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. 2016 Apr 12;185(1):98–106. doi: 10.1111/cei.12792

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

F‐C Huang 1,✉
PMCID: PMC4908287  PMID: 26990648

Summary

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: human beta‐defensin‐2, interleukin, intestinal epithelia, Salmonella, vitamin D

Introduction

The incidence of food‐borne human infections caused by Salmonella enteritidis and by multi‐drug‐resistant strains of Salmonella enterica serovar Typhimurium (S. Typhimurium) has increased substantially 1, with similar trends being reported from Europe 2 and Taiwan 3. Intestinal epithelial cells serve as not only a barrier to bacteria colonizing the gut but rather as an integral and essential component of the innate mucosal immune system of the host through its secretion of inflammatory cytokines, chemokines [interleukin (IL)‐8] and anti‐microbial peptides (human β‐defensins) to defend against the invasion of Salmonella. IL‐8 recruits neutrophils from the circulation into the subepithelial region to defend against the invasion of Salmonella. However, the accumulation of neutrophils gives rise to characteristic pathological changes of colitis 4.

We have demonstrated previously that PI3K/Akt recruited to the Salmonella‐containing vesicle membrane played an anti‐inflammatory effect on Salmonella‐induced IL‐8 5. Nucleotide‐binding oligomerization domain‐containing protein 2 (NOD2) mediates induction of the human beta‐defensin‐2 (hBD‐2) in epithelial cells 6, leading to the killing of Salmonella 7.

Vitamin D deficiency has been correlated with increased rates of infection. Since the early 19th century, both environmental (i.e. sunlight) and dietary sources (cod liver) of vitamin D have been identified as treatments for TB. The recent discovery that vitamin D induces anti‐microbial peptide gene expression 8, 9, 10 explains, in part, the ‘antibiotic’ effect of vitamin D and has greatly renewed interest in the ability of vitamin D to improve immune function. In a study with sepsis patients, an association was found between severe illness and lower 25(OH)D, defensin and cathelicidin levels compared with healthy controls 11. 1,25‐dihydroxyvitamin D3 (1,25D3), the active form of vitamin D, can enhance significantly the production of LL37 from the urinary bladder epithelium during uropathogenic Escherichia coli infection 12 and may influence susceptibility to urinary tract infection. Vitamin D may play a role in protecting against infection during pregnancy and bacterial vaginosis 13. Thus, supplementation with active vitamin D could provide a novel strategy to reduce antibiotic use among patients at risk and indirectly prevent the emerging epidemic of bacterial resistance.

Vitamin D is an important mediator of intestinal epithelial defence against infectious agents. Vitamin D deficiency predisposes to more severe intestinal injury in an infectious model of colitis 14; however, the mechanism is unknown. Recent research has begun to unravel important roles of vitamin D in the regulation of innate immunity 15. In response to bacterial pathogens, the innate immune response includes the production and release of anti‐microbial peptides 16. 1,25D3 induces corresponding increases in anti‐microbial peptides and secretion of anti‐microbial activity against Pseudomonas aeruginosa 16. Calcitriol, active vitamin D, inhibits IL‐6 and IL‐8 expression in human nasal polyp fíbroblast cultures 17. Induction by 1,25D3 of the NOD2‐defensin‐2 has been demonstrated in human monocytic and epithelial cells 10, leading to anti‐bacterial responses. Therefore, we investigated the effects of active vitamin D on Salmonella‐induced IL‐8 and hBD‐2 expression in intestinal epithelial cells (IECs).

Materials and methods

Bacterial strains

The wild‐type S. Typhimurium strain used in this work was SL1344. Salmonella inoculum was prepared as described previously 18.

Cell culture and infection

SW480 and SW620 cells were purchased from the American Type Culture Collection (Manassas, VA, USA) and were cultured as described previously 7 or as recommended by the manufacturer. Aliquots of bacterial suspension (25 or 50 μl; (1–2) × 108 bacteria) were used to infect the cells. The bacterial inoculum was adjusted to a bacteria to cell ratio of 200 : 1.

Reagents

Standard laboratory reagents were obtained from Sigma (St Louis, MO, USA) or Fisher Scientific (Pittsburgh, PA, USA). 1,25‐dihydroxyvitamin D3 (1,25D3) (Biomol Research Laboratories, Plymouth, PA, USA) was stored as a stock solution in pure ethanol at −20°C in the dark.

Cytokine assays

IL‐8 and hBD‐2 were measured in the culture supernatants by enzyme‐linked immunosorbent assay (ELISA), according to the manufacturer's instructions, and modified as described previously 19.

Cell fractionation

Cytosolic, membranous and nuclear extracts from untreated and treated cultured cells were prepared by the method described previously 18. Protein concentrations in cell fractions were determined using a Bio‐Rad assay kit and normalized before loading for Western blot.

Western blotting

Equal amounts of total protein from cultured cells were separated by sodium dodecyl sulphate‐polymerase gel electrophoresis (SDS‐PAGE) and then transferred to nitrocellulose membranes by semi‐dry blotting as described previously 18. After blocking the membranes with 5% non‐fat dry milk, they were probed with antibodies to either phosphorylated Akt (Cell Signaling Technology, Danvers, MA, USA) or NOD2 (Labome Org., Princeton, NJ, USA), and then developed with horseradish peroxidase‐conjugated second antibodies (Zymed Laboratories, San Francisco, CA, USA) and enhanced chemiluminescence (Pierce Chemical Co., Rockford, IL, USA). Appropriate exposures to X‐ray film were made, and the filters then stripped and reprobed with antibodies to total glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) or E‐cadherin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), as appropriate.

RNA isolation and cDNA synthesis

Total RNA was prepared from control or infected cells with the Trizol reagent (Invitrogen Corporation, Carlsbad, CA, USA), following the manufacturer's directions. The RNA was reverse‐transcribed with random hexamers using the GeneAmp kit (Roche Diagnostics, Nutley, NJ, USA), as described in detail previously 19.

Real‐time reverse transcription–polymerase chain reaction (RT–PCR)

Real‐time RT–PCR analyses were performed in a fluorescence temperature cycler (LightCycler; Roche Diagnostics), as described previously 19, 20, to determine the IL‐8 and hBD2 mRNA expression levels using the comparative threshold cycle (△△Ct) method of relative quantitation.

RNA interference (RNAi) in cultured cells

RNAi experiments in cultured cells were performed as described previously 19, 20. Cells were transfected according to the manufacturer's protocol, which was modified in our laboratory. Briefly, cells were transfected with protein kinase B (Akt) siRNA (Santa Cruz Biotechnology, Dallas, TX, USA), NOD2 siRNA (Invitrogen Corporation, CA, USA) or control RNA duplex (Santa Cruz Biotechnology, TX, USA) by lipofectamine RNAiMAX (Invitrogen Corporation, CA, USA). After 48–72 h incubation in a 37°C incubator, the cells were then infected by bacteria. Then, the cells were lysed and RNA or proteins extracted over ice for further experiments. All siRNA were tested and verified as reducing expression by > 80% protein reduction in cells by immunoblot analysis or reducing expression of > 50% of mRNA by real‐time PCR.

Gentamicin protection assay

SW480 cells were pretreated and infected, and gentamicin protection assays were undertaken as described previously 20.

Cell viability and morphological features

Representative cell populations from each condition were examined under light microscopy. No significant morphological change was observed under any condition.

Cell viability in untreated or treated cells was found to be > 90% as analysed by trypan blue exclusion (data not shown).

Statistical analysis

All the above experiments were carried out at least in triplicate with similar results. Statistical analysis was performed using the paired Student's t‐test and analyses of variance (anovas) (StatView; SAS Institute, Cary, NC, USA). P‐values < 0·05 were considered significant.

Results

1,25D3 suppressed Salmonella‐induced IL‐8 secretion while it enhanced hBD‐2 secretion in SW480 cells

SW480 cells, similar to SW620 cells, originate from human colorectal adenocarcinoma. Because their physiology is similar to IECs, they are usually used as IECs for in‐vitro study. The cultured cells were uninfected or infected by wild‐type S. Typhimurium strain SL1344 for 1 h in the presence or absence of 1,25D3. Supernatant of cultured cells was analysed by ELISA for IL‐8 and hBD‐2 secretion. As shown in Fig. 1, SL1344 infection induced IL‐8 and hBD‐2 protein secretion in SW480 cells. 1,25D3 suppressed Salmonella‐induced IL‐8 production while it enhanced hBD‐2 production.

Figure 1.

Figure 1

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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).

1,25D3 suppressed Salmonella‐induced IL‐8 mRNA expression while it enhanced hBD‐2 mRNA expression in SW480 cells

We then examined the effect of 1,25D3 on Salmonella‐induced IL‐8 and hBD‐2 mRNA levels. SW480 cells were either uninfected or infected by wild‐type S. Typhimurium strain SL1344 for 1 h in the presence or absence of 1,25D3. Total RNA was analysed by real‐time quantitative PCR (RT–qPCR) for IL‐8 and hBD‐2 mRNA expression. As shown in Fig. 2, SL1344 infection induced IL‐8 and hBD‐2 mRNA expression after 1 h of infection in SW480 cells. As in protein secretion, Salmonella‐induced IL‐8 mRNA expression in SW480 cells was suppressed by 1,25D3 while hBD‐2 mRNA was enhanced in the presence of 1,25D3.

Figure 2.

Figure 2

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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).

Intracellular bacterial count is diminished in Salmonella‐infected SW480 cells in the presence of 1,25D3

To investigate if 1,25D3 enhances the bactericidal effect and decreases the intracellular bacteria in Salmonella‐infected IECs, SW480 cells were infected by S. typhimurium wild‐type strain SL1344 in the presence or absence of 1,25D3. Gentamicin protection assay was performed as in the Experimental section. As demonstrated in Supporting information, Fig. S1, 1,25D3 diminished the intracellular bacterial count in SW480 cells compared to the infection‐only or vehicle‐treated cells.

The involvement of PI3K/Akt signalling pathway in negative regulation of 1,25D3 on Salmonella‐induced IL‐8 in SW480 cells

To evaluate further which signalling pathway was involved in regulatory effects of vitamin D on Salmonella‐induced IL‐8 and hBD‐2 regulation, we investigated the intracellular signal pathways. Based on our previous study, showing that PI3K/Akt plays a negative regulator on Salmonella‐induced IL‐8 expression in IECs 5, we investigated the involvement of PI3K/Akt on the negative regulation of vitamin D on Salmonella‐induced IL‐8. SW480 cells were uninfected or infected wild‐type S. Typhimurium strain SL1344, and activation of the Akt, a downstream effector of PI3K, was analysed in whole cell protein by Western blot. Western blot data showed that Salmonella‐induced phosphorylated Akt (p‐Akt) was up‐regulated significantly by 1,25D3 (Fig. 3a & 3b), suggesting the involvement of Akt in the regulatory effect of vitamin D. To verify the role of PI3K/Akt on the regulatory effects of 1,25D3 on Salmonella‐induced IL‐8 expression, Akt siRNA‐transfected SW480 cells were infected by wild‐type S. Typhimurium strain SL1344 in the presence or absence of 1,25D3. Knock‐down of Akt was confirmed by Western blot (Fig. 3a). Following knock‐down of Akt, we detected that the 1,25D3‐mediated suppression of Salmonella‐induced IL‐8 mRNA was abolished almost completely in Akt‐silenced cells compared to control siRNA‐silenced cells (Fig. 3b). This suggests the involvement of Akt in the negative regulation of 1,25D3 on Salmonella‐induced IL‐8 expression.

Figure 3.

Figure 3

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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).

Involvement of NOD2 in enhancement of 1,25D3 on Salmonella‐induced hBD‐2 expression in cultured cells

NOD2 serves as an intracellular pattern recognition receptor to enhance host defence by inducing the production of anti‐microbial peptides such as hBD‐2 6. It was demonstrated that the membranous recruitment of NOD2 was induced by wild‐type S. Typhimurium strain SL1344 infection and 1,25D3 enhanced membranous NOD2 protein expression (Fig. 4a & 4b). Based on our observation that membranous NOD2 protein was up‐regulated after 1,25D3 treatment, we investigated if NOD2 was involved in the regulation of 1,25D3 on Salmonella‐induced hBD‐2 expression by NOD2 siRNA. Knock‐down of NOD2 was confirmed by Western blot (Fig. 4a). Control siRNA‐ or NOD2 siRNA‐transfected SW480 cells were left uninfected (CON) or infected with the wild‐type S. Typhimurium strain SL1344 in the presence or absence of 1,25D3. Following knock‐down of NOD2, we observed that the enhancement of 1,25D3 on Salmonella‐induced hBD‐2 mRNA expression in SW480 cells was abolished almost completely in NOD2‐silenced cells (Fig. 4b), but not in control non‐silenced cells. Therefore, specific suppression by siRNA targeting NOD2 diminished the enhancement of 1,25D3 on Salmonella‐induced hBD‐2 expression.

Figure 4.

Figure 4

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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).

1,25D3 up‐regulates NOD2 mRNA expressions in Salmonella‐infected SW480 cells

In order to explore if 1,25D3 up‐regulated NOD2 mRNA expression in Salmonella‐infected SW480 cells, cultured cells were left uninfected or infected with the wild‐type S. Typhimurium strain SL1344 for 1 h in the presence or absence of 1,25D3 and RT–PCR was performed to detect NOD2 mRNA expression. We demonstrate clearly in Fig. 5 that 1,25D3 had a synergistic effect on Salmonella‐induced NOD2 mRNA expression in SW480 cells, although 1,25D3 itself could induce NOD2 mRNA expression.

Figure 5.

Figure 5

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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).

A general phenomenon observed in SW620 intestinal epithelial cell lines

To determine whether the above finding was a general phenomenon among different intestinal epithelial cell lines, the same experiments were undertaken in SW620 cells. SW620 cells were untreated or pretreated by 1,25D3 and infected with wild‐type S. Typhimurium strain SL1344, using the same experiments as above. The isolated total RNA was analysed for mRNA expression in SW620 cells. The proteins extracted from the cells were analysed for signalling pathways involved in the regulation. Similar to SW480 cells, the same results were observed in SW620 cells in response to infection. Pretreatment of 1,25D3 results in suppression of Salmonella‐induced IL‐8 but enhancement of hBD‐2 mRNA expression in SW620 cells (Supporting information, Fig. S2). 1,25D3 enhanced Salmonella‐induced membranous recruitment of NOD2 and activation of Akt (Supporting information, Fig. S3).

Discussion

Active vitamin D was effective in ameliorating dextran sulphate sodium (DSS)‐induced acute colitis, gauged by reducing clinical symptoms, decreasing macroscopic and histological inflammation, enhancing epithelial cell resistance to injury and suppressing proinflammatory responses to luminal antigens 21 and mediating intestinal epithelial defences against infectious agents 10.

However, the effect of active vitamin D on Salmonella infection and its underlined mechanisms has been rarely reported. Clinically, gastrointestinal infections with pathogens, like Salmonella, can trigger or increase the likelihood that a patient will subsequently develop inflammatory bowel disease 22. The colon is relatively insensitive to circulating concentrations of 1,25D3 and the strongest gene enhancement occurs when the hormone reaches the colon via the lumen of the intestinal tract 23. These findings have broad implications for the use of oral active vitamin D compounds in colon disorders.

1,25D3 down‐regulates the expression of many proinflammatory cytokines, such as IL‐1, IL‐6, IL‐8 and tumour necrosis factor (TNF)‐α, in a variety of cell types 24. Human microvessel endothelial cells treated with 1,25D3 showed inhibited lipopolysaccharide (LPS) activation of nuclear factor (NF)‐κB and expression of IL‐6 and IL‐8 25. In contrast, 1,25D3 increases the expression of IL‐8 in a human proximal tubule cell line. However, there is no known report on the regulatory effect of vitamin D on Salmonella‐induced innate immunity in IECs. We demonstrated that active 1,25D3 suppressed Salmonella‐induced IL‐8 in IECs, which may prevent the detrimental effect of IL‐8 on intestinal mucosa leading to colitis. 1,25D3 has also been found to up‐regulate human β‐defensin 2 in a variety of human cells 16. Pretreatment with 1,25D3 induced synergistically NF‐κB function and expression of genes encoding DEFB2/HBD2 and anti‐microbial peptide cathelicidin in the presence of muramyl dipeptide 10. We observed that active 1,25D3 enhances anti‐microbial peptide hBD‐2 but decreases proinflammatory IL‐8 expression in IECs. This suggests that 1,25D3 may play a protective role in mucosal Salmonella‐induced inflammatory injury by defending against the bacterial infection and suppressing proinflammatory responses to luminal bacterial infection. Our observation suggests that oral 1,25D3 may have therapeutic potential for Salmonella infection and inflammatory bowel disease.

Using peripheral mononuclear cells and monocyte‐derived dendritic cells from Crohn's disease (CD), differential effects of vitamin D on NOD2‐ and Toll‐like receptor (TLR)‐induced cytokines in Crohn's disease were observed 26. TLR‐5‐mediated PI3K/Akt activation negatively regulates flagellin‐induced proinflammatory gene expression, including IL‐6 and IL‐8 27. TLR‐2‐induced PI3K/Akt‐mediated anti‐apoptosis controls mucosal inflammation by regulating epithelial barrier function 28. The observation in this study that 1,25D3 enhanced Salmonella‐induced phosphorylation of Akt in cultured cells, along with our previous study 5 demonstrating PI3K/Akt mediated anti‐inflammatory effect on Salmonella‐induced IL‐8 production in IECs, suggests strongly the involvement of PI3K/Akt in the suppressing effect of 1,25D3 on Salmonella‐induced IL‐8 in cultured cells. Additionally, PI3K/Akt mediated the anti‐apoptotic effect on intestinal epithelial cells to enhance epithelial cell resistance to injury 29. A healthy and intact mucosal barrier prevents bacterial invasion and thus reduces mucosal inflammation. Excess IEC apoptosis causes focal disruption of the mucosal barrier independently of the tight junction. Vitamin D restrains macrophage‐mediated inflammatory processes by suppressing the Akt/NF‐κB/cyclooxygenase 2 (COX‐2) pathway 30, suggesting that vitamin D supplementation might be utilized for adjunctive therapy for inflammatory disease. Indeed, increased IEC apoptosis has been reported in patients with ulcerative colitis (UC) and CD 31, 32, as well as in murine models of colitis 33. Conversely, similar to the reports that 1,25D3 is both a direct and indirect inducer of the NOD2–HBD2 innate immune pathway 10, 16, we observed that 1,25D3 itself up‐regulated synergistically the Salmonella‐induced NOD2 mRNA expression and recruitment of the protein into membrane in IECs, even though Salmonella infection only did not obviously induce NOD2 mRNA expression. We observed that active 1,25D3 provides the different effects on Salmonella‐infected IECs via NOD2 and TLR downstream signal pathway PI3K/Akt, which is novel for clinical use.

Intestinal epithelial vitamin D receptor (VDR) signalling protects the integrity of the mucosal barrier by inhibiting inflammation‐induced epithelial cell apoptosis. Dysfunction of the barrier leads to increased translocation of luminal substances to the lamina propria, triggering an inflammatory response 34. Colonic epithelial VDR levels are markedly reduced in patients with inflammatory bowel diseases or in experimental colitis models, whereas vitamin D analogue therapy that ameliorates colitis up‐regulates epithelial VDR. Calcitriol could up‐regulate VDR expression in podocytes in diabetic nephropathy rats, leading to the up‐regulation of the nephrin‐PI3K/Akt signalling pathway and the renoprotective effect 35. Currently we are investigating the role of VDR in mucosal innate immunity under invasive Salmonella infection.

Higher vitamin D concentrations have been proposed as a protective ‘seasonal stimulus’ against influenza, and there are suggestions for associations with other aspects of respiratory health. A study 36 has suggested that vitamin D status has a linear association with seasonal infections and lung function in British adults. Endoscopic studies in humans have demonstrated that β‐defensin is secreted in the gastric mucosa after infection by Helicobacter pylori 37. According to our observation that higher concentration of 1,25D3 enhanced higher levels of Salmonella‐induced hBD‐2 expression and epithelial anti‐microbial peptides play an important role in determining the outcome of the host–pathogen interaction at the mucosal barrier 38, oral intake of vitamin D may up‐regulate hBD‐2 secretion in the gastric mucosa against H. pylori infection. Low serum vitamin D levels may further increase a CD manifestation by compromising the efficacy of vitamin‐D‐induced NOD2 expression in the gastrointestinal tract. However, patients receiving vitamin D supplementation might not have a significant rise in serum‐active vitamin D levels 39. Additionally, an increased risk of hypercalcaemia may result from excessive vitamin D intake and the effective dose required to elicit an effect on the immune system in vivo remains to be determined. Nevertheless, this pivotal observation may lead to important future applications in anti‐bacterial treatment.

Conclusively, we observed the differential regulation of active vitamin D on Salmonella‐induced cytokine and anti‐microbial peptide in IECs via different proteins expression and signal pathways. Active vitamin D‐induced anti‐microbial peptide in IECs protects the host against infection, while modulation of proinflammatory response by active vitamin D prevents the host from the detrimental effects of overwhelming inflammation. Thus, active vitamin D‐induced innate immunity in IECs may provide an alternative therapy for invasive Salmonella infection. However, well‐controlled clinical trials are needed to determine the administration of active vitamin D as adjunctive treatment in Salmonella colitis or sepsis.

Disclosure

The authors declare that there are no financial and commercial disclosures.

Author contributions

F. C. H. conceived and designed the study, analysed and interpreted the data and wrote the manuscript.

Supporting information

Additional Supporting information may be found in the online version of this article at the publisher's web‐site:

Fig. S1. The effect of 1,25‐dihydroxyvitamin D3 (1,25D3) on the intracellular proliferation of Salmonella Typhimurium in cultured intestinal epithelial cells (IECs). SW480c cells were left untreated or treated with 1,25D3 (D3) or phosphate‐buffered saline (PBS) (vehicle), and then infected with wild‐type S. Typhimurium strain SL1344 (SL), and the levels of bacterial proliferation were examined 60 min after infection, as indicated in the Experimental section. Lysed cell cultures were plated on Luria broth (LB) to count colony‐forming units (CFUs). Values represent the percentage of intracellular bacteria compared with the wild type without any treatment (assigned as 100%). Each value represents the mean ± standard error of the mean (s.e.m.) of three independent experiments. An asterisk indicates a significant difference (P < 0·05).

Click here for additional data file. (97.2KB, pdf)

Fig. S2. 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 SW620 cells. SW620 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 amounts of the IL‐8 and hBD‐2 transcript. The amounts of IL‐8 and hBD‐2 mRNA produced and normalized to the corresponding amount of glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) transcript are 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 for IL‐8; #P < 0·05 for hBD‐2, compared to Salmonella infection only).

Click here for additional data file. (95.3KB, pdf)

Fig. S3. The protein expression of intracellular signalling pathway in Salmonella‐infected SW620 cells. SW620 cells were left uninfected (CON) or infected with wild‐type S. Typhimurium strain SL1344 (SL) for the indicated times in the presence or absence of 1,25‐dihydroxyvitamin D3 (1,25D3) (D3). Activation of the protein kinase B (Akt) (p‐Akt) and nucleotide oligomerization domain (NOD2) protein expression were analysed in whole cell and membrane protein by immunoblotting with antibodies to phosphorylated (p) Akt and NOD2, respectively. The results shown are representative of three separate experiments. Glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) and E‐cadherin worked as a normalization of cytosolic and membranous proteins.

Click here for additional data file. (114.7KB, pdf)

Acknowledgements

This work was supported in part by Ministry of Science and Technology grant MOST 103‐2314‐B‐182‐032 and Chang Gung Memorial Hospital grants CMRPG8B1431, CMRPG8B1481, and CMRPG880443. We thank Stem Cell Research Core Laboratory (grant CLRPG8B0052) for technical support.

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Associated Data

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Supplementary Materials

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Fig. S1. The effect of 1,25‐dihydroxyvitamin D3 (1,25D3) on the intracellular proliferation of Salmonella Typhimurium in cultured intestinal epithelial cells (IECs). SW480c cells were left untreated or treated with 1,25D3 (D3) or phosphate‐buffered saline (PBS) (vehicle), and then infected with wild‐type S. Typhimurium strain SL1344 (SL), and the levels of bacterial proliferation were examined 60 min after infection, as indicated in the Experimental section. Lysed cell cultures were plated on Luria broth (LB) to count colony‐forming units (CFUs). Values represent the percentage of intracellular bacteria compared with the wild type without any treatment (assigned as 100%). Each value represents the mean ± standard error of the mean (s.e.m.) of three independent experiments. An asterisk indicates a significant difference (P < 0·05).

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Fig. S2. 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 SW620 cells. SW620 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 amounts of the IL‐8 and hBD‐2 transcript. The amounts of IL‐8 and hBD‐2 mRNA produced and normalized to the corresponding amount of glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) transcript are 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 for IL‐8; #P < 0·05 for hBD‐2, compared to Salmonella infection only).

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Fig. S3. The protein expression of intracellular signalling pathway in Salmonella‐infected SW620 cells. SW620 cells were left uninfected (CON) or infected with wild‐type S. Typhimurium strain SL1344 (SL) for the indicated times in the presence or absence of 1,25‐dihydroxyvitamin D3 (1,25D3) (D3). Activation of the protein kinase B (Akt) (p‐Akt) and nucleotide oligomerization domain (NOD2) protein expression were analysed in whole cell and membrane protein by immunoblotting with antibodies to phosphorylated (p) Akt and NOD2, respectively. The results shown are representative of three separate experiments. Glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) and E‐cadherin worked as a normalization of cytosolic and membranous proteins.

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