and stagnant loops (10). Butyrate concentration is also increased in intestinal inflammation as seen in infants with necrotizing enterocolitis (11) and in patients with Crohn’s disease (9). Because short-chain fatty acid concentrations reflect bacterial colonization, they may alter the secretion of cytokines that control inflammation. Intestinal epithelial cells synthesize and secrete a number of chemokines, including interleukin-8 (IL-8) (3) and monocyte che-moattractant protein-1 (MCP-1) (12). IL-8 and MCP-1 are two potent members of a family of at least 30 distinct 8–14 kD chemokines. Their genes are located at chromosomes 4 and 17, respectively. IL-8 has chemotactic and activating properties for neutrophils. MCP-1 attracts and activates monocytes. We have shown that butyrate enhances the secretion of IL-8 by intestinal epithelial (Caco-2) cells stimulated with IL-1β (13). Its action on MCP-1 secretion has not been reported.
Changes in histone acetylation affect gene transcription by altering the charge on histones such that nucleosomes become less compact (14–16). Butyrate inhibits histone deacetylase activity, resulting in the increased acetylation of histones. The consequent destabilization on certain genes has been examined using DNase I sensitivity of defined regions of chromatin (17,18). We hypothesized, therefore, that short-chain fatty acids regulate IL-8 and MCP-1 expression and that this regulation involves histone acetylation.
Materials and Methods
Culture of Intestinal Epithelial (Caco-2) Cell Monolayers
Caco-2 cells are epithelial cells derived from human colon carcinoma which differentiate in vitro after reaching confluence (19). Caco-2 cells (American Type Culture Collection, Rockville, MD, passages 20–40) were plated into 12-well plates (Falcon, Becton Dickinson, Franklin Lakes, NJ) at an initial density of 5 × 104 cells/cm2. Caco-2 cells were grown in Dulbecco’s modified Eagle medium (DMEM) (Gibco-BRL, Gaithersburg, MD) containing 10% fetal bovine serum (FBS) (Hyclone Laboratories, Logan, UT), 2 mM glutamine (Gibco), 100,000 U/liter penicillin, and 100 mg/liter streptomycin (Gibco), 0.1 mM MEM nonessential amino acid (Gibco), and 10 mM N-2-hydroxyethylpiperazine-N’2-ethanesulfonic acid (HEPES) (Gibco) buffer solution in a humid atmosphere at 37°C and 5% CO2.
On day 7 after plating, the media were changed from media containing serum to media without serum (DTS). DTS medium was DMEM (Gibco) supplemented with 5 mg/liter iron-saturated human transferrin (Sigma, St. Louis, MO) and 5 µg/liter selenous acid (Na2SeO3; Collaborative Biomedical, Bedford, MA) but contained no serum (20). Caco-2 cells grow and differentiate normally in DTS medium (20). Experimental protocols were initiated on day 14, when cells were confluent.
Protocol of Incubation with Butyrate and Stimulation with IL-1β or Lipopolysaccharide
IL-8 AND MCP-1 SECRETION BY INTESTINAL EPITHELIAL CELLS IN DTS MEDIA CONTAINING BOTH SODIUM BUTYRATE AND/OR LIPOPOLYSACCHARIDE OR IL-1β. Caco-2 cells were incubated with varying concentrations of butyrate (0–20 mM) in DTS media without serum for 48 hr. No changes in the pH of the media were observed with the addition of butyrate. The epithelial cells were stimulated with 100 µg/ml lipopolysaccharide (LPS) (from Escherichia coli serotype 055:85, Sigma) or 1 ng/ml IL-1β (R&D Systems, Minneapolis, MN) during the last 24 hr. Experiments were also performed to examine IL-8 and MCP-1 mRNA accumulation within Caco-2 cells incubated with sodium butyrate (0–5 mM) for 24 hr and subsequently stimulated for 2 hr with 1 ng/ml IL-1 β or 100 µg/ml LPS.
To determine whether butyrate’s action on histone acetylation and MCP-1 production was reversible, butyrate was removed from the culture media after 24 hr incubation and replaced with fresh serum-free media. Histone acetylation and MCP-1 production in Caco-2 cells were determined at 0, 2, and 6 days after the removal of butyrate. At these times IL-1β was given and MCP-1 secretion was measured 24 hr later.
Assay of IL-8 and MCP-1 Secretion and mRNA Accumulation
MEASUREMENT OF IL-8 AND MCP-1 SECRETION. IL-8 and MCP-1 levels were quantitated in cultured supernatants by enzyme linked immunosorbent assay (ELISA) for IL-8 and MCP-1 (Quantikine™, R&D Systems). The culture supernatants from Caco-2 cells were collected, centrifuged at 800 rpm, and stored frozen at −20°C. The amount of IL-8 was quantified within each supernatant in duplicates. The maximum variation between duplicates was 2%. Colorimetric results were read on a Titertek Multiscan II 96-well plate reader at a wavelength of 450 nm. Quantitation of IL-8 and MCP-1 was determined using a standard calibration curve for each chemokine.
MEASUREMENT OF TOTAL CACO-2 CELL PROTEIN. The total protein content of cell monolayers was measured to express chemokine production per unit weight of cellular material. Deionized water was placed into wells containing adherent Caco-2 cells to detach the cells. They were immediately sonicated with an ultrasonic cell disrupter (Kontes microultrasonic cell disrupter, Vineland, NJ) to release cellular proteins. Protein was measured with the Bicinchonic Acid Protein Assay (Pierce BCA Protein Assay, Rockford, IL). Colorimetric results were read on a DU-70 spectrophotometer (Beckman Instrument, Wakefield, MA) at a wavelength of 562 nm and converted into protein concentration using an albumin standard calibration curve. The protein content within each well was measured in duplicates with an experimental variation of <5%.
CACO-2 CELL mRNA COLLECTION. CaCO-2 cells were cultured with sodium butyrate (0-–5 mM) for 24 hr followed by IL-1β for 2 hr. Caco-2 cells were homogenized and the RNA extracted, precipitated, washed, and collected according to the manufacturer’s recommendations as described in the procedures for RNA isolation using the Trizol Reagent (Gibco).
RNA TRANSFER BLOT ANALYSIS OF CACO-2 CELL mRNA. For RNA transfer blot analysis, 30 µg of purified total RNA from each well was electrophoresed in a 2% agarose gel containing 1.8% formaldehyde (Mallinckrodt, Paris, KY) and 20% 3-[N-morpholino]propanesulfonic acid (MOPS) (Sigma) and transferred to Gene Screen Plus™ membranes (Dupont/NEN, Boston, MA) by capillary action. The membranes were then baked, prehybridized, hybridized, with labeled IL-8 cDNA (a gift from Dr. Joost Oppenheim) or MCP-1 cDNA (12). Blots were washed in 0.5 × SSC and 0.5% SDS (Bio-Rad Laboratories, Hercules, CA) at 65°C. Washed blots were autoradiographed between intensifying screens at −70°C (13).
HISTONE EXTRACTION AND SEPARATION. Histones were extracted from Caco-2 cells according to Cousens and Alberts (21). Cells were plated at 5 × 104 cells/cm2 in 75-cm2 flasks for 14 days. After 24 hr incubation with either butyrate (5 mM), trichostatin-A (10 µM), or media alone, cells were removed and the nuclear protein harvested by centrifuging in MLB buffer (60 mM KCl/15 mM NaCl/3 mM MgCl2/15 mM piperazine-N/N’ bis|2 ethanesulfonic acid}, pH 6.5/0.1% Nonidet P-40/0.5 mM phenyl methyl sulfonyl fluoride/1 mM tetrathionate). The nuclear pellet was suspended in H2SO4 to a final concentration of 0.2 M for 2 hr. The suspension was centrifuged and the supernatants containing the histones removed. The dissolved histones were precipitated with alcohol at −20°C. The precipitant was suspended in water, quantified, and 50 µg of each sample loaded onto a triton X-acetic acid-urea gel for histone separation. Gels were stained with Coumassie blue and destained in methanol and acetic acid. The position of the histone-4 was identified with a histone-4 marker (Boehringer Mannheim). In addition to butyrate, the effect of other short-chain fatty acids and that of trichostatin-A, a histone deacetylase inhibitor (22), on histone acetylation were determined. Each of these reagents was given for 24 hr before adding IL-1β to the media.
Differences between sets of wells were examined by analysis of variance, using the Gabriel test for multiple comparisons. Results with a probability of <0.05 were considered significant.
Butyrate Differentially Regulates Secretion of IL-8 and MCP-1 from Caco-2 Cells
The intestinal epithelial cell line Caco-2 does not normally secrete IL-8, but it expresses MCP-1 (Fig. 1). This reflects the secretion of chemokines from epithelial cells in vivo (12). Butyrate increased the secretion of IL-8 in a dose-dependent manner. However, butyrate decreased the production of MCP-1 (Fig. 1). Thus, butyrate, a bacterial fermentation product, regulated the expression of IL-8 and MCP-1 by intestinal epithelial cells in different directions. In the absence of butyrate, LPS had no effect on IL-8 and MCP-1 secretion by Caco-2 cells (Fig. 2). However, with butyrate, LPS induced the secretion of IL-8, as previously described (13). In cells stimulated with LPS, butyrate also increased IL-8 and decreased MCP-1 secretion (Fig. 2).
IL-1β is a proinflammatory cytokine synthesized and secreted by macrophages in the intestinal mucosa during inflammation. IL-1β activated intestinal epithelial cells to secrete IL-8 and MCP-1 in increased amounts (Fig. 3). Again, butyrate increased IL-8 secretion and decreased MCP-1 secretion. Butyrate had a marked effect on chemokine secretion because IL-1β produced a strong stimulatory response on both chemokines. The differential regulation of butyrate on IL-8 and MCP-1, therefore, did not depend on whether cells were stimulated by LPS or IL-1β or had received no proinflammatory stimuli.
Butyrate Differentially Alters IL-8 and MCP-1 mRNA Accumulation in IL-1β-Stimulated Caco-2 Cells
To exclude the possibility that the differential effects of butyrate acted on the translation of chemokine mRNA or on a post-translational mechanism (such as chemokine secretion), we examined MCP-1 and IL-8 mRNA accumulation (Fig. 4). RNA was collected after 24 hr of butyrate (0–5 mM) followed by 2 hr of IL-1β. Chemokine mRNA accumulation mirrored the changes seen in chemokine secretion: IL-8 mRNA was increased by butyrate, whereas MCP-1 mRNA was decreased. Butyrate, therefore, differentially regulated the pattern of IL-8 and MCP-1 mRNA accumulation in intestinal epithelial (Caco-2) cells.
Trichostatin-A Induces Histone Acetylation in Caco-2 Cells and Increases IL-8 Secretion While Decreasing MCP-1 Secretion
Butyrate increased both the acetylation of histones and the secretion of IL-8 by epithelial cells when stimulated with BL-1β (Fig. 5). Butyrate down-regulated MCP-1 production at the same time (Fig. 5). We hypothesized that butyrate acted on IL-8 and MCP-1 secretion through its ability to induce histone acetylation by inhibiting histone deacetylase. To determine if histone acetylation increased IL-8 secretion and decreased MCP-1 production, we inhibited histone deacetylase by a separate mechanism. Trichostatin-A is a specific inhibitor of histone deacetylase and is 1000-fold more potent than butyrate. Addition of trichostatin-A to Caco-2 cells increased the acetylation of histones (Fig. 5). At the concentrations required to cause the degree of histone acetylation comparable to that of butyrate, trichostatin-A increased the secretion of IL-8 and decreased that of MCP-1 in cells stimulated with IL-lβ. Therefore, the differential effects of butyrate on the regulation of IL-8 and MCP-1 secretion can be explained by its action on histone acetylation.
Histone Acetylation and Down-Regulation of MCP-1 Induced by Butyrate Are Both Reversible
Fourteen days after plating, Caco-2 cells were incubated with butyrate for 24 hr, washed, and the media replaced with fresh DTS media. IL-1β was added, in different arms of the experiment, on days 1, 3, and 7 after butyrate incubation. Histone acetylation was also examined at these times. MCP-1 mRNA and MCP-1 secretion were examined at 2 and 24 hr, respectively, after IL-1β. As in earlier experiments, butyrate induced histone acetylation and down-regulated MCP-1 production in Caco-2 cells (Fig. 6). However, butyrate’s effect on histone acetylation had disappeared by day 3, 2 days after its removal from the culture media. At this time, MCP-1 protein secretion and MCP-1 mRNA were no longer inhibited. On day 7, stimulation with IL-lβ also induced MCP-1. Thus, the effects of butyrate on MCP-1 expression and histone expression were completely reversible.
Hexanoate (C6) were added to Caco-2 cells for 24 hr. The relative effect of butyrate on histone acetylation was quantified by measuring the densitometry of the acetylated histone-4 as a ratio of unacetylated histone-4 in the sample (Fig. 7). The effect of each short-chain fatty acid on chemokine expression was simultaneously studied. Propionate, butyrate, and valerate altered the pattern of IL-8 and MCP-1 secretion (Fig. 7). Moreover, they altered chemokine secretion according to the degree by which histones were acetylated (Fig. 7). There was no relation between the energy content of any particular short-chain fatty acid and its effects on chemokine expression.
The intestinal lumen contains a multitude of antigenic and proinflammatory agents derived from the diet and from the microflora. Cells of the mucosal immune system play a crucial role in initiating, activating, and perpetuating inflammatory and immune mechanisms. The epithelium, therefore, serves as an important barrier separating the luminal contents from immunocytes (1). However, an impenetrable barrier would inhibit the mucosal immune system from surveying the constituents of the lumen. M cells, which allow the controlled passage of antigens and microbial agents, permit sampling of the intestinal lumen (23). Release of cytokines by the epithelium in response to luminal factors represents an additional process by which intestinal contents can be surveyed (24). Although the microflora alter the expression of enzymes that regulate epithelial cell surface molecules (25), little attention has been paid to the effect of normal luminal bacteria on cytokine expression. It seems likely that the intestine responds to variations in resident bacteria by subtle changes in its immunoregulation. Such changes would be smaller than inflammatory responses to invasive pathogens.
In our experiments, we examined the effects of the bacterial product butyrate on IL-8 and MCP-1 production in the cultured intestinal epithelial cell line Caco-2. Butyrate increased the secretion of IL-8 by Caco-2 cells and decreased the production of MCP-1 (Fig. 1) in a dose-dependent fashion. Moreover, when the cells were subsequently stimulated with IL-1β, a proinflammatory cytokine secreted by macrophages in response to inflammatory stimuli, a marked differential response of IL-8 and MCP-1 was observed (Fig. 3). Similar patterns were observed in the accumulation of mRNA for both of these chemokines (Fig. 4). These observations were not restricted to Caco-2 cells. In H4 cells, a non-malignant human intestinal epithelial cell line derived from the fetus, butyrate inhibited MCP-1 (R.D. Fusunyan et al., unpublished data) and increased IL-8 secretion (13) in cells stimulated with IL-1β.
In our experiments (Fig. 1), the changes in IL-8 and MCP-1 were small in the absence of a proinflammatory stimulus. This is consistent with the observation that short-chain fatty acids do not normally induce neutrophil in vivo [except at high concentrations (26) where, in mice, inflammatory changes were observed]. On the other hand, the increase in IL-8 production in cells (Fig. 3) receiving a proinflammatory stimulus was large in the presence of butyrate. Thus, only in the presence of inflammation would butyrate markedly alter epithelial cell IL-8 expression. The increased butyrate concentrations reported in both Crohn’s colitis (9) and necrotizing enterocolitis (11) may therefore contribute to the inflammatory changes.
We examined IL-8 secretion into the lumen in normal subjects with no evidence of intestinal inflammation (27). Rectal dialysates were collected by inserting bags made from dialysis tubing for 4 hr. Low levels of IL-8 were detected in the dialysate. However, similar experiments performed on patients with colitis showed greater IL-8 secretion. The IL-8 collected induced neutrophil activation. Short-chain fatty acid enemas have been used therapeutically in patients with diversion colitis (28) and distal ulcerative colitis with limited success (29), but they have not been beneficial to patients with Crohn’s disease. Instead, elemental diets that are low in fiber (an important substrate for short-chain fatty acids) trophil activation. Short-chain fatty acid enemas have been used therapeutically in patients with diversion colitis (28) and distal ulcerative colitis with limited success (29), but they have not been beneficial to patients with Crohn’s disease. Instead, elemental diets that are low in fiber (an important substrate for short-chain fatty acids) decrease Crohn’s disease activity (30) and reverse growth retardation in children (31). This observation is consistent with the assertion that butyrate may enhance inflammation in Crohn’s disease. Furthermore, the benefits of short-chain fatty acids in ulcerative colitis do not conflict with the data presented in this report. Butyrate is a nutrient of the colonic epithelial cell; it reduces epithelial permeability (32) and facilitates salt and water absorption in the colon. Ulcerative colitis is characterized by changes in epithelial cell morphology and often loss of surface epithelial cells. At early stages of the disease, when injury to epithelial cells is greatest, the benefit of butyrate is statistically significant (33).
The effects of butyrate on cultured epithelial cells were reversible (Fig. 6), both in terms of histone acetylation and their effects on the inhibition of MCP-1. The stimulation of IL-8 by butyrate was also reversed (R.D. Fusunyan et al., unpublished data). These data support the assertion that the changes observed with short-chain fatty acids were an active response, and not the result of cell damage. Furthermore, the distinct direction in which the two chemokines alter makes apoptosis (34) or other causes of cell death an unlikely trigger for these phenomena.
The switching of chemokines from IL-8 to MCP-1 by butyrate was mimicked by trichostatin-A (Fig. 5). This compound has no chemical similarity to short-chain fatty acids. It is 1000-fold more potent than butyrate in inhibiting histone deacetylase (22). At the concentrations at which trichostatin-A induced the same effect on histone acetylation as butyrate, it induced a similar response in chemokine regulation (Fig. 5). In addition, other short-chain fatty acids varied the expression of chemokines according to their ability to induce histone acetylation (Fig. 7). We also investigated the effect of glutamine, another intestinal epithelial cell nutrient. Glutamine had no effect on IL-8 production by Caco-2 cells. The effects of short-chain fatty acids, therefore, were specific and not related to the delivery of energy to the epithelial cell. Furthermore, IL-1β alone did not induce histone acetylation (unpublished data). These data, therefore, support the hypothesis that short-chain fatty acids switch chemokine expression through their effects on histone acetylation. Nevertheless, demonstrating that histone acetylation alters chemokine secretion does not exclude other mechanisms of gene regulation involving regulatory elements of chemokine promoters. Stability of RNA could, in theory, also be altered by butyrate; however, butyrate did not alter the stability of mRNA for another chemokine, macrophage inflammatory protein-2, in a rat intestinal epithelial cell line (35).
Evidence is accumulating that chromosomal proteins are important in regulating transcription (36). Recently, the involvement of histone tails in binding to DNA has been inferred from experiments in which histones have been thermally denatured (37). In vivo, changes in the charges of histones are normally the result of acetylation. The hydrogen atom in the free lysine group of the histones is normally substituted by an acetyl group. This reduces the net positive charge on the histone molecule, resulting indirectly in the destabilization of the chromatin structure (38).
The question arises as to how butyrate can have distinct effects on different genes. The variability of butyrate in increasing transcription in any particular gene can be explained by the proximity of the gene to a“scaffold attachment region”(39). Such regions, rich in AT sequences, form the basis by which DNA is bound to non-chromosomal structures in the nucleus. Studies on plasmids containing the IFNβ gene (40) linked to a scaffold attachment sequence showed that close proximity to such a sequence results in a much greater sensitivity of transcription to butyrate. In this case, adding scaffold attachment sequences to the DNA containing the IFNβ gene increased the enhancing effect from 2-fold to 80-fold. Inhibition of gene expression, however, cannot be explained by the involvement of scaffold attachment regions, and different mechanisms responsible for the down-regulation of MCP-1 must be inferred. It has been suggested that butyrate may inhibit genes whose promoters include a κB sequence (41), by up-regulating the inhibitor IκB. However, changes in IκB cannot explain the distinct effects of butyrate on IL-8 and MCP-1 because the promoters of both genes contain a κB element. Regulation through NFκB should, therefore, induce changes in expression in the same direction. IL-1β, for example, acts through the κB site; and both IL-8 and MCP-1 are up-regulated by its actions (Figs. 1 and 3). It seems likely, therefore, that an as-yet unidentified inhibitor is up-regulated by butyrate and acts on the MCP-1 promoter.
In conclusion, we have shown for the first time that butyrate switches the expression of chemokines by intestinal epithelial cells. Its effects are mediated through histone acetylation. We believe we have defined a mechanism in human intestinal epithelial cells by which resident bacteria may regulate inflammatory processes within the intestine.
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This work was supported by grants DK47753, AI 43472, DK21474, DK40561, DK43351, DK08894, and DK02481 from the National Institutes of Health; the K.C. Irving Family Research Fellowship Fund; Lahey-Hitchcock Clinic Foundation; and the Hood Foundation. Dr. Ian Sanderson was also supported by the Crohn’s and Colitis Foundation of America. The authors are grateful to Dr. David Schoenfeld for his advice with statistical analyses.
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Fusunyan, R.D., Quinn, J.J., Fujimoto, M. et al. Butyrate Switches the Pattern of Chemokine Secretion by Intestinal Epithelial Cells through Histone Acetylation. Mol Med 5, 631–640 (1999). https://doi.org/10.1007/BF03402075
- Induce Histone Acetylation
- Chemokine Secretion
- Intestinal Epithelial Cells
- Short-chain Fatty Acids
- Resident Bacteria