Effects of high doses of vitamin D₃ on mucosa-associated gut microbiome vary between regions of the human gastrointestinal tract

The human gut is home to about 10¹⁴ microorganisms, which are collectively referred to as the gut microbiome. These microorganisms work as biochemical factories, helping the host in nutrient acquisition, vitamin production, and toxin degradation. Bacterial enzymes are also involved in an array of metabolic pathways [1] and can even affect the central nervous system by converting amino acids, carbohydrates, and other ingested substances into bioactive compounds [2]. When it comes to energy acquisition, especially carbohydrate metabolism, the human genome is quite limited and a healthy gut microbiome is necessary to effectively digest the diverse carbohydrates [3].

The human gut microbiome is known to have a major impact on colonisation resistance against intestinal pathogens [4], on modulation of the intestinal immune system [5], and on host metabolism [6, 7]. At the current stage of knowledge, some factors, which influence the composition of the intestinal microbiome, have been identified, including diet [8‐10], host genetics [11], environmental circumstances [12], and drugs, such as antibiotics or laxatives [13, 14]. Impairment of gut homoeostasis has been linked to many gastrointestinal diseases, such as inflammatory bowel diseases (IBD), intestinal infections, irritable bowel syndrome (IBS), and colon cancer [15‐18] but also to extra-intestinal diseases, such as obesity, diabetes mellitus, autoimmune diseases, and neurological disorders [19]. For general health benefits but also for a number of diseases, supplementation with vitamin D has shown to be beneficial. Vitamin D is well known for its role on bone mineralisation but has also major effects on the immune and the cardiovascular system as well as on host defence against pathogenic microorganisms [15, 20‐22]. In many parts of the world, about half of the population is assumed to have insufficient vitamin D levels (lower than 20 ng/mL serum) due to a lack of UV exposure and a diet-lacking vitamin D [23]. Sufficient levels of vitamin D have been associated with a lower risk of autoimmune diseases such as IBD, type 1 diabetes mellitus, and rheumatic diseases, but also neoplasia and infections such as tuberculosis and hepatitis C [21]. Elevating serum vitamin D levels to 42 ng/mL has been estimated to decrease disease rates in various cancers, cardiovascular diseases, diabetes mellitus, and infections by 10–50 % and the overall mortality rate by 18 % per year [24].

The hormonal activity of vitamin D has been attributed to the expression of the vitamin D receptor (VDR), which has its highest expression in CD8⁺ T cells compared with other immune cells [25], and to the vitamin D activating enzyme CYP27B1. VDR and CYP27B1 are found in several different cell types including kidneys, muscle, or prostate, but also in cells of the immune system [25, 26], supporting a prominent role of vitamin D in gut homoeostasis and immunity [25]. Given the beneficial effects of vitamin D, many people are using vitamin D supplementation as part of their regular diet, but effects of vitamin D on the human intestinal microbiome have not yet been studied. We hypothesised that some of the beneficial effects attributed to vitamin D might be mediated by the gastrointestinal microbiome. We recruited healthy human volunteers for an interventional study to assess whether vitamin D₃ (vitD₃) affects the human microbiome. Most microbiome studies are limited to easily accessible stool samples, but studies have indicated that there can be a pronounced difference between mucosal and stool microbiomes [13, 27]. Therefore, we sampled seven gastrointestinal regions in addition to stools to assess the mucosa-associated microbiome before and after 8 weeks of oral vitD₃ supplementation.

Fifteen volunteers completed the whole study. One volunteer could not attend the second colonoscopy due to personal reasons but completed all other study procedures. Vitamin D levels (25(OH)D, calcidiol) increased significantly from 22.3 ± 13.1 ng/mL (before) to 55.2 ± 13.3 ng/mL after vitD₃ supplementation (P < .0001). In all volunteers, all safety parameters, including serum and urine calcium and phosphate, as well as urine calcium/creatinine ratio, remained within a normal range and were not significantly altered (Table 1 and supplementary table S2). Throughout the study, we generated 1003,488 high-quality 16S amplicon reads in the range of 211–380 bp (average 264 bp) from mucosal and stool samples. Five microbiome samples were excluded from sequence data analysis as they were poorly covered (<900 sequences/sample). In a subgroup of 3 volunteers, a gastric Helicobacter pylori infection was found during routine clinical processing of biopsies and in the sequencing data. This subgroup was analysed separately as up to 90 % of the observed phylotypes in the GC were classified as Helicobacter spp. In total, we analysed 242 well-covered samples, resulting in 13 paired samples (before/after) for the upper GI tract (GC, GA, and DD), 12 paired samples for AC, 11 for TI, AO, and SC, and 8 paired stool samples (Fig. 1b).

The results of our study demonstrate the effect of an 8-week high-dose vitD₃ supplementation on the human microbiome of the upper GI tract. These changes included a reduction in opportunistic pathogens and an increase in bacterial richness.

To the best of our knowledge, the effects of vitD₃ on the human microbiome have not yet been studied. So far, the effects of a vitamin D supplementation on the gut microbiome have only been studied in murine colitis model. Vitamin D₃ protected these mice from dextran sodium sulphate-induced colitis, which is a common model of IBD. The severity of colitis was significantly higher in VDR and CYP27B1 knockout mice [37]. Because the composition of the gut microbiome of mice is substantially different from the human microbiome [38], any interventional effects that are found in mouse studies cannot be translated directly to humans.

By taking a series of biopsies from the major regions of the human GI tract, we found an overall increase in phylotype richness together with a decrease in Proteobacteria mostly in the mucosa of the upper GI tract. In particular, the class of Gammaproteobacteria decreased significantly, including genera of typical opportunistic pathogens like Pseudomonas spp. and Escherichia/Shigella spp. Stools and mucosal regions of the lower GI tract showed only minor changes and support recent findings that mucosa-associated microbiomes differ significantly from stools [13] and that only biopsies offer the chance to analyse changes in the mucosa-associated microbiome in sufficient detail. Most human intestinal microbiome studies are based on data obtained from stools [8, 9, 16, 17, 39] or rectal/colonic mucosal samples [13, 18, 40]. These studies have investigated external influences, such as the effects of diet [8, 9, 41, 42], physical exercise [39], or drugs [14], and have identified associations with certain disease like IBD, IBS, or obesity [8, 18, 40]. But some interventional studies using therapeutics which affect the microbiome (e.g. prebiotics, omega-3 fatty acids) were not able to identify significant changes in stools [43, 44] since some effects might be limited to the upper GI tract. With our comprehensive sampling of the whole GI tract, we were able to show that the major effects of vitamin D on the human gastrointestinal microbiome were only present in the upper GI tract but not in mucosal samples of the colon, ileum, or stools. Future microbiome studies need to be aware that interventions such as vitamin D supplementation can have significantly different effects on different regions of the GI tract and that these effects cannot necessarily be detected in stools.

In general, the assessment of the microbial composition of samples from the gastrointestinal system is often affected by high inter-individual variation [45] and the methods used for sample processing (e.g. sample storage, DNA extraction, and primer choice). Previous studies have shown that freezing of stools prior to DNA extraction can lead to an increased ratio of Firmicutes to Bacteroidetes [46]. The proportion of Firmicutes reported in the literature varies considerably. Some studies report a microbial composition dominated by Firmicutes [27, 47, 48], whereas other studies report similar levels compared to the results in our study [45, 49, 50].

Our study also showed that the intestinal microbiome varies along the human GI tract and thus supports a previous study which found a similar bacterial distribution pattern in the ileum and colon of healthy humans [51]. The bacterial composition along the GI tract is determined by different niche factors (e.g. pH, oxygen, tension, nutrients), and microbiome data do not necessarily reflect the anatomical categorisation. In our study, the microbiome of the duodenum was more similar to the stomach rather than to the terminal ileum, which was more similar to colon samples. Different regions of the human GI tract vary in function, permeability, type, and number of immune cells [52, 53].

A significantly higher number of CD8⁺ T cells in the upper GI (GC and GA) compared with lower GI sites [52] is part of the first defence line against foodborne pathogens. The significant decrease in Gammaproteobacteria that we found was limited to the mucosa of the upper GI tract and might have been driven by the increased CD8⁺ T cell fraction found after 8 weeks of vitD₃ supplementation. CD8⁺ T cells have the highest expression level of VDR compared with other immune cells [25], which suggests that CD8⁺ T cell function is also regulated by vitamin D. High vitamin D levels have been associated with a decline in naïve CD8⁺ T cells and an increase in CD8⁺ effector T cells [54]. After vitD₃ supplementation, we observed a trend towards elevated CD8⁺ T cell levels in all seven assessed GI regions, but we did not analyse the different CD8⁺ T cell subpopulations in this study. The stimulation of mucosal CD8⁺ T cells by vitD₃ could contribute for the effects on Gammaproteobacteria together with other known effects of vitamin D, such as the induction of antimicrobial peptides (e.g. cathelicidin [55]) and the stimulation of certain cytokine production by mucosal dendritic cells [56]. In addition, murine CD8⁺ T cells [57] and human activated T cells [58] have been found to express 1α-hydroxylase (CYP27B1) which enables the production of calcitriol, the active hormonal state of vitD₃. Calcitriol influences cytokine production in human CD8⁺ T cells [59] and is known to act as a stop signal in inflammatory processes [60, 61]. CD8⁺ T cells are also capable of directly destroying harmed or infected host cells, thus lowering the number of proinflammatory cells [62].

Reducing such an inflammatory environment by vitD₃ could diminish the competitive advantage of opportunistic pathogens, such as Escherichia/Shigella spp. or Pseudomonas spp. which are evolutionary better adapted to inflammation and can outcompete commensal bacteria [63]. In return, a low inflammatory environment allows beneficial bacteria such as Bacteroidetes to outcompete opportunistic pathogens, resulting in increased phylotype richness which we found in this study.

An increase in bacterial richness could be particularly beneficial for gastrointestinal problems such non-alcoholic steatohepatitis or IBD which is characterised by decreased bacterial richness, lower vitamin D levels, and an overgrowth of pathogens [17, 50, 64‐68]. Studies have reported beneficial effects of a vitamin D treatment in IBD patients [69] which could be to some extent due to the restoration of gut microbiome homoeostasis indicated by a reduction in opportunistic pathogens such as Enterobacteriaceae, which belong to the class of Gammaproteobacteria [17, 65‐67, 70]. 16S rRNA amplicon sequencing as used in our study yields only relative abundances which cannot be used to differentiate between an increase in Bacteroidia and a decrease in Gammaproteobacteria. We assume that a decrease in Gammaproteobacteria is more likely because we found a decrease in several genera of Gammaproteobacteria, whereas no single genus of Bacteroidia was significantly altered after 8 weeks of vitD₃ supplementation.

After vitD₃ supplementation, we also found a decrease in overall abundance of Helicobacter spp. in the H. pylori-positive subgroup where approximately 90 % of all bacteria in the stomach were classified as Helicobacter spp. These are the first data to describe an effect of vitD₃ on H. pylori infections and support the finding that CYP27B1 knockout mice which cannot produce calcitriol show a significantly higher relative abundance of Helicobacteriaceae compared to wild-type mice [37]. In these knockout mice, a calcitriol supplementation successfully decreased Helicobacteriaceae levels. The relevant role of vitD₃ in H. pylori infections is also supported by the finding that H. pylori itself induces increased expression of the VDR [71].

VitD₃ dosing in our study was above currently recommended dosing for vitamin D supplementation [72] and resulted in mean vitamin D levels of 55.2 ng/mL. The safety of such a high vitamin D supplementation has previously been reported [73]. We did not observed any adverse effects and no safety parameters, including serum and urine calcium and phosphate were significantly changed during 8 weeks of a high-dose vitD₃ supplementation. At present, it is not advised to generally administer such high vitD₃ doses for longer time periods in humans. Randomized intervention long-term trials are needed to evaluate the risks and benefits of such high vitD₃ doses for human health, because this dose is not what the international guidelines state as recommended daily allowance. One limitation of our study is the relatively small sample size mainly because of the invasive procedures. Despite the relatively small study cohort and a high degree of inter-individual variation of the intestinal microbiome, we were able to observe a significant modulatory effect of vitD₃ in the upper GI tract.

The marked reduction in Gammaproteobacteria, which include typical opportunistic pathogens and the increase in phylotype richness, supports the beneficial effect of a high-dose vitD₃ supplementation on the human gut microbiome. This might in part explain the effects of a vitamin D-rich diet on IBD or bacterial infections and encourage studying the effects of vitamin D in these patients. The fact that the vitamin D effect on the microbiome was only evident in the upper GI tract should guide future microbiome studies in humans to also assess the upper GI tract in addition to stools and colonic mucosal samples.