The differential effects of 1,25-dihydroxyvitamin D3 on Salmonella-induced interleukin-8 and human beta-defensin-2 in intestinal epithelial cells

Abstract

Salmonellosis or Salmonella, one of the most common food-borne diseases, remains a major public health problem worldwide. Intestinal epithelial cells (IECs) play an essential role in the mucosal innate immunity of the host to defend against the invasion of Salmonella by interleukin (IL)-8 and human β-defensin-2 (hBD-2). Accumulated research has unravelled important roles of vitamin D in the regulation of innate immunity. Therefore, we investigated the effects of 1,25-dihydroxyvitamin D3 (1,25D3) on Salmonella-induced innate immunity in IECs. We demonstrate that pretreatment of 1,25D3 results in suppression of Salmonella-induced IL-8 but enhancement of hBD-2, either protein secretion and mRNA expression, in IECs. Furthermore, 1,25D3 enhanced Salmonella-induced membranous recruitment of nucleotide oligomerization domain (NOD2) and its mRNA expression and activation of protein kinase B (Akt), a downstream effector of phosphoinositide 3-kinase (PI3K). Inhibition of the PI3K/Akt signal counteracted the suppressive effect of 1,25D3 on Salmonella-induced IL-8 expression, while knock-down of NOD2 by siRNA diminished the enhanced hBD-2 expression. These data suggest differential regulation of 1,25D3 on Salmonella-induced IL-8 and hBD-2 expression in IECs via PI3K/Akt signal and NOD2 protein expression, respectively. Active vitamin D-enhanced anti-microbial peptide in Salmonella-infected IECs protected the host against infection, while modulation of proinflammatory responses by active vitamin D prevented the host from the detrimental effects of overwhelming inflammation. Thus, active vitamin D-induced innate immunity in IECs enhances the host's protective mechanism, which may provide an alternative therapy for invasive Salmonella infection.

Keywords: Salmonella; human beta-defensin-2; interleukin; intestinal epithelia; vitamin D.

Figure 1

The effect of 1,25‐dihydroxyvitamin D3 (1,25D3) on Salmonella‐induced interleukin (IL)−8 and human β‐defensin‐2 (hBD‐2) protein secretion in SW480 cells. SW480 cells were left uninfected (CON) or infected with the wild‐type S. Typhimurium strain SL1344 (SL) for 1 h in the presence or absence of 20 and 100 nM 1,25D3 (20D3 and 100D3). Supernatant was analysed by enzyme‐linked immunosorbent assay (ELISA) for IL‐8 and hBD‐2. The amount of IL‐8 and hBD‐2 produced is shown as the fold increase over uninfected control cells. Results are represented as means ± standard error of the mean (s.e.m.) for at least three determinations from independent experiments (*P < 0·05 for IL‐8; #P < 0·05 for hBD‐2, compared to Salmonella infection only).

Figure 2

The effect of 1,25‐dihydroxyvitamin D3 (1,25D3) on Salmonella‐induced interleukin (IL)−8 and human β‐defensin‐2 (hBD‐2) mRNA expression in SW480 cells. SW480 cells were left uninfected (CON) or infected with the wild‐type S. Typhimurium strain SL1344 (SL) for 1 h in the presence or absence of 20 and 100 nM 1,25D3 (20D3 and 100D3). Total RNA was prepared after infection and analysed by real‐time quantitative polymerase chain reaction (PCR) to estimate the amounts of IL‐8 and hBD‐2 transcript. The amounts of IL‐8 and hBD‐2 mRNA produced and normalized to the corresponding amount of the glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) transcript are shown as the fold increase over uninfected control cells. Results are represented as means ± standard error of the mean (s.e.m.) for at least three determinations from independent experiments (*P < 0·05 for IL‐8; #P < 0·05 for hBD‐2, compared to Salmonella infection only).

Figure 3

The involvement of phosphoinositide 3‐kinase/protein kinase B (PI3K/Akt) signal pathways in the negative regulation of 1,25‐dihydroxyvitamin D3 (1,25D3) on Salmonella‐induced interleukin (IL)−8 in SW480 cells. SW480 cells were transfected with control siRNA and Akt siRNA (siRNA = non‐target control siRNA; siAkt = siRNA to Akt) for 48 h. Knockdown of Akt was confirmed by Western blot (a). The transfected cells were left uninfected (CON) or infected with the wild‐type S. Typhimurium strain SL1344 (SL) for 1 h in the presence or absence of 1,25D3 (D3). Activation of Akt was analysed in whole cell protein by immunoblotting with antibodies to phosphorylated (p) Akt. The results shown are representative of three separate experiments. Glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) worked as a normalization of cytosolic protein (a). The relative band intensities of p‐Akt (white) and Akt (black) were quantified as fold increases compared with the control cells (b). Total RNA was prepared after infection and analysed by real‐time quantitative polymerase chain reaction (PCR) to estimate amounts of IL‐8 transcript (c). The amount of mRNAs produced and normalized to the corresponding amount of GAPDH transcript is shown as the fold increase over uninfected control cells. Results are represented as mean ± standard error of the mean (s.e.m.) for at least three determinations from independent experiments (*P < 0·05; #P < 0·01).

Figure 4

Involvement of nucleotide oligomerization domain (NOD2) in the enhancement of 1,25‐dihydroxyvitamin D3 (1,25D3) on Salmonella‐induced human β‐defensin‐2 (hBD‐2) expression in SW480 cells. SW480 cells were transfected with control siRNA and NOD2 siRNA (siRNA = non‐target control siRNA; siNOD2 = siRNA to NOD2) for 48 h. Knock‐down of NOD2 was confirmed by Western blot (a). The transfected cells were left uninfected (CON) or infected with the wild‐type S. Typhimurium strain SL1344 (SL) for 1 h in the presence or absence of 1,25D3 (D3). The Western blots illustrate the expression of NOD2 protein in membranous extracts of SW480 cells. The results shown are representative of three separate experiments. E‐cadherin worked as a normalization of membranous protein (a). The relative band intensities of NOD2 were quantified as fold increases compared with the control cells (b). Total RNA was prepared after infection and analysed by real‐time quantitative polymerase chain reaction (PCR) to estimate amounts the of hBD‐2 transcript. The amount of hBD‐2 mRNA produced and normalized to the corresponding amount of glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) transcript is shown as the fold increase over uninfected control cells (c). Results are represented as means ± standard error of the mean (s.e.m.) for at least three determinations from independent experiments (*P < 0·05).

Figure 5

1,25‐dihydroxyvitamin D3 (1,25D3) up‐regulates the nucleotide oligomerization domain (NOD2) mRNA expression on Salmonella‐infected SW480 cells. SW480 cells were uninfected (CON) or infected by wild‐type S. Typhimurium strain SL1344 for 1 h in the presence or absence of 1,25D3. Total RNA was prepared after infection and analysed by real‐time quantitative polymerase chain reaction (PCR) to estimate amounts of NOD2 transcript. The amount of NOD2 mRNA produced and normalized to the corresponding amount of glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) transcript is shown as the fold increase over uninfected control cells. Results are represented as means ± standard error of the mean (s.e.m.) for at least three determinations from independent experiments (*P < 0·05).