The gut microbiota, also known as the intestinal flora, is a complex ecosystem of microorganisms such as bacteria, fungi, viruses, parasites, and archaea that live in symbiosis in the human intestinal lumen [1, 2]. These microorganisms are mainly host–specific, and their composition and numbers vary depending on the host species and microenvironment [3]. The gut microflora plays an important direct and/or indirect role in a variety of physiological processes, including regulation of the immune system, modulation of neurotransmitters and hormones, and production of metabolites and antioxidants. Emerging evidence suggests that the gut microbiota plays a critical role in alleviating various systemic diseases [3]. The gut microbiome is considered a new active organ due to its ability to interact with host biology and integrate systemically [4, 5]. Changes in the composition or number of the gut microbiota disrupt the commensal relationship with the host and have negative consequences for human health [6, 7, 8].

Vitamin D/vitamin D receptor (VDR) signaling contributes significantly to the immunological, genetic, environmental, and microbial aspects of human health and disease [8, 9]. The human VDR is the first identified gene that functions as an essential host factor and genetically influences the modulation of the gut microbiome [10]. Several studies have demonstrated the crucial function of vitamin D and its receptor in maintaining a healthy gut [11, 12].

The aim of this review is to provide an overview of the data linking the gut microbiome to vitamin D and VDR, with a focus on data from experimental models and translational data from human studies related to changes in gut microbiota composition caused by vitamin D.

Vitamin D, a fat-soluble vitamin, occurs in two forms, ergocalciferol or vitamin D${}_{\text{2}}$ and cholecalciferol or vitamin D${}_{\text{3}}$, which are chemically distinguished by their side chains [13]. These forms are either formed in the skin by the photochemical conversion of 7-dehydrocholesterol to pre-vitamin D or are absorbed in the small intestine (Fig. 1) [14, 15].

Fig. 1.

Biosynthesis and metabolism of vitamin D. This figure was created using BioRender (https://biorender.com, accessed February 13, 2023). UVB, ultraviolet B; CYP2R1, cytochrome P450, family 2, subfamily R, polypeptide 1; CYP27A1, cytochrome P450, family 27, subfamily A, polypeptide 1; CYP27B1, cytochrome P450, family 27, subfamily B, polypeptide 1; VDR, vitamin D receptor; VDRE, vitamin D response element; RXR, retinoid X receptor; DHCR7, 7-dehydrocholesterol reductase; VDBP, vitamin D-binding protein.

Until their enzymatic hydroxylation, these forms remain biologically inactive [16]. After vitamin D${}_{\text{3}}$ binds to vitamin D-binding protein (VDBP), this complex is transported to the liver. The liver and other tissues are able to convert the vitamin D–DBP complex to 25-hydroxyvitamin D or 25(OH)D, the predominant circulating form of vitamin D. Several enzymes have a 25-hydroxylase function; however, the cytochrome P450, family 2, subfamily R, polypeptide 1 (CYP2R1) enzyme is most important [16, 17]. Subsequently, 25(OH)D is further metabolized, mainly in the kidney, by the enzyme 1-$\alpha$-hydroxylase cytochrome P450, family 27, subfamily B, polypeptide 1 (CYP27B1); this produces 1,25(OH)${}_{\text{2}}$D${}_{\text{3}}$, the biologically active form of vitamin D responsible for most of its biological effects (Fig. 1) [16, 17]. Synthesis of 1,25(OH)${}_{\text{2}}$D${}_{\text{3}}$ in the kidney is a highly regulated process that is suppressed by calcium, phosphate, and fibroblast growth factor 23 (FGF23) [14] and activated by parathyroid hormone [18]. In addition to renal tissues, 1,25(OH)${}_{\text{2}}$D${}_{\text{3}}$ synthesis by CYP27B1 also occurs in extrarenal tissues, such as intestinal macrophages and cells of the immune system [19].

In addition to the classical effects of vitamin D in regulating bone mineralization and maintaining systemic calcium homeostasis, non-classical effects such as regulation of cell proliferation and differentiation, hormone secretion, and immune system function have recently been reported [16, 17].

The VDR acts as a transcription factor and controls the expression of genes involved in the biological effects of vitamin D. Binding of 1,25(OH)${}_{\text{2}}$D${}_{\text{3}}$ to the VDR activates its transcription (Fig. 1) [20, 21]. The VDR belongs to the superfamily of nuclear hormone receptors [22] and exerts its functions in various genes of mammalian cells and tissues [23]. Therefore, 1,25(OH)${}_{\text{2}}$D${}_{\text{3}}$-induced changes in the transcriptome of VDR-expressing cells are closely related to the biological effects of vitamin D${}_{\text{3}}$ [24]. After binding to 1,25(OH)${}_{\text{2}}$D${}_{\text{3}}$, the VDR heterodimerizes with the retinoid X receptor and is transported to the nucleus. After binding to vitamin D response elements (VDREs) in the promoter region of target genes, this complex regulates their transcription (Fig. 1). Several additional factors, called coregulators, form complexes with the VDR to stimulate (coactivators) or repress (corepressors) the transcriptional activity of the VDR. These coregulators are specific for different genes, and different cells express these coregulators differently to ensure the specificity of vitamin D and VDR actions [13]. Healthy intestinal epithelial cells (IECs) show high VDR expression, especially in crypts [25] and the vast majority of immune cells [26]. Interestingly, the bacterial microbiome does not express the VDR gene, suggesting that vitamin D likely exerts its effects via VDR signaling in immune cells and IECs [27].

The protective barriers of the mucus layer and intestinal epithelium prevent bacteria and their products from entering the interstitial space, thus preventing the development of intestinal inflammation [28]. However, disruption of the homeostasis of the gut microbiota and the subsequent entry of immunogenic byproducts into the interstitial space can trigger an inflammatory response by activating the immune system [29].

In the human body, the total number of bacterial cells ranges from 10${}^{13}$ to 10${}^{14}$, with a ratio of microbial cells to human cells of approximately 1:1 [30]. It is estimated that the gastrointestinal tract harbors between 200 and more than 1000 bacterial species [31, 32]. The extent and composition of the gut microbiota vary widely throughout the gastrointestinal tract [2]. The colon is colonized by a large number of bacterial species that exhibit a variety of microbiological characteristics [33]. The predominant bacterial phyla in the gastrointestinal microbiota include Bacteroidetes, Firmicutes, Actinobacteria, and Proteobacteria [34]. The phylum Firmicutes consists of aerobic and anaerobic Gram-positive bacteria, Bacteroidetes of Gram-negative bacteria, Actinobacteria of Gram-positive bacteria with beneficial effects, and Proteobacteria of Gram-negative bacteria commonly found in the family Enterobacteriaceae [2]. While the gut microbiota community is generally stable in adulthood, changes may occur over time-related to diet and general health [35, 36]. It is estimated that the gut microbiome encodes approximately 3,000,000 genes, which is about 100 times more genes than in the host. In humans, the gut microbiome is relatively unique and stable but also constantly changes over time [37, 38]. It also appears to be critical to human body functions, contributing to the breakdown of otherwise indigestible polysaccharides from the diet and to the synthesis of important amino acids and vitamins [39]. Gut dysbiosis leads to impaired immunoregulatory function of the intestinal mucosa, which may result in the development of various inflammatory and immune-mediated diseases [2, 40]. Emerging evidence suggests that a modified diet has the potential to partially reverse dysbiosis, which may ultimately improve human health [41].

There is increasing evidence that bacteria play an important role in altering vitamin D metabolism. Several enzymes expressed by bacteria are involved in the hydroxylation of steroids; consequently, these enzymes can induce vitamin D in a similar manner as in humans [59]. The bacterial CYP105A1 (Streptomyces griseolus) is responsible for the conversion of vitamin D${}_{\text{3}}$ to the biologically active form of vitamin D by two successive hydroxylations. This suggests that the bacterial function corresponds to the CYP27A1, CYP27B1, and CYP2R1 enzymes associated with vitamin D [60]. The National Center for Biotechnology Information BioSystems database showed the existence of homologous proteins for CYP27A1 and CYP27B1 in bacterial populations, and in particular, in Ruminococcus torques from the phylum Firmicutes and in Mycobacterium tuberculosis, respectively [61]. A patent (US5474923) describes a technique for incorporating hydroxyl groups into vitamin D compounds at the 1$\alpha$-position in the kidney and/or at the 25-position in the liver in the presence of a cyclodextrin using a mixture containing a microorganism (Actinomycetales such as Nocardia, Streptomyces, Sphinogmonas, and Amycolata) capable of hydroxylating vitamin D compounds or an enzyme produced by a microorganism. In addition, factors such as FGF23, which play a crucial role in the regulation of vitamin D metabolism by the microbiota, are key elements for further studies to understand how they influence this process [62].

The presence of dysbiosis and alterations in the gut microbiota in autoimmune diseases is becoming increasingly clear [63]. Nevertheless, the exact way in which the immune system and the gut microbiota interact in the development of disease is not yet fully understood. Although the extent of dysbiosis may vary among autoimmune diseases, there is evidence that certain bacteria may promote or inhibit immune responses in different ways, suggesting that the microbiota may have a multifaceted influence on inflammatory diseases [63].

Commensal bacteria, toxins, and food antigens are involved in stimulating the immune response, which in turn leads to the initiation of inflammatory response; persistent inflammation can lead to the development of autoimmune diseases [64]. Recent evidence has shown that vitamin D signaling has a major impact on a variety of aspects of intestinal physiology and plays a critical role in maintaining intestinal homeostasis [65]. Vitamin D contributes to the activation and synthesis of pattern recognition receptors, various cytokines, and antimicrobial peptides (AMPs). It also plays an important role in modulating the gut microbiota, preventing bacterial overgrowth, and strengthening the integrity of the gut barrier [65]. In parallel, vitamin D positively affects intestinal tissues by promoting innate immune responses and attenuating T-cell-mediated inflammatory adaptive immunity, functions that are closely associated with the development of autoimmunity [66].

The intestinal mucosa serves as both a physical and functional barrier separating host cells from the microenvironment. It consists of several components that work together to fulfill its function, such as the commensal gut microbiota, an outer mucus layer, secretory immunoglobulin A (sIgA), and AMPs secreted into the mucus layer as immune-sensing molecules and regulatory proteins. The specialized epithelial cell layer and inner lamina propria also contribute to this function (Fig. 2) [67].

Fig. 2.

Schematic representation of the changes associated with the intestinal barrier between a vitamin D-sufficient and vitamin D-deficient dysbiotic gut. This figure was created using BioRender (https://biorender.com, accessed February 13, 2023). DC, dendritic cell; IL-12, interleukin 12; Th, T helper; TNF-$\alpha$, tumor necrosis factor alpha; IFN-$\gamma$; interferon gamma; Treg, regulatory T cell; TJ, tight junction; AMPs, antimicrobial peptides; AJ, adherent junction; ZO-1, zonula occluden-1; VDR, vitamin D receptor; RXR, retinoid X receptor; IECs, intestinal epithelial cells; NOD2, nucleotide-binding oligomerization domain protein 2; CAMP, cathelicidin antimicrobial peptide; TLR, Toll-like receptor; DEFB2/HBD2, antimicrobial peptide defensin $\beta$2/human beta-defensin-2; NF-$\kappa$B, nuclear factor kappa-light-chain-enhancer of activated B cells; IgA, immunoglobulin A; JAM, junctional adhesion molecule.

The IECs form a monolayer and are tightly connected by symmetrical structures called junctional complexes. The tight junctions (TJs) are located on the apical side of the cells and are responsible for ion transport and small molecule regulation (Fig. 2). The gut-associated lymphoid tissue is involved in both adaptive and innate immunity and consists of B cells, T cells, macrophages, and dendritic cells, which contribute critically to the immunological defense mechanisms of the gut barrier [68]. Immunoregulators such as sIgA and AMPs are secreted into the mucus layer to enhance the gradient separation of the microbiota from the epithelium to the lumen [69]. The sIgAs are produced by plasma cells, and their functions are diverse: they coat bacteria to facilitate their interaction with host immune cells, downregulate inflammation-associated epitopes, or neutralize toxins [70]. The composition of the mucus layer can influence the gut microbiota, and in parallel, the gut microbiota determines the properties of the mucus layer [71]. The IEC layer preserves the property of not being penetrated by pathogens and toxins through the function of TJs. Impairment of any aspect of the intestinal barrier, whether physical or functional, renders the host vulnerable to pathogen invasion, triggers a local immune response, and stimulates an inflammatory response in the gut (Fig. 2) [72].

Vitamin D–VDR signaling is critical for maintaining the integrity of the intestinal barrier by regulating apical junctions, adherent junctions, and proteins associated with TJs [73]. Defensin beta 4/human beta-defensin-2 (HBD-2) and cathelicidin antimicrobial peptide are stimulated directly and indirectly by vitamin D in human intestinal and monocyte cell lines through stimulation of the intracellular pattern recognition receptor nucleotide-binding oligomerization domain protein 2 (NOD2) [74, 75]. Stimulation of NOD2 by its ligand muramyl dipeptide induces HBD-2 gene expression (Fig. 2) [76]. These data support the hypothesis that AMPs have a major impact on the composition of the gut microbiota, whereas AMPs, in combination with IgAs, exert protective effects on the mucus layer [77].

Vitamin D–VDR signaling helps maintain intestinal barrier integrity by regulating myosin light chain kinase (MLCK) and provides protection by preventing nuclear factor kappa-light-chain-enhancer of activated B cells (NF-$\kappa$b)-mediated MLCK stimulation [78]. In VDR-deficient mice, ileal Paneth cell production of $\alpha$-defensins and matrix metalloproteinase 7 is impaired, possibly leading to the development of dysbiosis (Fig. 2) [79]. VDR KO Paneth cells inhibit the growth of pathogenic bacteria and enhance the autophagic response [80]. Following infection with Salmonella increased inflammation and susceptibility to small intestinal damage were observed in this experimental model, suggesting that deficiency of VDR in Paneth cells may lead to decreased antibacterial activity and increased inflammation [80]. Vitamin D supplementation and the production of AMPs, such as $\alpha$-defensins by Paneth cells and HBD-2 and cathelicidin by IECs, exert antimicrobial effects [81].

There is growing evidence that the microbiome plays an important role in both health and disease. Variations in the human microbiome are closely related to a variety of physiological functions. In parallel, new data show that the effect of vitamin D on health is mediated in part by the microbiome. Vitamin D exerts its modulatory effects on gastrointestinal health by either regulating the composition and metabolic activity of the gut microbiome or modulating the functions of the physiological gut barrier and immune system. However, the effects of vitamin D on the gut microbiome are also closely related to VDR activation, as VDR is a crucial host factor that can influence the gut microbiome at the genetic level [82]. Several studies have demonstrated the association between vitamin D deficiency and the development of dysbiosis, while intervention studies have demonstrated vitamin D-induced changes in the composition of the microbiome, mainly through the induction of a health-promoting microbiota and the reduction of the Firmicutes/Bacteroides ratio.

Future research should focus on elucidating the mechanisms involved in the changes in gut microbiota composition associated with vitamin D status or supplementation and/or genetic impairment of VDR expression/activity. Thus, there is a great need for well-designed animal and human studies that allow consistent analyses of gut microbiota and assessment of vitamin D or VDR. In addition, the question of what dose of vitamin D supplementation results in specific changes in the gut microbiome that affect host health remains unresolved. Considering that the gut microbiome can provide valuable information to assess individual responses to vitamin D supplementation, future research should focus on intra- and inter-individual variability using multi-omics strategies. In parallel, the design of experimental animal studies is necessary to understand causality, as it is possible that microbial changes precede disease onset, suggesting a potential causal relationship or passive response to disease.

VDR, vitamin D receptor; VDBP, vitamin D binding protein; CYP2R1, cytochrome P450, family 2, subfamily R, polypeptide 1; CYP27B1, cytochrome P450, family 27, subfamily B, polypeptide 1; FGF23, fibroblast growth factor 23; VDRE, vitamin D response elements; IEC, intestinal epithelial cell; KO, knock out; WT, wild type; ZO-1, zonula occluden-1; PICRUST, phylogenetic investigation of communities by reconstruction of unobserved states; AMP, antimicrobial peptide; sIgA, secretory immunoglobulin A; TJ, tight junction; NOD2, nucleotide-binding oligomerization domain protein 2; MLCK, myosin light chain kinase; NF-$\kappa$b, nuclear factor kappa-light-chain-enhancer of activated B cells.

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IA and ET: conducting research and investigation process, specifically performing the data/evidence collection; contributing to the visualization preparation, creation and presentation of the published work; contributing to the preparation, creation and presentation of the published work, specifically writing the initial draft (including substantive translation) and AM and CT: contributing to conceptualization ideas; formulation and evolution of review aims; contributing to management and coordination responsibility for the research activity planning and execution; contributing to preparation, creation and presentation of the published work, specifically critical review, commentary and revision—including pre- or post-publication stages. All authors read and approved the final manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work.

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This research received no external funding.

The authors declare no conflict of interest.