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Vitamin D Receptor Negatively Regulates Bacterial-Stimulated NF-κB Activity in Intestine

      Vitamin D receptor (VDR) plays an essential role in gastrointestinal inflammation. Most investigations have focused on the immune response; however, how bacteria regulate VDR and how VDR modulates the nuclear factor (NF)-κB pathway in intestinal epithelial cells remain unexplored. This study investigated the effects of VDR ablation on NF-κB activation in intestinal epithelia and the role of enteric bacteria on VDR expression. We found that VDR−/− mice exhibited a pro-inflammatory bias. After Salmonella infection, VDR−/− mice had increased bacterial burden and mortality. Serum interleukin-6 in noninfected VDR+/+ mice was undetectable, but was easily detectable in VDR−/− mice. NF-κB p65 formed a complex with VDR in noninfected wild-type mouse intestine. In contrast, deletion of VDR abolished VDR/P65 binding. P65 nuclear translocation occurred in colonic epithelial cells of untreated VDR−/− mice. VDR deletion also elevated NF-κB activity in intestinal epithelia. VDR was localized to the surface epithelia of germ-free mice, but to crypt epithelial cells in conventionalized mice. VDR expression, distribution, transcriptional activity, and target genes were regulated by Salmonella stimulation, independent of 1,25-dihydroxyvitamin D3. Our study demonstrates that commensal and pathogenic bacteria directly regulate colonic epithelial VDR expression and location in vivo. VDR negatively regulates bacterial-induced intestinal NF-κB activation and attenuates response to infection. Therefore, VDR is an important contributor to intestinal homeostasis and host protection from bacterial invasion and infection.
      Vitamin D receptor (VDR) is a nuclear receptor
      • Haussler MR
      • Whitfield GK
      • Haussler CA
      • Hsieh J-C
      • Thompson PD
      • Selznick SH
      • Dominguez CE
      • Jurutka PW
      The nuclear vitamin D receptor: biological and molecular regulatory properties revealed.
      that mediates most known functions of 1,25-dihydroxyvitamin D (1,25[OH]2D3), the hormonal form of vitamin D. VDR and 1,25(OH)2D3 are involved in calcium homeostasis,
      • Van Cromphaut SJ
      • Dewerchin M
      • Hoenderop JG
      • Stockmans I
      • Van Herck E
      • Kato S
      • Bindels RJ
      • Collen D
      • Carmeliet P
      • Bouillon R
      • Carmeliet G
      Duodenal calcium absorption in vitamin D receptor-knockout mice: functional and molecular aspects.
      electrolyte and blood pressure regulation,
      • Li YC
      • Kong J
      • Wei M
      • Chen ZF
      • Liu SQ
      • Cao LP
      1,25-Dihydroxyvitamin D(3) is a negative endocrine regulator of the renin-angiotensin system.
      • Demay MB
      Mechanism of vitamin D receptor action.
      immune response,
      • Nagpal S
      • Na S
      • Rathnachalam R
      Noncalcemic actions of vitamin D receptor ligands.
      and anti-inflammation activity.
      • Liu PT
      • Stenger S
      • Li H
      • Wenzel L
      • Tan BH
      • Krutzik SR
      • Ochoa MT
      • Schauber J
      • Wu K
      • Meinken C
      • Kamen DL
      • Wagner M
      • Bals R
      • Steinmeyer A
      • Zugel U
      • Gallo RL
      • Eisenberg D
      • Hewison M
      • Hollis BW
      • Adams JS
      • Bloom BR
      • Modlin RL
      Toll-like receptor triggering of a vitamin D-mediated human antimicrobial response.
      • Hewison M
      Vitamin D and innate immunity.
      Different ligand shapes of 1, 25(OH)2D3 act through VDR in different cellular locations.
      • Norman AW
      Minireview: vitamin D receptor: new assignments for an already busy receptor.
      • Bouillon R
      • Carmeliet G
      • Verlinden L
      • van Etten E
      • Verstuyf A
      • Luderer HF
      • Lieben L
      • Mathieu C
      • Demay M
      Vitamin D and human health: lessons from vitamin D receptor null mice.
      The target genes of the VDR signal pathway include the enzyme Cyp24 and antimicrobial peptides β-defensin
      • Schauber J
      • Dorschner RA
      • Yamasaki K
      • Brouha B
      • Gallo RL
      Control of the innate epithelial antimicrobial response is cell-type specific and dependent on relevant microenvironmental stimuli.
      • Wang TT
      • Nestel FP
      • Bourdeau V
      • Nagai Y
      • Wang Q
      • Liao J
      • Tavera-Mendoza L
      • Lin R
      • Hanrahan JW
      • Mader S
      • White JH
      Cutting edge: 1,25-dihydroxyvitamin D3 is a direct inducer of antimicrobial peptide gene expression.
      and cathelicidin.
      • Gombart AF
      • Borregaard N
      • Koeffler HP
      Human cathelicidin antimicrobial peptide (CAMP) gene is a direct target of the vitamin D receptor and is strongly up-regulated in myeloid cells by 1,25-dihydroxyvitamin D3.
      Nuclear factor-κB (NF-κB) is a family of transcription factors that play an essential role in innate and adaptive immune responses. NF-κB is active in the nucleus, and its activity is inhibited by the inhibitor of κBα (IκBα).
      • Bonizzi G
      • Karin M
      The two NF-kappaB activation pathways and their role in innate and adaptive immunity.
      VDR physically interacts with NF-κB subunit p65 in human osteoblasts
      • Lu X
      • Farmer P
      • Rubin J
      • Nanes MS
      Integration of the NfkappaB p65 subunit into the vitamin D receptor transcriptional complex: identification of p65 domains that inhibit 1,25-dihydroxyvitamin D3-stimulated transcription.
      and mouse embryonic fibroblast cells,
      • Sun J
      • Kong J
      • Duan Y
      • Szeto FL
      • Liao A
      • Madara JL
      • Li YC
      Increased NF-kappaB activity in fibroblasts lacking the vitamin D receptor.
      but the functional relevance of this VDR/p65 interaction in regulating intestinal inflammation remains unclear.
      Human intestinal epithelial cells are constitutively exposed to commensal microbiota and pathogenic bacteria. Enteric commensal bacteria play a crucial role in the pathogenesis of many diseases such as inflammatory bowel diseases.
      • Packey CD
      • Sartor RB
      Interplay of commensal and pathogenic bacteria, genetic mutations, and immunoregulatory defects in the pathogenesis of inflammatory bowel diseases.
      • Xavier RJ
      • Podolsky DK
      Unravelling the pathogenesis of inflammatory bowel disease.
      • Sartor RB
      Microbial influences in inflammatory bowel diseases.
      and colon cancer.
      • Fukata M
      • Abreu MT
      TLR4 signalling in the intestine in health and disease.
      • Rhodes JM
      • Campbell BJ
      Inflammation and colorectal cancer: IBD-associated and sporadic cancer compared.
      Several studies have implicated vitamin D in inflammatory bowel disease (IBD). Low vitamin D levels have been reported in patients with IBD.
      • Sentongo TA
      • Semaeo EJ
      • Stettler N
      • Piccoli DA
      • Stallings VA
      • Zemel BS
      Vitamin D status in children, adolescents, and young adults with Crohn disease.
      • Abreu MT
      • Kantorovich V
      • Vasiliauskas EA
      • Gruntmanis U
      • Matuk R
      • Daigle K
      • Chen S
      • Zehnder D
      • Lin YC
      • Yang H
      • Hewison M
      • Adams JS
      Measurement of vitamin D levels in inflammatory bowel disease patients reveals a subset of Crohn's disease patients with elevated 1,25-dihydroxyvitamin D and low bone mineral density.
      In animal models, 1,25(OH)2D3 suppressed the development of experimental intestinal inflammation.
      • Cantorna MT
      • Zhu Y
      • Froicu M
      • Wittke A
      Vitamin D status, 1,25-dihydroxyvitamin D3, and the immune system.
      • Cantorna MT
      Vitamin D and its role in immunology: multiple sclerosis, and inflammatory bowel disease.
      Both local and endocrine synthesis of 1,25(OH)2D3 affect murine colitis
      • Kong J
      • Qiao G
      • Zhang Z
      • Liu SQ
      • Li YC
      Targeted vitamin D receptor expression in juxtaglomerular cells suppresses renin expression independent of parathyroid hormone and calcium.
      and VDR status affects the development of murine colitis.
      • Kong J
      • Zhang Z
      • Musch MW
      • Ning G
      • Sun J
      • Hart J
      • Bissonnette M
      • Li YC
      Novel role of the vitamin D receptor in maintaining the integrity of the intestinal mucosal barrier.
      VDR mediates T cell homing to the gut.
      • Cantorna MT
      • Zhu Y
      • Froicu M
      • Wittke A
      Vitamin D status, 1,25-dihydroxyvitamin D3, and the immune system.
      VDR expression is significantly decreased in IBD patients.
      • Abreu MT
      • Kantorovich V
      • Vasiliauskas EA
      • Gruntmanis U
      • Matuk R
      • Daigle K
      • Chen S
      • Zehnder D
      • Lin YC
      • Yang H
      • Hewison M
      • Adams JS
      Measurement of vitamin D levels in inflammatory bowel disease patients reveals a subset of Crohn's disease patients with elevated 1,25-dihydroxyvitamin D and low bone mineral density.
      • Wada K
      • Tanaka H
      • Maeda K
      • Inoue T
      • Noda E
      • Amano R
      • Kubo N
      • Muguruma K
      • Yamada N
      • Yashiro M
      • Sawada T
      • Nakata B
      • Ohira M
      • Hirakawa K
      Vitamin D receptor expression is associated with colon cancer in ulcerative colitis.
      However, the majority of studies of vitamin D, VDR, and inflammation are focused on immunoregulation, with little emphasis on assessing the effects of VDR in intestinal epithelial cells. It is unknown how intestinal VDR signaling responds to commensal and pathogenic bacterial stimulation.
      In the present study, we hypothesize that Salmonella typhimurium infection can induce colonic VDR expression, alter location of VDR in vivo, and stimulate VDR expression, transcription, and signaling in colonic epithelial cell lines. We found that mice lacking VDR are in a pre-inflammatory or pro-inflammatory state. Hence, our current study investigated the effect of VDR ablation on NF-κB activation using a VDR knockout mouse model infected with pathogenic Salmonella or human commensal Escherichia coli. F18. We also examined intestinal VDR expression in normal, germ-free (GF), and conventionalized wild-type mice. Our data demonstrate that intestinal VDR is directly involved in suppression of bacteria-induced NF-κB activation and that commensal bacterial colonization affects intestinal epithelial VDR expression and distribution.

      Materials and Methods

      Bacterial Strains and Growth Condition

      S. typhimurium wild-type ATCC14028, commensal E. coli F18, and nonpathogenic Salmonella mutant strain PhoPc
      • Miller SI
      • Mekalanos JJ
      Constitutive expression of the phoP regulon attenuates Salmonella virulence and survival within macrophages.
      were used in this study. Non-agitated micro-aerophilic bacterial cultures were as follows: nonagitated micro-aerophilic bacterial cultures were prepared by inoculation of 10 ml of Luria-Bertani broth with 0.01 ml of a stationary phase culture, followed by overnight incubation (∼18 hours) at 37°C, as previously described.
      • Sun J
      • Hobert ME
      • Rao AS
      • Neish AS
      • Madara JL
      Bacterial activation of beta-catenin signaling in human epithelia.
      Bacterial overnight cultures were concentrated 33-fold in Hanks’ balanced salt solution (HBSS) supplemented with 10 mmol/L HEPES, pH 7.4.

      Streptomycin Pretreated Mouse Model

      Animal experiments were performed by using specific-pathogen-free female C57BL/6 mice (Taconic, Hudson, NY) that were 6 to 7 weeks old
      • Duan Y
      • Liao AP
      • Kuppireddi S
      • Ye Z
      • Ciancio MJ
      • Sun J
      Beta-catenin activity negatively regulates bacteria-induced inflammation.
      and VDR knock out C57BL/6 mice (Jackson Laboratory, Bar Harbor, Maine) as previously described.
      • Li YC
      • Pirro AE
      • Amling M
      • Delling G
      • Baron R
      • Bronson R
      • Demay MB
      Targeted ablation of the vitamin D receptor: an animal model of vitamin D-dependent rickets type II with alopecia.
      The protocol was approved by the University of Rochester University Committee on Animal Resources. Water and food were withdrawn 4 hours before oral gavage with 7.5 mg/mouse of streptomycin (100 μl of sterile solution or 100 μl of sterile water as control). Afterward, animals were supplied with water and food. Twenty hours after streptomycin treatment, water and food were withdrawn again for 4 hours before the mice were infected with 1 × 107 colony forming units (CFU) of S. typhimurium (100-μl suspension in HBSS) or treated with sterile HBSS (control) by oral gavage as previously described.
      • McCormick BA
      • Colgan SP
      • Delp-Archer C
      • Miller SI
      • Madara JL
      Salmonella typhimurium attachment to human intestinal epithelial monolayers: transcellular signalling to subepithelial neutrophils.
      For the mouse survival experiment, 1 × 108 CFU of S. typhimurium was used.

      GF and Conventionalized Mouse Colon

      GF mice (129/SvEv) were obtained from the Center for Gastrointestinal Biology and Disease Gnotobiotic Core Facility and the National Gnotobiotic Rodent Resource Center, University of North Carolina, Chapel Hill. Mice were analyzed at the University of North Carolina National Gnotobiotic Rodent Resource Center. Sterility of germ-free mice was documented on a monthly basis by fecal Gram stains and aerobic and anaerobic cultures of the feces and bedding. For selected mice, sterility of cecal contents was documented Gram stain and cultures at the time of necropsy. Conventionalized mice were originally GF mice that were infected with a commensal enteric bacteria by orally swabbing fecal slurries from specific pathogen-free (SPF) mice, placing fecal slurries on their food and then housing them in SPF conditions.

      Cell Culture

      Caco2BBE and HCT116 cells were grown in Dulbecco’s Modified Eagle Medium (high glucose, 4.5g/L) containing 5% (v/v) fetal bovine serum, 50 μg/ml streptomycin, and 50 U/ml penicillin. Monolayers of Caco2BBE cells were grown on permeable supports (0.33 or 4.67 cm2, 0.4 μm pore. Costar, Cambridge, MA) and used 6 to 14 days after being plated.

      Mouse Embryonic Fibroblasts

      Mouse embryonic fibroblasts (MEFs) were isolated from embryonic day 13.5 embryos generated from VDR+/− × VDR+/− mouse breeding as previously described.
      • Sun J
      • Kong J
      • Duan Y
      • Szeto FL
      • Liao A
      • Madara JL
      • Li YC
      Increased NF-kappaB activity in fibroblasts lacking the vitamin D receptor.
      Cells from each embryo were genotyped by PCR using genomic DNA isolated from the cells. VDR+/− and VDR−/− MEFs were used in experiments after more than 15 passages when they had been immortalized.

      Bacterial Colonization in Cultured Cells in Vitro

      Human colonic epithelial cells were colonized with equal numbers of the indicated bacteria for 30 minutes, washed with HBSS, and incubated in Dulbecco’s Modified Eagle Medium containing gentamicin (500 μg/ml) for the times indicated in our previous studies.
      • Sun J
      • Hobert ME
      • Rao AS
      • Neish AS
      • Madara JL
      Bacterial activation of beta-catenin signaling in human epithelia.
      • Sun J
      • Hobert ME
      • Duan Y
      • Rao AS
      • He TC
      • Chang EB
      • Madara JL
      Crosstalk between NF-kappaB and beta-catenin pathways in bacterial-colonized intestinal epithelial cells.
      The first 30-minute incubation allowed bacteria to contact the surface of the epithelial cells and inject the effectors in the host cells. After extensive HBSS washing, the extracellular bacteria were washed away. Incubation with gentamicin inhibited the growth of bacteria. In this way, we focused on the effects of the bacterial effectors injected to the host cells.

      Immunoblotting

      Mouse colonic epithelial cells were collected by scraping from mouse colon including proximal and distal regions as previously described.
      • Duan Y
      • Liao AP
      • Kuppireddi S
      • Ye Z
      • Ciancio MJ
      • Sun J
      Beta-catenin activity negatively regulates bacteria-induced inflammation.
      Mouse epithelial cells were lysed in lysis buffer (1% Triton X-100, 150 mmol/L NaCl, 10 mmol/L Tris pH 7.4, 1 mmol/L EDTA, 1 mmol/L EGTA pH 8.0, 0.2 mmol/L sodium ortho-vanadate, and protease inhibitor cocktail). MEFs were rinsed twice in ice-cold HBSS, lysed in protein loading buffer (50 mmol/L Tris, pH 6.8, 100 mmol/L dithiothreitol, 2% SDS, 0.1% bromophenol blue, and 10% glycerol). Immunoblot was performed with primary antibodies: anti-VDR, anti-p65, anti-IκBα (Santa Cruz Biotechnology Inc., Santa Cruz, CA), anti-phospho-IκBα, anti-phospho-p65 (ser536) (Cell Signal, Beverly, MA) or anti-β-actin (Sigma-Aldrich, Milwaukee, WI) antibodies and visualized by enhanced chemiluminescence.
      • Sun J
      • Kong J
      • Duan Y
      • Szeto FL
      • Liao A
      • Madara JL
      • Li YC
      Increased NF-kappaB activity in fibroblasts lacking the vitamin D receptor.
      • Sun J
      • Mustafi R
      • Cerda S
      • Chumsangsri A
      • Xia YR
      • Li YC
      • Bissonnette M
      Lithocholic acid down-regulation of NF-kappaB activity through vitamin D receptor in colonic cancer cells.

      Co-Immunoprecipitation Assay

      Cells were rinsed twice in ice-cold HBSS and lysed in cold immunoprecipitation buffer (1% Triton X-100, 150 mmol/L NaCl, 10 mmol/L Tris pH 7.4, 1 mmol/L EDTA, 1 mmol/L EGTA pH 8.0, 0.2 mmol/L sodium ortho-vanadate, protease inhibitor cocktail (Boehringer Mannheim). Samples were immunoprecipitated with the anti-VDR antibody (Santa Cruz) as previously described.
      • Sun J
      • Kong J
      • Duan Y
      • Szeto FL
      • Liao A
      • Madara JL
      • Li YC
      Increased NF-kappaB activity in fibroblasts lacking the vitamin D receptor.
      Blots were probed with anti-VDR antibody (Transduction Laboratories), stripped, and reprobed with anti-p50 and anti-p65 antibodies (Santa Cruz Biotechnology, CA) and visualized by enhanced chemiluminescence.

      Nuclear Protein Fraction

      Mouse colon epithelial mucosa was washed with HBSS three times and lysed. The nuclear proteins were extracted using Nuclear Extract Kit (Active Motif, Carlsbad, CA). Protein fraction was used for the co-immunoprecipitation assay.

      S. typhimurium Invasion of MEFs Monolayers

      Infection of MEFs was performed by the method described previously.
      • McCormick BA
      • Colgan SP
      • Delp-Archer C
      • Miller SI
      • Madara JL
      Salmonella typhimurium attachment to human intestinal epithelial monolayers: transcellular signalling to subepithelial neutrophils.
      Bacterial solution (∼20 bacteria/epithelial cell) was added and bacterial invasion was assessed after 1 hour. Cell-associated bacteria represent bacteria adhered to and/or internalized into the monolayers were released by incubation with 100 μl of 1% Triton X-100 (Sigma). For cell-associated bacteria, 0.9 ml of Luria broth was added, mixed, and quantified by plating on MacConkey agar medium for CFU.

      Salmonella Burden in Intestine

      Cecum was dissected from mouse, and put into 14 ml tube with 5 ml sterile PBS, cut into pieces with scissors, then homogenized adequately using homogenizer (Polytron PT2100, Kinematica, Switzerland). Homogenate was diluted at 1000 to 10,000 with Luria-Bertani broth. One hundred μl of diluted homogenate was plated out on MacConkey agar plates and incubated at 37°C overnight. CFU were quantified.

      Histology of Mouse Colon

      Mouse colons were harvested, fixed in 10% formalin (pH 7.4), processed, and paraffin embedded. “Swiss rolls” were prepared as described.
      • Park CM
      • Reid PE
      • Walker DC
      • MacPherson BR
      A simple, practical ‘swiss roll’ method of preparing tissues for paraffin or methacrylate embedding.
      Sections (5 μm) were stained with H&E. For immunostaining, antigens were retrieved by 10-minute boiling in 10 mmol/L citrate (pH 6.0). The slides were stained with anti-VDR antibody (Santa Cruz) as previously described.
      • Duan Y
      • Liao AP
      • Kuppireddi S
      • Ye Z
      • Ciancio MJ
      • Sun J
      Beta-catenin activity negatively regulates bacteria-induced inflammation.
      Salmonella distribution in intestine was determined by immunostaining using anti-Salmonella lipopolysaccharide antibody (Santa Cruz). Blinded histological inflammatory scores were performed by a validated scoring system
      • Sellon RK
      • Tonkonogy S
      • Schultz M
      • Dieleman LA
      • Grenther W
      • Balish E
      • Rennick DM
      • Sartor RB
      Resident enteric bacteria are necessary for development of spontaneous colitis and immune system activation in interleukin-10-deficient mice.
      by a trained pathologist.

      Salmonella-Induced Mouse Interleukin-6 Secretion

      Mouse blood samples were collected by cardiac puncture and placed in tubes containing EDTA (10 mg/ml).
      • Liao AP
      • Petrof EO
      • Kuppireddi S
      • Zhao Y
      • Xia Y
      • Claud EC
      • Sun J
      Salmonella type III effector AvrA stabilizes cell tight junctions to inhibit inflammation in intestinal epithelial cells.
      • Ye Z
      • Petrof EO
      • Boone D
      • Claud EC
      • Sun J
      Salmonella effector AvrA regulation of colonic epithelial cell inflammation by deubiquitination.
      Mouse interleukin (IL)-6 was measured using the TiterZyme Enzyme Immunometric Assay kits (Assay Designs, Inc., Ann Arbor, MI) according to the manufacturer’s instructions.

      Vitamin D-Responsive Element Transcriptional Activity

      Cells were grown in triplicate and transfected with Cignal Vitamin D Reporter (luc) Kit (SABiosciences, Frederick, MD) using Surefect reagent (SABiosciences, Frederick, MD). The plasmid for the VDR reporter is a mixture of inducible Vitamin D-responsive firefly luciferase construct and constitutively expressing Renilla luciferase construct (40:1). The negative control is a mixture of noninducible firefly luciferase construct and constitutively expressing Renilla luciferase construct (40:1). After transfection for 24 hours, cells were colonized with Salmonella for 30 minutes, washed, incubated in Dulbecco’s Modified Eagle Medium with gentamicin (500 μg/ml) for 12 hour or 16 hours. Luciferase activity was determined using the Dual Luciferase Reporter Assay System (Promega) with a TD-20/20 luminometer (Turner Designs, Sunnyvale, CA).

      Quantitative Real-Time PCR Analysis

      Total RNA was extracted from epithelial cell monolayers using TRIzol reagent (Invitrogen, Carlsbad, CA). The reverse transcription cDNA reaction products were subjected to quantitative real-time PCR using the MyiQ single-color real-time PCR detection system (Bio-Rad) and iQ SYBR green supermix (Bio-Rad) according to the manufacturer’s directions.
      • Ye Z
      • Petrof EO
      • Boone D
      • Claud EC
      • Sun J
      Salmonella effector AvrA regulation of colonic epithelial cell inflammation by deubiquitination.
      All expression levels were normalized to the β-actin levels of the same sample. Percent expression was calculated as the ratio of the normalized value of each sample to that of the corresponding untreated control cells. All real-time PCR reactions were performed in triplicate. All PCR primers (Table 1) were designed using Lasergene (DNAStar, Madison, WI).
      Table 1Real-Time PCR Primers
      Gene NamePrimer
      hVDR F5′-GGACTGCCGCATCACCAA-3′
      hVDR R5′-TCATCTCCCGCTTCCTCT-3′
      hCathe F5′-TGCCCAGGTCCTCAGCTAC-3′
      hCathe R5′-GTGACTGCTGTGTCGTCCT-3′
      hCyp24 F5′-GCCTGGCAGAGCTTGAATT-3′
      hCyp24 R5′-ACAGTCCGGGTCTTGGGT-3′
      hDEFB4 F5′-ATTCCTGATGCCTCTTCC-3′
      hDEFB4 R5′-GTGCCAATTTGTTTATACCT-3′
      h β-actin F5′-AGAGCAAGAGAGGCATCCTC-3′
      h β-actin R5′-CTCAAACATGATCTGGGTCA-3′
      m β-actin F5′-TGTTACCAACTGGGACGACA-3′
      m β-actin R5′-CTGGGTCATCTTTTCACGGT-3′
      F: forward; R: reverse.

      Statistical Analysis

      Data are expressed as mean ± SD. Differences between two samples were analyzed by Student’s t-test. P values of 0.05 or less were considered significant.

      Results

      The Pre-Inflammatory State of the VDR−/− Mouse

      Because NF-κB is a key regulator involved in the synthesis of inflammatory cytokines, we measured the production of IL-6, a well-known NF-κB target gene, as a read-out of the biological effect of VDR expression on NF-κB pathway in vivo. The synthesis of IL-6 was compared in the serum of the VDR+/+ and VDR−/− mice under unstimulated (noninfected) and stimulated (pathogenic Salmonella infection) conditions. Whereas serum IL-6 in specific pathogen-free VDR+/+ mice was undetectable (<0. 01pg/ml), it was easily detectable in VDR−/− mice (IL-6 200 pg/ml, P < 0.0001 vs. wild-type mice) (Figure 1, VDR−/− noninfected control). When the mice were infected with S. typhimurium, serum levels of IL-6 in VDR−/− mice (IL-6 1200 pg/ml) was increased from baseline sixfold compared with VDR+/+ mice (200 pg/ml, P < 0.0001) (Figure 1A).
      Figure thumbnail gr1
      Figure 1VDR-null mutant mice have worse outcomes with Salmonella-induced infection. A: Serum IL-6 increased in VDR deficient mice. Normal mice (VDR+/+) and VDR-deficient mice were infected with wild-type S. typhimurium for six hours. Mouse serum was collected for IL-6 enzyme-linked immunosorbent assay. Data are presented as the mean ± SD from a single experiment assayed in triplicate. *P < 0.05. B: Cecum was dissected out from the indicated mouse and photographed. Cecum shortening was found in the mouse intestine with Salmonella infection. C: H&E staining and scores of the mouse intestine with or without Salmonella infection. D: Relative body weight change in mice with or without Salmonella infection. E: Survival percentage of the VDR+/+ and VDR−/− mice administrated S. typhimurium. wild-type group n = 10, VDR−/− group n = 12. *P < 0.05, **P < 0.01.

      VDR Null Mutant Mice Have Worse Outcomes Following Salmonella-Induced Infection

      We used the Salmonella infection model to investigate the role of VDR in intestinal homeostatic responses to infection. We compared colitis development in VDR+/+ and VDR −/− mice infected with Salmonella. Figure 1, B–E show experiments in which VDR+/+ and VDR−/− mice were infected with S. typhimurium. Cecal inflammation is the key feature in the Salmonella-colitis mouse model.
      • Barthel M
      • Hapfelmeier S
      • Quintanilla-Martinez L
      • Kremer M
      • Rohde M
      • Hogardt M
      • Pfeffer K
      • Russmann H
      • Hardt WD
      Pretreatment of mice with streptomycin provides a Salmonella enterica serovar Typhimurium colitis model that allows analysis of both pathogen and host.
      We found that ceca of the VDR−/− mice infected with S. typhimurium looked smaller and more red compared with the wild-type mice infected with S. typhimurium. The length of ceca in VDR−/− group infected with Salmonella was significantly decreased comparing to the wild-type mice (Figure 1B). H&E staining data showed more severe intestinal inflammation in VDR−/− mice infected with S. typhimurium compared with the wild-type mice (Figure 1C). Next, the level of intestinal inflammation was quantified histologically in a blinded fashion by a trained pathologist. The inflammation score in the Salmonella-infected VDR−/− mice was significantly higher than the Salmonella-infected VDR+/+ mice (Figure 1D). Moreover, the body weight of the Salmonella-infected VDR−/− mice dropped much earlier than the VDR+/+ mice (Figure 1D). Ninety percent of VDR−/− mice infected with Salmonella died within 5 days and 100% by 6 days (Figure 1E). In contrast, VDR+/+ mice were relatively resistant at Salmonella; with higher survival rates (60%) seen within the 7-day observation period (Figure 1E). Hence, these data indicate that VDR contributes to host protection against detrimental outcomes to pathogenic invasive enteric bacterial infection.

      VDR Expression Protects against Salmonella Colonization and Mucosal Invasion

      We also examined whether VDR expression contribute to protect cells from Salmonella invasion. Immunofluorescence assay showed more Salmonella staining (green) in the VDR−/− mouse intestine. We further counted the numbers of Salmonella invading the intestine. VDR−/− mice had increased Salmonella invasion of the intestine, whereas the VDR+/+ mouse had significantly fewer Salmonella (Figure 2A). Culture of the cecum showed significantly higher concentrations of Salmonella in the VDR−/− mice than in VDR+/+ mice (Figure 2B). Furthermore, we used MEFs to investigate the effect of VDR ablation on Salmonella invasion. We used VDR+/− and VDR−/− MEFs, as we wanted to compare the difference between VDR-null and one allele of the Vdr gene. Salmonella invasion was increased in the VDR−/− MEFs (Figure 2C). These in vitro data are consistent with our in vivo findings (Figure 2, A and B).
      Figure thumbnail gr2
      Figure 2VDR expression reduces Salmonella invasion in cells. A: Salmonella location (green) in the VDR+/+ and VDR−/− mouse cecum. Normal mice and VDR deficient mice were infected with wild-type Salmonella typhimurium for five days. B: Salmonella concentrations in the VDR+/+ and VDR mouse ceca. C: Salmonella invasion in infected VDR+/− and VDR−/− MEFs. MEFs were stimulated with wild-type Salmonella for 30 minutes, washed, and incubated in fresh Dulbecco’s Modified Eagle Medium for 30 minutes. The mean ± SD is from three replicate experiments. **P < 0.01.

      Absence of VDR Permits Translocation of NF-κB p65 to the Nucleus

      We then assessed the level of NF-κB p65 protein, the most abundant NF-κB subunit, in epithelial cells. It is known that VDR physically binds to p65.
      • Lu X
      • Farmer P
      • Rubin J
      • Nanes MS
      Integration of the NfkappaB p65 subunit into the vitamin D receptor transcriptional complex: identification of p65 domains that inhibit 1,25-dihydroxyvitamin D3-stimulated transcription.
      • Sun J
      • Kong J
      • Duan Y
      • Szeto FL
      • Liao A
      • Madara JL
      • Li YC
      Increased NF-kappaB activity in fibroblasts lacking the vitamin D receptor.
      Total p65 was reduced in colonic epithelial cells of noninfected VDR−/− mice (Figure 3, A and B). To examine the possibility that cross-regulation of the VDR and NF-κB signaling pathways may occur through physical interaction, we determined whether VDR can form a complex with NF-κB in vivo. Using a co-immunoprecipitation assay, we found that the NF-κB p65 subunit formed a complex with VDR in unstimulated wild-type mouse colonic epithelial cells. VDR deletion totally abolished the VDR/p65 binding in freshly isolated VDR−/− mouse colonic epithelial cells, whereas there is no interaction of VDR with nonspecific IgG (Figure 3C). Additionally, we found no apparent interaction of VDR with p50, a NF-κB subunit (data not shown) in wild-type mice indicating that the physical interaction between VDR and NF-κB p65 is specific.
      Figure thumbnail gr3
      Figure 3The expression of NFκB p65 in intestinal epithelial cells in VDR knockout mice. A: NF-κB p65 expression in normal mice and VDR-deficient mice. Colonic epithelial cells were collected and lysates were immunoblotted with antibody against NF-κB p65. B: Relative NF-κB p65 band intensity in normal and VDR−/− mice. Data are presented as the mean ± SD from three repeated experiments. C: VDR physically interacts with NF-κB p65 in intestinal epithelial cells in vivo. VDR deletion abolishes this interaction. Mouse intestinal epithelial cells were collected. Cell lysates were immunoprecipitated with anti-VDR antibody, then the precipitated complex was probed with anti-p65 antibody by Western blot (W.B. left). Note that the background level of p65 was detectable in VDR−/− cells. An aliquot of the cell lysates was probed with antibodies against VDR, villin, and β-actin to confirm equal input (right). D: VDR/p65 binding in mouse colon using cell fraction. Mouse colon epithelial mucosa was lysed and the nuclear proteins were extracted. Cell fraction was used for co-immunoprecipitation assay using anti-VDR antibody. N: nuclei; C: cytoplasm. **P < 0.01.
      The IP results were also confirmed by cell fraction of VDR/p65 levels in the cytosolic and nuclear compartments of cells in mouse colon. As shown in Figure 3D, VDR/p65 binding was found in cytosolic compartments in normal mouse colon. The VDR/p65 physical interaction was undetectable in the nuclear fraction of mouse colon epithelial cells. Thus our data demonstrate a clear physical interaction between VDR and NF-κB in basal colonic epithelia.

      Absence of VDR Permits Translocation of NF-κB p65 to the Nucleus

      Because of the lack of VDR, the physical interaction between VDR and p65 was absent in VDR−/− cells. VDR knockout also reduced the levels of the NF-κB inhibitor, IκBα, in colonic epithelial cells.
      • Wu S
      • Xia Y
      • Liu X
      • Sun J
      Vitamin D receptor deletion leads to reduced level of IkappaBalpha protein through protein translation, protein-protein interaction, and post-translational modification.
      Consequently, these alterations combined may led to free p65 and increase p65 translocation to the nuclei. Immunostaining data showed a marked increase in nuclear p65 in VDR−/− mouse colon without any stimulation (Figure 4A). In contract, p65 was located in the cytosol and absent in the nuclei in the VDR+/+ mouse colon. Moreover, we examined the cell fraction of p65 level in the cytosolic and nuclear compartments of cells in the mouse colon. There was detectable nuclear p65 in the VDR−/− colon, whereas no p65 was found in the nuclei of the VDR+/+ mice (Figure 4B). Equal loading of the epithelial cells was by Villin, the epithelial cell marker. The loading of nuclear proteins was confirmed by the level of Sp1, a nuclear protein absent in the cytosol. In the MEFs, the expression level of p65 in the nuclear extracts was higher in VDR−/− cells than in VDR+/− cells (Figure 4C). These data are also consistent with our previous observation in VDR−/− MEFs.
      • Sun J
      • Kong J
      • Duan Y
      • Szeto FL
      • Liao A
      • Madara JL
      • Li YC
      Increased NF-kappaB activity in fibroblasts lacking the vitamin D receptor.
      Taken together, our in vivo and in vitro data show that absence of VDR permits translocation of NF-κB p65 to the nucleus.
      Figure thumbnail gr4
      Figure 4The location and activity of NFκB p65 in intestinal epithelial cells in VDR knockout mice. A: NF-κB p65 location in colon in normal mice (VDR+/+) and VDR−/− mice without any treatment. Arrows indicate the p65 nuclear location (green). B: Western blot analysis of p65 levels in cytosolic (C) and nuclear (N) extracts isolated from VDR+/+ and VDR−/− mouse colons. C: The p65 level in cytosolic and nuclear extracts isolated from VDR+/− and VDR−/− MEFs. The nuclear protein Sp1, which is absent in the cytosolic fraction, serves as a nuclear protein loading control. D: Higher phospho-p65 level in the VDR−/− MEFs without any treatment. *P < 0.05. E: Increased phospho-p65 level in the VDR−/− mouse colonic epithelial cells without any treatment. Relative phospho-p65 band intensity in normal and VDR−/− mice is shown. Villin is used as a marker for epithelial cells and an internal control. β-actin is also used as an internal control. The mean ± SD is from three repeats. **P < 0.01.

      VDR Expression Negatively Regulates Transcriptional NF-κB Activity

      We further hypothesize that the NF-κB activity is high in cells lacking VDR expression. Transcriptional activity of NF-κB is controlled by phosphorylation of p65 at serine 536.
      • Hayden MS
      • Ghosh S
      Signaling to NF-kappaB.
      • Sakurai H
      • Chiba H
      • Miyoshi H
      • Sugita T
      • Toriumi W
      IkappaB kinases phosphorylate NF-kappaB p65 subunit on serine 536 in the transactivation domain.
      Constitutive phospho-p65 levels were also significantly increased in VDR−/− MEFs without stimulation (Figure 4D). We found that VDR−/− mouse colonic epithelial cells had significantly elevated phospho-p65, over 2 times of the p-p65 concentrations in VDR+/+ epithelial cells (Figure 4E). Overall, absence of VDR led to elevated levels of phospho-p65 in the fibroblasts and colonic epithelial cells without any bacterial stimulation.

      VDR Distribution in the Normal Mouse Colon

      There has been very little information on the VDR distribution in colonic epithelial cells, which are consistently exposed to the enteric bacteria. We investigated the distribution of VDR from the proximal colon to the distal colon in the normal mouse intestine by making a colonic “Swiss roll” (Figure 5A). We demonstrated less VDR staining (green) in the distal colon with heavier staining in the proximal colon. In contrast, 4,6-diamidino-2-phenylindole staining (blue) is even throughout the colon. Under high magnification, we identified VDR distributed at the top of the epithelial cell crypts in the proximal colon. Most of the enhanced VDR staining is in the cytosol; some of the staining is in the nuclei (indicated with white arrows in Figure 5B).
      Figure thumbnail gr5
      Figure 5The distribution of VDR in normal mouse colon. A: VDR distribution in the “Swiss roll” made with the SPF mouse colon. Arrow indicates the direction of the proximal and distal colon. B: Higher magnification of VDR distribution in the normal mouse proximal colon. White arrow indicates the nuclear staining of VDR at the top of colonic crypts. C: VDR protein expression in the proximal and distal colon determined by Western blot. DC: distal colon; PC: proximal colon. D: Relative VDR band intensity in normal mice. Data are presented as the mean ± SD; n = four mice per group. *P < 0.05.
      Western blot analysis of VDR levels in colon also confirmed the immunostaining observation (Figure 5C). Data from 4 individual mouse colon samples showed that the VDR expression is significantly higher in the proximal colon than that in the distal colon (Figure 5D).

      Salmonella Infection Increased the VDR Protein Expression in Mouse Colon in Vivo

      Furthermore, we examined the expression of VDR in pathogenic bacteria-induced inflammation using normal mice. Interestingly, we found that the total amount of VDR protein increased in the colonic epithelium of mice 6 hours after Salmonella infection (Figure 6A). To confirm that the increased VDR expression is induced specifically by pathogenic wild-type Salmonella infection, we gavaged the mice with wild-type Salmonella or commensal E. coli F18. Western blot (Figure 6B) showed that only wild-type Salmonella was able to increase the expression of VDR, whereas commensal E. coli F18 colonization did not change the VDR expression in the mouse colonic epithelial cells. These results indicate that colonic epithelial responses to specific pathogenic bacteria such as wild-type Salmonella are determined in part through the VDR signaling pathway.
      Figure thumbnail gr6
      Figure 6The expression and location of VDR protein in mouse intestinal epithelial cells infected with or without bacteria. A: Total VDR protein in mouse colonic epithelial cells increased after infection with pathogenic Salmonella typhimurium for six hours. B: Normal mice were gavaged without (−) or with either wild-type Salmonella typhimurium or E. coli F18 for six hours. Colonic epithelial cell lysates were immunoblotted with antibodies against VDR or β-actin. Data are from a single experiment and are representative of three separate mouse cells. *P < 0.05. C: Relocation of VDR after Salmonella for six hours. D: VDR distribution in germ-free and conventionalized mouse intestine seven days after colonization with SPF slurries. E: Total VDR protein in mouse colonic epithelial cells increased after GF mice mono-associated with E.coli F18 for six days. **P < 0.01.

      VDR Relocation in the Mouse Colon after Bacterial Infection Colonization

      To further examine the distribution of VDR in the mouse colon with bacterial infection, we used the Salmonella infection model in normal mice. Histological examination demonstrates that VDR was located on the surface of colonic epithelial cells in normal mice without any treatment (Figure 6C control mouse). Pathogenic Salmonella colonization in mice increased VDR staining at the surface of the crypts, but also induced VDR expression in the middle and at the bottom of the crypts in mice infected with wild-type Salmonella (Figure 6C Salmonella). Our data indicate that pathogenic Salmonella infection changed the location of VDR in colonic epithelial cells.
      We also compared GF and conventionalized mice (GF mice colonized with SPF fecal bacteria) to explore the effect of bacteria on modulating VDR distribution. Our data showed that VDR was localized at the top of the crypts of the GF mouse intestine by immunostaining (Figure 6D). Bacterial colonization of ex-GF conventionalized mice for 7 days induced VDR relocation to the middle and even at the bottom of the crypts. In addition, mono-association of GF mice with E. coli F18 increased the epithelial VDR protein expression just like the Salmonella infection in the SPF mice (Figure 6E).

      Salmonella Directly Increased VDR Protein Expression and VDR Transcriptional Activity Independent of Vitamin D3

      The classical VDR pathway is initiated on 1,25-dihydroxyvitamin D3 binding to VDR. Recent studies demonstrate that VDR was activated through increased protein expression independent of classical transcriptional regulation.
      • Li QP
      • Qi X
      • Pramanik R
      • Pohl NM
      • Loesch M
      • Chen G
      Stress-induced c-Jun-dependent vitamin D receptor (VDR) activation dissects the non-classical VDR pathway from the classical VDR activity.
      • Qi X
      • Pramanik R
      • Wang J
      • Schultz RM
      • Maitra RK
      • Han J
      • DeLuca HF
      • Chen G
      The p38 and JNK pathways cooperate to trans-activate vitamin D receptor via c-Jun/AP-1 and sensitize human breast cancer cells to vitamin D(3)-induced growth inhibition.
      • Qi X
      • Tang J
      • Pramanik R
      • Schultz RM
      • Shirasawa S
      • Sasazuki T
      • Han J
      • Chen G
      p38 MAPK activation selectively induces cell death in K-ras-mutated human colon cancer cells through regulation of vitamin D receptor.
      Therefore, it is possible that bacteria alone modulate VDR expression and subsequent activity in the absence of 1,25-dihydroxyvitamin D3. We cultured human intestinal epithelial cells in medium with or without 1,25-dihydroxyvitamin D3, then exposed them to Salmonella typhimurium. We found that bacterial colonization of Caco2 BBE cells for only 1 hour increased VDR expression in the absence of 1,25-dihydroxyvitamin D3 (Figure 7A). In cells stimulated by 1,25-dihydroxyvitamin D3 for 24 hours, the level of VDR increased, but there was no additional increase with Salmonella stimulation. We further investigated VDR transcription activity under bacterial stimulation in two intestinal epithelial cell lines, Caco2 BBE and HCT116. In cells transfected with the VDR reporter plasmid, Salmonella colonization for 12 hours and 16 hours induced the VDR transcriptional activity significantly compared with cells without bacteria. The relative fold increase was compared with cells transfected with the negative control plasmid (Negative) (Figure 7B). Additionally, the expression of VDR target genes Cyp24, β-defensin, and cathelicidin increased significantly after Salmonella infection in the human epithelial cells without 1,25-dihydroxyvitamin D3 in media (Figure 7C). Taken together, these data indicate that S. typhimurium increased VDR protein expression, VDR transcriptional activity and VDR- mediated gene transcription independent of 1,25-dihydroxyvitamin D3.
      Figure thumbnail gr7
      Figure 7Salmonella directly increases VDR expression and transcriptional activity in human intestinal epithelial cells. A: Salmonella directly increases VDR protein expression in human intestinal epithelial CaCo2 BBE cells in the absence of 1,25-dihydroxyvitamin D3 (−). 1,25-Dihydroxyvitamin D3 (20 nmol/L) stimulated VDR protein was measured by Western blot. B: Salmonella colonization increased the VDR transcription activity in human epithelial cells. The cells were transfected with Cignal Vitamin D Reporter (luc) Kit. After transfection for 24 hours, cells were colonized with Salmonella for indicated time and lysed, and luciferase activity was determined. Firefly luciferase activity was normalized to Renilla luciferase activity, and the activity was expressed as relative luminescence units. C: Salmonella colonization increased the mRNA level of the VDR target genes in human epithelial cells. *P < 0.05; **P < 0.001. Data are from a single experiment and are representative of two to three separate experiments.

      Discussion

      In the current study, we demonstrate that VDR contributes to host protection against detrimental outcomes of pathogenic enteric bacterial infection (Figure 8). In the wild-type mouse with VDR expression in the intestine, VDR is able to reduce Salmonella invasion comparing to the VDR−/− cells. Therefore, there is less bacterial burden in the wild-type intestine. Moreover, VDR expression increases after Salmonella infection in intestine. VDR plays a role as a negative regulator of the NF-κB activity and modulates inflammatory responses of the host, thus allowing mice to survive the enteric bacterial infection (Figure 8A). In contract, VDR−/− cells have more Salmonella invasion in the intestine. Mice lacking VDR have a pro-inflammatory state due to intrinsically high NF-κB activity. VDR deletion leads to more aggressive gross and histological colonic injury, increases serum IL-6 levels as a marker of systemic inflammation, and enhances mortality after Salmonella infection (Figure 8B). Hence, VDR is an important contributor to host protection and intestinal homeostasis.
      Figure thumbnail gr8
      Figure 8Working model of VDR in regulation of bacterial invasion, NF-κB activity, host protection, and intestinal homeostasis.
      We postulate that increased NF-κB activity in VDR−/− intestinal cells is a consequence of the absence of VDR, which leads to free p65 and elevated phospho-p65, in part due to the reduction in IκBα.
      • Wu S
      • Xia Y
      • Liu X
      • Sun J
      Vitamin D receptor deletion leads to reduced level of IkappaBalpha protein through protein translation, protein-protein interaction, and post-translational modification.
      Consistent with these findings, the induction of serum IL-6 by pathogenic bacterial infection was much more robust in VDR−/− than in VDR+/+ mice. Interestingly, basal IL-6 serum levels in noninfected VDR−/− mice were comparable to those of Salmonella infected VDR+/+ mice. This is consistent with our demonstration that NF-κB activity is increased in MEFs lacking the vitamin D receptor, leading to a pre-inflammatory or pro-inflammatory state.
      • Sun J
      • Kong J
      • Duan Y
      • Szeto FL
      • Liao A
      • Madara JL
      • Li YC
      Increased NF-kappaB activity in fibroblasts lacking the vitamin D receptor.
      Take together; our in vitro and in vivo data suggest that VDR negatively regulates NF-κB activity and suppresses inflammatory responses to a common invasive enteric bacterial pathogen.
      We demonstrate intracellular complexes of VDR and p65 in freshly isolated colonic epithelial cells. Likewise, VDR physically interacts with NF-κB p65 in osteoblasts
      • Lu X
      • Farmer P
      • Rubin J
      • Nanes MS
      Integration of the NfkappaB p65 subunit into the vitamin D receptor transcriptional complex: identification of p65 domains that inhibit 1,25-dihydroxyvitamin D3-stimulated transcription.
      and fibroblasts
      • Sun J
      • Kong J
      • Duan Y
      • Szeto FL
      • Liao A
      • Madara JL
      • Li YC
      Increased NF-kappaB activity in fibroblasts lacking the vitamin D receptor.
      . In tubular cells, VDR/p65 formed a complex, thus inhibiting the ability of p65 to transactivate gene transcription.
      • Tan X
      • Wen X
      • Liu Y
      Paricalcitol inhibits renal inflammation by promoting vitamin D receptor-mediated sequestration of NF-{kappa}B signaling.
      In the present study, we show that VDR is able to negatively modulate NF-κB activity through physical binding with NF-κB p65, decreasing translocation of p65 in the nucleus, and reducing p65 phosphorylation. Intestinal VDR concentrations are increased by enteric Salmonella infection and are directly involved in the regulation of the NF-κB pathway in intestinal epithelial inflammation in vivo. Consistent with a host protective role of intestinal VDR during intestinal infection, VDR null mutant mice have worse outcomes following Salmonella-induced infection.
      Our study also demonstrates that enteric bacterial infection directly increases VDR protein expression and distribution in the colon and that commensal enteric bacteria also influence VDR distribution in epithelial cells. In the normal mouse colon, VDR locates to differentiated colonic epithelial cells at the top of crypts. Pathogenic Salmonella infection increases VDR expression and changes VDR distribution in mouse colonic epithelial cells in vivo to cells lower in the crypt. VDR was localized at the top of the crypts of the GF mouse colon. Commensal bacterial colonization of GF mice (conventionalization) induced VDR relocation in the colon and similar to cecum (data not shown) that seen with pathogenic Salmonella infection in conventional mice. The small intestine contains relatively few bacteria compared with the colon; therefore, bacterial colonization in GF mice did not change the VDR distribution in small intestine (data not shown). VDR exerts multiple effects, differentiation,
      • Bouillon R
      • Carmeliet G
      • Verlinden L
      • van Etten E
      • Verstuyf A
      • Luderer HF
      • Lieben L
      • Mathieu C
      • Demay M
      Vitamin D and human health: lessons from vitamin D receptor null mice.
      growth inhibitory
      • Li YC
      • Pirro AE
      • Amling M
      • Delling G
      • Baron R
      • Bronson R
      • Demay MB
      Targeted ablation of the vitamin D receptor: an animal model of vitamin D-dependent rickets type II with alopecia.
      • Yoshizawa T
      • Handa Y
      • Uematsu Y
      • Takeda S
      • Sekine K
      • Yoshihara Y
      • Kawakami T
      • Arioka K
      • Sato H
      • Uchiyama Y
      • Masushige S
      • Fukamizu A
      • Matsumoto T
      • Kato S
      Mice lacking the vitamin D receptor exhibit impaired bone formation, uterine hypoplasia and growth retardation after weaning.
      and anti-inflammatory actions.
      • Hewison M
      Vitamin D and innate immunity.
      • Lu X
      • Farmer P
      • Rubin J
      • Nanes MS
      Integration of the NfkappaB p65 subunit into the vitamin D receptor transcriptional complex: identification of p65 domains that inhibit 1,25-dihydroxyvitamin D3-stimulated transcription.
      The relocation of VDR in intestine may relate to transient activation of NFκB and other signaling pathways by colonization of the GF colon by commensal bacteria
      • Ruiz PA
      • Shkoda A
      • Kim SC
      • Sartor RB
      • Haller D
      IL-10 gene-deficient mice lack TGF-beta/Smad-mediated TLR2 degradation and fail to inhibit proinflammatory gene expression in intestinal epithelial cells under conditions of chronic inflammation.
      or infection in specific-pathogen-free mice
      • Eckmann L
      • Nebelsiek T
      • Fingerle AA
      • Dann SM
      • Mages J
      • Lang R
      • Robine S
      • Kagnoff MF
      • Schmid RM
      • Karin M
      • Arkan MC
      • Greten FR
      Opposing functions of IKKbeta during acute and chronic intestinal inflammation.
      or possibly by replacement of the infected epithelial cells and host defense against the Salmonella-induced damage.
      Using normal mice, we also identified a gradient for distribution of VDR from the proximal colon to the distal colon. In the normal proximal colon, bacterial fermentation is very intense with high production of short-chain fatty acids, an acid environment (pH 5 to 6), and rapid bacterial growth.
      • Guarner F
      • Malagelada JR
      Gut flora in health and disease.
      By contrast, the distal colon has less substrate availability, neutral pH and slow bacterial growth.
      • Guarner F
      • Malagelada JR
      Gut flora in health and disease.
      Interestingly, we found that in the proximal colon where the bacterial growth and metabolism is high, VDR expression is enhanced, whereas in the distal colon with less commensal bacterial growth, less VDR expression was detected. Our data on the distribution of VDR expression in the gut are correlated with bacterial density. VDR plays a critical role in protecting the colon from injury and maintaining intestinal integrity.
      • Kong J
      • Zhang Z
      • Musch MW
      • Ning G
      • Sun J
      • Hart J
      • Bissonnette M
      • Li YC
      Novel role of the vitamin D receptor in maintaining the integrity of the intestinal mucosal barrier.
      • Froicu M
      • Cantorna MT
      Vitamin D and the vitamin D receptor are critical for control of the innate immune response to colonic injury.
      Previous studies indicated that bacterial products, such as butyrate, regulate VDR expression.
      • Schwab M
      • Reynders V
      • Loitsch S
      • Steinhilber D
      • Schröder O
      • Stein J
      The dietary histone deacetylase inhibitor sulforaphane induces human beta-defensin-2 in intestinal epithelial cells.
      • Gaschott T
      • Stein J
      Short-chain fatty acids and colon cancer cells: the vitamin D receptor–butyrate connection.
      • Gaschott T
      • Werz O
      • Steinmeyer A
      • Steinhilber D
      • Stein J
      Butyrate-induced differentiation of Caco-2 cells is mediated by vitamin D receptor.
      • Schwab M
      • Reynders V
      • Loitsch S
      • Steinhilber D
      • Stein J
      • Schroder O
      Involvement of different nuclear hormone receptors in butyrate-mediated inhibition of inducible NFKB signalling.
      • Schwab M
      • Reynders V
      • Shastri Y
      • Loitsch S
      • Stein J
      • Schroder O
      Role of nuclear hormone receptors in butyrate-mediated up-regulation of the antimicrobial peptide cathelicidin in epithelial colorectal cells.
      Therefore, bacterial growth and metabolic products may contribute to the gradient distribution of VDR in the colonic epithelium in normal mice and be responsible for redistribution of VDR to the base and middle of colonic epithelial crypts after colonization of GF mice with complex enteric commensal microbiota.
      Toll-like receptors are key contributors to bacterial-induced inflammation and commensal bacteria- driven epithelial homeostasis.
      • Sartor RB
      • Muehlbauer M
      Microbial host interactions in IBD: implications for pathogenesis and therapy.
      • Rakoff-Nahoum S
      • Paglino J
      • Eslami-Varzaneh F
      • Edberg S
      • Medzhitov R
      Recognition of commensal microflora by toll-like receptors is required for intestinal homeostasis.
      • Otte JM
      • Cario E
      • Podolsky DK
      Mechanisms of cross hyporesponsiveness to Toll-like receptor bacterial ligands in intestinal epithelial cells.
      Recent studies show that activation of toll-like receptor-2 on human macrophages up-regulated the expression of VDR and induced the human antimicrobial peptide CAMP and killing of intracellular Mycobacterium tuberculosis.
      • Liu PT
      • Stenger S
      • Li H
      • Wenzel L
      • Tan BH
      • Krutzik SR
      • Ochoa MT
      • Schauber J
      • Wu K
      • Meinken C
      • Kamen DL
      • Wagner M
      • Bals R
      • Steinmeyer A
      • Zugel U
      • Gallo RL
      • Eisenberg D
      • Hewison M
      • Hollis BW
      • Adams JS
      • Bloom BR
      • Modlin RL
      Toll-like receptor triggering of a vitamin D-mediated human antimicrobial response.
      It is known that the CAMP gene is strongly up-regulated in myeloid cells by 1,25(OH)2D3.
      • Gombart AF
      • Borregaard N
      • Koeffler HP
      Human cathelicidin antimicrobial peptide (CAMP) gene is a direct target of the vitamin D receptor and is strongly up-regulated in myeloid cells by 1,25-dihydroxyvitamin D3.
      This process was dependent on both the endogenous production and action of 1,25(OH)2D3 through the VDR. It will be interesting to investigate the influence of intestinal toll-like receptor signaling on VDR expression in a future study.
      Our data indicate that VDR expression, location, transcriptional activity, and target gene expression are regulated by intestinal bacteria, which may be independent of 1,25-dihydroxyvitamin D3. Classically, VDR is considered to be a ligand-activated transcription factor. VDR binds to the vitamin D-response element in the target gene promoter to regulate gene transcription.
      • Farmer PK
      • He X
      • Schmitz ML
      • Rubin J
      • Nanes MS
      Inhibitory effect of NF-kappaB on 1,25-dihydroxyvitamin D(3) and retinoid X receptor function.
      Several studies demonstrate that VDR signaling can be activated independent of 1,25-dihydroxyvitamin D3.
      • Sun J
      • Mustafi R
      • Cerda S
      • Chumsangsri A
      • Xia YR
      • Li YC
      • Bissonnette M
      Lithocholic acid down-regulation of NF-kappaB activity through vitamin D receptor in colonic cancer cells.
      • Qi X
      • Tang J
      • Pramanik R
      • Schultz RM
      • Shirasawa S
      • Sasazuki T
      • Han J
      • Chen G
      p38 MAPK activation selectively induces cell death in K-ras-mutated human colon cancer cells through regulation of vitamin D receptor.
      • Makishima M
      • Lu TT
      • Xie W
      • Whitfield GK
      • Domoto H
      • Evans RM
      • Haussler MR
      • Mangelsdorf DJ
      Vitamin D receptor as an intestinal bile acid sensor.
      Our study suggested that bacteria directly activate VDR signaling by increasing protein level of VDR. We have not addressed this issue in vivo, but did show 1,25-dihydroxyvitamin D3 independent induction of VDR by Salmonella in colonic cell lines in vitro.
      In summary, we demonstrate that an invasive enteric pathogen, S. typhimurium, can induce colonic VDR expression and location in vivo, and stimulate VDR expression, transcription, and signaling in colonic epithelial cell lines and MEFs. Our current study also highlights the importance of VDR in negatively regulating bacterial-stimulated NF-κB activity in intestine. The presence of VDR in various tissues along with its ability to exert differentiation,
      • Bouillon R
      • Carmeliet G
      • Verlinden L
      • van Etten E
      • Verstuyf A
      • Luderer HF
      • Lieben L
      • Mathieu C
      • Demay M
      Vitamin D and human health: lessons from vitamin D receptor null mice.
      growth inhibitory
      • Li YC
      • Pirro AE
      • Amling M
      • Delling G
      • Baron R
      • Bronson R
      • Demay MB
      Targeted ablation of the vitamin D receptor: an animal model of vitamin D-dependent rickets type II with alopecia.
      • Yoshizawa T
      • Handa Y
      • Uematsu Y
      • Takeda S
      • Sekine K
      • Yoshihara Y
      • Kawakami T
      • Arioka K
      • Sato H
      • Uchiyama Y
      • Masushige S
      • Fukamizu A
      • Matsumoto T
      • Kato S
      Mice lacking the vitamin D receptor exhibit impaired bone formation, uterine hypoplasia and growth retardation after weaning.
      and anti-inflammatory actions,
      • Hewison M
      Vitamin D and innate immunity.
      • Lu X
      • Farmer P
      • Rubin J
      • Nanes MS
      Integration of the NfkappaB p65 subunit into the vitamin D receptor transcriptional complex: identification of p65 domains that inhibit 1,25-dihydroxyvitamin D3-stimulated transcription.
      set the stage for therapeutic exploitation of VDR ligands for the treatment of various intestinal inflammatory conditions and cancers.
      • Yee YK
      • Chintalacharuvu SR
      • Lu J
      • Nagpal S
      Vitamin D receptor modulators for inflammation and cancer.
      Our results and other investigators’ studies demonstrate that VDR has multiple critical functions in regulating response to enteric invasive pathogen, intestinal homeostasis,
      • Kong J
      • Zhang Z
      • Musch MW
      • Ning G
      • Sun J
      • Hart J
      • Bissonnette M
      • Li YC
      Novel role of the vitamin D receptor in maintaining the integrity of the intestinal mucosal barrier.
      • Froicu M
      • Cantorna MT
      Vitamin D and the vitamin D receptor are critical for control of the innate immune response to colonic injury.
      tight junction structure,
      • Kong J
      • Zhang Z
      • Musch MW
      • Ning G
      • Sun J
      • Hart J
      • Bissonnette M
      • Li YC
      Novel role of the vitamin D receptor in maintaining the integrity of the intestinal mucosal barrier.
      bacterial infection, and commensal bacterial colonization. Given the crucial role of enteric microbiota in the pathogenesis of IBD
      • Packey CD
      • Sartor RB
      Interplay of commensal and pathogenic bacteria, genetic mutations, and immunoregulatory defects in the pathogenesis of inflammatory bowel diseases.
      • Xavier RJ
      • Podolsky DK
      Unravelling the pathogenesis of inflammatory bowel disease.
      • Sartor RB
      Microbial influences in inflammatory bowel diseases.
      • Sartor RB
      • Muehlbauer M
      Microbial host interactions in IBD: implications for pathogenesis and therapy.
      and the therapeutic and pharmacological potential of probiotics in the treatment of inflammatory bowl disease,
      • Isaacs K
      • Herfarth H
      Role of probiotic therapy in IBD.
      • Vanderpool C
      • Yan F
      • Polk DB
      Mechanisms of probiotic action: Implications for therapeutic applications in inflammatory bowel diseases.
      understanding how pathogenic bacteria and probiotics regulate the VDR pathway may facilitate the development of targeted interventions to control intestinal inflammation.

      Acknowledgements

      We thank Yingli Duan, Juan Kong, and Yun Zhao for their technical support and Maureen Bower and Bo Liu for technical assistance with gnotobiotic mice.

      References

        • Haussler MR
        • Whitfield GK
        • Haussler CA
        • Hsieh J-C
        • Thompson PD
        • Selznick SH
        • Dominguez CE
        • Jurutka PW
        The nuclear vitamin D receptor: biological and molecular regulatory properties revealed.
        J Bone and Mineral Research. 1998; 13: 325-349
        • Van Cromphaut SJ
        • Dewerchin M
        • Hoenderop JG
        • Stockmans I
        • Van Herck E
        • Kato S
        • Bindels RJ
        • Collen D
        • Carmeliet P
        • Bouillon R
        • Carmeliet G
        Duodenal calcium absorption in vitamin D receptor-knockout mice: functional and molecular aspects.
        Proc Natl Acad Sci USA. 2001; 98: 13324-13329
        • Li YC
        • Kong J
        • Wei M
        • Chen ZF
        • Liu SQ
        • Cao LP
        1,25-Dihydroxyvitamin D(3) is a negative endocrine regulator of the renin-angiotensin system.
        J Clin Invest. 2002; 110: 229-238
        • Demay MB
        Mechanism of vitamin D receptor action.
        Ann NY Acad Sci. 2006; 1068: 204-213
        • Nagpal S
        • Na S
        • Rathnachalam R
        Noncalcemic actions of vitamin D receptor ligands.
        Endocr Rev. 2005; 26: 662-687
        • Liu PT
        • Stenger S
        • Li H
        • Wenzel L
        • Tan BH
        • Krutzik SR
        • Ochoa MT
        • Schauber J
        • Wu K
        • Meinken C
        • Kamen DL
        • Wagner M
        • Bals R
        • Steinmeyer A
        • Zugel U
        • Gallo RL
        • Eisenberg D
        • Hewison M
        • Hollis BW
        • Adams JS
        • Bloom BR
        • Modlin RL
        Toll-like receptor triggering of a vitamin D-mediated human antimicrobial response.
        Science. 2006; 311: 1770-1773
        • Hewison M
        Vitamin D and innate immunity.
        Curr Opin Investig Drugs. 2008; 9: 485-490
        • Norman AW
        Minireview: vitamin D receptor: new assignments for an already busy receptor.
        Endocrinology. 2006; 147: 5542-5548
        • Bouillon R
        • Carmeliet G
        • Verlinden L
        • van Etten E
        • Verstuyf A
        • Luderer HF
        • Lieben L
        • Mathieu C
        • Demay M
        Vitamin D and human health: lessons from vitamin D receptor null mice.
        Endocr Rev. 2008; 29: 726-776
        • Schauber J
        • Dorschner RA
        • Yamasaki K
        • Brouha B
        • Gallo RL
        Control of the innate epithelial antimicrobial response is cell-type specific and dependent on relevant microenvironmental stimuli.
        Immunology. 2006; 118: 509-519
        • Wang TT
        • Nestel FP
        • Bourdeau V
        • Nagai Y
        • Wang Q
        • Liao J
        • Tavera-Mendoza L
        • Lin R
        • Hanrahan JW
        • Mader S
        • White JH
        Cutting edge: 1,25-dihydroxyvitamin D3 is a direct inducer of antimicrobial peptide gene expression.
        J Immunol. 2004; 173: 2909-2912
        • Gombart AF
        • Borregaard N
        • Koeffler HP
        Human cathelicidin antimicrobial peptide (CAMP) gene is a direct target of the vitamin D receptor and is strongly up-regulated in myeloid cells by 1,25-dihydroxyvitamin D3.
        FASEB J. 2005; 19: 1067-1077
        • Bonizzi G
        • Karin M
        The two NF-kappaB activation pathways and their role in innate and adaptive immunity.
        Trends Immunol. 2004; 25: 280-288
        • Lu X
        • Farmer P
        • Rubin J
        • Nanes MS
        Integration of the NfkappaB p65 subunit into the vitamin D receptor transcriptional complex: identification of p65 domains that inhibit 1,25-dihydroxyvitamin D3-stimulated transcription.
        J Cell Biochem. 2004; 92: 833-848
        • Sun J
        • Kong J
        • Duan Y
        • Szeto FL
        • Liao A
        • Madara JL
        • Li YC
        Increased NF-kappaB activity in fibroblasts lacking the vitamin D receptor.
        Am J Physiol Endocrinol Metab. 2006; 291: E315-E322
        • Packey CD
        • Sartor RB
        Interplay of commensal and pathogenic bacteria, genetic mutations, and immunoregulatory defects in the pathogenesis of inflammatory bowel diseases.
        J Intern Med. 2008; 263: 597-606
        • Xavier RJ
        • Podolsky DK
        Unravelling the pathogenesis of inflammatory bowel disease.
        Nature. 2007; 448: 427-434
        • Sartor RB
        Microbial influences in inflammatory bowel diseases.
        Gastroenterology. 2008; 134: 577-594
        • Fukata M
        • Abreu MT
        TLR4 signalling in the intestine in health and disease.
        Biochem Soc Trans. 2007; 35: 1473-1478
        • Rhodes JM
        • Campbell BJ
        Inflammation and colorectal cancer: IBD-associated and sporadic cancer compared.
        Trends Mol Med. 2002; 8: 10-16
        • Sentongo TA
        • Semaeo EJ
        • Stettler N
        • Piccoli DA
        • Stallings VA
        • Zemel BS
        Vitamin D status in children, adolescents, and young adults with Crohn disease.
        Am J Clin Nutr. 2002; 76: 1077-1081
        • Abreu MT
        • Kantorovich V
        • Vasiliauskas EA
        • Gruntmanis U
        • Matuk R
        • Daigle K
        • Chen S
        • Zehnder D
        • Lin YC
        • Yang H
        • Hewison M
        • Adams JS
        Measurement of vitamin D levels in inflammatory bowel disease patients reveals a subset of Crohn's disease patients with elevated 1,25-dihydroxyvitamin D and low bone mineral density.
        Gut. 2004; 53: 1129-1136
        • Cantorna MT
        • Zhu Y
        • Froicu M
        • Wittke A
        Vitamin D status, 1,25-dihydroxyvitamin D3, and the immune system.
        Am J Clin Nutr. 2004; 80: 1717S-1720S
        • Cantorna MT
        Vitamin D and its role in immunology: multiple sclerosis, and inflammatory bowel disease.
        Prog Biophys Mol Biol. 2006; 92: 60-64
        • Kong J
        • Qiao G
        • Zhang Z
        • Liu SQ
        • Li YC
        Targeted vitamin D receptor expression in juxtaglomerular cells suppresses renin expression independent of parathyroid hormone and calcium.
        Kidney Int. 2008; 74: 1577-1581
        • Kong J
        • Zhang Z
        • Musch MW
        • Ning G
        • Sun J
        • Hart J
        • Bissonnette M
        • Li YC
        Novel role of the vitamin D receptor in maintaining the integrity of the intestinal mucosal barrier.
        Am J Physiol Gastrointest Liver Physiol. 2008; 294: G208-G216
        • Wada K
        • Tanaka H
        • Maeda K
        • Inoue T
        • Noda E
        • Amano R
        • Kubo N
        • Muguruma K
        • Yamada N
        • Yashiro M
        • Sawada T
        • Nakata B
        • Ohira M
        • Hirakawa K
        Vitamin D receptor expression is associated with colon cancer in ulcerative colitis.
        Oncol Rep. 2009; 22: 1021-1025
        • Miller SI
        • Mekalanos JJ
        Constitutive expression of the phoP regulon attenuates Salmonella virulence and survival within macrophages.
        J Bacteriol. 1990; 172: 2485-2490
        • Sun J
        • Hobert ME
        • Rao AS
        • Neish AS
        • Madara JL
        Bacterial activation of beta-catenin signaling in human epithelia.
        Am J Physiol Gastrointest Liver Physiol. 2004; 287: G220-G227
        • Duan Y
        • Liao AP
        • Kuppireddi S
        • Ye Z
        • Ciancio MJ
        • Sun J
        Beta-catenin activity negatively regulates bacteria-induced inflammation.
        Lab Invest. 2007; 87: 613-624
        • Li YC
        • Pirro AE
        • Amling M
        • Delling G
        • Baron R
        • Bronson R
        • Demay MB
        Targeted ablation of the vitamin D receptor: an animal model of vitamin D-dependent rickets type II with alopecia.
        Proc Natl Acad Sci USA. 1997; 94: 9831-9835
        • McCormick BA
        • Colgan SP
        • Delp-Archer C
        • Miller SI
        • Madara JL
        Salmonella typhimurium attachment to human intestinal epithelial monolayers: transcellular signalling to subepithelial neutrophils.
        J Cell Biol. 1993; 123: 895-907
        • Sun J
        • Hobert ME
        • Duan Y
        • Rao AS
        • He TC
        • Chang EB
        • Madara JL
        Crosstalk between NF-kappaB and beta-catenin pathways in bacterial-colonized intestinal epithelial cells.
        Am J Physiol Gastrointest Liver Physiol. 2005; 289: G129-G137
        • Sun J
        • Mustafi R
        • Cerda S
        • Chumsangsri A
        • Xia YR
        • Li YC
        • Bissonnette M
        Lithocholic acid down-regulation of NF-kappaB activity through vitamin D receptor in colonic cancer cells.
        J Steroid Biochem Mol Biol. 2008; 111: 37-40
        • Park CM
        • Reid PE
        • Walker DC
        • MacPherson BR
        A simple, practical ‘swiss roll’ method of preparing tissues for paraffin or methacrylate embedding.
        J Microsc. 1987; 145: 115-120
        • Sellon RK
        • Tonkonogy S
        • Schultz M
        • Dieleman LA
        • Grenther W
        • Balish E
        • Rennick DM
        • Sartor RB
        Resident enteric bacteria are necessary for development of spontaneous colitis and immune system activation in interleukin-10-deficient mice.
        Infect Immun. 1998; 66: 5224-5231
        • Liao AP
        • Petrof EO
        • Kuppireddi S
        • Zhao Y
        • Xia Y
        • Claud EC
        • Sun J
        Salmonella type III effector AvrA stabilizes cell tight junctions to inhibit inflammation in intestinal epithelial cells.
        PLoS ONE. 2008; 3: e2369
        • Ye Z
        • Petrof EO
        • Boone D
        • Claud EC
        • Sun J
        Salmonella effector AvrA regulation of colonic epithelial cell inflammation by deubiquitination.
        Am J Pathol. 2007; 171: 882-892
        • Barthel M
        • Hapfelmeier S
        • Quintanilla-Martinez L
        • Kremer M
        • Rohde M
        • Hogardt M
        • Pfeffer K
        • Russmann H
        • Hardt WD
        Pretreatment of mice with streptomycin provides a Salmonella enterica serovar Typhimurium colitis model that allows analysis of both pathogen and host.
        Infect Immun. 2003; 71: 2839-2858
        • Wu S
        • Xia Y
        • Liu X
        • Sun J
        Vitamin D receptor deletion leads to reduced level of IkappaBalpha protein through protein translation, protein-protein interaction, and post-translational modification.
        Int J Biochem Cell Biol. 2010; 42: 329-336
        • Hayden MS
        • Ghosh S
        Signaling to NF-kappaB.
        Genes Dev. 2004; 18: 2195-2224
        • Sakurai H
        • Chiba H
        • Miyoshi H
        • Sugita T
        • Toriumi W
        IkappaB kinases phosphorylate NF-kappaB p65 subunit on serine 536 in the transactivation domain.
        J Biol Chem. 1999; 274: 30353-30356
        • Li QP
        • Qi X
        • Pramanik R
        • Pohl NM
        • Loesch M
        • Chen G
        Stress-induced c-Jun-dependent vitamin D receptor (VDR) activation dissects the non-classical VDR pathway from the classical VDR activity.
        J Biol Chem. 2007; 282: 1544-1551
        • Qi X
        • Pramanik R
        • Wang J
        • Schultz RM
        • Maitra RK
        • Han J
        • DeLuca HF
        • Chen G
        The p38 and JNK pathways cooperate to trans-activate vitamin D receptor via c-Jun/AP-1 and sensitize human breast cancer cells to vitamin D(3)-induced growth inhibition.
        J Biol Chem. 2002; 277: 25884-25892
        • Qi X
        • Tang J
        • Pramanik R
        • Schultz RM
        • Shirasawa S
        • Sasazuki T
        • Han J
        • Chen G
        p38 MAPK activation selectively induces cell death in K-ras-mutated human colon cancer cells through regulation of vitamin D receptor.
        J Biol Chem. 2004; 279: 22138-22144
        • Tan X
        • Wen X
        • Liu Y
        Paricalcitol inhibits renal inflammation by promoting vitamin D receptor-mediated sequestration of NF-{kappa}B signaling.
        J Am Soc Nephrol. 2008; 19: 1741-1752
        • Yoshizawa T
        • Handa Y
        • Uematsu Y
        • Takeda S
        • Sekine K
        • Yoshihara Y
        • Kawakami T
        • Arioka K
        • Sato H
        • Uchiyama Y
        • Masushige S
        • Fukamizu A
        • Matsumoto T
        • Kato S
        Mice lacking the vitamin D receptor exhibit impaired bone formation, uterine hypoplasia and growth retardation after weaning.
        Nat Genet. 1997; 16: 391-396
        • Ruiz PA
        • Shkoda A
        • Kim SC
        • Sartor RB
        • Haller D
        IL-10 gene-deficient mice lack TGF-beta/Smad-mediated TLR2 degradation and fail to inhibit proinflammatory gene expression in intestinal epithelial cells under conditions of chronic inflammation.
        Ann NY Acad Sci. 2006; 1072: 389-394
        • Eckmann L
        • Nebelsiek T
        • Fingerle AA
        • Dann SM
        • Mages J
        • Lang R
        • Robine S
        • Kagnoff MF
        • Schmid RM
        • Karin M
        • Arkan MC
        • Greten FR
        Opposing functions of IKKbeta during acute and chronic intestinal inflammation.
        Proc Natl Acad Sci USA. 2008; 105: 15058-15063
        • Guarner F
        • Malagelada JR
        Gut flora in health and disease.
        Lancet. 2003; 361: 512-519
        • Froicu M
        • Cantorna MT
        Vitamin D and the vitamin D receptor are critical for control of the innate immune response to colonic injury.
        Bmc Immunology. 2007; 8: 11
        • Schwab M
        • Reynders V
        • Loitsch S
        • Steinhilber D
        • Schröder O
        • Stein J
        The dietary histone deacetylase inhibitor sulforaphane induces human beta-defensin-2 in intestinal epithelial cells.
        Immunology. 2008; 125: 241-251
        • Gaschott T
        • Stein J
        Short-chain fatty acids and colon cancer cells: the vitamin D receptor–butyrate connection.
        Recent Results Cancer Res. 2003; 164: 247-257
        • Gaschott T
        • Werz O
        • Steinmeyer A
        • Steinhilber D
        • Stein J
        Butyrate-induced differentiation of Caco-2 cells is mediated by vitamin D receptor.
        Biochem Biophys Res Commun. 2001; 288: 690-696
        • Schwab M
        • Reynders V
        • Loitsch S
        • Steinhilber D
        • Stein J
        • Schroder O
        Involvement of different nuclear hormone receptors in butyrate-mediated inhibition of inducible NFKB signalling.
        Molecular Immunology. 2007; 44: 3625-3632
        • Schwab M
        • Reynders V
        • Shastri Y
        • Loitsch S
        • Stein J
        • Schroder O
        Role of nuclear hormone receptors in butyrate-mediated up-regulation of the antimicrobial peptide cathelicidin in epithelial colorectal cells.
        Mol Immunol. 2007; 44: 2107-2114
        • Sartor RB
        • Muehlbauer M
        Microbial host interactions in IBD: implications for pathogenesis and therapy.
        Curr Gastroenterol Rep. 2007; 9: 497-507
        • Rakoff-Nahoum S
        • Paglino J
        • Eslami-Varzaneh F
        • Edberg S
        • Medzhitov R
        Recognition of commensal microflora by toll-like receptors is required for intestinal homeostasis.
        Cell. 2004; 118: 229-241
        • Otte JM
        • Cario E
        • Podolsky DK
        Mechanisms of cross hyporesponsiveness to Toll-like receptor bacterial ligands in intestinal epithelial cells.
        Gastroenterology. 2004; 126: 1054-1070
        • Farmer PK
        • He X
        • Schmitz ML
        • Rubin J
        • Nanes MS
        Inhibitory effect of NF-kappaB on 1,25-dihydroxyvitamin D(3) and retinoid X receptor function.
        Am J Physiol Endocrinol Metab. 2000; 279: E213-E220
        • Makishima M
        • Lu TT
        • Xie W
        • Whitfield GK
        • Domoto H
        • Evans RM
        • Haussler MR
        • Mangelsdorf DJ
        Vitamin D receptor as an intestinal bile acid sensor.
        Science. 2002; 296: 1313-1316
        • Yee YK
        • Chintalacharuvu SR
        • Lu J
        • Nagpal S
        Vitamin D receptor modulators for inflammation and cancer.
        Mini Rev Med Chem. 2005; 5: 761-778
        • Isaacs K
        • Herfarth H
        Role of probiotic therapy in IBD.
        Inflamm Bowel Dis. 2008; 14: 1597-1605
        • Vanderpool C
        • Yan F
        • Polk DB
        Mechanisms of probiotic action: Implications for therapeutic applications in inflammatory bowel diseases.
        Inflamm Bowel Dis. 2008; 14: 1585-1596