Effects of Vitamin D Supplementation on Inflammation, Colonic Cell Kinetics, and Microbiota in Colitis: A Review

Vitamin D is widely known to regulate bone health, but there is increasing evidence that it may also ameliorate colitis through inflammation, cell proliferation and apoptosis, and the microbiota. The purpose of this review is to systematically examine the mechanisms by which vitamin D reduces colitis. PubMed and Web of Science were searched for articles published between 2008 and 2019 using key words such as “vitamin D,” “colitis,” “inflammatory bowel disease,” “inflammation,” “apoptosis,” “cell proliferation,” and “gut bacteria”. Retrieved articles were further narrowed and it was determined whether their title and abstracts contained terminology pertaining to vitamin D in relation to colitis in human clinical trials, animal studies, and cell culture/biopsy studies, as well as selecting the best match sorting option in relation to the research question. In total, 30 studies met the established criteria. Studies consistently reported results showing that vitamin D supplementation can downregulate inflammatory pathways of COX-2, TNF-α, NF-κB, and MAPK, modify cell kinetics, and alter gut microbiome, all of which contribute to an improved state of colitis. Although vitamin D and vitamin D analogs have demonstrated positive effects against colitis, more randomized, controlled human clinical trials are needed to determine the value of vitamin D as a therapeutic agent in the treatment of colitis.


Introduction
Vitamin D is a fat-soluble vitamin deemed essential for human health. Besides being known for maintaining bone homeostasis, Vitamin D has been implicated in innate and adaptive immunity, as well as having anti-inflammatory properties [1,2]. The Dietary Reference Intakes (DRIs) for vitamin D depend on age: 400 IU vitamin D/day for children <1 year old, 600 IU vitamin D/day for people ages 1-70 years old, and 800 IU vitamin D/day for people >70 years old [2,3]. Vitamin D is obtained through sunlight, dietary supplements, and vitamin D-rich foods such as cod liver oil, tuna, sardines, milk, eggs, certain mushrooms and fortified orange juice and dairy products [2]. Supplementation with vitamin D 3 (5000 IU) improves serum concentrations of 25-hydroxy vitamin D , the precursor to the physiologically-active metabolite 1,25-duhydroxyvitamin D (1,, in deficient (<30 ng/mL) individuals [4].
Colitis is a chronic, uncontrolled inflammation of the colon and destruction of the colonic crypt affecting the gastrointestinal tract; it causes malabsorption and diarrhea, and increased risk of developing colorectal adenocarcinoma [5][6][7][8][9]. In 2015, an estimated 3.1 million adults in the United States were diagnosed with either ulcerative colitis (UC) or Crohn's disease; most were ≥45 years old, and non-Hispanic white (estimated 345,000 people) or Hispanic (estimated 395,000 people) [10].
According to the American Cancer Society, colorectal cancer is the third most common cancer diagnosed in the United States: 1 in 22 men, and 1 in 24 women [11]. History of UC for ≥8 years is one of the major risk factors for developing colorectal cancer [11,12]. Genetic, immune, environmental, and microbial factors may also play a role in the development of colitis [9,13,14].
Vitamin D status is negatively associated with inflammatory bowel disease (IBD) activity and clinical outcomes. A systematic review and meta-analysis of 27 studies found low vitamin D status, measured via 25-OHD, to be associated with increased disease activity, mucosal inflammation, and future clinical relapse, as well as decreased quality of life among IBD patients [15]. In subgroup analyses, these associations remained significant for patients with Crohn's disease, but only associations for disease activity and future clinical relapse remained significant among patients with ulcerative colitis [15].
Many studies have demonstrated that vitamin D improves colitis in human and animal models. In two animal studies, vitamin D deficiency contributed to increased severity and mortality in experimental colitis and even in experimental colon cancer [7,8]. Mice fed increased dietary vitamin D had significantly lower pro-inflammatory cytokines associated with colon tumor growth [8]. It has been shown that proinflammatory cytokines, such as interleukin-6 (IL-6) and tumor necrosis alpha (TNF-α), inhibit vitamin D metabolism in colon cancer cells [16]. The one-time administration of 300,000 IU vitamin D 3 reduced inflammation in UC subjects 90 days after intervention [17]. Associations between low serum vitamin D and the incidence of UC have been posited, but the exact mechanisms have not been well defined [7,9,17]. This review paper explores potential underlying mechanisms for the effects of vitamin D on colitis in terms of inflammation, colon cell kinetics, and the microbiota.

Dose-Dependent Effects of Vitamin D Supplementation on Ulcerative Colitis and Crohn's Disease
Supplementation of Vitamin D 2 (ergocalciferol) and Vitamin D 3 (cholecalciferol) improves serum vitamin D levels, with the latter having both higher potency and efficacy [18]. The optimal dose for Vitamin D supplementation in UC patients remains to be elucidated.
Vitamin D 3 supplementation for 90 days at 4000 IU/day outperformed supplementation at 2000 IU/day in a prospective, double-blind, randomized trial of adults with hypovitaminosis D and UC [9]. Serum Vitamin D levels rose in both groups but to a larger degree in those receiving 4000 IU/day (16.8 ng/dL) than in those receiving 2000 IU/day (5.00 ng/dL). In the 4000 IU/day group, four of the ten patients achieved normal Vitamin D levels (>30 ng/mL) following supplementation compared to only one of the eight patients in the 2000 IU/day group. Quality of life measured via the Short IBD Questionnaire (SIBDQ) increased after 90 days to a statistically significant degree in the 4000 IU/day group, but not in the 2000 IU/day group [9].
In another double blind, randomized clinical trial, adults with hypovitaminosis D and mild to moderate UC were supplemented with either 1000 IU/day or 2000 IU/day of Vitamin D for 12 weeks. Measures included serum 25-hydroxy vitamin D (25-OHD), inflammatory bowel disease questionnaire-9 (IBDQ-9) scores, and a simple clinical colitis activity index questionnaire (SCCAI) scores. At the end of the study, supplementing with 2000 IU/day compared to 1000 IU/day resulted in a larger mean change in 25-OHD (6.7 ng/mL vs 0.2 ng/mL), larger improvement in mean IBDQ-9 score, and larger improvement in mean SCCAI scores [19].
A double blind, randomized, controlled trial compared the efficacy of two doses of weekly Vitamin D 3 supplementation, 5000 IU and 10,000 IU/10 kg bodyweight/week for six weeks in pediatric IBD patients (N = 32). Serum 25-OHD was significantly higher compared to baseline in both groups at week 8 (24.0 to 41.5 ng/mL and 23.7 to 49.2 ng/mL, respectively), but only in the higher dose group at week 12 (30.8 ng/mL, p < 0.132 and 35.1 ng/mL, p < 0.001 respectively). No patient in either group experienced hypercalcemia or serious adverse events [20].

Cyclooxygenase-2
Vitamin D has been shown to reduce inflammation and attenuate autoimmune disease by affecting both adaptive and innate immunity [21,22]. The anti-inflammatory properties of Vitamin D may work in part through the inhibition of the prostaglandin (PG) pathway. This inhibition is mediated by inhibiting the expression of cyclooxygenase-2 (COX-2), the enzyme responsible for synthesizing PGs, promoting the expression of 15-prostaglandin dehydrogenase (15-PGDH), the enzyme responsible for deactivating PGs, and disrupting PG signaling by decreasing EP and FP PG receptor expression [16,23,24] (Table 1). A human biopsy study examining COX-2 immunostaining among subjects with long-standing colitis revealed that over 90% of the biopsies from dysplastic lesions were positive for COX-2 expression [25]. The overexpression of COX-2 may accelerate tumor growth and the development of colitis to colorectal cancer through the induction of pro-oncogenic colonic crypt mutations [24,26]. Treatment of an adenocarcinoma colon cancer cell line, COGA-1A, with 10 nM 1,25-dihydroxyvitamin D (1,25-OHD) inhibited the upregulation of COX-2 mediated by TNF-α by 37% after 12 h [16]. Notably, 1,25-OHD has been found to inhibit COX-2 expression in a dose-dependent manner in murine macrophages through upregulation of thioesterase superfamily member 4 (THEM4) [27].
A Vitamin D analog, Ro26-2198, was found to inhibit COX-2 signaling and delay the onset of clinical colitis in an azoxymethane, dextran sulfate sodium mouse model, as well as to inhibit COX-2 induction and decrease proliferation in HCA-7 colon cancer cells. Ro26-2198 also inhibited increases in phosphoactive extracellular signal related kinase (ERK) and c-Myc intrinsic to the model [28]. Activation of Vitamin D in the body is regulated by two genes, CYP27B1 and CYP24A1, with the former being responsible for activation and the latter for deactivation. Regulation of these enzymes is found to be important for colitis-associated colorectal cancer prevention by ensuring appropriate serum vitamin D to maintain normal cellular growth and differentiation [39]. The expression of these genes has been shown to be altered in the inflamed colonic tissue of IBD patients, resulting in a reduction of 25-OHD and impaired Vitamin D status [40]. Unsurprisingly, altered expression and polymorphisms of these genes, as well as low serum levels of 25-OHD, are associated with colorectal cancer [39,41].
A vitamin D-deficient, CYP27B1 knockout mouse model was shown to have shorter colon length, decreased body weight, thinner intestinal mucosa, and increased colonic crypt damage than wild-type counterparts [29]. The overexpression of CYP24A1 may play a role in the low serum 25-OHD levels seen in IBD patients, and modulating its expression may allow for better regulation of serum levels. In contrast, vitamin D analog BXL-62 showed significant reduction in proinflammatory cytokine expression such as TNF-α, and induced CYP24A1 expression in peripheral blood mononuclear cells in DSS-induced colitis in mice [30]. In the first known DSS-induced colitis mice study using gene therapy, CD11b + /Gr1 + monocytes were used as the cell vehicle as well as the macrophage-specific promoter (Mac1) to deliver and upregulate CYP27B1 expression in the colon to treat colitis [31]. It has been suggested that Gr1 + migrates to inflamed tissues first; however, Mac1 was only highly expressed in activated macrophages [31]. These activated macrophages were engineered to express the 1α-hydroxylase gene under the promoter of the Mac1 gene, so that CYP27B1 could be transported directly to the inflamed colon [31]. Mice injected with this treatment were found to have better survival, longer colon lengths, significantly reduced proinflammatory cytokines, and even mucosal regeneration compared to controls [31]. The upregulation of CYP24A1 following Vitamin D supplementation may be disadvantageous and should be considered when treating colitis [42].
Some studies have found that high CYP24A1 is prevalent in cancerous lesions in the colon compared to a normal colon. In a human biopsy study, CYP24A1 expression was 20 to 35 times higher and CYP27B1 was 4 times higher among low-and high-grade cancerous lesions in the colon compared to the normal colon [24]. Unbalanced, high basal levels of CYP24A1, however, may promote the progression of tumor growth, even in the colon [39]. How high the CYP24A1 expression has to be before tumorigenesis occurs remains unknown. Feedback mechanisms may be involved, but they are not well-defined at this time.
Single nucleotide polymorphisms (SNPs) are genetic variations that may potentially become biomarkers to predict risk of certain disease states [32]. Some studies explored CYP24A1 polymorphisms and risk for UC and colorectal cancer. One such study explored the association between CYP24A1 polymorphisms and risk of colonic polyps, colon cancer, and colitis in 144 subjects with colonic polyps, 96 subjects with colon cancer, and 44 subjects with UC [31]. SNPs identified and genotyped in CYP24A1 via an assay were rs4809957, rs6068816, rs6091822, and rs8124792 [36]. It was revealed that only SNPs rs4809957 and rs6068816 were significantly associated with risk of colonic polyps, colon cancer, and UC [31]. Similar studies also explored CYP24A1 polymorphisms for colorectal cancer risk, but risks for colitis were not discussed [43,44]. More research into the role of various Vitamin D activation and deactivation gene polymorphisms in UC treatment is warranted.

Tumor Necrosis Factor Alpha
TNF-α is a proinflammatory marker mediated by T-cells. It is one of the major pathogenic cytokines involved in the development of inflammatory bowel diseases, with elevated levels found in both CD and UC [16,45]. It has been shown that TNF-α promotes dysregulation of tight junction structures with remission following anti-TNF-α therapy [45][46][47]. If TNF-α-initiated inflammation is left unresolved, it may lead to colon cancer through angiogenesis and cell invasion [47,48]. Vitamin D may interfere with immune cells and suppress inflammatory cytokines, such as TNF-α, impeding intestinal inflammation [49].
A vitamin D analog, KH 1060, was shown to significantly increase serum vitamin D levels and decrease TNF-α levels among inflammatory bowel disease (IBD) patients. This analogue worked synergistically when combined with anti-TNF-α, resulting in lower TNF-α than either treatment alone [33]. Mice treated with vitamin D in a DSS-induced colitis model showed significantly reduced TNF-α expression compared to the control group. It was speculated that this reduction was due to a direct suppressive effect on cytokine expression or the maintenance of epithelial barrier function, thus averting innate immune system activation [34]. Similar results were seen in two other studies examining the effects of Vitamin D analogs, ZK191784 and TX527, on TNF-α expression in DSS-induced colitis murine models [35,36].

Nuclear Factor Kappa B
NF-κB is a key transcription factor that is crucial in various immune, physiological, and pathological responses; it is a major mediator of intestinal inflammation [50][51][52][53][54]. Among IBD patients, the expression of many inflammatory cytokines, such as NF-κB, IL-1 and IL-6, is increased in the intestinal mucosa [55]. In a bacterial-driven colitis and colon cancer murine model, the effects of two different Vitamin D doses were assessed: a maintenance dose of 1 IU/g of food and a high dose of 5 IU/g of food [8]. At 1 week postinfection, mice on the high-dose diet had decreased NF-κB activation in colonic epithelial cells. At 16 weeks, 41% of the mice on the maintenance-dose diet had cancer compared to only 11% of the mice on the high-dose diet. Decreased MAPK activation in the lamina propia leukocytes, inflammatory cell infiltrates, expression of proinflammatory cytokines, and IBD scores were also noted in the high-dose diet group [8].
Suppression of the NF-κB pathway and subsequent downstream expression of pro-inflammatory cytokines with 25-OHD has previously been confirmed in vitro [37]. A study on colon cancer-derived cells, HCT116, Caco-2, and SW480, treated with 20 nM 1,25-OHD for 24 h and followed by 100 ng/mL TNF-α for 12 h, revealed that vitamin D may maintain the intestinal epithelial barrier permeability by suppressing NF-κB activation [38].

Mitogen Activated Protein Kinase
The mitogen-activated protein kinase (MAPK) is a family of kinases that convert a variety of signals from the cell membrane to the nucleus in response to stimuli [56]. MAPK consists of three subunits, namely the extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK), as well as the p38 MAPK [56]. ERK is activated in response to growth stimuli, whereas JNK and p38 MAPK are activated in response to many cellular stresses such as inflammatory cytokines and DNA damage [56,57]. A subunit of p38 MAPK, p38α, was shown to regulate cellular response to stress and play a role in maintaining intestinal tissue homeostasis by cell differentiation [58,59]. Mice with downregulated p38α in intestinal epithelial cells in an AOM/DSS-colitis study were found to develop 29 low-grade tumors and 13 high-grade colonic tumors compared to 18 low-grade tumors and 7 high-grade tumors in wild-type mice [58]. Mice on 5 IU/g vitamin D showed a 2-fold decrease of JNK activation and 7-fold decrease of p38 activation in the colon compared to controls, suggesting that vitamin D may be used to treat IBD early on and prevent progression to colon cancer [8].

Retinoid X Receptor Alpha
RXRα belongs to the steroid nuclear receptor super family and is downregulated in murine colitis, colorectal cancer, and colon cancer cell lines, although the exact mechanism is yet to be identified [13,60]. RXRα +/− heterozygous mice treated with AOM/DSS showed significantly reduced RXRα expression and had a shorter colon length of 57.6 ± 9.4 mm compared to 67.8 ± 6.1 mm in the control group [60,61]. In the same study, heterozygous mice increased Snail1 and Snail2 expressions, which are transcription factors upregulated by inflammatory cytokines [60,61].
RXRα is also involved in the vitamin D signaling pathway via heterodimerization with VDR [60,62,63]. The biological activity of vitamin D may influence the immune system and inflammation by inducing VDR expression in the colon [6,9,14,64]. The VDR may play an anti-inflammatory role, especially in people with UC, as reduced VDR increases mucosal inflammation and barrier dysfunction [14]. In an experimental colitis mouse model, mice fed a vitamin D-deficient diet showed a lower serum vitamin D level of 20.1 ± 5.1 ng/mL, shortened colon length of 44.1 ± 4.6 mm, higher mortality rate at 7-10%, and lower VDR and RXRα expressions compared to control mice with a serum vitamin D level of 36.5 ± 4.7 ng/mL and colon length of 63.3 ± 3.8 mm [13].
Together with vitamin D and its proposed anti-inflammatory properties, vitamin D may reduce colitis by regulating COX-2, TNF-α, NF-κB through IkBa expression, RXR signaling, and vitamin D-activating enzymes CYP24A1 and CYP27B1.

Cell Proliferation
Normal colonic mucosa are made up of epithelial cells with folds called crypts, which are consistent throughout the colon [65,66]. Crypt renewal occurs at the bottom of the crypts where intestinal stem cells generate cell proliferation and differentiation, and thus replenish the epithelium [65][66][67]. In colitis, however, inflammation ultimately causes crypt disruption and architectural irregularities [65,66]. Vitamin D has been shown to promote mucosal regeneration through the modulation of signaling pathways, including VDR [65] ( Table 2). Treatment of 10 −8 M 1,25-OHD (calcitriol) in a cell study for 24 hr revealed increased expression of tight junction proteins, suggesting that vitamin D may play a role in maintaining the integrity of the tight junction complex [65]. VDR knockout mice showed impaired mucosal wound healing after DSS treatment, to the point where severe ulceration caused total crypt loss compared to wild-type mice [65].
Ki-67 is a nuclear protein constitutively expressed in mammalian cells, and is widely used as a cell proliferation marker in colitis and colon cancer [68]. DSS-treated rats had a significantly increased Ki-67 proliferation index compared to mice without DSS treatment [67]. Mice treated with vitamin D 3 at 15, 30 and 60 IU·g −1 ·w −1 had significantly inhibited colonic cell proliferation through Ki-67 staining by 17%, 35%, and 41%, respectively, in an AOM/DSS-treated colitis model compared to controls [69]. Patients with colitis have significantly increased Ki-67 expression in low-grade dysplasia, high-grade dysplasia, and carcinoma compared to normal epithelium [70]. A longstanding UC biopsy study also revealed elevated Ki-67 expression in both low-and high-grade dysplasia [25].
Another biopsy study showed an inverse relationship between low serum vitamin D and Ki-67 expression among patients with precancerous and cancerous colon cells, as high proliferation is associated with cell malignancy [71]. Patients who had ≥50% Ki-67 expression in colon cells had a mean serum vitamin D level of 6.27 ng/mL; patients who had ≤50% Ki-67 expression had a mean serum vitamin D level of 13.42 ng/mL; and patients who had <15% Ki-67 expression and a mean serum vitamin D level of 20.53 ng/mL [71]. Treatment of 800 IU vitamin D/day for six months increased p21 expression by 242% compared to placebo [72]. A cyclin-dependent kinase inhibitor, p21 plays a critical role in cell differentiation, cell cycle control, apoptosis modulation, and tumorigenesis [72]. Vitamin D reduces colitis by promoting colonic mucosa regeneration and regulating cell proliferation via modulation of p21 expression.

E-Cadherin
E-cadherin is a transmembrane linker protein and calcium-dependent cell adhesion molecule for colon crypt structure and function, making it important for mediating and maintaining intestinal homeostasis and epithelial architecture [74]. The loss of E-cadherin expression transitions adenoma to carcinoma, is a hallmark of colon carcinogenesis, and is prevalent in colitis [75,76]. The deletion of E-cadherin in mice resulted in severe loss of body weight, severe loss of intestinal epithelial structure, an increased number of apoptotic cells, impaired bacterial defense, and length reduction of the small intestine compared to controls [76]. In an AOM/DSS-colitis mice study, vitamin D 3 supplementation (15, 30, and 60 IU·g −1 ·w −1 3x/week) showed significant preventive effects in high doses, decreased the number of neoplasms by 33-63%, and increased E-cadherin expression, suggesting inhibition of colonic carcinogenesis progression [69]. E-cadherin in epithelial and immune cells was found to be expressed less in CYP27B1 knockout mice compared to their wild type counterparts in this DSS-induced colitis study [77]. These studies suggest that vitamin D improves colitis and prevents colon cancer progression by enhancing E-cadherin expression.

Wnt/β-Catenin
Wnt signaling is crucially involved in stem cell maintenance and epithelial cell proliferation [78][79][80]. Wnt is activated in most colon cancers; therefore, it can be used as a potential biomarker for colon health [78,81,82]. Wnt may also contribute to carcinogenesis from colitis to colon cancer [78,81,82]. Wnt is capable of signaling in a paracrine and autocrine manner, and the absence of Wnt promotes the degradation of β-catenin [79]. β-catenin is a key component of the Wnt pathway, and the activation of β-catenin predisposes humans and mice to both colitis and colon cancer [78,79,82,83]. In a biopsy study, 100% of the patients in the colitis-associated colorectal cancer group demonstrated β-catenin expression compared to none of the patients in the control group [81]. In another biopsy study, there were significantly more colonic Wnt and β-catenin stained cells in colitis patients (2.5-fold) and colorectal cancer patients (4.5-fold increase) compared to colon tissue from healthy controls [82]. Tumor samples extracted from patients with chronic UC and colon cancer were found to have elevated β-catenin expression after staining compared to noncancerous colonic tissues [83].
Supplementation of vitamin D 3 (15,30, and 60 IU·g −1 ·w −1 3x/week) in an AOM/DSS-colitis mouse model revealed decreased β-catenin, number and burden of colorectal neoplasms, and shortening of colon length, all in a dose-dependent manner [69]. Many other studies have explored the effects of vitamin D on the Wnt signaling pathway in the colon, but few have done so specifically in colitis [84,85].

Cell Apoptosis
Apoptosis is a process of natural cell death by eliminating genetically-mutated cells [86][87][88]. Homeostasis of the colonic epithelium requires a balance among cell proliferation, necrosis, and apoptosis [86][87][88][89]. Excessive intestinal epithelial cell apoptosis is a major cause of loss of epithelial cells and mucosal permeability, contributing to the development of colitis [7,90,91]. In a DSS-induced colitis rat model, the epithelial apoptotic index was significantly increased, at 7.2% ± 1.2% compared to 2.8% ± 1.2% in the control group [90]. Increased apoptosis in the DSS-treated colitis model showed a significantly decreased number of intact colonic crypts [67] VDR signaling has been shown to prevent apoptosis by downregulating p53-upregulated modulator of apoptosis (PUMA) by blocking NF-κB in TNBS-induced colitis [91]. A tumor suppressing gene, p53 is crucial for cell fate and prevents the spread of damaged cells [91]. Along with p53, PUMA is a potent inducer of apoptosis that is correlated with the severity of colitis, and is crucial to mediating intestinal epithelial cell apoptosis [7,92]. In an experimental colitis mouse model, VDR knockout mice developed more severe colitis with significant colonic epithelial cell apoptosis compared to control mice [91]. In another study, VDR knockout mice had significantly increased apoptotic epithelial cells compared to wild-type mice [7]. Mice treated with a vitamin D analog, paricalcitol, in a 2,4,6-trinitrobenzene sulfonic acid (TNBS) induced colitis model, had decreased severity of colitis compared to controls mediated in part through an inhibition of intestinal epithelial cell (IEC) apoptosis from a downregulation of PUMA expression [73]. The absence of PUMA was found to relieve DSSand TNBS-induced colitis, as well as to inhibit IEC apoptosis in PUMA-knockout mice compared to wildtype mice [92]. Human biopsies have revealed elevated levels of p53 and PUMA in the inflamed mucosal tissues of UC patients compared to those of healthy controls [93]. Vitamin D protects against colitis through the prevention of apoptosis via downregulation of PUMA.

Vitamin D and Microbiota
The gastrointestinal tract houses a variety of microbiota that are important for nutrient absorption, immunity, and intestinal epithelial barrier homeostasis [94,95]. Dysbiosis or abnormal interactions between the host and the gut microbiota lead to gastrointestinal diseases such as colitis [94][95][96][97]. UC patients have an unstable microbiota profile and reduced bacterial species richness compared to healthy patients [98]. Probiotics aid in intestinal homeostasis, and studies have shown potential therapeutic benefits of probiotics in colitis [94][95][96]. Probiotics have been shown to enhance VDR transcriptional activity, induce remission, reduce clinical activity scores, and prevent pouchitis, a complication in colitis following ileal pouch-anal anastomosis surgery [94][95][96]. In an experimental Salmonella colitis mouse model, probiotics Lactobacillus rhamnosus strain GG and Lactobacillus plantarum increased VDR expression and signaling, suggesting that VDR is necessary to prevent pathogenic invasion in the colon [96] (Table 3).
Serum vitamin D levels are associated with the function and composition of the intestinal microbiome [99,100]. Clostridium difficile (C. difficile) is a Gram-positive, anaerobic, spore-forming bacillus associated with diarrhea, sepsis, megacolon, and pseudomembranous colitis [101]. C. difficile is associated with a four-fold increase in mortality, increased hospitalization, and risk for colectomy up to 5 years after the first episode [102]. C. difficile-induced colitis is a costly and common healthcare-associated infection following antibiotic treatment, causing approximately 29,000 annual deaths in the United States [103]. C. difficile infection (CDI) is correlated with low serum vitamin D in IBD patients (mean of 20.4 ng/mL) versus non-CDI IBD patients (mean of 27.1 ng/mL), with a 4% reduction in CDI risk associated with each 1 ng/mL increase in plasma 25-OHD [102]. One innate line of defense against the toxic effects of C. difficile is cathelicidin, an anti-inflammatory, antimicrobial peptide activated by plasma 25-OHD [98,102,104]. Cathelicidin levels are higher in UC patients than healthy controls, but decrease with disease severity [98,102,104] A randomized, controlled trial in UC patients found a 3-fold increase in the cathelicidin precursor, human cationic antimicrobial protein 18 (hCAP18), gene expression following a single intramuscular injection containing 300,000 IU Vitamin D 3 [17]. Serum levels of 25-OHD are positively associated with serum and colonic cathelicidin among UC patients. Serum cathelicidin is negatively associated with histologic inflammation, and clinical relapse risk though these associations did not remain significant in multivariate analyses. In these multivariate analyses, only 25-OHD levels ≤35 ng/mL were independently associated with an increase in histologic inflammation and baseline histologic inflammation independently with an increase in clinical relapse risk [105].
It has been suggested that VDR acts as a master regulator of intestinal inflammation by regulating both autophagy and antimicrobial peptide production [106]. VDR KO mice have a higher susceptibility to DSS-induced colitis and subsequent dysbiosis with fewer butyrate-producing bacteria and increased E. coli and Bacteriocides [106]. Decreased levels of butyrate-producing bacteria and butyrate itself have been noted in UC patients relative to healthy controls [107]. The pretreatment of human IEC with butyrate was found to increase VDR expression in a dose-dependent manner [106].
Angiogenin-4 (Ang4) is an antimicrobial protein involved in host containment of enteric bacteria [108]. The expression of Ang4 is downregulated in the colon after DSS treatment, and even lower in vitamin D-deficient mice receiving either tap water or DSS [108]. Decreased Ang4 expression in vitamin D-deficient mice leads to homeostasis disturbance in the colon compared to vitamin D sufficient mice [108]. It is possible that vitamin D may regulate enteric bacteria by promoting Ang4 expression, but the mechanisms have not yet been identified [108].
The administration of vitamin D to patients with UC has been shown to reduce intestinal inflammation. These reductions coincided with increases in Enterobacteriaceae and a trend of reduction of the mucolytic species Ruminococcus gnavus, but not with changes in the overall fecal microbial diversity [98]. The supplementation of vitamin D in individuals with Crohn's disease has also been shown to influence microbial communities. One week of supplementation increased the abundance of Alistipes (Bacteroidetes); however, by the second week prevalence of Bacteroidetes decreased, while Blautia, Firmicutes, Fusicatenibacter, Intestinibacter, and Veillonella increased. By the third week Parabacteroides were at their highest. By week 4, Lactobacillus and Megasphera were at their highest. From week 0 to week 4 of vitamin D supplementation, the number of bacterial taxa decreased [111].
There are bacterial changes observed in feces between Cyp KO mice and Cyp wild-type mice [77]. Cyp KO and VDR KO mice had more Bacteroidetes and Proteobacteria phyla, but fewer Firmicutes and Deferribacteres phyla than wild-type mice [77]. However, vitamin D treatment reduced the numbers of the Helicobacteraceae family from the Proteobacteria phylum in Cyp KO mice [77]. Vitamin D protects against colitis through VDR by regulating the gut microbiota and decreasing pathogenic infections in the colon.  Vitamin D has a specific influence on the bacterial communities in CD, but not in HC. Administration of vitamin D may have a positive effect in CD by modulating the intestinal bacterial composition and also by increasing the abundance of potential beneficial bacterial strains. Vitamin D did not change the bacterial communities in HC.

Discussion
Evidence from tissue biopsies, cell line studies, murine models, and clinical human trials suggest that vitamin D may be beneficial against colitis by reducing inflammation, inhibiting cell proliferation and apoptosis, and regulating the gut microbiota. Furthermore, these benefits are often exerted in a dose-dependent manner with no clearly established ceiling. The reduction in inflammation appears to be multifactorial, resulting from a modification of the expression of COX-2, CYP24A1 and CYP27B1, MAPK, and RXRα, and a reduction of cytokines including TNF-α and NF-kB. A decrease in cell proliferation and β-catenin and increase in cell apoptosis and E-cadherin expression also helps explain the beneficial effect of Vitamin D. Pathogenic alterations in the composition of gut microbiota associated with colitis may be mitigated by Vitamin D supplementation through increased VDR signaling and Ang4 expression. Most current evidence supporting the benefit of Vitamin D supplementations to colitis stems from cell line studies and murine models. While the evidence from human-based genetic studies and tissue biopsies suggest similar underlying mechanisms, the success of Vitamin D in treating colitis in murine models needs to be confirmed in human clinical trials. Furthermore, while Vitamin D analogs can be effective in certain models, evidence of their efficacy in humans is lacking. Mechanistic evidence from animal and cell models, such as that cited within this review, should be considered when developing much needed human clinical trials.
The beneficial effects of vitamin D on IBD also appear to be mediated through actions on inflammatory cytokines including TNF-α, IL-6, and IL-12 and anti-inflammatory cytokines including IL-4, IL-10, and TGB-β. The ratio of these anti-inflammatory to inflammatory cytokines is improved by restoring vitamin D status via supplementation in IBD patients who otherwise often have insufficient levels [112].
According to our findings, VDR plays a critical role in the action of vitamin D against colitis. Inflammation-related signaling including E-cadherin, RXRα, and wnt/β-catenin seems to work via VDR. VDR KO mice generally suffer from impaired mucosal healing, severe crypt loss, and higher apoptotic ratio. These studies indicate that VDR status is crucial and appears to be some sort of defense mechanism in colitis. Colitis decreases cell proliferation and increases apoptosis; a lack of VDR in several mice studies was shown to affect cell proliferation and apoptosis. VDR is involved in maintaining the tight junction and intestinal epithelial barriers that regulate cell proliferation.
The role of the microbiome is very important in colitis prevention or treatment. However, more studies are needed on the potential mechanisms of vitamin D supplementation on the microbiota in IBD patients.
Due to the short half-life and lower circulating levels of the active form, 1,25-OHD, its precursor, 25-OHD, is often used to assess the vitamin D status in the body [113]. However, in order for 25-OHD to accurately reflect 1,25-OHD levels, the 1α-hydroxylating enzyme CYP27B1, which converts the former to the latter, would need to be optimally expressed [29]. Similarly, optimal levels of the catabolizing hydroxylase CYP24A1, which degrades 1,25-OHD, would be essential. CYP24A1 is induced mainly by 1,25-OHD, and CYP24A1 reduces elevated local 1,25-OHD levels in a negative feedback manner [39]. In those with colitis, the ratio of CYP27B1 to CYP24A1 may prove to be important for successful treatment [39].
In addition, CYP27B1 polymorphisms can lead to Vitamin D insufficiency and supplementation of Vitamin D increases CYP24A1 expression. Some studies have shown that high CYP24A1 can induce colon carcinogenesis, but no current studies have indicated how high it would have to be to do so. The role of CYP27B1 and CYP24A1 in regulating serum Vitamin D levels is both complex and consequential. Future studies should include the dose effects of vitamin D on CYP24A1 expression to determine the threshold at which CYP24A1 promotes colon carcinogenesis.
It was observed that the criteria for defining Vitamin D statuses varied between studies [9,17,29,38,99]. While the Institute of Medicine has specified serum vitamin D thresholds indicative of insufficiency and deficiency with regards to bone health [114], levels which optimize human immune function remain unclear and require further investigation.
Vitamin D analogs have shown promising results, and their utility in treating colitis should continue to be explored. Administration of 1,24(OH) 2 D 2 was found to have similar effects compared to 1,25(OH) 2 D 2 on reducing colitis symptoms, but 1,24(OH) 2 D 2 presents less risk in raising serum calcium levels [63]. Having high serum calcium, or hypercalcemia, is a potentially life-threatening condition that can cause progressive kidney failure, dehydration, and granulomatous diseases such as tuberculosis and sarcoidosis [106]. The relationship between calcium and vitamin D, specifically concerning the various types of vitamin D, should be further investigated as potential therapeutic options for colitis.
Animal studies remain popular, as genetic techniques can optimize disease models to better understand the pathology of ulcerative colitis. There are limited human clinical trials on the effect of vitamin D on colitis. More randomized, controlled human clinical trials are needed. In conclusion, vitamin D may benefit sufferers of colitis by reducing inflammatory cytokines, maintaining intestinal epithelial cell integrity by modulation of cell proliferation and apoptosis, and reducing pathogenic infections.

Materials and Methods
PubMed and Web of Science were the primary databases used to search for articles-published between 2008 and 2019-using key words such as "vitamin D and colitis," "vitamin D and inflammatory bowel disease," "inflammation," "apoptosis," "cell proliferation," and "gut bacteria". PubMed initially yielded 447 articles that were then evaluated further. Secondary inclusion criteria were based on whether or not titles and abstracts included "vitamin D" in relation to colitis in human subjects, animal models, and cell culture studies, as well as selecting the best match sorting option on PubMed according to key words. Upon further evaluation, 251 articles met the secondary criteria. After further checking based on the criteria of original studies and mechanism-based vitamin D intervention on colitis, 30 articles were finally chosen that were most relevant to vitamin D and colitis on inflammation (Table 1), cell kinetics (Table 2), and the microbiota (Table 3).