Recombinant human IL-1β and TNF-α were purchased from R&D Systems (Minneapolis, MN, USA), and human IFN-γ was obtained from Pepro Tech (Rocky Hill, NJ, USA). SCFAs (sodium acetate, sodium propionate and sodium butyrate) were purchased from Sigma Chemical Co. (Dorset, UK). Biotinylated anti-human IL-32α antibodies were purchased from R&D Systems, and horseradish peroxidase (HRP)-conjugated streptavidin was purchased from Dako Japan (Kyoto, Japan). Antibodies against phosphorylated and total Akt were obtained from Cell Signaling Technology (Beverly, MA, USA). Trichostatin-A was purchased from Tocris Cookson (St. Louis, MO, USA).

The human intestinal epithelial cell lines HT-29[16], SW480[17], and T84[18] were obtained from the American Type Culture Collection (Manassas, VA, USA). The cells were cultured as described previously[16-18].

Total RNA was isolated by the acid guanidinium thiocyanate-phenol-chloroform method, and was then reverse-transcribed into cDNA using a PrimeScript RT reagent kit (TAKARA-BIO, Shiga, Japan). The expression of human IL-32α mRNA was assessed by real-time polymerase chain reaction (PCR) analyses. Real-time PCR was performed using a LightCycler 2.0 system (Roche Applied Science, Tokyo, Japan). The oligonucleotide primers used in this study were specific for human IL-32α as follows: 5'-AGCTG- GAGGACGACTTCAAA [nucleotides 192-211, GenBank accession no. BC018782[19,20] and 3'-AGGTGGTGTCAGTATCTTCA (nucleotides 642-623)]. The PCR was conducted using a SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA). β-actin was used as an endogenous control to normalize for differences in the amount of total RNA in each sample.

The intracellular expression of IL-32α protein was determined by Western blotting. Briefly, the cells were washed and lysed in sodium dodecyl sulphate (SDS) sample buffer containing 100 μmol/L orthovanadate. The lysates were homogenized, and the protein concentration was measured by the Bradford method. For Western blotting, 10 μg of protein from each sample was subjected to SDS-PAGE on a 4%-20% gradient gel under reducing conditions. The proteins were then electrophoretically transferred onto a nitrocellulose membrane, and the membrane was blocked with 5% skimmed milk. After washing with PBS containing 0.1% Tween-20 (PBST), the membrane was incubated with a biotinylated anti-human IL-32α antibody at 4°C overnight. Next, the membrane was reacted with HRP-conjugated streptavidin at room temperature for 1 h. The detection was performed using enhanced chemiluminescence (ECL) Western blotting systems (Amersham Biosciences).

Histone extraction and the detection of global histone H3 acetylation were performed using the Epiquik global histone H3 acetylation assay kit (Epigentek; Brooklyn, NY, USA). The histone protein content was detected by the Bradford method.

Statistical significance of the differences was determined using unpaired t test (Statview version 4.5). Differences resulting in P values less than 0.05 were considered to be statistically significant.

To evaluate the effects of SCFAs on IL-32α mRNA expression, intestinal epithelial cells (HT-29, Caco-2, T84 cells) were stimulated with 10 mmol/L of each SCFA (acetate, propionate, and butyrate) for 12 h, and then the expression of IL-32α mRNA was determined by real-time PCR. Acetate had no effect, but butyrate significantly increased IL-32α mRNA expression in all cell lines. Propionate increased the expression of IL-32α mRNA only in T84 cells, but in the other cell lines it had no effect (Figure 1).

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Figure 1 Effects of short-chain fatty acids (SCFAs) on IL-32α mRNA expression in colon cancer cell lines. The cell lines (HT-29, SW480, T84) were incubated for 12 h with each SCFA (10 mmol/L). In another experiment, the cells were stimulated with IL-1β (50 ng/mL) in the presence or absence of each SCFA (10 mmol/L). The IL-32α mRNA expression was determined by real-time PCR. The data are expressed as mean ± SD (n = 4). ^aP < 0.05, ^bP < 0.01.

Next, we investigated the effects of SCFAs on cytokine-induced IL-32α mRNA expression. We recently reported that IL-1β, IFN-γ and TNF-α stimulate IL-32α mRNA expression in intestinal epithelial cells[8], and the cells were stimulated with cytokines in the presence or absence of each SCFA (10 mmol/L). In all cell lines, butyrate by itself stimulated IL-32α mRNA expression, and enhanced the IL-1β-induced IL-32α mRNA expression. SCFAs did not modulate IFN-γ- and TNF-α-induced IL-32α mRNA expression in any of the cell lines (data not shown).

To confirm the effects of SCFAs on IL-32α protein expression, the cells were stimulated for 48 h with IL-1β and/or SCFAs. Intracellular IL-32α was detected as a 25 kDa protein[8,21]. The effects of the SCFAs were similar to those observed at the mRNA level. The combination of IL-1β and butyrate enhanced IL-32α protein expression additively (Figure 2).

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Figure 2 Effects of SCFAs on IL-32α protein secretion in colon cancer cell lines. The cell lines (HT-29, SW480, T84) were stimulated for 48 h with IL-1β (50 ng/mL) in the presence or absence of butyrate (10 mmol/L), and the intracellular IL-32α protein levels were detected by Western blotting.

To examine the effects of butyrate on IL-1β-induced IL-32α expression more precisely, HT-29 cells were incubated for 12 h with IL-1β (50 ng/mL) plus increasing concentrations of butyrate. The effects of butyrate on IL-1β-induced IL-32α secretion appeared at the concentration of 5.0 mmol/L, and the most significant difference was observed at 10 mmol/L (Figure 3).

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Figure 3 Dose-dependent effects of butyrate on IL-1β-induced IL-32α mRNA expression in colon cancer cell lines. The cell lines (HT-29, SW480, T84) were stimulated for 12 h with increasing concentrations (0-10 mmol/L) of butyrate in the presence or absence of IL-1β (50 ng/mL). IL-32α mRNA expression was then analyzed by real-time PCR. The data are expressed as mean ± SD (n = 4). ^aP < 0.05, ^cP < 0.005.

We tested the effects of butyrate on the IL-1β-induced phosphorylation of Akt, which plays a major role in IL-32α induction[8,19]. As shown in Figure 4, there were no effects of butyrate on IL-1β-induced Akt phosphorylation. These observations suggest that the modulation of Akt activity is not involved in the actions of butyrate on IL-1β-induced IL-32α expression.

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Figure 4 Effects of butyrate on Akt phosphorylation. The cells were stimulated in the presence of butyrate (5 mmol/L) and/or IL-1β (50 ng/mL) for 15 min, and then Akt phosphorylation was determined by Western blotting.

Previous studies have demonstrated that butyrate exerts its biological effects through the induction of histone hyperacetylation[15]. To examine the relationship between histone acetylation and the effects of butyrate on IL-32α mRNA expression, we tested the effects of trichostatin-A (TSA), a potent histone deacetylase inhibitor[22-24].

The cells were stimulated with cytokines in the presence or absence of TSA (5 μmol/L), and the expression of IL-32α mRNA was then analyzed by real-time PCR. As shown in Figure 5, TSA enhanced the IL-1β-induced IL-32α mRNA expression in all cell lines.

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Figure 5 Effects of trichostatin-A (TSA) on IL-32α mRNA expression. The cells (HT-29, SW480, T84) were incubated for 12 h with IL-1β (50 ng/mL) in the presence or absence of TSA (5 μmol/L), and then the IL-32α mRNA expression was determined by real-time PCR. The data are expressed as mean ± SD (n = 4). ^aP < 0.05.

We tested the effects of butyrate and IL-1β on histone H3 hyperacetylation in HT-29 cells. As shown in Figure 6, butyrate induced histone H3 hyperacetylation in HT-29 cells (Figure 6).

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Figure 6 Effects of butyrate on histone H3 acetylation. HT-29 cells were stimulated for 12 h with IL-1β (50 ng/mL) in the presence or absence of butyrate (10 mmol/L). Histone H3 acetylation was then detected by histone H3 acetylation assay kits (Epigentek; Brooklyn, NY, USA). Histone H3 acetylation was expressed as a value relative to medium alone. The data are expressed as mean ± SD (n = 4). ^aP < 0.05, ^bP < 0.01.

Recently, interleukin (IL)-32 was defined as a proinflammatory cytokine characterized by the stimulation of secretion of IL-1β, tumor necrosis factor (TNF)-α, IL-6, and IL-8. Dietary fiber (nonstarch polysaccharides) and resistant starch escape digestion in the upper gastrointestinal tract, and undergo anaerobic bacterial fermentation in the colon. This process produces short-chain fatty acids (SCFAs), predominantly acetate, propionate, and butyrate, as the major by-products.

The authors have previously reported that IL-32α is overexpressed in inflammatory bowel disease (IBD). IL-32α was overexpressed in the inflamed mucosa of IBD patients and was strongly induced by IL-1β, IFN-γ and TNF-α. However, the role of IL-32α is yet to be elucidated. In this study, they investigated the in vitro effects of butyrate on IL-32α expression in human intestinal epithelial cell lines.

The authors observed that butyrate stimulates IL-32α expression in intestinal epithelial cells. Since IL-32α is considered to be a proinflammatory cytokine, their finding might be a part of the proinflammatory picture of butyrate in mucosal inflammatory responses in the intestine.

Clinical application of this study is limited because the role of IL-32α in IBD is still obscure. A recent report demonstrated that IL-32 augments the production of IL-1α, TNF-α, IL-6 and IL-8 by means of the nucleotide-binding oligomerization domain proteins (NOD1 and NOD2) through a caspase-1-dependent mechanism. NODs are a family of intracytoplasmic bacterial peptidoglycans which subsequently induce NF-κB activation. Mutations in NOD2 have been implicated in the pathogenesis of Crohn’s disease (CD). Recently, it has been shown that the NOD2 mutation in CD patients potentiates NF-κB activity and IL-1α processing. Thus, these findings suggest a pivotal role of IL-32 in the pathophysiology of IBD, and CD in particular. This study also augments the proinflammatory aspect of butyrate.

IL-32 was originally reported as natural killer (NK) transcript 4, and has been described as a cytokine produced mainly by T-lymphocytes, NK cells, epithelial cells, and blood monocytes. The gene encoding IL-32 is located on human chromosome 16p13.3 and is organized into eight exons. There are four splice variants, and IL-32α is reported as the most abundant transcript. Recently, IL-32 was defined as a proinflammatory cytokine characterized by the stimulation of secretion of IL-1β, TNF-α, IL-6, and IL-8 via the activation of p38 mitogen-activated protein kinases, nuclear factor (NF)-κB and activating protein-1 signal transduction pathways. Recently, the authors reported the overexpression of IL-32α in the inflamed mucosa of IBD. In vitro experiments on human intestinal epithelial cell lines showed that IL-32α expression was induced by IL-1β, IFN-γ and TNF-α through the activation of Akt-phosphatidylinositol 3-kinase.

Kobori et al present an interesting study of a novel pro-inflammatory cytokine IL-32. They investigated the effects of butyrate on IL-32α expression in epithelial cell lines.