Effect of Vitamin D Supplementation on Inflammatory Bowel Disease in Children: A Meta-Analysis

Background Apart from its role in bone health, recent developments have shown that vitamin D also has anti-inflammatory properties, and therefore may have a role in inflammatory bowel disease (IBD) in children. Objectives To determine the effect of vitamin D supplementation on the disease activity of pediatric patients with IBD.


INTRODUCTION
Vitamin D, as it is widely known, has an important role in calcium homeostasis. Following the discovery of vitamin D receptors on peripheral blood mononuclear cells, [1] vitamin D was also acknowledged to have a role in regulation of the immune system. [2] It has been shown to have functions including anti-inflammatory, cell differentiation, and apoptotic effects. [3] Inflammatory bowel disease (IBD), which includes Crohn's Disease (CD) and ulcerative colitis (UC), is a well-known gastrointestinal tract disease with a chronic and relapsing course that requires lifelong treatment. [4] The exact cause remains largely unknown, but it is hypothesized that genetic, environmental, and microbial influences converge and result in a dysregulated mucosal immune response resulting in disease. [5] Children with IBD are known to be at greater risk for having nutritional consequences than adults. In particular, due to vitamin D deficiency, they are more likely to have decreased bone mineral density. [5] Recent studies have focused on the potential link between vitamin D and severity of IBD in children; proposing that a deficiency state of vitamin D is associated with a more severe course of disease. [6,7] While there are several studies on vitamin D therapy in children with IBD, there has not yet been a meta-analysis of these published studies. The aim therefore of this study was to determine the effect of vitamin D supplementation on disease activity of IBD in children, by analyzing results of published studies that have evaluated the supplementation of vitamin D in this population.

METHODOLOGY Search Strategy
Articles were searched on the following search engines and research databases: Cochrane Library, PubMed, EBSCO Host, ScienceDirect, Google Scholar, and Wiley Online. Keyword search and Boolean logic search were used as search techniques. Using these search strategies, the following phrases were searched: ("Vitamin D" OR "25-hydroxy vitamin D" OR "25(OH)D" OR "vitamin D2" OR "vitamin D3") AND ("Inflammatory Bowel Disease" OR "Crohn's" OR "Ulcerative Colitis") AND ("Child") AND ("Treat" OR "Therapy") AND ("Randomized Trial"). The references of included articles were searched for other relevant articles. Duplicates were subsequently removed.

Eligibility Criteria
Trials conducted on pediatric patients with IBD, CD or UC in which vitamin D supplementation was instituted and where serum 25(OH)D concentrations and disease activity parameters such as Pediatric Crohn Disease Activity Index (PCDAI)/Pediatric Ulcerative Colitis Activity Index (PUCAI), and inflammatory markers [ie, C-reactive protein (CRP), and erythrocyte sedimentation rate (ESR)] reported from baseline and follow-ups were included in this meta-analysis. Studies were excluded if: (a) they included adult patients; (b) they were not in the English language; and (c) they were only available in abstract.

Selection Process
Two reviewers independently screened titles and abstracts of all identified articles and assessed fulltext articles of each potentially eligible study for inclusion. Disagreement between the reviewers was resolved by discussion and with a third reviewer. The Cochrane Collaboration's Grading of Recommendations, Assessment, Development, and Evaluations (GRADE) assessment tool was utilized to identify and categorize the quality of studies as low, unclear, and high risk. The following elements of a randomized controlled trial were appraised: sequence generation, blinding, allocation concealment, incomplete outcome data, selective outcome reporting, and other sources of bias. Low risk of bias indicates that plausible bias is unlikely to seriously alter results, while unclear bias denotes the presence of plausible bias which raises doubts about results. High risk of bias, on the other hand, implies that there is plausible bias which seriously weakens results. [8] Data Collection Process The following information were obtained from each study and encoded in a standardized Excel file: authors, year of publication, country, study design, sample size, demographic characteristics of patients (age, sex), disease of study population (IBD/CD/ UC), vitamin D supplementation (generic, dose and duration), vitamin D levels (mean, standard deviation, or interquartile range of serum 25-OHD) and vitamin D cut-off points, disease activity of IBD, outcomes of the study, and any conclusions regarding the effect of vitamin D therapy. (ie, risk ratio, odds ratio). Vitamin D levels reported in nmol were adjusted to and reported in ng/mL to allow accurate comparisons.

Data Analysis
Statistical analyses were conducted using STATA MP Statistical Software, Version 13, College Station, TX: StataCorp LP. A p-value ≤0.05 was considered statistically significant. Since primary studies did not assume one effect size among all the studies, the overall effect of meta-analysis was derived using a random-effects model (REM), which took within-study and between-study variation into account. Two summary effect measures were utilized according to the type of outcome data: pooled odds ratio for categorical outcomes and standardized mean difference for continuous-level outcomes. Pooled odds ratio was utilized for disease activity between the interventional and control groups, while standardized mean difference was employed for serum 25(OH)D and different inflammatory markers (ESR and CRP). Statistical heterogeneity between studies was evaluated using Q statistics test, I 2 statistics, and tau squared (τ 2 ) statistics. [9] I 2 values greater than 50% imply substantial heterogeneity. [9] Publication bias was assessed using both graphical and statistical approaches. Graphical approach for publication bias involved contour-enhanced funnel plots, while statistical assessment of funnel plot asymmetry was performed using Egger's test and Begg's asymmetry test. [9]

ETHICAL CONSIDERATIONS
The study involved the use of data from results of previously published studies; thus, no ethical approval and patient consent was needed.

Study Selection
The search strategy retrieved a total of 1,001 articles. Upon initial screening, 764 duplicates were removed leaving 237 papers. The abstract and title of the remaining 237 articles were screened, and 219 of these were removed due to reasons enumerated in Figure 1. The full-text articles of the remaining 18 papers were then reviewed and 14 of these were removed as these involved a different population (adult population); hence, a total of four studies were included in the analyses.

Study Characteristics of Individual Studies
Four experimental studies were included in this meta-analysis. Table 1 presents a summary of characteristics and the population of included studies.

Risk of Bias Analysis and Quality of Evidence of Included Studies
The risk of bias analysis and quality of evidence of included studies are depicted in Figure 2. All studies have low risk for random sequence generation, allocation concealment bias, attrition bias, reporting bias, and other biases. However, 25% of the included studies have high risk for performance and detection biases due to the absence of blinding in the studies.
Pooled Estimate of Disease Activity between the Experimental and Control Groups The pooled estimate for disease activity (remission/ inactivity), measured using PCDAI or PUCAI, between the experimental and control group are presented in Figure 4. A total of three trials were included, which enrolled 96 respondents in the experimental    The change in ESR was not different between the two groups (SMD = -2.35, z = 1.24, p = 0.215, 95%CI = -6.06 to 1.37). There was a substantially high heterogeneity among the included studies (χ 2 = 47.90, p = 0.001, I 2 = 97.90%, τ 2 = 7.03).

Pooled Estimate of CRP and Mean Change in CRP Between the Experimental and Control Groups
The pooled estimates of mean CRP and change in CRP between the experimental and control groups, which included two articles and a total of 110 respondents, are presented in Figure 6. There was no evidence that the mean CRP (SMD = -0.25, z = 1.10, p = 0.242, 95%CI = -0.69 to 0.19) and mean change in CRP (SMD = 0.00, z = 0.00, p = 1.000, 95%CI = -0.37 to 0.37) were statistically different. There was also negligible heterogeneity for studies included in the analysis for both mean CRP (χ 2 = 1.37, p = 0.242, I 2 = 27.00%, τ 2 = 0.03) and mean change in CRP (χ 2 = 0.00, p = 1.000, I 2 = 0.00 τ 2 = 0.01).

Publication Bias Assessment
Graphical analysis using contour-enhanced funnel plots indicated the likelihood of funnel asymmetry with a left-side predominance for mean serum     Table 2).

DISCUSSION
IBD is a chronic, relapsing intestinal inflammatory disorder with unidentified causes. [10] However, it is believed to be due to a result of inappropriate and ongoing activation of the immune system against environmental triggers among genetically predisposed individuals. [11] Studies in animal models of colitis and in vitro human studies support the role of vitamin D in regulation of the immune system of the gut and its potential as therapeutic adjuncts in IBD. [12][13][14] Therefore, this study aimed to analyze the effect of vitamin D supplementation in disease activity of IBD among pediatric patients and also provide evidence-based recommendation for future clinical application. The serum 25(OH)D levels vary among the IBD population, and the effect of vitamin D supplementation is also not consistent. Currently, the optimal serum 25(OH)D level for children with IBD has not been established, and there is no optimal vitamin D regimen for the maintenance of serum 25(OH)D in children with IBD. In this study, pooled analysis showed that mean serum 25(OH) D concentration post-vitamin D therapy and mean change in serum 25(OH)D concentration from baseline was significantly higher among those who received high doses of vitamin D, a result consistent with previous studies. [15][16][17] The effect of vitamin D on serum 25(OH)D was more evident among pediatric patients who had received it for at least 6 months. [16] There are limited studies that have focused on the relationship between vitamin D and disease activity. Some suggested an association of vitamin D deficiency with a more severe course of disease, while other studies do not report the same. [18][19][20] In this study, remission or inactivity of IBD using PCDAI or PUCAI was not significantly different between those who received high-dose and lowdose or standard recommended dose of vitamin D. Likewise, the change of serum CRP and ESR were not significantly different between those who received high-dose and low-dose or standard recommended dose of vitamin D. Nevertheless, the included articles reported improvement with reduction in inflammatory markers from baseline and a lower incidence of elevated inflammatory markers among participants receiving post-vitamin D treatment.

Strengths and Limitations
A limitation of this study is the low number of included articles and accumulated sample size from the articles, which may have affected the current results. Although an exhaustive search of eligible articles was conducted, there were only a few articles which fit into the study's criteria. Another limitation of this study was the amount of risk for performance and detection biases due to the lack of blinding in approximately 25% of included studies. The high heterogeneity of several study outcomes is also a limitation, which prevented comparison of treatment regimens and making a conclusion for optimal dose. Although further analyses such as subgroup analyses and meta-regression should be conducted to explore the source of heterogeneity, the low number of included articles makes these statistical treatments not feasible. Nevertheless, this study has strengths attributable to high quality of evidence, with low risk of bias in almost all potential sources of bias and meticulous and exhaustive search strategy.

Clinical Implications
The acquired results of this meta-analysis may be attributed to various effects of vitamin D on the immune system and immune response of individuals with IBD. Across literature, several researches have suggested that IBD develops because of three primary mechanisms, namely, imbalance in intestinal flora, excessive inflammatory response of affected individuals, and injury of the intestinal mucosal barrier. [21][22][23] The antibiosis effect of 1,25(OH) 2 D is associated with its ability to bind and activate vitamin D receptors of the body, which induces monocyte-induced anti-bacterial protein expression and thus, enhancing the antimicrobial effect. [24] Furthermore, 1,25(OH) 2 D induces multiple types of cells to express nucleotide-binding oligomerization domain protein 2 (NOD2), which causes the expression of gene-encoding antimicrobial peptide defensin beta 2, which further improves bactericidal effects. [25] The anti-inflammatory activity of vitamin D, on the other hand, is a result of its effect on CD4+ T cells to instigate the proliferation and differentiation of Th2 cells and to inhibit Th1 cells which act on the dendritic cells of the gastrointestinal tract. [26] Vitamin D also increases the level of interleukin-10 (IL-10), an anti-inflammatory cytokine; decreases the levels of interleukin-12 (IL-12), a pro-inflammatory cytokine which enhances the cytotoxic activity of natural killer cells and cytotoxic T lymphocytes; and reduces the production of tumor necrotic factoralpha (TNF-a), a mediator of inflammatory functions, by increasing mitogen-activated protein kinase phosphatase-1 (MAPKP-1) and inhibiting activation of mitogen-activated protein kinase (MAPK). [27][28][29] Finally, vitamin D promotes intestinal mucosal repair by increasing the expression of tight junction proteins such as ZO-1 (zona occludens 1), claudin-1 and occludin between epithelial cells of the intestinal lining, thus maintaining mucosal barrier function. [21][22]30] CONCLUSION This meta-analysis highlighted the efficacy of vitamin D supplementation, wherein high doses of vitamin D resulted in a significant change in disease activity among children with IBD as manifested by a lower ESR level and higher serum 25(OH)D concentration at follow-up compared to those who were given vitamin D at low doses.
Cognizant of the functions of vitamin D in enhancing intestinal flora balance, regulating immunologic response and improving intestinal mucosal barrier, vitamin D supplementation can be considered as an adjunct in treatment of IBD among the pediatric population. Nevertheless, it should be noted that there is still insufficient evidence for the cut-off level of adequate levels of serum 25(OH)D among pediatric patients with IBD. Hence, further research should be conducted recognizing the various limitations of this meta-analysis study.
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