The Effect of Vitamin D on Intestinal Inflammation and Faecal Microbiota in Patients with Ulcerative Colitis

The Effect of Vitamin D on Intestinal Inflammation and Faecal Microbiota in Patients with... Abstract Background and Aims Vitamin D may be immunomodulatory and alter faecal microbiota, but results from clinical studies in humans to date have been inconclusive. This study aimed to assess the effect of vitamin D replacement in vitamin D-deficient patients with and without ulcerative colitis [UC] on inflammation and faecal microbiota. Methods Vitamin D was replaced over 8 weeks in patients with active UC [defined by faecal calprotectin ≥ 100 µg/g], inactive UC [faecal calprotectin < 100 µg/g] and non-inflammatory bowel disease [IBD] controls with baseline serum 25[OH] vitamin D <50 nmol/l, and markers of inflammation and faecal microbiota were analysed. Results Eight patients with active UC, nine with inactive UC and eight non-IBD controls received 40000 units cholecalciferol weekly for 8 weeks. Mean baseline 25[OH] vitamin D increased from 34 [range 12–49] to 111 [71–158] nmol/l [p < 0.001], with no difference across the groups [p = 0.32]. In patients with active UC, faecal calprotectin levels decreased from a median 275 to 111 µg/g [p = 0.02], platelet count decreased [mean 375 to 313 × 109/l, p = 0.03] and albumin increased [mean 43 to 45 g/l, p = 0.04]. These parameters did not change in patients with inactive UC or non-IBD controls. No changes in overall faecal bacterial diversity were noted although a significant increase in Enterobacteriaceae abundance was observed in patients with UC [p = 0.03]. Conclusions Vitamin D supplementation was associated with reduced intestinal inflammation in patients with active UC, with a concomitant increase in Enterobacteriaceae but no change in overall faecal microbial diversity. Basic science, experimental models and pathophysiology, clinical trials 1. Introduction With expanding therapeutic options for inflammatory bowel diseases [IBD], the costs associated with medical therapies have risen disproportionately to those associated with disease complications.1 Numerous epidemiological and laboratory-based immunological studies support the role of vitamin D as a potential inexpensive immunomodulator, and serum 25[OH] vitamin D (25[OH]D) status has been shown to be inversely proportional to intestinal inflammation in patients with IBD.2–8 However, there remains a paucity of interventional data supporting vitamin D as a treatment for patients with IBD. Dysbiosis, or dysregulation of the gut microbiota, is a recognized feature of IBD, and is thought to play a role in the pathogenesis and perpetuation of inflammation.9 Patients with ulcerative colitis [UC] have reduced bacterial species richness, as well as temporal instability of the microbiota profile in clinical remission and in active disease, compared with healthy controls.10–12 Members of the phyla Firmicutes and Bacteroidetes have been demonstrated to be reduced in patients with IBD.10 Increases in pathobiont bacterial species including Fusobacterium nucleatum and Escherichia coli have been shown in the mucosa and faeces of patients with UC,13,14 whilst the immunoregulatory species Faecalibacterium prausnitzii has been shown to be under-represented.15 Mucolytic bacterial species including Ruminococcus gnavus and Ruminococcus torques are also disproportionately increased in abundance in patients with IBD, with the suggestion that increased numbers contributes towards the gut environment changes seen as the disease progresses.16 Therapeutically targeting the microbiota using the broad approach of faecal microbiota transfusion has been shown to improve outcomes in patients with UC.17–19 There is evidence that vitamin D may modify the gut microbiota. Specifically, vitamin D supplementation has been shown to suppress intra-macrophage Escherichia coli survival in in vitro studies.20 Vitamin D has also been shown to regulate anti-microbial peptide production.20–22 Vitamin D-deficient and vitamin D receptor [VDR] knockout mice have reduced ileal Paneth cell alpha defensin secretion, increased abundance of Helicobacter hepaticus and reduced abundance of Akkermansia muciniphila, compared with control or wild-type mice.23 Studies have also shown that VDR negatively regulates bacterial-induced intestinal epithelial NFκB activation and response to infection.24 Conversely, a cross-sectional study of 150 young healthy adults found an inverse correlation between 25[OH]D status and faecal abundance of butyrate producing bacterium Coprococcus, as well as Bifidobacterium, both of which may theoretically mediate an anti-inflammatory effect.25 It is currently unknown whether vitamin D supplementation in patients with UC affects pro-inflammatory or anti-inflammatory gut microbiota as part of a strategy to influence disease activity. This prospective pilot study aimed to evaluate changes in subjective and objective markers of intestinal inflammation, and within the faecal microbiota, following vitamin D replacement in patients with active and inactive UC, and in non-IBD controls. 2. Materials and Methods 2.1. Subjects and study protocol Consecutive patients with vitamin D deficiency (defined by 25[OH]D < 50 nmol/l) attending outpatient clinics at St Mark’s Hospital were invited to participate. Three groups were studied: [1] those without IBD or other known gastrointestinal malabsorptive conditions, [2] those with inactive UC [defined as faecal calprotectin < 100 µg/g] and [3] those with active UC [faecal calprotectin ≥ 100 µg/g].26,27 Inclusion criteria for patients with UC comprised Partial Mayo Index of ≤ 4, and stable therapy including mesalazine [≥ 2 months] and immunomodulatory or anti-tumour necrosis factor therapy [≥ 3 months] with no change in therapy planned for at least 12 weeks as per the patient’s treating clinician. Exclusion criteria included other significant gastrointestinal disease, pregnancy [current or planned within 6 months], hypercalcaemia or evidence of primary or tertiary hyperparathyroidism, chronic kidney or severe cardiovascular disease, antibiotics within the previous 2 months or bowel preparation within the previous 4 weeks. Demographic and disease characteristics and activity as assessed by Simple Clinical Colitis Activity Index [SCCAI]28,29 and Partial Mayo Index30 were recorded, patients were asked to complete a food diary, and blood tests collected for markers of inflammation. Serum 25[OH] levels were quantified using liquid chromatography tandem mass spectrometry. Patients were asked to provide two faecal specimens with the assistance of StoolcatcherTM [TagHemi] as per the manufacturer’s instructions, and supplied with an ice pack for transport to the hospital within 2 h. One container was analysed for calprotectin [by enzyme linked immunosorbent assay, ELISA, Schottdorf Laboratories], and the second stored at −80°C for analysis of microbiota. Patients were prescribed vitamin D replacement according to the London North West Healthcare NHS Trust guidelines, at a dose of 40000 IU once weekly for 8 weeks using two capsules of 20000 IU vitamin D3 [Plenachol, Encap]. Following replacement, patients were re-assessed symptomatically and by objective markers of inflammation, with repeat faecal microbiota analysis. Adherence was checked by direct patient questioning of number of capsules remaining. 2.2. Faecal microbiota analysis 2.2.1. DNA extraction All samples were extracted within 1 month of collection using the Stratech PSP Spin Stool DNA kit following the manufacturer’s instructions. 2.2.2. PCR amplification and sequencing The V3-4 region of the 16S rRNA gene was amplified using primers Bakt_341F and Bakt_805R, as described previously,31 then pooled and purified using AMPure XP [Beckman Coulter]. The samples were then indexed using the Nextera XT Index Kit V2 [Illumina] and KAPA HiFi Hotstart ReadyMix [Kapa Biosystems] with libraries quantified using a Quant-iT dsDNA Assay Kit HS [Thermo Fisher Scientific]. Sequencing was performed using an Illumina MiSeq sequencer using Illumina V3 chemistry and paired-end 2 × 300-bp reads. Further details regarding PCR amplification are presented in Supplementary Material 1. 2.3. Bioinformatic analysis Sequence quality was assessed using FastQC [version 0.11.3].32 The V3–V4 primer sequences at the 5′ end of reads were hard trimmed using TrimGalore! [version 0.4.0].33 Sequences were analysed using DADA2 [version 1.3.1] to produce sequence variants. Taxonomy was assigned against the GreenGenes 13.8 database.34 The outcome sequence variant table was converted to biom format using biomformat [version 2.1.3].35 Further details regarding bioinformatic analysis are presented in Supplementary Material 1. Diversity analyses including Simpson Index for alpha diversity and Bray–Curtis statistic for beta diversity were performed using the core_diversity_analyses.py script from QIIME [version 1.9.0] with a subsampling level of 19505 to ensure that all samples were included.36 Taxon numbers at each taxonomic level were also produced. LEfSe analysis was carried out using the Huttenhower Galaxy Server [http://huttenhower.sph.harvard.edu/galaxy/] to identify any potential biomarkers associated with sample types.37 DESeq2 analysis,38 Wilcoxon Rank sum tests and Kruskal–Wallis tests for significant changes in abundance in relation to sample type were carried out in R. A p-value of ≤ 0.05 was considered statistically significant, with the exception of DESeq2 analysis where an adjusted p-value of ≤ 0.05 was used. Figures were made using ggplot2 in RStudio. Principal co-ordinate analysis [PCoA] plots were visualized using Emperor.39 2.4. Statistical considerations Statistical analyses for non-microbiota results were performed using SPSS v23 [IBM Corporation] and GraphPad Prism v5.04 [Graphpad software]. Dependent and independent samples t-tests, Mann–Whitney U test, analysis of variance [ANOVA] and Kruskal–Wallis tests were used where appropriate. Associations with increases in 25[OH]D were examined by bivariate correlations. A p-value of ≤ 0.05 was considered statistically significant. 2.5. Ethical statement The protocol for this study was approved by the Office of Research and Ethics at London Northwest Healthcare NHS Trust, and was performed in accordance with UK regulations and the principles of the Declaration of Helsinki 1954 and its later amendments. Informed consent was obtained from all individual participants included in this study. 3. Results Twenty-five patients participated in this study from August to December 2015 [late Summer to Winter at 52°N], with baseline characteristics outlined in Table 1. No significant demographic differences were noted across the three groups. A trend to higher body mass index [BMI] and waist circumference in non-IBD controls was seen. Most patients with UC had left-sided or extensive colitis of variable duration. Table 1. Baseline participant characteristics. Non-IBD controls [n = 8] Inactive UC [n = 9] Active UC [n = 8] p value Age, years (mean [range]) 51 [35–66] 45 [28–72] 45 [30–68] 0.541a Female:male 3:5 3:6 4:4 0.793a Ethnicity, n  British White 3 1 1  Southern European 0 0 2  Indian Subcontinental 5 7 5  Arab & Middle Eastern 0 1 0 Fitzpatrick skin type, n  I 0 0 0  II 1 1 3  III 2 1 0  IV 0 1 0  V 5 6 5  VI 0 0 0 Co-morbid illnesses, n  Hypertension 3 1 3  Hyperlipidaemia 2 1 1  Type 2 DM 1 0 1  Asthma 0 3 1  Congestive cardiac failure 1 0 0 Smoking status, n  Never smoked 5 6 6  Ex-smokers 2 3 1  Current smokers 1 0 1 BMI, kg/m2 (mean [range]) 28.9 [23.5–36.9] 25.8 [20.5–29.7] 24.6 [21.5–28.4] 0.077a Waist circumference, cm (mean [range]) 104 [93–119] 92 [84–99] 91 [70–109] 0.052a Vitamin D supplementation, n 1 4 3 0.205b Montreal Classification  Disease extent: E1:E2:E3 NA 0:3:6 1:2:4 UC disease duration, years [range] NA 11 [0.8–36] 12 [1–40] SCCAI [median, range] NA 2 [0–6] 3 [0–5] Partial Mayo Index [median, range] NA 0 [0–3] 1 [0–4] Medical therapy for UC NA  Nil 1 1  Mesalazine only 6 3  Thiopurine +/- mesalazine 1 1  Anti-TNF +/- mesalazine 1 0  Vedolizumab +/- mesalazine 0 1  Anti-TNF +/- thiopurine 0 2 Non-IBD controls [n = 8] Inactive UC [n = 9] Active UC [n = 8] p value Age, years (mean [range]) 51 [35–66] 45 [28–72] 45 [30–68] 0.541a Female:male 3:5 3:6 4:4 0.793a Ethnicity, n  British White 3 1 1  Southern European 0 0 2  Indian Subcontinental 5 7 5  Arab & Middle Eastern 0 1 0 Fitzpatrick skin type, n  I 0 0 0  II 1 1 3  III 2 1 0  IV 0 1 0  V 5 6 5  VI 0 0 0 Co-morbid illnesses, n  Hypertension 3 1 3  Hyperlipidaemia 2 1 1  Type 2 DM 1 0 1  Asthma 0 3 1  Congestive cardiac failure 1 0 0 Smoking status, n  Never smoked 5 6 6  Ex-smokers 2 3 1  Current smokers 1 0 1 BMI, kg/m2 (mean [range]) 28.9 [23.5–36.9] 25.8 [20.5–29.7] 24.6 [21.5–28.4] 0.077a Waist circumference, cm (mean [range]) 104 [93–119] 92 [84–99] 91 [70–109] 0.052a Vitamin D supplementation, n 1 4 3 0.205b Montreal Classification  Disease extent: E1:E2:E3 NA 0:3:6 1:2:4 UC disease duration, years [range] NA 11 [0.8–36] 12 [1–40] SCCAI [median, range] NA 2 [0–6] 3 [0–5] Partial Mayo Index [median, range] NA 0 [0–3] 1 [0–4] Medical therapy for UC NA  Nil 1 1  Mesalazine only 6 3  Thiopurine +/- mesalazine 1 1  Anti-TNF +/- mesalazine 1 0  Vedolizumab +/- mesalazine 0 1  Anti-TNF +/- thiopurine 0 2 DM, diabetes mellitus; BMI, body mass index; SCCAI, Simple Clinical Colitis Activity Index; Fitzpatrick skin types: I - pale white skin, blue/hazel eyes, blond/red hair, II - fair skin, blue eyes, III - darker white skin, IV - light brown skin, V - brown skin, VI - dark brown or black skin. aANOVA bChi-square, UC vs non-IBD controls. View Large Table 1. Baseline participant characteristics. Non-IBD controls [n = 8] Inactive UC [n = 9] Active UC [n = 8] p value Age, years (mean [range]) 51 [35–66] 45 [28–72] 45 [30–68] 0.541a Female:male 3:5 3:6 4:4 0.793a Ethnicity, n  British White 3 1 1  Southern European 0 0 2  Indian Subcontinental 5 7 5  Arab & Middle Eastern 0 1 0 Fitzpatrick skin type, n  I 0 0 0  II 1 1 3  III 2 1 0  IV 0 1 0  V 5 6 5  VI 0 0 0 Co-morbid illnesses, n  Hypertension 3 1 3  Hyperlipidaemia 2 1 1  Type 2 DM 1 0 1  Asthma 0 3 1  Congestive cardiac failure 1 0 0 Smoking status, n  Never smoked 5 6 6  Ex-smokers 2 3 1  Current smokers 1 0 1 BMI, kg/m2 (mean [range]) 28.9 [23.5–36.9] 25.8 [20.5–29.7] 24.6 [21.5–28.4] 0.077a Waist circumference, cm (mean [range]) 104 [93–119] 92 [84–99] 91 [70–109] 0.052a Vitamin D supplementation, n 1 4 3 0.205b Montreal Classification  Disease extent: E1:E2:E3 NA 0:3:6 1:2:4 UC disease duration, years [range] NA 11 [0.8–36] 12 [1–40] SCCAI [median, range] NA 2 [0–6] 3 [0–5] Partial Mayo Index [median, range] NA 0 [0–3] 1 [0–4] Medical therapy for UC NA  Nil 1 1  Mesalazine only 6 3  Thiopurine +/- mesalazine 1 1  Anti-TNF +/- mesalazine 1 0  Vedolizumab +/- mesalazine 0 1  Anti-TNF +/- thiopurine 0 2 Non-IBD controls [n = 8] Inactive UC [n = 9] Active UC [n = 8] p value Age, years (mean [range]) 51 [35–66] 45 [28–72] 45 [30–68] 0.541a Female:male 3:5 3:6 4:4 0.793a Ethnicity, n  British White 3 1 1  Southern European 0 0 2  Indian Subcontinental 5 7 5  Arab & Middle Eastern 0 1 0 Fitzpatrick skin type, n  I 0 0 0  II 1 1 3  III 2 1 0  IV 0 1 0  V 5 6 5  VI 0 0 0 Co-morbid illnesses, n  Hypertension 3 1 3  Hyperlipidaemia 2 1 1  Type 2 DM 1 0 1  Asthma 0 3 1  Congestive cardiac failure 1 0 0 Smoking status, n  Never smoked 5 6 6  Ex-smokers 2 3 1  Current smokers 1 0 1 BMI, kg/m2 (mean [range]) 28.9 [23.5–36.9] 25.8 [20.5–29.7] 24.6 [21.5–28.4] 0.077a Waist circumference, cm (mean [range]) 104 [93–119] 92 [84–99] 91 [70–109] 0.052a Vitamin D supplementation, n 1 4 3 0.205b Montreal Classification  Disease extent: E1:E2:E3 NA 0:3:6 1:2:4 UC disease duration, years [range] NA 11 [0.8–36] 12 [1–40] SCCAI [median, range] NA 2 [0–6] 3 [0–5] Partial Mayo Index [median, range] NA 0 [0–3] 1 [0–4] Medical therapy for UC NA  Nil 1 1  Mesalazine only 6 3  Thiopurine +/- mesalazine 1 1  Anti-TNF +/- mesalazine 1 0  Vedolizumab +/- mesalazine 0 1  Anti-TNF +/- thiopurine 0 2 DM, diabetes mellitus; BMI, body mass index; SCCAI, Simple Clinical Colitis Activity Index; Fitzpatrick skin types: I - pale white skin, blue/hazel eyes, blond/red hair, II - fair skin, blue eyes, III - darker white skin, IV - light brown skin, V - brown skin, VI - dark brown or black skin. aANOVA bChi-square, UC vs non-IBD controls. View Large Routine laboratory indices and circulating components of the vitamin D axis amongst participants at baseline are outlined in Table 2. As expected, faecal calprotectin was significantly higher amongst patients with active disease, and platelet counts higher, with a trend towards higher C-reactive protein [CRP]. No significant differences across the groups in serum 25[OH]D, calcium, phosphate or parathyroid hormone were noted. Baseline dietary patterns of the participants are outlined in Supplementary Table 1. Seven of the 25 participants had a vegetarian diet, with self-reported vegetable intake reported as > 35 standard serves per week by the majority [14 of 25] of participants. Table 2. Routine laboratory indices and components of the vitamin D axis in the patient groups and healthy controls Non-IBD controls [n = 8] Inactive UC [n = 9] Active UC [n = 8] p value Haemoglobin [g/l, mean, range] 140 [118–155] 145 [122–167] 124 [87–154] 0.07a White cell count [×109/l, mean, range] 7.4 [4.9–10.0] 6.5 [4.9–8.0] 7.2 [5.3–10.6] 0.53a Platelet count [×109/l, mean, range] 266 [200–321] 241 [160–313] 375 [255–509] 0.001a Serum albumin [g/l, mean, range] 45 [42–50] 46 [43–51] 43 [38–49] 0.09a Serum C-reactive protein [mg/l, median, range] 1.0 [< 1.0–5.0] 1.0 [< 1.0–8.0] 4.0 [1.0–28.0] 0.054b Faecal calprotectin [µg/g, median, range] 16.4 [12.2–50.9] 34.2 [< 5.3–87.1] 257 [110->2000] 0.002b 25[OH]D [nmol/l, mean, range] 31 [12–49] 33 [17–49] 34 [16–43] 0.90a Serum calcium [corrected, mmol/l, mean, range] 2.42 [2.29–2.57] 2.44 [2.30–2.55] 2.46 [2.36–2.58] 0.67a Serum phosphate [mmol/l, mean, range] 1.06 [0.83–1.42] 1.02 [0.73–1.65] 0.99 [0.55–1.33] 0.85a Serum PTH [pmol/l, mean, range] 3.3 [2.2–4.7] 4.6 [3.7–5.7] 4.5 [2.7–5.6] 0.20a Non-IBD controls [n = 8] Inactive UC [n = 9] Active UC [n = 8] p value Haemoglobin [g/l, mean, range] 140 [118–155] 145 [122–167] 124 [87–154] 0.07a White cell count [×109/l, mean, range] 7.4 [4.9–10.0] 6.5 [4.9–8.0] 7.2 [5.3–10.6] 0.53a Platelet count [×109/l, mean, range] 266 [200–321] 241 [160–313] 375 [255–509] 0.001a Serum albumin [g/l, mean, range] 45 [42–50] 46 [43–51] 43 [38–49] 0.09a Serum C-reactive protein [mg/l, median, range] 1.0 [< 1.0–5.0] 1.0 [< 1.0–8.0] 4.0 [1.0–28.0] 0.054b Faecal calprotectin [µg/g, median, range] 16.4 [12.2–50.9] 34.2 [< 5.3–87.1] 257 [110->2000] 0.002b 25[OH]D [nmol/l, mean, range] 31 [12–49] 33 [17–49] 34 [16–43] 0.90a Serum calcium [corrected, mmol/l, mean, range] 2.42 [2.29–2.57] 2.44 [2.30–2.55] 2.46 [2.36–2.58] 0.67a Serum phosphate [mmol/l, mean, range] 1.06 [0.83–1.42] 1.02 [0.73–1.65] 0.99 [0.55–1.33] 0.85a Serum PTH [pmol/l, mean, range] 3.3 [2.2–4.7] 4.6 [3.7–5.7] 4.5 [2.7–5.6] 0.20a PTH, parathyroid hormone. Bold type indicates statistical significance defined by p < 0.05. aANOVA bKruskal–Wallis test View Large Table 2. Routine laboratory indices and components of the vitamin D axis in the patient groups and healthy controls Non-IBD controls [n = 8] Inactive UC [n = 9] Active UC [n = 8] p value Haemoglobin [g/l, mean, range] 140 [118–155] 145 [122–167] 124 [87–154] 0.07a White cell count [×109/l, mean, range] 7.4 [4.9–10.0] 6.5 [4.9–8.0] 7.2 [5.3–10.6] 0.53a Platelet count [×109/l, mean, range] 266 [200–321] 241 [160–313] 375 [255–509] 0.001a Serum albumin [g/l, mean, range] 45 [42–50] 46 [43–51] 43 [38–49] 0.09a Serum C-reactive protein [mg/l, median, range] 1.0 [< 1.0–5.0] 1.0 [< 1.0–8.0] 4.0 [1.0–28.0] 0.054b Faecal calprotectin [µg/g, median, range] 16.4 [12.2–50.9] 34.2 [< 5.3–87.1] 257 [110->2000] 0.002b 25[OH]D [nmol/l, mean, range] 31 [12–49] 33 [17–49] 34 [16–43] 0.90a Serum calcium [corrected, mmol/l, mean, range] 2.42 [2.29–2.57] 2.44 [2.30–2.55] 2.46 [2.36–2.58] 0.67a Serum phosphate [mmol/l, mean, range] 1.06 [0.83–1.42] 1.02 [0.73–1.65] 0.99 [0.55–1.33] 0.85a Serum PTH [pmol/l, mean, range] 3.3 [2.2–4.7] 4.6 [3.7–5.7] 4.5 [2.7–5.6] 0.20a Non-IBD controls [n = 8] Inactive UC [n = 9] Active UC [n = 8] p value Haemoglobin [g/l, mean, range] 140 [118–155] 145 [122–167] 124 [87–154] 0.07a White cell count [×109/l, mean, range] 7.4 [4.9–10.0] 6.5 [4.9–8.0] 7.2 [5.3–10.6] 0.53a Platelet count [×109/l, mean, range] 266 [200–321] 241 [160–313] 375 [255–509] 0.001a Serum albumin [g/l, mean, range] 45 [42–50] 46 [43–51] 43 [38–49] 0.09a Serum C-reactive protein [mg/l, median, range] 1.0 [< 1.0–5.0] 1.0 [< 1.0–8.0] 4.0 [1.0–28.0] 0.054b Faecal calprotectin [µg/g, median, range] 16.4 [12.2–50.9] 34.2 [< 5.3–87.1] 257 [110->2000] 0.002b 25[OH]D [nmol/l, mean, range] 31 [12–49] 33 [17–49] 34 [16–43] 0.90a Serum calcium [corrected, mmol/l, mean, range] 2.42 [2.29–2.57] 2.44 [2.30–2.55] 2.46 [2.36–2.58] 0.67a Serum phosphate [mmol/l, mean, range] 1.06 [0.83–1.42] 1.02 [0.73–1.65] 0.99 [0.55–1.33] 0.85a Serum PTH [pmol/l, mean, range] 3.3 [2.2–4.7] 4.6 [3.7–5.7] 4.5 [2.7–5.6] 0.20a PTH, parathyroid hormone. Bold type indicates statistical significance defined by p < 0.05. aANOVA bKruskal–Wallis test View Large The follow-up visit took place following 8 weeks of vitamin D replacement [mean duration 58 days]. Vitamin D replacement resulted in an increase in serum 25[OH]D across all participants from a mean of 34 [range 12–49] to 111 [71–183] nmol/l [p < 0.001, paired t-test] [Figure 1]. There was no significant difference in the increase in 25 [OH]D between the three groups [p = 0.316]. All patients reported completion of the full course of supplementation [320000 IU vitamin D] except two non-IBD controls [240000 IU each], one patient each with inactive and active UC [280000 IU each]. One patient with active UC ceased mesalazine tablets [taken at 3.2 g daily] during the course of therapy in the context of symptomatic improvement. All other patients continued usual therapy. No significant change in dietary patterns over the study period were observed across most subjects. Figure 1. View largeDownload slide View largeDownload slide Change in clinical and laboratory indices following vitamin D supplementation in participants without IBD, and those with inactive and active UC. Figure 1. View largeDownload slide View largeDownload slide Change in clinical and laboratory indices following vitamin D supplementation in participants without IBD, and those with inactive and active UC. Symptomatic disease activity indices declined significantly amongst patients with inactive and active colitis following vitamin D supplementation, reaching significance for SCCAI [p = 0.04 and p = 0.01, respectively] but not for the Partial Mayo Index [p = 0.10 and p = 0.09, respectively]. In patients with active UC, objective markers of disease activity improved significantly following vitamin D supplementation: faecal calprotectin (median 257 [range 110 to > 2000] to median 111 [5–2000] µg/g, p = 0.02); platelet count (mean 375 [255–509] to mean 313 [243–461] × 109/l, p = 0.03); and albumin (mean 43 [38–49] to mean 45 [41–50] g/l, p = 0.04) [Figure 1]. No effect on faecal calprotectin, CRP, white cell count, platelet count or albumin was observed amongst patients with inactive colitis or non-IBD controls. Baseline overall dietary pattern, cereal and bread, vegetable or fruit intake did not influence response to faecal calprotectin, or circulating markers of inflammation, of vitamin D replacement [data not shown]. Among patients with active UC, there was no significant correlation between the change in serum 25[OH]D and change in faecal calprotectin [Spearman r = −0.21, p = 0.61]. There was no significant alteration in serum calcium, phosphate or alkaline phosphatase. Serum parathyroid hormone levels decreased significantly across the whole cohort (mean 4.0 [range 2.2–5.7] to 3.4 [1.8–6.5] pmol/l, p = 0.02). No patients were hospitalized or required surgery. 3.1. Change in faecal microbiota All 25 patients submitted a faecal sample at baseline, with 23 patients also providing a follow-up sample. One patient with active colitis and one non-IBD control did not submit follow-up samples for microbiota analysis. Between 50228 and 189688 raw sequences were produced per sample following amplicon sequencing. Following filtering and DADA2 analysis, each sample had between 19505 and 98075 sequence variant counts with an average of 45734 counts [Supplementary Table 2]. 3.2. Diversity analyses No differences in alpha diversity as assessed by the Simpson, Shannon, Chao or Observed species diversity indices were noted across the three patient groups at baseline. No differences in alpha diversity were noted in samples following vitamin D replacement across the patient groups. PCoA plots using the Bray–Curtis beta diversity metric demonstrated that patients without IBD clustered together distinct from UC patients [Figure 2a; p = 0.003, PERMANOVA]. When the same analysis approach was applied to both the data for before and after vitamin D supplementation, no difference between patients with inactive and active UC was noted [p = 1.0, Figure 2b]. Figure 2. View largeDownload slide Principal co-ordinate analysis [PCoA] plots at [a] baseline and [b] before and following vitamin D supplementation. Figure 2. View largeDownload slide Principal co-ordinate analysis [PCoA] plots at [a] baseline and [b] before and following vitamin D supplementation. 3.3. Taxonomic profiling Changes in the relative abundance of sequence variants showed statistically significant differences between the three groups at baseline [Table 3]. Abundance of the mucus-associated bacterium Ruminococcus gnavus was marginally but not significantly higher in patients with UC than non-IBD controls [p = 0.068, Kruskal—Wallis test, Supplementary Figure 1]. Table 3. Significant DESeq2 results comparing the relative abundance of sequence variants across sample types. Fold change values are given to three significant figures Sequence variant taxonomy Log fold change Adjusted p value Inactive UC > non-IBD controls  E. coli 5.34 0.00448 Non-IBD controls > active UC  Prevotella copri 8.48 0.0442  Coprococcus genus 8.76 0.0294 Inactive > active UC  Prevotella copri 8.46 0.00640  Bacteroides plebeius 9.08 0.0131  Bacteroides fragilis 8.79 0.0158  Bacteroides genus 7.83 0.0116  Ruminococcaceae family 8.73 0.00927  Bacteroides caccae 8.30 0.00640  Coprococcus genus 6.62 0.0284 Active > inactive UC  Lachnospira genus 6.03 0.00927  Sutterella genus 8.75 0.0158  Coprococcus genus 6.62 0.0284 Sequence variant taxonomy Log fold change Adjusted p value Inactive UC > non-IBD controls  E. coli 5.34 0.00448 Non-IBD controls > active UC  Prevotella copri 8.48 0.0442  Coprococcus genus 8.76 0.0294 Inactive > active UC  Prevotella copri 8.46 0.00640  Bacteroides plebeius 9.08 0.0131  Bacteroides fragilis 8.79 0.0158  Bacteroides genus 7.83 0.0116  Ruminococcaceae family 8.73 0.00927  Bacteroides caccae 8.30 0.00640  Coprococcus genus 6.62 0.0284 Active > inactive UC  Lachnospira genus 6.03 0.00927  Sutterella genus 8.75 0.0158  Coprococcus genus 6.62 0.0284 View Large Table 3. Significant DESeq2 results comparing the relative abundance of sequence variants across sample types. Fold change values are given to three significant figures Sequence variant taxonomy Log fold change Adjusted p value Inactive UC > non-IBD controls  E. coli 5.34 0.00448 Non-IBD controls > active UC  Prevotella copri 8.48 0.0442  Coprococcus genus 8.76 0.0294 Inactive > active UC  Prevotella copri 8.46 0.00640  Bacteroides plebeius 9.08 0.0131  Bacteroides fragilis 8.79 0.0158  Bacteroides genus 7.83 0.0116  Ruminococcaceae family 8.73 0.00927  Bacteroides caccae 8.30 0.00640  Coprococcus genus 6.62 0.0284 Active > inactive UC  Lachnospira genus 6.03 0.00927  Sutterella genus 8.75 0.0158  Coprococcus genus 6.62 0.0284 Sequence variant taxonomy Log fold change Adjusted p value Inactive UC > non-IBD controls  E. coli 5.34 0.00448 Non-IBD controls > active UC  Prevotella copri 8.48 0.0442  Coprococcus genus 8.76 0.0294 Inactive > active UC  Prevotella copri 8.46 0.00640  Bacteroides plebeius 9.08 0.0131  Bacteroides fragilis 8.79 0.0158  Bacteroides genus 7.83 0.0116  Ruminococcaceae family 8.73 0.00927  Bacteroides caccae 8.30 0.00640  Coprococcus genus 6.62 0.0284 Active > inactive UC  Lachnospira genus 6.03 0.00927  Sutterella genus 8.75 0.0158  Coprococcus genus 6.62 0.0284 View Large Changes in the abundance of specific bacteria following vitamin D administration were analysed using LEfSe analysis. Across all participants, an increase in Clostridium colinae [p = 0.03; driven by two non-IBD controls and two patients with inactive UC] and Enterobacteriacae [p = 0.03; driven by five patients with inactive UC and three with active UC] was noted. Ruminococcus gnavus decreased marginally but not significantly following vitamin D supplementation across the whole cohort [p = 0.15; Wilcoxon Rank sum, Supplementary Figure 1]. No significant change in abundances of other mucus-associated bacteria Ruminococcus torques or Akkermansia muciniphila, butyrate-producing bacteria from the Clostridium Cluster IV or Cluster XIVa groups, or of lactic acid-producing bacteria [Lactobacilli or Bifidobacteria], or of the invasive bacteria Fusobacterium nucleatum and E. coli were noted [data not shown]. 4. Discussion The role of vitamin D as a potential immunomodulator in patients with IBD has been investigated extensively for over a decade. Numerous studies demonstrate involvement of the vitamin D axis in regulation of the epithelial barrier, and innate immune cell and T-cell function.20,21,23,24,40–47 Although there are some preliminary data suggesting that vitamin D may influence the intestinal microbiota in IBD, this has not been studied in humans. Furthermore, evidence for efficacy at the clinical level remains poor. This study is the first to show that vitamin D replacement in patients with active UC who are deficient in vitamin D improved objective markers of inflammation. Although this was associated with a significant increase in Enterobacteriacae in patients with UC, there was no change in overall diversity or other specific bacteria analysed. Previous studies have shown that vitamin D supplementation may be associated with reduced rates of relapse in patients with Crohn’s disease in remission when given at a dose of 1200 IU daily for 12 months,2 and improved Crohn’s disease activity index [CDAI] and quality of life when given at up to 5000 IU daily for 24 weeks.4 An alternative placebo-controlled randomized controlled trial showed no significant change in CDAI, quality of life, CRP or faecal calprotectin in patients given 2000 IU vitamin D daily for 3 months.5 In patients with UC, low vitamin D levels have been associated with greater disease activity, as assessed by symptoms, faecal calprotectin and endoscopic activity, as well as increased risk of subsequent relapse.7,8,48–50 A small pilot study demonstrated reduction in symptomatic disease activity indices but not intestinal inflammation as measured by faecal calprotectin in patients with UC and Crohn’s colitis.51 No placebo-controlled studies in patients with UC have been reported to date. The current study is the first to show an improvement in objective markers of inflammation [faecal calprotectin, albumin, platelet count] following vitamin D replacement, limited to a group of patients with active UC defined by faecal calprotectin ≥ 100 µg/g at baseline. The reason for the faecal calprotectin reduction in these patients warrants further consideration. Although it has been postulated that a 25[OH]D level higher than 75 nmol/l, or closer to 100–125 nmol/l, may be required for an immunomodulatory effect,4,48,51–53 such a level was not specifically targeted in this study. Rather, high-dose oral weekly supplementation according to local institutional guidelines was administered at the same dose in all patients, as opposed to daily supplementation in most previous studies. Significant inter-individual variation in response to vitamin D supplementation exists, particularly in diseased states such as IBD,54 and unsurprisingly the serum 25[OH]D level achieved varied from 75 to 183 nmol/l across the patients with UC. Five of eight patients with active UC achieved a 25[OH]D of ≥ 100 nmol/l, all of whom had a reduction in faecal calprotectin to varying extents; however, there was no clear correlation between the rise in 25[OH]D and reduction in faecal calprotectin. Therefore, the findings in this study raise the prospect that it may not be the final serum 25[OH]D level but the administration of a higher dose of vitamin D itself that potentially confers an immunomodulatory effect. This concept requires further investigation in an appropriately powered prospective controlled trial. The VDR is expressed in colonic intestinal epithelial cells, dendritic cells and macrophages.21,42 Vitamin D has been shown to potently stimulate cathelicidin, an anti-microbial peptide produced by macrophages22 which plays an important role in defence against intracellular organisms such as mycobacteria.21 VDR expression is significantly increased in inflamed and non-inflamed mucosal biopsies from patients with UC.55 Vitamin D supplementation suppressed intra-macrophage E. coli survival in in vitro studies,20 and vitamin D-deficient and VDR knockout mice had impaired ileal Paneth cell alpha defensin secretion and increased abundance of the colitogenic Helicobacter hepaticus, compared with control or wild-type mice.23 Therefore, there is biological plausibility for an interaction between the vitamin D axis and intestinal microbiota in the pathogenesis and perpetuation of inflammation in patients with IBD, especially UC. In the current study, no overall change in faecal microbial diversity occurred following vitamin D supplementation. Although an increase in the abundance of Enterobacteriaecae was noted following vitamin D supplementation in patients with UC, this large family comprises a large proportion of harmless and commensal as well as potentially pathogenic bacteria in the human gut, and therefore the significance of such a change is uncertain. Ruminococcus gnavus is a Gram-positive anaerobic mucolytic bacterium belonging to Cluster XIVa of the class Clostridia, which is increased in abundance in patients with IBD.16 The intestinal mucus layer provides a protective barrier between the luminal environment and mucosa, comprising dense glycoproteins interspersed with antimicrobial peptides produced by Paneth cells and other epithelial cells.56 A previous study has shown reduced abundance of Ruminococcus gnavus in mucosal biopsies from patients with active UC defined symptomatically,57 and this trend was confirmed in the current study, albeit without statistical significance. Furthermore, the abundance of Ruminococcus gnavus decreased non-significantly across all patients after vitamin D supplementation. Whether vitamin D supplementation mediates regulation of intestinal mucus antimicrobial composition and therefore susceptibility to specific mucolytic bacteria warrants further investigation. Nonetheless, an absence of a significant effect on the faecal microbiota across the whole cohort of patients studied is of note. It is possible that vitamin D does not alter human microbiota, despite laboratory data from mouse studies.23 Other possible explanations include a differential effect on faecal and mucosa-associated microbiota. Faecal microbiota was assessed during this study rather than mucosal-associated microbiota as this is less invasive and is not subject to variation as a result of bowel preparation.58 However, given the intimate relationship between vitamin D-induced anti-microbial peptide secretion and the mucosal microbiota, one may postulate that significant changes in the latter may be more reflective of the effect of vitamin D in this setting. An absence of significant alteration of the faecal microbiota by vitamin D supplementation, however, is not an isolated finding: despite widespread use, there remains a paucity of published data regarding the effect of conventional therapies such as 5-aminosalicylates, thiopurines and anti-tumour necrosis factor α agents on the faecal microbiota independent of changes in mucosal inflammation in patients with IBD.59,60 Conversely, the absence of a change in microbiota composition despite reduction in inflammation in the active UC group is also notable, and may reflect only a mild reduction in inflammation in these patients. It is important to note that there are few robust data regarding change in microbiota composition in patients with UC in the absence of medical therapy.61 Furthermore, patients with UC in this study had a relatively long disease duration, with a median of 11–12 years. Data regarding the effect of duration of UC on temporal variability of microbiota are also limited.61 Longer disease duration has previously been described as a risk factor for vitamin D deficiency,62 but no influence of disease activity on initial 25[OH]D level or response to supplementation was noted in the current study [data not shown]. There are multiple other limitations in this small study. Although no overt toxicity as measured by serum calcium and phosphate was noted, long-term potential effects of the supplementation strategy in this study were not able to be elucidated, particularly the risk of hypercalciuria or nephrocalcinosis.51 Dietary assessment of patients at baseline and follow-up visits showed no clear changes across most patients, but specific effects of change in diet as confounders were difficult to elicit. In conclusion, vitamin D supplementation at a dose of 40000 IU weekly for 8 weeks reduced objective circulating and intestinal markers of inflammation in patients with active UC. A significant increase in abundance of Enterobacteriaceae in patients with UC, and a trend to reduction in the mucolytic species Ruminococcus gnavus, was noted, but overall microbiota diversity was unchanged. Vitamin D may therefore reduce intestinal inflammation, but independently of any change in faecal bacterial composition. A larger placebo-controlled clinical trial incorporating immunological and extended microbiota analyses, including functional assessment, will shed further light upon this effect. Funding This work was supported by the European Crohn’s and Colitis Organisation Fellowship awarded to Dr Mayur Garg, and St Mark’s Foundation Research Grant 2015 awarded to Prof. Ailsa Hart and Dr Mayur Garg. Conflict of Interest The authors declare that they have no conflict of interest with respect to this manuscript. Author Contributions M.G.: conception and design of the study, acquisition of data, analysis and interpretation of data, writing and drafting the article and its final approval; P.H., J.N.D.: acquisition of data, critical appraisal of manuscript for important intellectual content, final approval of submitted version; S.S., G.H.: analysis and interpretation of data, critical appraisal of manuscript for important intellectual content, final approval of submitted version; A.H.: conception and design of the study, critical appraisal of manuscript for important intellectual content, final approval of submitted version. Supplementary Data Supplementary data to this article can be found online at: ECCO-JCC Conference: Australian Gastroenterology Week 2017 [Oral], Gold Coast, Australia. 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Ablation of tumor necrosis factor is associated with decreased inflammation and alterations of the microbiota in a mouse model of inflammatory bowel disease . PLoS One 2015 ; 10 : e0119441 . Google Scholar CrossRef Search ADS PubMed 61. Fite A , Macfarlane S , Furrie E , et al. Longitudinal analyses of gut mucosal microbiotas in ulcerative colitis in relation to patient age and disease severity and duration . J Clin Microbiol 2013 ; 51 : 849 – 56 . Google Scholar CrossRef Search ADS PubMed 62. Tajika M , Matsuura A , Nakamura T , et al. Risk factors for vitamin D deficiency in patients with Crohn’s disease . J Gastroenterol 2004 ; 39 : 527 – 33 . Google Scholar CrossRef Search ADS PubMed Copyright © 2018 European Crohn’s and Colitis Organisation (ECCO). Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Crohn's and Colitis Oxford University Press

The Effect of Vitamin D on Intestinal Inflammation and Faecal Microbiota in Patients with Ulcerative Colitis

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Copyright © 2018 European Crohn’s and Colitis Organisation (ECCO). Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com
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Abstract

Abstract Background and Aims Vitamin D may be immunomodulatory and alter faecal microbiota, but results from clinical studies in humans to date have been inconclusive. This study aimed to assess the effect of vitamin D replacement in vitamin D-deficient patients with and without ulcerative colitis [UC] on inflammation and faecal microbiota. Methods Vitamin D was replaced over 8 weeks in patients with active UC [defined by faecal calprotectin ≥ 100 µg/g], inactive UC [faecal calprotectin < 100 µg/g] and non-inflammatory bowel disease [IBD] controls with baseline serum 25[OH] vitamin D <50 nmol/l, and markers of inflammation and faecal microbiota were analysed. Results Eight patients with active UC, nine with inactive UC and eight non-IBD controls received 40000 units cholecalciferol weekly for 8 weeks. Mean baseline 25[OH] vitamin D increased from 34 [range 12–49] to 111 [71–158] nmol/l [p < 0.001], with no difference across the groups [p = 0.32]. In patients with active UC, faecal calprotectin levels decreased from a median 275 to 111 µg/g [p = 0.02], platelet count decreased [mean 375 to 313 × 109/l, p = 0.03] and albumin increased [mean 43 to 45 g/l, p = 0.04]. These parameters did not change in patients with inactive UC or non-IBD controls. No changes in overall faecal bacterial diversity were noted although a significant increase in Enterobacteriaceae abundance was observed in patients with UC [p = 0.03]. Conclusions Vitamin D supplementation was associated with reduced intestinal inflammation in patients with active UC, with a concomitant increase in Enterobacteriaceae but no change in overall faecal microbial diversity. Basic science, experimental models and pathophysiology, clinical trials 1. Introduction With expanding therapeutic options for inflammatory bowel diseases [IBD], the costs associated with medical therapies have risen disproportionately to those associated with disease complications.1 Numerous epidemiological and laboratory-based immunological studies support the role of vitamin D as a potential inexpensive immunomodulator, and serum 25[OH] vitamin D (25[OH]D) status has been shown to be inversely proportional to intestinal inflammation in patients with IBD.2–8 However, there remains a paucity of interventional data supporting vitamin D as a treatment for patients with IBD. Dysbiosis, or dysregulation of the gut microbiota, is a recognized feature of IBD, and is thought to play a role in the pathogenesis and perpetuation of inflammation.9 Patients with ulcerative colitis [UC] have reduced bacterial species richness, as well as temporal instability of the microbiota profile in clinical remission and in active disease, compared with healthy controls.10–12 Members of the phyla Firmicutes and Bacteroidetes have been demonstrated to be reduced in patients with IBD.10 Increases in pathobiont bacterial species including Fusobacterium nucleatum and Escherichia coli have been shown in the mucosa and faeces of patients with UC,13,14 whilst the immunoregulatory species Faecalibacterium prausnitzii has been shown to be under-represented.15 Mucolytic bacterial species including Ruminococcus gnavus and Ruminococcus torques are also disproportionately increased in abundance in patients with IBD, with the suggestion that increased numbers contributes towards the gut environment changes seen as the disease progresses.16 Therapeutically targeting the microbiota using the broad approach of faecal microbiota transfusion has been shown to improve outcomes in patients with UC.17–19 There is evidence that vitamin D may modify the gut microbiota. Specifically, vitamin D supplementation has been shown to suppress intra-macrophage Escherichia coli survival in in vitro studies.20 Vitamin D has also been shown to regulate anti-microbial peptide production.20–22 Vitamin D-deficient and vitamin D receptor [VDR] knockout mice have reduced ileal Paneth cell alpha defensin secretion, increased abundance of Helicobacter hepaticus and reduced abundance of Akkermansia muciniphila, compared with control or wild-type mice.23 Studies have also shown that VDR negatively regulates bacterial-induced intestinal epithelial NFκB activation and response to infection.24 Conversely, a cross-sectional study of 150 young healthy adults found an inverse correlation between 25[OH]D status and faecal abundance of butyrate producing bacterium Coprococcus, as well as Bifidobacterium, both of which may theoretically mediate an anti-inflammatory effect.25 It is currently unknown whether vitamin D supplementation in patients with UC affects pro-inflammatory or anti-inflammatory gut microbiota as part of a strategy to influence disease activity. This prospective pilot study aimed to evaluate changes in subjective and objective markers of intestinal inflammation, and within the faecal microbiota, following vitamin D replacement in patients with active and inactive UC, and in non-IBD controls. 2. Materials and Methods 2.1. Subjects and study protocol Consecutive patients with vitamin D deficiency (defined by 25[OH]D < 50 nmol/l) attending outpatient clinics at St Mark’s Hospital were invited to participate. Three groups were studied: [1] those without IBD or other known gastrointestinal malabsorptive conditions, [2] those with inactive UC [defined as faecal calprotectin < 100 µg/g] and [3] those with active UC [faecal calprotectin ≥ 100 µg/g].26,27 Inclusion criteria for patients with UC comprised Partial Mayo Index of ≤ 4, and stable therapy including mesalazine [≥ 2 months] and immunomodulatory or anti-tumour necrosis factor therapy [≥ 3 months] with no change in therapy planned for at least 12 weeks as per the patient’s treating clinician. Exclusion criteria included other significant gastrointestinal disease, pregnancy [current or planned within 6 months], hypercalcaemia or evidence of primary or tertiary hyperparathyroidism, chronic kidney or severe cardiovascular disease, antibiotics within the previous 2 months or bowel preparation within the previous 4 weeks. Demographic and disease characteristics and activity as assessed by Simple Clinical Colitis Activity Index [SCCAI]28,29 and Partial Mayo Index30 were recorded, patients were asked to complete a food diary, and blood tests collected for markers of inflammation. Serum 25[OH] levels were quantified using liquid chromatography tandem mass spectrometry. Patients were asked to provide two faecal specimens with the assistance of StoolcatcherTM [TagHemi] as per the manufacturer’s instructions, and supplied with an ice pack for transport to the hospital within 2 h. One container was analysed for calprotectin [by enzyme linked immunosorbent assay, ELISA, Schottdorf Laboratories], and the second stored at −80°C for analysis of microbiota. Patients were prescribed vitamin D replacement according to the London North West Healthcare NHS Trust guidelines, at a dose of 40000 IU once weekly for 8 weeks using two capsules of 20000 IU vitamin D3 [Plenachol, Encap]. Following replacement, patients were re-assessed symptomatically and by objective markers of inflammation, with repeat faecal microbiota analysis. Adherence was checked by direct patient questioning of number of capsules remaining. 2.2. Faecal microbiota analysis 2.2.1. DNA extraction All samples were extracted within 1 month of collection using the Stratech PSP Spin Stool DNA kit following the manufacturer’s instructions. 2.2.2. PCR amplification and sequencing The V3-4 region of the 16S rRNA gene was amplified using primers Bakt_341F and Bakt_805R, as described previously,31 then pooled and purified using AMPure XP [Beckman Coulter]. The samples were then indexed using the Nextera XT Index Kit V2 [Illumina] and KAPA HiFi Hotstart ReadyMix [Kapa Biosystems] with libraries quantified using a Quant-iT dsDNA Assay Kit HS [Thermo Fisher Scientific]. Sequencing was performed using an Illumina MiSeq sequencer using Illumina V3 chemistry and paired-end 2 × 300-bp reads. Further details regarding PCR amplification are presented in Supplementary Material 1. 2.3. Bioinformatic analysis Sequence quality was assessed using FastQC [version 0.11.3].32 The V3–V4 primer sequences at the 5′ end of reads were hard trimmed using TrimGalore! [version 0.4.0].33 Sequences were analysed using DADA2 [version 1.3.1] to produce sequence variants. Taxonomy was assigned against the GreenGenes 13.8 database.34 The outcome sequence variant table was converted to biom format using biomformat [version 2.1.3].35 Further details regarding bioinformatic analysis are presented in Supplementary Material 1. Diversity analyses including Simpson Index for alpha diversity and Bray–Curtis statistic for beta diversity were performed using the core_diversity_analyses.py script from QIIME [version 1.9.0] with a subsampling level of 19505 to ensure that all samples were included.36 Taxon numbers at each taxonomic level were also produced. LEfSe analysis was carried out using the Huttenhower Galaxy Server [http://huttenhower.sph.harvard.edu/galaxy/] to identify any potential biomarkers associated with sample types.37 DESeq2 analysis,38 Wilcoxon Rank sum tests and Kruskal–Wallis tests for significant changes in abundance in relation to sample type were carried out in R. A p-value of ≤ 0.05 was considered statistically significant, with the exception of DESeq2 analysis where an adjusted p-value of ≤ 0.05 was used. Figures were made using ggplot2 in RStudio. Principal co-ordinate analysis [PCoA] plots were visualized using Emperor.39 2.4. Statistical considerations Statistical analyses for non-microbiota results were performed using SPSS v23 [IBM Corporation] and GraphPad Prism v5.04 [Graphpad software]. Dependent and independent samples t-tests, Mann–Whitney U test, analysis of variance [ANOVA] and Kruskal–Wallis tests were used where appropriate. Associations with increases in 25[OH]D were examined by bivariate correlations. A p-value of ≤ 0.05 was considered statistically significant. 2.5. Ethical statement The protocol for this study was approved by the Office of Research and Ethics at London Northwest Healthcare NHS Trust, and was performed in accordance with UK regulations and the principles of the Declaration of Helsinki 1954 and its later amendments. Informed consent was obtained from all individual participants included in this study. 3. Results Twenty-five patients participated in this study from August to December 2015 [late Summer to Winter at 52°N], with baseline characteristics outlined in Table 1. No significant demographic differences were noted across the three groups. A trend to higher body mass index [BMI] and waist circumference in non-IBD controls was seen. Most patients with UC had left-sided or extensive colitis of variable duration. Table 1. Baseline participant characteristics. Non-IBD controls [n = 8] Inactive UC [n = 9] Active UC [n = 8] p value Age, years (mean [range]) 51 [35–66] 45 [28–72] 45 [30–68] 0.541a Female:male 3:5 3:6 4:4 0.793a Ethnicity, n  British White 3 1 1  Southern European 0 0 2  Indian Subcontinental 5 7 5  Arab & Middle Eastern 0 1 0 Fitzpatrick skin type, n  I 0 0 0  II 1 1 3  III 2 1 0  IV 0 1 0  V 5 6 5  VI 0 0 0 Co-morbid illnesses, n  Hypertension 3 1 3  Hyperlipidaemia 2 1 1  Type 2 DM 1 0 1  Asthma 0 3 1  Congestive cardiac failure 1 0 0 Smoking status, n  Never smoked 5 6 6  Ex-smokers 2 3 1  Current smokers 1 0 1 BMI, kg/m2 (mean [range]) 28.9 [23.5–36.9] 25.8 [20.5–29.7] 24.6 [21.5–28.4] 0.077a Waist circumference, cm (mean [range]) 104 [93–119] 92 [84–99] 91 [70–109] 0.052a Vitamin D supplementation, n 1 4 3 0.205b Montreal Classification  Disease extent: E1:E2:E3 NA 0:3:6 1:2:4 UC disease duration, years [range] NA 11 [0.8–36] 12 [1–40] SCCAI [median, range] NA 2 [0–6] 3 [0–5] Partial Mayo Index [median, range] NA 0 [0–3] 1 [0–4] Medical therapy for UC NA  Nil 1 1  Mesalazine only 6 3  Thiopurine +/- mesalazine 1 1  Anti-TNF +/- mesalazine 1 0  Vedolizumab +/- mesalazine 0 1  Anti-TNF +/- thiopurine 0 2 Non-IBD controls [n = 8] Inactive UC [n = 9] Active UC [n = 8] p value Age, years (mean [range]) 51 [35–66] 45 [28–72] 45 [30–68] 0.541a Female:male 3:5 3:6 4:4 0.793a Ethnicity, n  British White 3 1 1  Southern European 0 0 2  Indian Subcontinental 5 7 5  Arab & Middle Eastern 0 1 0 Fitzpatrick skin type, n  I 0 0 0  II 1 1 3  III 2 1 0  IV 0 1 0  V 5 6 5  VI 0 0 0 Co-morbid illnesses, n  Hypertension 3 1 3  Hyperlipidaemia 2 1 1  Type 2 DM 1 0 1  Asthma 0 3 1  Congestive cardiac failure 1 0 0 Smoking status, n  Never smoked 5 6 6  Ex-smokers 2 3 1  Current smokers 1 0 1 BMI, kg/m2 (mean [range]) 28.9 [23.5–36.9] 25.8 [20.5–29.7] 24.6 [21.5–28.4] 0.077a Waist circumference, cm (mean [range]) 104 [93–119] 92 [84–99] 91 [70–109] 0.052a Vitamin D supplementation, n 1 4 3 0.205b Montreal Classification  Disease extent: E1:E2:E3 NA 0:3:6 1:2:4 UC disease duration, years [range] NA 11 [0.8–36] 12 [1–40] SCCAI [median, range] NA 2 [0–6] 3 [0–5] Partial Mayo Index [median, range] NA 0 [0–3] 1 [0–4] Medical therapy for UC NA  Nil 1 1  Mesalazine only 6 3  Thiopurine +/- mesalazine 1 1  Anti-TNF +/- mesalazine 1 0  Vedolizumab +/- mesalazine 0 1  Anti-TNF +/- thiopurine 0 2 DM, diabetes mellitus; BMI, body mass index; SCCAI, Simple Clinical Colitis Activity Index; Fitzpatrick skin types: I - pale white skin, blue/hazel eyes, blond/red hair, II - fair skin, blue eyes, III - darker white skin, IV - light brown skin, V - brown skin, VI - dark brown or black skin. aANOVA bChi-square, UC vs non-IBD controls. View Large Table 1. Baseline participant characteristics. Non-IBD controls [n = 8] Inactive UC [n = 9] Active UC [n = 8] p value Age, years (mean [range]) 51 [35–66] 45 [28–72] 45 [30–68] 0.541a Female:male 3:5 3:6 4:4 0.793a Ethnicity, n  British White 3 1 1  Southern European 0 0 2  Indian Subcontinental 5 7 5  Arab & Middle Eastern 0 1 0 Fitzpatrick skin type, n  I 0 0 0  II 1 1 3  III 2 1 0  IV 0 1 0  V 5 6 5  VI 0 0 0 Co-morbid illnesses, n  Hypertension 3 1 3  Hyperlipidaemia 2 1 1  Type 2 DM 1 0 1  Asthma 0 3 1  Congestive cardiac failure 1 0 0 Smoking status, n  Never smoked 5 6 6  Ex-smokers 2 3 1  Current smokers 1 0 1 BMI, kg/m2 (mean [range]) 28.9 [23.5–36.9] 25.8 [20.5–29.7] 24.6 [21.5–28.4] 0.077a Waist circumference, cm (mean [range]) 104 [93–119] 92 [84–99] 91 [70–109] 0.052a Vitamin D supplementation, n 1 4 3 0.205b Montreal Classification  Disease extent: E1:E2:E3 NA 0:3:6 1:2:4 UC disease duration, years [range] NA 11 [0.8–36] 12 [1–40] SCCAI [median, range] NA 2 [0–6] 3 [0–5] Partial Mayo Index [median, range] NA 0 [0–3] 1 [0–4] Medical therapy for UC NA  Nil 1 1  Mesalazine only 6 3  Thiopurine +/- mesalazine 1 1  Anti-TNF +/- mesalazine 1 0  Vedolizumab +/- mesalazine 0 1  Anti-TNF +/- thiopurine 0 2 Non-IBD controls [n = 8] Inactive UC [n = 9] Active UC [n = 8] p value Age, years (mean [range]) 51 [35–66] 45 [28–72] 45 [30–68] 0.541a Female:male 3:5 3:6 4:4 0.793a Ethnicity, n  British White 3 1 1  Southern European 0 0 2  Indian Subcontinental 5 7 5  Arab & Middle Eastern 0 1 0 Fitzpatrick skin type, n  I 0 0 0  II 1 1 3  III 2 1 0  IV 0 1 0  V 5 6 5  VI 0 0 0 Co-morbid illnesses, n  Hypertension 3 1 3  Hyperlipidaemia 2 1 1  Type 2 DM 1 0 1  Asthma 0 3 1  Congestive cardiac failure 1 0 0 Smoking status, n  Never smoked 5 6 6  Ex-smokers 2 3 1  Current smokers 1 0 1 BMI, kg/m2 (mean [range]) 28.9 [23.5–36.9] 25.8 [20.5–29.7] 24.6 [21.5–28.4] 0.077a Waist circumference, cm (mean [range]) 104 [93–119] 92 [84–99] 91 [70–109] 0.052a Vitamin D supplementation, n 1 4 3 0.205b Montreal Classification  Disease extent: E1:E2:E3 NA 0:3:6 1:2:4 UC disease duration, years [range] NA 11 [0.8–36] 12 [1–40] SCCAI [median, range] NA 2 [0–6] 3 [0–5] Partial Mayo Index [median, range] NA 0 [0–3] 1 [0–4] Medical therapy for UC NA  Nil 1 1  Mesalazine only 6 3  Thiopurine +/- mesalazine 1 1  Anti-TNF +/- mesalazine 1 0  Vedolizumab +/- mesalazine 0 1  Anti-TNF +/- thiopurine 0 2 DM, diabetes mellitus; BMI, body mass index; SCCAI, Simple Clinical Colitis Activity Index; Fitzpatrick skin types: I - pale white skin, blue/hazel eyes, blond/red hair, II - fair skin, blue eyes, III - darker white skin, IV - light brown skin, V - brown skin, VI - dark brown or black skin. aANOVA bChi-square, UC vs non-IBD controls. View Large Routine laboratory indices and circulating components of the vitamin D axis amongst participants at baseline are outlined in Table 2. As expected, faecal calprotectin was significantly higher amongst patients with active disease, and platelet counts higher, with a trend towards higher C-reactive protein [CRP]. No significant differences across the groups in serum 25[OH]D, calcium, phosphate or parathyroid hormone were noted. Baseline dietary patterns of the participants are outlined in Supplementary Table 1. Seven of the 25 participants had a vegetarian diet, with self-reported vegetable intake reported as > 35 standard serves per week by the majority [14 of 25] of participants. Table 2. Routine laboratory indices and components of the vitamin D axis in the patient groups and healthy controls Non-IBD controls [n = 8] Inactive UC [n = 9] Active UC [n = 8] p value Haemoglobin [g/l, mean, range] 140 [118–155] 145 [122–167] 124 [87–154] 0.07a White cell count [×109/l, mean, range] 7.4 [4.9–10.0] 6.5 [4.9–8.0] 7.2 [5.3–10.6] 0.53a Platelet count [×109/l, mean, range] 266 [200–321] 241 [160–313] 375 [255–509] 0.001a Serum albumin [g/l, mean, range] 45 [42–50] 46 [43–51] 43 [38–49] 0.09a Serum C-reactive protein [mg/l, median, range] 1.0 [< 1.0–5.0] 1.0 [< 1.0–8.0] 4.0 [1.0–28.0] 0.054b Faecal calprotectin [µg/g, median, range] 16.4 [12.2–50.9] 34.2 [< 5.3–87.1] 257 [110->2000] 0.002b 25[OH]D [nmol/l, mean, range] 31 [12–49] 33 [17–49] 34 [16–43] 0.90a Serum calcium [corrected, mmol/l, mean, range] 2.42 [2.29–2.57] 2.44 [2.30–2.55] 2.46 [2.36–2.58] 0.67a Serum phosphate [mmol/l, mean, range] 1.06 [0.83–1.42] 1.02 [0.73–1.65] 0.99 [0.55–1.33] 0.85a Serum PTH [pmol/l, mean, range] 3.3 [2.2–4.7] 4.6 [3.7–5.7] 4.5 [2.7–5.6] 0.20a Non-IBD controls [n = 8] Inactive UC [n = 9] Active UC [n = 8] p value Haemoglobin [g/l, mean, range] 140 [118–155] 145 [122–167] 124 [87–154] 0.07a White cell count [×109/l, mean, range] 7.4 [4.9–10.0] 6.5 [4.9–8.0] 7.2 [5.3–10.6] 0.53a Platelet count [×109/l, mean, range] 266 [200–321] 241 [160–313] 375 [255–509] 0.001a Serum albumin [g/l, mean, range] 45 [42–50] 46 [43–51] 43 [38–49] 0.09a Serum C-reactive protein [mg/l, median, range] 1.0 [< 1.0–5.0] 1.0 [< 1.0–8.0] 4.0 [1.0–28.0] 0.054b Faecal calprotectin [µg/g, median, range] 16.4 [12.2–50.9] 34.2 [< 5.3–87.1] 257 [110->2000] 0.002b 25[OH]D [nmol/l, mean, range] 31 [12–49] 33 [17–49] 34 [16–43] 0.90a Serum calcium [corrected, mmol/l, mean, range] 2.42 [2.29–2.57] 2.44 [2.30–2.55] 2.46 [2.36–2.58] 0.67a Serum phosphate [mmol/l, mean, range] 1.06 [0.83–1.42] 1.02 [0.73–1.65] 0.99 [0.55–1.33] 0.85a Serum PTH [pmol/l, mean, range] 3.3 [2.2–4.7] 4.6 [3.7–5.7] 4.5 [2.7–5.6] 0.20a PTH, parathyroid hormone. Bold type indicates statistical significance defined by p < 0.05. aANOVA bKruskal–Wallis test View Large Table 2. Routine laboratory indices and components of the vitamin D axis in the patient groups and healthy controls Non-IBD controls [n = 8] Inactive UC [n = 9] Active UC [n = 8] p value Haemoglobin [g/l, mean, range] 140 [118–155] 145 [122–167] 124 [87–154] 0.07a White cell count [×109/l, mean, range] 7.4 [4.9–10.0] 6.5 [4.9–8.0] 7.2 [5.3–10.6] 0.53a Platelet count [×109/l, mean, range] 266 [200–321] 241 [160–313] 375 [255–509] 0.001a Serum albumin [g/l, mean, range] 45 [42–50] 46 [43–51] 43 [38–49] 0.09a Serum C-reactive protein [mg/l, median, range] 1.0 [< 1.0–5.0] 1.0 [< 1.0–8.0] 4.0 [1.0–28.0] 0.054b Faecal calprotectin [µg/g, median, range] 16.4 [12.2–50.9] 34.2 [< 5.3–87.1] 257 [110->2000] 0.002b 25[OH]D [nmol/l, mean, range] 31 [12–49] 33 [17–49] 34 [16–43] 0.90a Serum calcium [corrected, mmol/l, mean, range] 2.42 [2.29–2.57] 2.44 [2.30–2.55] 2.46 [2.36–2.58] 0.67a Serum phosphate [mmol/l, mean, range] 1.06 [0.83–1.42] 1.02 [0.73–1.65] 0.99 [0.55–1.33] 0.85a Serum PTH [pmol/l, mean, range] 3.3 [2.2–4.7] 4.6 [3.7–5.7] 4.5 [2.7–5.6] 0.20a Non-IBD controls [n = 8] Inactive UC [n = 9] Active UC [n = 8] p value Haemoglobin [g/l, mean, range] 140 [118–155] 145 [122–167] 124 [87–154] 0.07a White cell count [×109/l, mean, range] 7.4 [4.9–10.0] 6.5 [4.9–8.0] 7.2 [5.3–10.6] 0.53a Platelet count [×109/l, mean, range] 266 [200–321] 241 [160–313] 375 [255–509] 0.001a Serum albumin [g/l, mean, range] 45 [42–50] 46 [43–51] 43 [38–49] 0.09a Serum C-reactive protein [mg/l, median, range] 1.0 [< 1.0–5.0] 1.0 [< 1.0–8.0] 4.0 [1.0–28.0] 0.054b Faecal calprotectin [µg/g, median, range] 16.4 [12.2–50.9] 34.2 [< 5.3–87.1] 257 [110->2000] 0.002b 25[OH]D [nmol/l, mean, range] 31 [12–49] 33 [17–49] 34 [16–43] 0.90a Serum calcium [corrected, mmol/l, mean, range] 2.42 [2.29–2.57] 2.44 [2.30–2.55] 2.46 [2.36–2.58] 0.67a Serum phosphate [mmol/l, mean, range] 1.06 [0.83–1.42] 1.02 [0.73–1.65] 0.99 [0.55–1.33] 0.85a Serum PTH [pmol/l, mean, range] 3.3 [2.2–4.7] 4.6 [3.7–5.7] 4.5 [2.7–5.6] 0.20a PTH, parathyroid hormone. Bold type indicates statistical significance defined by p < 0.05. aANOVA bKruskal–Wallis test View Large The follow-up visit took place following 8 weeks of vitamin D replacement [mean duration 58 days]. Vitamin D replacement resulted in an increase in serum 25[OH]D across all participants from a mean of 34 [range 12–49] to 111 [71–183] nmol/l [p < 0.001, paired t-test] [Figure 1]. There was no significant difference in the increase in 25 [OH]D between the three groups [p = 0.316]. All patients reported completion of the full course of supplementation [320000 IU vitamin D] except two non-IBD controls [240000 IU each], one patient each with inactive and active UC [280000 IU each]. One patient with active UC ceased mesalazine tablets [taken at 3.2 g daily] during the course of therapy in the context of symptomatic improvement. All other patients continued usual therapy. No significant change in dietary patterns over the study period were observed across most subjects. Figure 1. View largeDownload slide View largeDownload slide Change in clinical and laboratory indices following vitamin D supplementation in participants without IBD, and those with inactive and active UC. Figure 1. View largeDownload slide View largeDownload slide Change in clinical and laboratory indices following vitamin D supplementation in participants without IBD, and those with inactive and active UC. Symptomatic disease activity indices declined significantly amongst patients with inactive and active colitis following vitamin D supplementation, reaching significance for SCCAI [p = 0.04 and p = 0.01, respectively] but not for the Partial Mayo Index [p = 0.10 and p = 0.09, respectively]. In patients with active UC, objective markers of disease activity improved significantly following vitamin D supplementation: faecal calprotectin (median 257 [range 110 to > 2000] to median 111 [5–2000] µg/g, p = 0.02); platelet count (mean 375 [255–509] to mean 313 [243–461] × 109/l, p = 0.03); and albumin (mean 43 [38–49] to mean 45 [41–50] g/l, p = 0.04) [Figure 1]. No effect on faecal calprotectin, CRP, white cell count, platelet count or albumin was observed amongst patients with inactive colitis or non-IBD controls. Baseline overall dietary pattern, cereal and bread, vegetable or fruit intake did not influence response to faecal calprotectin, or circulating markers of inflammation, of vitamin D replacement [data not shown]. Among patients with active UC, there was no significant correlation between the change in serum 25[OH]D and change in faecal calprotectin [Spearman r = −0.21, p = 0.61]. There was no significant alteration in serum calcium, phosphate or alkaline phosphatase. Serum parathyroid hormone levels decreased significantly across the whole cohort (mean 4.0 [range 2.2–5.7] to 3.4 [1.8–6.5] pmol/l, p = 0.02). No patients were hospitalized or required surgery. 3.1. Change in faecal microbiota All 25 patients submitted a faecal sample at baseline, with 23 patients also providing a follow-up sample. One patient with active colitis and one non-IBD control did not submit follow-up samples for microbiota analysis. Between 50228 and 189688 raw sequences were produced per sample following amplicon sequencing. Following filtering and DADA2 analysis, each sample had between 19505 and 98075 sequence variant counts with an average of 45734 counts [Supplementary Table 2]. 3.2. Diversity analyses No differences in alpha diversity as assessed by the Simpson, Shannon, Chao or Observed species diversity indices were noted across the three patient groups at baseline. No differences in alpha diversity were noted in samples following vitamin D replacement across the patient groups. PCoA plots using the Bray–Curtis beta diversity metric demonstrated that patients without IBD clustered together distinct from UC patients [Figure 2a; p = 0.003, PERMANOVA]. When the same analysis approach was applied to both the data for before and after vitamin D supplementation, no difference between patients with inactive and active UC was noted [p = 1.0, Figure 2b]. Figure 2. View largeDownload slide Principal co-ordinate analysis [PCoA] plots at [a] baseline and [b] before and following vitamin D supplementation. Figure 2. View largeDownload slide Principal co-ordinate analysis [PCoA] plots at [a] baseline and [b] before and following vitamin D supplementation. 3.3. Taxonomic profiling Changes in the relative abundance of sequence variants showed statistically significant differences between the three groups at baseline [Table 3]. Abundance of the mucus-associated bacterium Ruminococcus gnavus was marginally but not significantly higher in patients with UC than non-IBD controls [p = 0.068, Kruskal—Wallis test, Supplementary Figure 1]. Table 3. Significant DESeq2 results comparing the relative abundance of sequence variants across sample types. Fold change values are given to three significant figures Sequence variant taxonomy Log fold change Adjusted p value Inactive UC > non-IBD controls  E. coli 5.34 0.00448 Non-IBD controls > active UC  Prevotella copri 8.48 0.0442  Coprococcus genus 8.76 0.0294 Inactive > active UC  Prevotella copri 8.46 0.00640  Bacteroides plebeius 9.08 0.0131  Bacteroides fragilis 8.79 0.0158  Bacteroides genus 7.83 0.0116  Ruminococcaceae family 8.73 0.00927  Bacteroides caccae 8.30 0.00640  Coprococcus genus 6.62 0.0284 Active > inactive UC  Lachnospira genus 6.03 0.00927  Sutterella genus 8.75 0.0158  Coprococcus genus 6.62 0.0284 Sequence variant taxonomy Log fold change Adjusted p value Inactive UC > non-IBD controls  E. coli 5.34 0.00448 Non-IBD controls > active UC  Prevotella copri 8.48 0.0442  Coprococcus genus 8.76 0.0294 Inactive > active UC  Prevotella copri 8.46 0.00640  Bacteroides plebeius 9.08 0.0131  Bacteroides fragilis 8.79 0.0158  Bacteroides genus 7.83 0.0116  Ruminococcaceae family 8.73 0.00927  Bacteroides caccae 8.30 0.00640  Coprococcus genus 6.62 0.0284 Active > inactive UC  Lachnospira genus 6.03 0.00927  Sutterella genus 8.75 0.0158  Coprococcus genus 6.62 0.0284 View Large Table 3. Significant DESeq2 results comparing the relative abundance of sequence variants across sample types. Fold change values are given to three significant figures Sequence variant taxonomy Log fold change Adjusted p value Inactive UC > non-IBD controls  E. coli 5.34 0.00448 Non-IBD controls > active UC  Prevotella copri 8.48 0.0442  Coprococcus genus 8.76 0.0294 Inactive > active UC  Prevotella copri 8.46 0.00640  Bacteroides plebeius 9.08 0.0131  Bacteroides fragilis 8.79 0.0158  Bacteroides genus 7.83 0.0116  Ruminococcaceae family 8.73 0.00927  Bacteroides caccae 8.30 0.00640  Coprococcus genus 6.62 0.0284 Active > inactive UC  Lachnospira genus 6.03 0.00927  Sutterella genus 8.75 0.0158  Coprococcus genus 6.62 0.0284 Sequence variant taxonomy Log fold change Adjusted p value Inactive UC > non-IBD controls  E. coli 5.34 0.00448 Non-IBD controls > active UC  Prevotella copri 8.48 0.0442  Coprococcus genus 8.76 0.0294 Inactive > active UC  Prevotella copri 8.46 0.00640  Bacteroides plebeius 9.08 0.0131  Bacteroides fragilis 8.79 0.0158  Bacteroides genus 7.83 0.0116  Ruminococcaceae family 8.73 0.00927  Bacteroides caccae 8.30 0.00640  Coprococcus genus 6.62 0.0284 Active > inactive UC  Lachnospira genus 6.03 0.00927  Sutterella genus 8.75 0.0158  Coprococcus genus 6.62 0.0284 View Large Changes in the abundance of specific bacteria following vitamin D administration were analysed using LEfSe analysis. Across all participants, an increase in Clostridium colinae [p = 0.03; driven by two non-IBD controls and two patients with inactive UC] and Enterobacteriacae [p = 0.03; driven by five patients with inactive UC and three with active UC] was noted. Ruminococcus gnavus decreased marginally but not significantly following vitamin D supplementation across the whole cohort [p = 0.15; Wilcoxon Rank sum, Supplementary Figure 1]. No significant change in abundances of other mucus-associated bacteria Ruminococcus torques or Akkermansia muciniphila, butyrate-producing bacteria from the Clostridium Cluster IV or Cluster XIVa groups, or of lactic acid-producing bacteria [Lactobacilli or Bifidobacteria], or of the invasive bacteria Fusobacterium nucleatum and E. coli were noted [data not shown]. 4. Discussion The role of vitamin D as a potential immunomodulator in patients with IBD has been investigated extensively for over a decade. Numerous studies demonstrate involvement of the vitamin D axis in regulation of the epithelial barrier, and innate immune cell and T-cell function.20,21,23,24,40–47 Although there are some preliminary data suggesting that vitamin D may influence the intestinal microbiota in IBD, this has not been studied in humans. Furthermore, evidence for efficacy at the clinical level remains poor. This study is the first to show that vitamin D replacement in patients with active UC who are deficient in vitamin D improved objective markers of inflammation. Although this was associated with a significant increase in Enterobacteriacae in patients with UC, there was no change in overall diversity or other specific bacteria analysed. Previous studies have shown that vitamin D supplementation may be associated with reduced rates of relapse in patients with Crohn’s disease in remission when given at a dose of 1200 IU daily for 12 months,2 and improved Crohn’s disease activity index [CDAI] and quality of life when given at up to 5000 IU daily for 24 weeks.4 An alternative placebo-controlled randomized controlled trial showed no significant change in CDAI, quality of life, CRP or faecal calprotectin in patients given 2000 IU vitamin D daily for 3 months.5 In patients with UC, low vitamin D levels have been associated with greater disease activity, as assessed by symptoms, faecal calprotectin and endoscopic activity, as well as increased risk of subsequent relapse.7,8,48–50 A small pilot study demonstrated reduction in symptomatic disease activity indices but not intestinal inflammation as measured by faecal calprotectin in patients with UC and Crohn’s colitis.51 No placebo-controlled studies in patients with UC have been reported to date. The current study is the first to show an improvement in objective markers of inflammation [faecal calprotectin, albumin, platelet count] following vitamin D replacement, limited to a group of patients with active UC defined by faecal calprotectin ≥ 100 µg/g at baseline. The reason for the faecal calprotectin reduction in these patients warrants further consideration. Although it has been postulated that a 25[OH]D level higher than 75 nmol/l, or closer to 100–125 nmol/l, may be required for an immunomodulatory effect,4,48,51–53 such a level was not specifically targeted in this study. Rather, high-dose oral weekly supplementation according to local institutional guidelines was administered at the same dose in all patients, as opposed to daily supplementation in most previous studies. Significant inter-individual variation in response to vitamin D supplementation exists, particularly in diseased states such as IBD,54 and unsurprisingly the serum 25[OH]D level achieved varied from 75 to 183 nmol/l across the patients with UC. Five of eight patients with active UC achieved a 25[OH]D of ≥ 100 nmol/l, all of whom had a reduction in faecal calprotectin to varying extents; however, there was no clear correlation between the rise in 25[OH]D and reduction in faecal calprotectin. Therefore, the findings in this study raise the prospect that it may not be the final serum 25[OH]D level but the administration of a higher dose of vitamin D itself that potentially confers an immunomodulatory effect. This concept requires further investigation in an appropriately powered prospective controlled trial. The VDR is expressed in colonic intestinal epithelial cells, dendritic cells and macrophages.21,42 Vitamin D has been shown to potently stimulate cathelicidin, an anti-microbial peptide produced by macrophages22 which plays an important role in defence against intracellular organisms such as mycobacteria.21 VDR expression is significantly increased in inflamed and non-inflamed mucosal biopsies from patients with UC.55 Vitamin D supplementation suppressed intra-macrophage E. coli survival in in vitro studies,20 and vitamin D-deficient and VDR knockout mice had impaired ileal Paneth cell alpha defensin secretion and increased abundance of the colitogenic Helicobacter hepaticus, compared with control or wild-type mice.23 Therefore, there is biological plausibility for an interaction between the vitamin D axis and intestinal microbiota in the pathogenesis and perpetuation of inflammation in patients with IBD, especially UC. In the current study, no overall change in faecal microbial diversity occurred following vitamin D supplementation. Although an increase in the abundance of Enterobacteriaecae was noted following vitamin D supplementation in patients with UC, this large family comprises a large proportion of harmless and commensal as well as potentially pathogenic bacteria in the human gut, and therefore the significance of such a change is uncertain. Ruminococcus gnavus is a Gram-positive anaerobic mucolytic bacterium belonging to Cluster XIVa of the class Clostridia, which is increased in abundance in patients with IBD.16 The intestinal mucus layer provides a protective barrier between the luminal environment and mucosa, comprising dense glycoproteins interspersed with antimicrobial peptides produced by Paneth cells and other epithelial cells.56 A previous study has shown reduced abundance of Ruminococcus gnavus in mucosal biopsies from patients with active UC defined symptomatically,57 and this trend was confirmed in the current study, albeit without statistical significance. Furthermore, the abundance of Ruminococcus gnavus decreased non-significantly across all patients after vitamin D supplementation. Whether vitamin D supplementation mediates regulation of intestinal mucus antimicrobial composition and therefore susceptibility to specific mucolytic bacteria warrants further investigation. Nonetheless, an absence of a significant effect on the faecal microbiota across the whole cohort of patients studied is of note. It is possible that vitamin D does not alter human microbiota, despite laboratory data from mouse studies.23 Other possible explanations include a differential effect on faecal and mucosa-associated microbiota. Faecal microbiota was assessed during this study rather than mucosal-associated microbiota as this is less invasive and is not subject to variation as a result of bowel preparation.58 However, given the intimate relationship between vitamin D-induced anti-microbial peptide secretion and the mucosal microbiota, one may postulate that significant changes in the latter may be more reflective of the effect of vitamin D in this setting. An absence of significant alteration of the faecal microbiota by vitamin D supplementation, however, is not an isolated finding: despite widespread use, there remains a paucity of published data regarding the effect of conventional therapies such as 5-aminosalicylates, thiopurines and anti-tumour necrosis factor α agents on the faecal microbiota independent of changes in mucosal inflammation in patients with IBD.59,60 Conversely, the absence of a change in microbiota composition despite reduction in inflammation in the active UC group is also notable, and may reflect only a mild reduction in inflammation in these patients. It is important to note that there are few robust data regarding change in microbiota composition in patients with UC in the absence of medical therapy.61 Furthermore, patients with UC in this study had a relatively long disease duration, with a median of 11–12 years. Data regarding the effect of duration of UC on temporal variability of microbiota are also limited.61 Longer disease duration has previously been described as a risk factor for vitamin D deficiency,62 but no influence of disease activity on initial 25[OH]D level or response to supplementation was noted in the current study [data not shown]. There are multiple other limitations in this small study. Although no overt toxicity as measured by serum calcium and phosphate was noted, long-term potential effects of the supplementation strategy in this study were not able to be elucidated, particularly the risk of hypercalciuria or nephrocalcinosis.51 Dietary assessment of patients at baseline and follow-up visits showed no clear changes across most patients, but specific effects of change in diet as confounders were difficult to elicit. In conclusion, vitamin D supplementation at a dose of 40000 IU weekly for 8 weeks reduced objective circulating and intestinal markers of inflammation in patients with active UC. A significant increase in abundance of Enterobacteriaceae in patients with UC, and a trend to reduction in the mucolytic species Ruminococcus gnavus, was noted, but overall microbiota diversity was unchanged. Vitamin D may therefore reduce intestinal inflammation, but independently of any change in faecal bacterial composition. A larger placebo-controlled clinical trial incorporating immunological and extended microbiota analyses, including functional assessment, will shed further light upon this effect. Funding This work was supported by the European Crohn’s and Colitis Organisation Fellowship awarded to Dr Mayur Garg, and St Mark’s Foundation Research Grant 2015 awarded to Prof. Ailsa Hart and Dr Mayur Garg. Conflict of Interest The authors declare that they have no conflict of interest with respect to this manuscript. Author Contributions M.G.: conception and design of the study, acquisition of data, analysis and interpretation of data, writing and drafting the article and its final approval; P.H., J.N.D.: acquisition of data, critical appraisal of manuscript for important intellectual content, final approval of submitted version; S.S., G.H.: analysis and interpretation of data, critical appraisal of manuscript for important intellectual content, final approval of submitted version; A.H.: conception and design of the study, critical appraisal of manuscript for important intellectual content, final approval of submitted version. Supplementary Data Supplementary data to this article can be found online at: ECCO-JCC Conference: Australian Gastroenterology Week 2017 [Oral], Gold Coast, Australia. 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Journal of Crohn's and ColitisOxford University Press

Published: May 3, 2018

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