Background
The two forms of inflammatory bowel disease (IBD): Crohn's disease and ulcerative colitis are chronic diseases characterized by aberrant responses to luminal bacteria in genetically susceptible subjects [
1]. Although IBD are chronic diseases, the initiation of the inflammation and reactivations of the disease are associated with engagement of the innate immune response and progressive induction of IL-12, IL-1β, and TNF-α in the intestine [
2].
The vitamin D receptor (VDR) is a ligand inducible transcription factor that has been shown to be an important regulator of many experimental autoimmune diseases including IBD [
3]. A major source of vitamin D results from its manufacture via a photolysis reaction in the skin. Dietary intake of vitamin D is problematic since there are few foods, which are naturally rich in vitamin D. There is mounting evidence for a link between vitamin D availability either from sunshine or diet and the prevalence of IBD [
3]. In addition, vitamin D deficiency is common in patients with IBD [
4]. Vitamin D is biologically inactive and two hydroxylation reactions occurring in the liver and kidney result in production of active vitamin D (1,25(OH)
2D
3). 1,25(OH)
2D
3is the form of vitamin D that binds to the VDR and inhibits experimental autoimmunity.
Vitamin D deficiency and VDR deficiency have been shown to exacerbate chronic IBD in IL-10 KO mice [
5,
6]. Furthermore, treatment of IL-10 KO mice with 1,25(OH)
2D
3 resulted in the suppression of IBD symptoms [
5]. The effects of vitamin D and 1,25(OH)
2D
3 in IBD have begun to be explored and include direct effects of vitamin D on T cells and innate immune cells. Suppression of TNF-α is one mechanism underlying the efficacy of 1,25(OH)
2D
3 in vivo [
7]. In the gut it is likely that the targets of vitamin D will include epithelial cells, innate as well as acquired immune cells.
Macrophage are important vitamin D targets since they are a potential source of the 1 alpha hydroxylase enzyme (Cyp27B1) that converts the provitamin D hormone 25(OH)D
3 to active VDR binding 1,25(OH)
2D
3 [
8]. Macrophage expression of Cyp27B1 has been shown to be increased following TLR ligation in vitro [
9]. Macrophage and the TLR pathways are critical regulators of experimental IBD. The TLR-4, TLR-2 and MyD88 KO mice are extremely susceptible to dextran sodium sulfate (DSS) induced colitis [
10]. Little is known on whether vitamin D regulates these pathways to maintain gastrointestinal homeostasis in vivo.
DSS initiates mucosal epithelial cell damage by disrupting barrier function, leading to ulceration, and bleeding [
11]. A relatively slow mucosal repair process occurs following withdrawal of DSS in wildtype (WT) mice [
11]. DSS induced colitis results due to stimulation of the innate immune response since T and B cell-deficient animals such as SCID mice [
12], and also SCID mice depleted of NK cells develop DSS colitis [
13]. The model is characterized by macrophage production of IL-1β, IL-6, and TNF-α [
13]. Macrophage induction at mucosal surfaces are early triggers in an inflammatory cascade that leads to destruction of the intestinal wall.
The role of the VDR and 1,25(OH)2D3 in regulation of the early innate immune response to DSS was probed in mice. Expression of the VDR was found to be critical to control the innate immune response in the gut. In addition, VDR KO mice had a delayed recovery following DSS withdrawal. 1,25(OH)2D3 treatments were protective and controlled the early innate response in the colon. VDR KO mice were extremely susceptible to the TLR-4 ligand LPS and furthermore the early death of the VDR KO mice following DSS challenge was associated with bacterial recovery from the peritoneal cavity.
Discussion
Absence of the VDR results in mice that are extremely susceptible to chemical injury in the gut. DSS treated VDR KO mice are anemic, have high white blood cell counts, large amounts of blood in the colon, and many inflammatory cytokines expressed at high levels. A breach in the intestinal mucosa would lead to the entry of bacteria into the blood stream followed by an excessive, uncontrolled, systemic inflammation including, at the extreme, septic shock. Masubuchi et. al have shown that endotoxin levels detected in the portal blood of rats treated with DSS were higher than those in control rats [
15]. In humans, systemic endotoxemia has been described in ulcerative colitis [
16,
17] and Crohn's disease patients and shown to correlate positively with disease activity, pro-inflammatory cytokine production and the extent of intestinal ulceration [
18‐
23]. VDR deficient mice are extremely sensitive to intravenous or intraperitoneal administration of LPS, supporting the possibility that VDR KO mice with colitis die due to endotoxemia. In addition, bacteria was cultured from the peritoneal cavities of moribund VDR KO mice following DSS treatment. All of the data support the hypothesis that the early mortality of the VDR KO mice treated with DSS is due to perforation of the gut and resulting endotoxemia.
DSS induced colitis is a chemical that damages the colonic epithelium [
24] with subsequent recruitment and activation of inflammatory cells and upregulation of inflammatory mediators [
25]. During injury or inflammation, intestinal epithelial cells are rapidly proliferating and this process of mucosal repair and regeneration is critical for gut homeostasis [
26]. The VDR is highly expressed in human [
27] and mouse [
28] colonic mucosa and intestinal epithelial cells [
29]. 1,25(OH)
2D
3 has been shown to control normal villus and crypt development by regulating proliferation and differentiation of intestinal cells [
30]. Furthermore, 1,25(OH)
2D
3 is an important regulator of cell growth and differentiation in many tissues including the colon [
31]. In DSS colitis most of the pathological changes are localized to the distal colon which is a site of low proliferation of epithelial cells [
31]. Others have shown that the baseline proliferative state of colonic eptithelial cells in the crypts of VDR KO mice are elevated and showed increased expression of markers for cycling cells (proliferating cell nuclear antigen and cyclin D1) when compared to WTs [
31]. Although increased proliferation in the colon of VDR KO mice might be seen as beneficial, it has been shown that crypts with increased numbers of epithelial cells in cell cycle are more susceptible to radiation-induced injury as determined by their inability to repopulate the crypt [
32]. The effectiveness of 1,25(OH)
2D
3 in protecting the colon and the heightened susceptibility of the VDR KO gut to DSS colitis is likely due in part to the importance of vitamin D and VDR signaling in control of epithelial growth and proliferation.
Once the mucosal barrier is breached, the submucosa is exposed to a vast pool of luminal antigens, including foods and bacteria, and the innate immune responses are engaged to produce large amounts of cytokines. Analysis of cytokine production by colonic homogenates revealed significant elevation of TNF-α, IL-1α, IL-1β, IL-12p70, IFN-γ and IL-10 in VDR KO mice treated with DSS when compared with WT mice. Human and animal studies support the idea that TNF-α and IFN-γ are important pathological mediators of IBD [
33]. In humans with IBD approximately two thirds of the patients responded to anti-TNF-α treatments [
34], and in mice the intestinal inflammation was significantly attenuated by anti-IFN-γ and/or anti-TNF-α monoclonal antibodies [
35]. The production of IFN-γ, TNF-α, IL-1α and IL-1β in the colonic homogenates of VDR KO mice was substantially higher at 10 days post-DSS than in WT mice consistent with the observed delay in recovery from inflammation in these mice. The prolonged expression of inflammatory cytokines corresponded with the increased susceptibility and delayed recovery of the VDR KO mice.
During experimental colitis members of the α-chemokine family are involved in the recruitment of immune cells and the development of intestinal inflammation. Ajuebor et. al have shown in experimental IBD that colonic KC and MIP-1α expression led to leukocyte recruitment in the gut [
36]. Furthermore studies by Banks et. al showed that the expression of MIP-1α correlated with the severity of colonic inflammation in patients with IBD [
37]. The increased levels of KC and MIP-1α in the colons of VDR KO suggest that in the absence of the VDR many more inflammatory cells may be recruited to the site of injury and then produce inflammatory cytokines that result in severe and fatal form of colitis.
1,25(OH)
2D
3 treatments both in the food or locally reduced colitis symptoms in WT mice. 1,25(OH)
2D
3 treated WT mice had increased IL-10 production that might serve to inhibit other cytokine responses and lead to a dampening of the cytokine storm in the colon. Furthermore 1,25(OH)
2D
3 has the ability to directly induce antimicrobial gene expression and activity of antimicrobial peptide CAMP and defensin β2 genes [
38]. CAMP is a potent antisepsis agent that blocks macrophage induction, enhances the survival of mice treated with lethal doses of LPS [
39] and accelerates epithelial wound healing [
40]. The induction of CAMP and other antimicrobial genes suggested that 1,25(OH)
2D
3 might be protective against sepsis after injury and might accelerate epithelial wound healing [
38].
Methods
Mice
Weight (20–25 g) and sex matched 10–12 week old C57BL/6 WT and VDR KO (C57BL/6, gift from M. Demay, Harvard University, Cambridge, MA) were bred for use at the Pennsylvania State University (University Park, PA). All procedures were reviewed and approved by the Pennsylvania State University Institutional Animal Care and Use Committee.
Induction of colitis
Mice were administered 0.5%–3.5% DSS (MW = 40 kDa; ICN Biomedicals, Aurora, OH) dissolved in filter-purified and sterilized water ad libitum for 5 days, after which the mice were resumed on water for the remainder of the experiment. Animals were weighed daily and monitored clinically for rectal bleeding, diarrhea, and general signs of morbidity. Moribund mice or mice that had lost more than 25% of their body weight were sacrificed and listed as dead following induction of DSS colitis.
Colitis symptoms
The gross colonic blood scoring system previously described by Siegmund et al[
41] was used. The colonic bleeding score was as follows: 0- no visible blood in the entire colon, 1-blood detected in less than 1/3 of the colon, 2- blood detected in less than 2/3 of the colon, 3- blood visible throughout the entire colon. The entire colon from cecum to anus was removed and the length was measured and reported as colonic length as described [
11].
The distal colon were removed from the mice, fixed in 10% formalin and sent to the Pennslyvania State University Animal Diagnostic Laboratories (University Park, PA) for H&E staining. Histological analysis was performed blinded by 2 independent investigators on a scale from 0 to 40 as follows: severity of inflammation (0–3: none, slight, moderate, severe), extent of injury (0–3: none, mucosal, mucosal and submucosal, transmural), and crypt damage (0–4: none, basal 1/3 damaged, basal 2/3 damaged, only surface epithelium intact, entire crypt and epithelium lost). Each score was then multiplied by a factor equivalent with the percentage of tissue involvement (× 1: 0–25%, × 2: 26–50%, × 3: 51–75%, × 4: 76–100%)[
42].
Peripheral blood analysis
Blood was collected by cardiac puncture in tubes coated with EDTA (Becton Dickinson Vacutainer System, NJ) and analyzed using an ADVIA 120 Hematology System (Bayer Diagnostic, NY). For some experiments 100–200 μl of blood was collected by retroorbital sinus bleeding using heparinized microcapillary pipettes.
Colonic homogenate
The distal colon was weighed and the same amount of tissue was cut open and washed in 1XPBS containing penicillin (100 U/ml) and streptomycin (100 μg/ml). Tissue was then homogenized in 1 ml PBS using a razor blade. The homogenized colon tissue was centrifuged at 10,000 g at 4°C for 10 min. Cytokine concentrations were determined in the supernatant.
Cytokine and chemokine ELISA
Serum and supernatants were assayed for mouse TNF-α, IL-12p70, IFN-γ, IL-1α, IL-1β, IL-10 production using Ab pairs and standards provided in the BD Pharmingen kits ELISA (San Diego, CA) according to the manufacturer's instructions. For KC and MIP-1α the ELISA kits were from R&D Systems. (Minneapolis, MN). The limits of detection were 31 pg/ml TNF-α, 125 pg/ml IL-12 p70,125 pg/ml IFN-γ, 31 pg/ml IL-1α, 31 pg/ml IL-1β, 31 pg/ml IL-10, 16 pg/ml KC and 31 pg/ml MIP-1α.
Endotoxic shock
C57BL/6 mice were injected iv or ip with LPS from Escherichia coli 0111:B4 (Sigma, St Louis, MO) at a dose of 10 mg/kg body weight. Mice were monitored 3–4 times daily during endotoxic shock, and moribund animals were sacrificed.
1,25(OH)2D3 treatment
Mice received 50 ng/daily of 1,25(OH)
2D
3 in the diet as described [
5] elsewhere 1 week prior and throughout DSS administration. For local treatment, 50 ng of 1,25(OH)
2D
3 was dissolved in 20 uL corn oil and administered rectally 1 day prior to DSS administration and every other day thereafter for the duration of the experiment. Control mice received the corresponding amount of ethanol diluted in corn oil.
Statistical analysis
Statistical analysis was performed using the paired Student's t test and ANOVAs (StatView; SAS Institute, Cary, NC). P values < 0.05 were considered significant. Error bars represent +/- SEM. The log-rank test was used to compare Kaplan-Meier survival curves.
Authors' contributions
MF carried out the studies, participated in the design of the study, performed the analyses and drafted the manuscript. MTC conceived of the study, participated in its design and coordination and helped to draft the manuscript. Both authors read and approved the final manuscript.