Butyrate regulates the expression of pathogen-triggered IL-8 in intestinal epithelia - PubMed (original) (raw)
Butyrate regulates the expression of pathogen-triggered IL-8 in intestinal epithelia
Meiqian Weng et al. Pediatr Res. 2007 Nov.
Abstract
Inflammatory bowel disease (IBD) is characterized by an exaggerated immune response that involves pro-inflammatory cytokines including IL-8. Production of these pro-inflammatory cytokines is triggered by pathogen-associated molecular patterns (PAMP). Butyrate, a product of bacterial fermentation of carbohydrates, has been reported to modulate inflammation in IBD, possibly by regulating production of pro-inflammatory cytokines. However, this effect of butyrate is controversial. In this study, we used Pam3CSK4 (Pam3CysSerLys4), the acylated NH2-terminus of the bacterial lipoprotein (a PAMP), to mimic in vivo infection of pathogens. Butyrate transiently down-regulated expression of IL-8 stimulated by Pam3CSK4. Treatment of cells with butyrate before Pam3CSK4, however, enhanced production of IL-8. Furthermore, butyrate induced expression of A20, a negative regulator of the nuclear factor-kappaB pathway. Over-expression of A20 inhibited Pam3CSK4-triggered IL-8 expression. Our data suggest that the inflammatory modulation of butyrate in IBD is mediated by A20 and a short pulse rather than continuous administration of butyrate may provide a protective effect on IBD.
Figures
Figure 1
Pam3CSK4-evoked IL-8 secretion by Caco-2 cells mimics the in vivo response of intestinal epithelia to pathogens. Caco-2 cells were stimulated with Pam3CSK4 at a final concentration of 50 μg/mL for indicated times. Postnuclei supernatants were prepared for detection of pIκBα (A, n = 3), and culture media were collected for measurement of IL-8 by ELISA (B, n = 3). Data are represented as mean ± SD.
Figure 2
Butyrate transiently down-regulates Pam3CSK4-triggered IL-8 in intestinal epithelial cells. Caco-2 (A) or tsFHI (32°C, B) or SW480 (C) cells were stimulated with 50 μg/mL Pam3CSK4 in the presence (gray column) or absence (hatched column) of 5 mM (A and B) or 3 mM (C) butyrate for different times, or pretreated with 5 mM (A and B) or 3 mM (C) butyrate for 24 h before adding Pam3CSK4 stimulation (black column). After stimulation at the indicated times, the concentration of IL-8 in culture media was measured by ELISA and plotted. Data are represented as mean ± SD (n = 3, *p < 0.05).
Figure 3
A20 expression is altered by butyrate, and it inhibits IL-8 secretion. (A) Butyrate acts independently of pIκBα. Caco-2 cells were incubated with 5 mM butyrate for the indicated times. Postnuclei supernatants were prepared and analyzed by Western blotting with indicated antibodies. The result for 0 h is from the same experiment as in Figure 1_A_. (B) Butyrate induces the expression of A20. Caco-2 cells were treated with various concentrations of butyrate for 3 h. Total cell lysates were prepared for immunoblotting with antibody against A20 and normalized by GAPDH. (C-F) A20 gain- and loss-of-function regulate PamsCSK4-stimulated secretion of IL-8 from Caco-2 cells. (C) Expression of exogenous A20. Caco-2 cells were transfected with A20 plasmid or the corresponding empty vector (mock) for 24 h. Postnuclei supernatants were prepared for detection of exogenous A20 expression normalized by GAPDH. (D) Expression of exogenous A20 significantly inhibits Pam3CSK4-triggered IL-8 production. After transfection with A20 plasmid (gray column) or empty vector (white column) for 24 h, Caco-2 cells were stimulated with 50 μg/mL Pam3CSK4 for 18 h. The concentration of IL-8 in the culture medium was measured by ELISA and plotted (n = 3, mean ± SD, *p < 0.05). (E) Knockdown of endogenous A20 in Caco-2 cells. A20-specific or nonspecific siRNAs were delivered into Caco-2 cells by electroporation as described in “Materials and Methods.” After 24 h, postnuclei supernatants were prepared for Western blotting analysis with indicated antibodies. (F) A20 loss-of-function significantly increases Pam3CSK4-triggered IL-8 production, Caco-2 cells were transfected with A20-specific (gray column) or nonspecific siRNAs (white column) for 48 h and then stimulated by Pam3CSK4 for 18 h. The concentration of IL-8 in the culture medium was measured by ELISA and plotted (n = 3, mean ± SD, *p < 0.05).
Figure 4
Effect of butyrate on human fetal intestinal explants. (A) Butyrate decreases the expression of IL-8 in short incubations. Human fetal intestine explants were cultured in the presence or absence of 5 mM butyrate or 50 μg/mL Pam3CSK4 for 6 h. The culture medium was collected, and the secreted IL-8 was measured by ELISA and plotted (n = 3 pieces, mean ± SD, *p < 0.05; N/S, no significance). Shown are data from one patient. Similar results were observed in other two patients. (B) Butyrate enhances expression of A20 in human intestine explants. A segment of human fetal intestine (18 wk) was divided into three parts and cultured in the presence or absence of 5 mM butyrate for 6 h. Lysates were prepared and the post debris supernatants were analyzed by immunoblotting with antibody against A20, GAPDH was used as the loading control.
References
- Dotan I, Mayer L. Immunopathogenesis of inflammatory bowel disease. Curr Opin Gastroenterol. 2002;18:421–427. - PubMed
- Fusunyan RD, Quinn JJ, Ohno Y, Macdermott RP, Sanderson IR. Butyrate enhances interleukin(IL)-8 secretion by intestinal epithelial cells in response to IL-1β and lipopolysaccharide. Pediatr Res. 1998;43:84–90. - PubMed
- Huang N, Katz JP, Martin DR, Wu GD. Inhibition of IL-8 gene expression in Caco-2 cells by compounds which induce histone hyperacetylation. Cytokine. 1997;9:27–36. - PubMed
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