Vitamin D in Systemic Sclerosis: A Review

(1) Background: In the present paper we aimed to review the evidence about the potential implication of vitamin D in the pathogenesis and management of systemic sclerosis (SSc); (2) Methods: we performed a review of the literature looking for studies evaluating the potential role of vitamin D and its analogs in SSc. We searched the PubMed, Medline, Embase, and Cochrane libraries using the following strings: (vitamin D OR cholecalciferol) AND (systemic sclerosis OR scleroderma). We included cohort studies, case-control studies, randomized controlled trials, and observational studies. (3) Results: we identified nine pre-clinical and 21 clinical studies. Pre-clinical data suggest that vitamin D and its analogs may suppress fibrogenesis. Clinical data are concordant in reporting a high prevalence of hypovitaminosis D and osteoporosis in SSc patients; data about the association with clinical manifestations and phenotypes of SSc are, conversely, far less consistent; (4) Conclusions: in vitro data suggest that vitamin D may play an antifibrotic role in SSc, but clinical data confirming this finding are currently lacking. Hypovitaminosis D is common among SSc patients and should be treated to reduce the risk of osteoporosis.


Introduction
Vitamin D 3 , also known as cholecalciferol, is an inactive fat-soluble hormone precursor produced in the skin as a result of ultraviolet activation of 7-dehydrocholesterol, but also ingested, in a small amount, from food. Its active form is 1,25-dihydroxycholecalciferol (1,25(OH) 2 D 3 ) or calcitriol, which plays a paramount role in calcium and phosphate homeostasis by activation of calcium absorption in the small intestine and the stimulation of osteoclastic maturation [1,2].
The activation process of cholecalciferol consists of two sequential hydroxylations, the first occurring in the liver, and the second, mediated by CYP27B1 (also called 1α-hydroxylase), taking place in the kidney [3]. Immune cells can express CYP27B1, allowing paracrine and autocrine activity in addition to the endocrine activity of 1,25(OH) 2 D 3 [3][4][5]; however, while CYP27B1 expression is upregulated by the parathyroid hormone (PTH) and downregulated by calcium concentration in the kidney, its expression in immune cells is independent of endocrine modulation [5][6][7][8].
The pleiotropic effects of calcitriol are mediated through the vitamin D receptor (VDR), a nuclear receptor that heterodimerizes with retinoid X receptor (RXR), and translocates to the nucleus, where it binds Vitamin D response elements (VDREs) in the promoters of Vitamin D-responsive genes [9,10]. VDR is almost ubiquitous, which explains in part the complexity of 1,25(OH) 2 D 3 activity, going well beyond calcium and phosphate metabolism [11]. Indeed, Vitamin D regulates innate and adaptive immunity by the modulation of the production of pro-inflammatory and anti-inflammatory cytokines [10,[12][13][14][15], the suppression of T cells proliferation [10,16], the induction of the shift from a T-helper (Th)1 and Th17 to Th2 phenotype [14,15,17]. Vitamin D also regulates myeloid differentiation towards monocytes and granulocytes while inhibiting the differentiation towards dendritic cells; by doing it, vitamin D reduces the inflammatory activity of antigen-presenting cells (APC), counteracting antigen presentation and the production of cathelicidin antimicrobial peptide (CAMP) and defensin β2 [16,18,19]. Vitamin D also acts on endothelial proliferation, stimulating angiogenesis [20]. Indeed, historically, before antibiotic discovery, vitamin D was used as an antitubercular treatment, due to the enhancement of innate immunity against mycobacterium rather than to a direct antitubercular activity [21,22].
In consideration of its immunoregulatory properties, Vitamin D has been studied over the past years to evaluate its use in the modulation of immune system activity in clinical practice [9,[23][24][25]. Specifically referring to systemic sclerosis (SSc), Vitamin D has regulatory activities on different pathogenetic mechanisms of this rare condition: immunity, peripheral vasculopathy, and fibrosis [26]. This is why its application in this context has been explored in the last years; in this study, we aim to review the current evidence about the potential implication of Vitamin D in the pathogenesis and treatment of systemic sclerosis (SSc), starting from pre-clinical data and then moving to the clinical setting.

Materials and Methods
This review was conducted following the requirements of the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA). The data extraction and data synthesis were performed independently by the authors (M.P.), (E.G.) and (M.B.) based on the PICO components (Population; Intervention; Comparison; Outcome). We performed a review of the literature, looking for studies evaluating the potential role of vitamin D and its analogs in SSc. On 18 March 2022, we searched the following databases: PubMed, Medline, Embase, and Cochrane library. We used the following strings: (vitamin D OR cholecalciferol) AND (systemic sclerosis OR scleroderma). We restricted the search to the last 10 years and we included papers that fulfilled the following inclusion criteria: - The availability of the full version of the paper online; -English language; -Study design: either cohort study (prospective or retrospective), case-control study, randomized controlled trial, or observational study; -Studies addressing preclinical and clinical effects of vitamin D in the context of SSc.
Therefore, we excluded letters, abstracts, conference abstracts, comments, case reports, reviews, papers with no English version available, and studies not directly assessing the role of vitamin D in SSc.
Based on inclusion and exclusion criteria, we were able to retrieve 605 papers. After careful, independent screening by two investigators, we finally selected 30 studies, as shown in Figure 1.

Preclinical and Experimental Studies
Among the 30 selected studies, nine reported pre-clinical findings on mouse models and fibroblast cultures. We summarized the main laboratory findings in Table 1.  Figure 1. Flowchart of the study selection process. We reported the selection process of the studies included in this review.

Preclinical and Experimental Studies
Among the 30 selected studies, nine reported pre-clinical findings on mouse models and fibroblast cultures. We summarized the main laboratory findings in Table 1. In topical calcipotriol-treated mice, the dermal collagen area and the dermal thickness were significantly reduced. Taking together the current body of evidence derived from in vitro studies, consistent data support the hypothesis that vitamin D can modify the fibrogenic activity of fibroblasts, suppressing the pro-fibrotic tissue growth factor (TGF)-β/small mother against decapentaplegic (SMAD) signalling [27,29]; paricalcitol, a noncalcemic analog of vitamin D, was shown to suppress the expression of periostin and collagen 1A1 in dermal fibroblasts exposed to TGF-β and Th2 cytokines [30]. Similarly, 17,20S(OH) 2 pD and calcitriol induce the production of metalloproteinases, such as MMP-1 and BMP-7, involved in matrix degradation [35].
Consistent with these data, the anti-fibrogenic activity of vitamin D has been reported in murine models of scleroderma. Specifically, in mice with bleomycin-induced skin fibrosis, the treatment with systemic topical vitamin D analogs reduced the amount of fibrosis [27][28][29][30] by down-regulating TGF-β signalling and by modulating cytokine mediators, such as IL-13, TNF-α, IL-6, IL-17, and IL-12p70 [34]. These findings suggest that the effect of vitamin D in this context is in part directly related to its activity on crucial pathways of fibrosis development and in part to the modulation of the immune system; indeed, in blood samples collected from SSc patients, vitamin D supplementation increased IL-10 production by T-reg lymphocytes providing a "suppressive" cytokine milieu able to modulate immune response [31].
The activity of vitamin D on fibrogenesis is VDR-dependent; indeed, in VDR knockout mice, skin fibrosis is enhanced after exposure to pro-fibrotic stimuli [33]. Moreover, VDR expression is decreased in fibroblasts of SSc patients and murine models of SSc and VDR downregulates TGF-β/SMAD signalling [29].

Clinical Studies
In Table 2 we report the clinical studies included in the present review. We identified a total of 21 papers; out of them, 15 were case-control studies, three were retrospective cohort studies, and three were cross-sectional studies.   Looking at the current literature, there is a concordance about the high prevalence of hypovitaminosis D among SSc patients [43,56]. When compared to healthy controls, patients affected by SSc showed generally lower vitamin D plasma concentrations, even in the case of cholecalciferol supplementation [38,39,42,[47][48][49][50][51]54,55]. Interestingly, when the levels of 25(OH)D were compared among patients with different disease phenotypes, the results were controversial. Indeed, Corrado et al. [39] found that, in patients with the diffuse cutaneous form (dcSSc), 25(OH)D levels were significantly lower than in limited cutaneous form (lcSSc); moreover, vitamin D levels were inversely correlated to Rodnan skin score in other papers [42,51]. However, other authors did not confirm these findings [43,44,50], reporting that the extent of skin involvement was not associated with vitamin D levels in SSc. This discrepancy is also evident in the relationship between vitamin D concentration and autoantibody profile: anti-Scl70 positivity was associated to lower vitamin D levels by some authors [37,40], while others failed to disclose this association [43,44,49]. Interestingly, Giuggioli et al. reported that SSc patients who undergo vitamin D supplementation show higher vitamin D plasma concentrations and are more likely to be anticentromere positive [45].
As expected, Vitamin D plasma concentration is inversely correlated with bone mass density in SSc patients [37,38,40] and, therefore, vitamin D deficiency is also paralleled by an impairment of bone mineral density (BMD), which is significantly lower in SSc patients than in healthy controls [37][38][39]54]. According to Rios-Fernandez et al., the prevalence of osteopenia/osteoporosis in a cohort of SSC patients was 77%, higher than that observed in an age-and sex-matched control group (50%; p < 0.0001) [36].
The investigations assessing the possible association of vitamin D levels with specific SSc clinical manifestations led to conflicting results; while some authors failed to disclose any association [39,47], others showed a weak association with some specific domain of disease. In particular, Caimmi et al. [53] and Park et al. [46] reported that vitamin D deficiency is an independent risk factor for the development of digital ulcers. Moreover, some reports suggest a potential association with the degree of lung involvement, since lower vitamin D levels were reported in patients with bibasal interstitial fibrotic changes in the lung [44], with vitamin D levels bearing a weak direct association with diffusing lung capacity of the lung for carbon monoxide (DLCO), according to other papers [36,43].
Finally, two papers assessed the potential additional risk of developing SSc in patients with specific VDR polymorphisms. The results are, once more, controversial. Indeed, while Kamal et al. [41] found that the ApaI and TaqI polymorphisms did not significantly affect SSc susceptibility, according to Li et al. [52], ApaI and BglI polymorphism genotypes were significantly associated with the risk of SSc in a case-control study on 100 SSc patients and 100 healthy controls.

Discussion
Vitamin D is a pleiotropic molecule that became the subject of intense scrutiny in the last decades because of its novel and putative activities in previously unexpected fields of human physiology and disease. A hot topic is the possible implication of vitamin D in the pathogenesis of autoimmune diseases [57,58]; indeed, vitamin D has a wide and well-characterized activity on the immune system in vitro, the real relevance of which, in vivo, is highly debated. In the present paper, we reviewed the current literature about vitamin D and SSc.
According to our review, pre-clinical data would strongly support the potential use of vitamin D and its non-calcemic analogs in the treatment of fibrosis in SSc. Indeed, different compounds have been used in the past, both in vitro and in murine models, showing an anti-fibrotic effect. This activity is VDR-related and is associated with the suppression of fibrogenic pathways and the modulation of the immune system. However, these findings are not supported by in vivo data, since there is a lack of trials specifically assessing the impact of vitamin D supplementation on SSc-related endpoints. Vitamin D use for the management of autoimmune diseases has been explored in the past, particularly in the context of inflammatory arthritis and systemic lupus erythematosus, with conflicting results [59][60][61]. More generally, the results obtained in vivo are far less promising than the ones suggested by in vitro or murine models. Indeed, differences in the concentration and the type of compounds used may account for many of these discrepancies; moreover, we should consider that in vitro models are very simple and far from being representative of the high complexity of a biological system. Taking all of these considerations into account, although the current body of evidence supports the need for clinical trials assessing the effectiveness of vitamin D supplementation on SSc, the chance of demonstrating real effectiveness is not high.
What is instead well described in vivo is the very high proportion of SSc patients showing inappropriately low levels of vitamin D. Hypovitaminosis D is a common issue in rheumatic patients [62][63][64]; although vitamin D deficiency is highly prevalent in the general population, rheumatic patients are at even higher risk, for several reasons: the chronic use of drugs affecting vitamin D metabolism, a reduced sun exposure, and inappropriate food intake or malabsorption. Malabsorption might also affect the metabolism of other fat soluble vitamins, such as vitamin A, K and E, although, to the best of our knowledge, no data are available in the literature in the specific context of SSc patients. However, it must be noted that the first step of vitamin D metabolism takes place in the skin, which is almost invariably involved along the course of SSc. The fibrotic changes observed in SSc patients, along with reduced sun exposure, may justify the increased risk of hypovitaminosis observed in the disease; in support of this hypothesis is the fact that some authors reported an inverse correlation between skin involvement and vitamin D levels [42,51]. Moreover, lower vitamin D levels have been observed in patients showing anti-topoisomerase positivity, which is generally associated with the diffuse variant of SSc. It should be acknowledged, however, that these observations have not been confirmed by other authors and would be better investigated in appropriately designed cohort studies.
Certainly, the huge prevalence of hypovitaminosis D puts SSc patients at a particularly high risk for osteoporosis; it is well known that osteoporosis is a common comorbidity of rheumatic conditions [65,66]. Many different factors contribute to bone loss in rheumatic conditions: first of all, chronic inflammation has an impact on the pathways involved in the regulation of the physiological bone turnover, but also immobility, vitamin D de-ficiency, and the chronic use of drugs such as glucocorticoids, with a detrimental effect on bone health have a major role in the pathogenesis of osteoporosis. Looking at SSc patients in particular, it should be recognized that osteoporosis is quite common, with an estimated prevalence ranging from 6.7 to 51.1% [67]; such a high prevalence accounts for the increased risk of osteoporotic fractures, which has been described in SSc [68]. In a Taiwanese cohort, out of 1712 SSc patients, 54 patients developed vertebral fractures, 17 developed hip fractures, and seven developed radius fractures along a median followup of approximately five years. The incidence rate ratios were increased in comparison to a group of controls; older age, female gender, using daily prednisolone equivalent to >7.5 mg, and bowel dysmotility treated with intravenous metoclopramide were all risk factors for osteoporotic fractures [69]. These findings reinforce the need for a systematic assessment of bone health in SSc patients with the implementation of all those interventions required to prevent bone loss and osteoporotic fractures, among which are the correction of vitamin D status. Whether SSc patients may require a specific vitamin D supplementation regimen has not been evaluated before. It has been previously reported that patients with autoimmune/inflammatory conditions may show an impairment of the vitamin D/PTH axis possibly related to the chronic inflammatory state [58,70]; this aspect and the possibly defective cutaneous activation of cholecalciferol suggest that SSc patients may need a higher dosage of vitamin D to correct cholecalciferol deficiency. This is still an open issue that should be elucidated with appropriately designed clinical trials.
We also examined data about the potential association of vitamin D levels with specific disease domains; this is probably the most controversial aspect, with the highest heterogeneity in the current literature. Data suggest that vitamin D may be protective against the development of digital ulcers and that patients with decreasing vitamin D concentrations over time are at higher risk for this complication, thus low vitamin D levels seem to play a role in risk factors rather than being a consequence of SSc disease activity [53]. This is possibly related to a direct beneficial effect of vitamin D on microcirculation, which has been demonstrated previously in healthy subjects [71,72]. The data on lung involvement are somehow conflicting; for example, Trombetta et al. reported an association between lower vitamin D levels and severe lung involvement at the chest CT scan, while vitamin D levels did not correlate with DLCO, as conversely reported by Rios-Fernandez and Groseanu, with weak associations. Vitamin D has already been associated with lung fibrosis in other clinical settings, and this led to the postulation of a potential role for this hormone in the management of interstitial lung diseases [73]. To further support this hypothesis, vitamin D was reported to mitigate the development of lung fibrosis in a well-described model of idiopathic pulmonary fibrosis and interstitial lung disease: bleomycin-induced lung fibrosis [74]. Moreover, vitamin D deficiency exacerbates the development of lung fibrosis in this murine model through the overactivation of TGF-β/Smad signalling, the same fibrogenic pathway which is suppressed by vitamin D activity in dermal fibroblasts of SSc patients [75]. Taking these findings together, low vitamin D levels might represent a potential risk factor for ILD development. Once more, this preclinical observation may suggest a beneficial effect of cholecalciferol supplementation in SSc patients which goes beyond the skeletal effect of this hormone, although specific interventional studies should assess this topic.

Conclusions
While in vitro data suggest that vitamin D may play an antifibrotic role which may be promising in the management of SSc, clinical data confirming this finding in vivo are currently lacking. However, vitamin D deficiency is particularly common in SSc patients, and its status should be carefully assessed and corrected in order to reduce the risk of osteoporosis and fractures, which are high among SSc patients.