Vitamin D, Autoimmune Disease and Rheumatoid Arthritis

Vitamin D Status and RA Disease Risk and Progress

In a recent meta-analysis and systematic review, Lin J et al. analysed 24 studies published prior to May 2015 whose focus was the relationship between serum 25-OHD3 and clinical/laboratory indices of RA disease activity. Overall, they reported an inverse relationship between serum 25-OHD3 and RA disease activity [117]. However, they also identified important differences between patient subgroups, including a stronger inverse relationship between RA disease activity and serum 25-OHD3 in studies from developing countries, and in low-latitude climates. Since the publication of this meta-analysis, further studies have been conducted. Therefore, to establish a clearer picture of whether vitamin D levels are indeed significantly lower in RA, and are linked to disease activity, all studies of vitamin D levels in RA patients currently listed on Pubmed are summarised in Table 1. The search terms “vitamin D levels” and “rheumatoid arthritis” were used, and no restrictions on study date were applied. In addition, studies included in the meta-analysis (referenced above) were also included. The collected studies listed in Table 1 underline the significant heterogeneity between studies and their findings, making it difficult to determine a clear role for vitamin D deficiency in the onset, progression and/or severity of RA. Studies to date have been conducted in over 20 countries, which invariably means that there will be confounding differences in environmental factors including sunlight exposure and diet, and genetic factors. Moreover, the overwhelming majority of the reported studies were observational or cross-sectional by design, and as such can only report on the association between RA disease and vitamin D, rather than a causal role.

RA is a heterogeneous disease, yet few studies have considered analysing RA subgroups based on antibody status, disease severity or duration. Where subgroups have been defined, this is often based on disease activity score 28 (DAS-28); a composite score that captures different subjective and objective measures of disease activity, hence the potential for skewing of results by one component. One study that did address this reported no correlation between vitamin D levels and DAS-28, with or without the inclusion of the patient global visual analogue scale score (Patient global VAS)—a scale for evaluating the patient’s overall perception of their RA activity. However, patient global VAS on its own did correlate with serum vitamin D levels [118]. In 2015, Cooles et al. reported data showing the relationship between serum 25-OHD3 levels and various clinical parameters in early RA (median symptom duration 12 weeks, range 4–104 weeks) [119]. They found no clear association between 25-OHD3 and early RA, but in early osteoarthritis (OA) 25-OHD3 was inversely associated with global health visual analogue scale scores (GH-VAS). This suggests that OA, which commonly co-exists with RA, may act as a confounder in interpreting the relationship between vitamin D and RA. These observations are intriguing, and need to be further explored to determine the role, if any, for vitamin D in pain and fatigue associated with RA.

Relatively few studies have assessed the impact of RA treatment regimens on the apparent inverse correlation between serum 25-OHD3 and RA disease activity [120–122]. Treatment for RA is aimed at reducing disease activity, and therefore comparing serum 25-OHD3 levels with measures of disease activity after the initiation of treatment could mask prior effects of vitamin D deficiency on treatment naïve disease. Since the 1990s, an early and aggressive approach to the management of new RA has been widely used in order to maximise chances of inducing remission [123]. Today, this strategy is fully integrated in clinical practice, but its application in the context of low vitamin D, and vitamin D replacement, for treatment naive, newly diagnosed RA patients, is still unclear.

As well as clinical and biochemical differences in subgroups of RA, there are also likely genetic differences which may influence the relative importance of vitamin D in the immunopathogenesis of disease. For example, Dennis et al. identified four distinct synovial tissue gene expression profiles in a cohort of RA patients [124]. Although some of these differences in gene expression may be related to the patient’s disease duration or treatment regimen, it seems unlikely that this sufficiently explains all the observed differences, and that distinctly different gene signatures may indeed characterise different subsets of RA. Accordingly, it seems plausible that vitamin D deficiency may play slightly different roles in the aetiopathogenesis and progression of disease in different groups of patients. Ultimately, the role of vitamin D in RA appears far too complex to be understood through simple observational methods.

To date, studies of RA disease and vitamin D have focused on the link between RA and serum levels of the main circulating form of vitamin D, 25-OHD3. However, 25OHD3 is an inactive precursor for 1,25-(OH)₂D3 and therefore has limited functional relevance for immunomodulation. Consequently, there has been a revival of interest in the role of other vitamin D metabolites both in serum and disease-affected synovial fluid as potentially more informative markers for vitamin D function in RA. Research on this subject began more than 25 years ago with seminal studies of vitamin D metabolism in RA tissues [125–127]. These reports indicated that concentrations of 25-OHD3 were significantly lower in synovial fluid relative to paired patient blood, but also revealed significant capacity for the generation of 1,25-(OH)₂D3 by macrophages isolated from synovial fluid [127]. In more recent studies, we have measured multiple vitamin D metabolites—the vitamin D ‘metabolome’ in paired serum and synovial fluid from patients with established RA, reactive arthritis and healthy controls using the current gold standard for vitamin D analysis, liquid chromatography–tandem mass spectrometry (LC–MS/MS) [128]. These studies showed that for markers such as swollen joint count (SJC), synovial fluid levels of vitamin D metabolites correlated better with RA disease activity than their circulating serum counterparts [128].

Another area of contention for vitamin D and RA concerns the definition of vitamin D deficiency itself (Table 1). In the UK, the National Institute of Clinical Excellence (NICE) recommends diagnosis of vitamin D deficiency when serum 25-OHD3 is < 30 ng/mL, and states that for some people 30-50 ng/mL may be insufficient, citing recommendations from the national osteoporosis society guidelines [129, 130]. Hence, there appears to be an ambiguity around whether or not some patients need more vitamin D than others. Current NICE guidance does not define what constitutes adequate vitamin D status in patients of different ages, sex, ethnicities or disease states; for example, patients at risk of RA vs patients with established RA, or in other inflammatory diseases. To date, few studies have attempted to define what adequate supplementation means in RA. A cohort study published in 2012 observed that even supplementation 800-880 IU/day did not achieve adequate repletion of vitamin D (defined as > 20 ng/mL) in 27.7% of RA patients who were vitamin D-deficient, although duration of therapy was not reported [131]. This suggests that different levels of vitamin D replacement may be required in different RA patients, depending on pre-supplementation of vitamin D levels, sunlight exposure and skin colour (darker skin absorbs less UV light to make vitamin D). Failure to adequately replete RA patients could also be related to poor treatment compliance or inadequate duration of supplementation. Importantly, inadequate repletion was related to higher HAQ scores for RA patients, implying that inadequate improvement of vitamin D status following supplementation had poorer outcomes [131]. Alternatively higher disease activity may simply be associated with less time spent outdoors, indirectly impacting sunlight exposure and skin synthesis of vitamin D. Further RCTs are needed to more clearly define the optimal levels of vitamin D for patients with RA, and how to achieve and maintain these levels. The next section of the review describes the reported supplementation trials for vitamin D and RA.

Vitamin D Supplementation Trials in RA

Ultimately, defining vitamin D deficiency in the context of RA is of clinical interest only if replacing vitamin D in RA patients who are deficient is likely to improve disease symptoms, or even prevent the onset of RA in those at risk. To date, vitamin D supplementation trials for RA have varied appreciably in terms of patient numbers, characteristics, disease duration and severity, concomitant medication regimen, type of/duration of supplementation regimen, number of outcomes and period over which these outcomes were measured (Table 2). A recent meta-analysis identified 9 RCTs of vitamin D supplementation for ≥ 3 months in rheumatic diseases, including 5 studies of RA patients [132]. In RA, there was a decrease in the rate of disease flare, VAS and DAS-28 with vitamin D supplementation; however, all failed to reach statistical significance. Similar findings have also been reported in previous meta-analyses on this subject [133]. Conversely, a meta-analysis conducted in 2012 found that all but one of the 11 studies included in the meta-analysis suggested low vitamin D intake was linked with both increased risk of RA and greater disease activity [134]; however, the studies included in that analysis were cohort/association by design, and not RCTs. Challenges associated with identifying whether vitamin D supplementation has a beneficial effect in RCTs to date include inter-study heterogeneity and relatively small sample numbers for a meta-analysis, with only 5 RCTs included, thus emphasising the need for larger RCTs in different subsets of RA patients to fully elucidate the role, if any, for vitamin D supplementation in the management of RA. In addition, there may be differences in vitamin D-binding protein levels, and other genetic variants, which influence the efficacy of vitamin D supplementation [135]. Vitamin D supplementation in low/moderate doses is not thought to be harmful to patients, has wider health benefits, is relatively inexpensive and has fewer side effects/interactions compared with many other commonly used treatments for RA, such as non-steroidal anti-inflammatory drugs (NSAIDs), or conventional synthetic or biological disease-modifying anti-rheumatic drugs (DMARDs). Evidence is also emerging that vitamin D may augment certain therapies in RA. In one in vitro study, vitamin D 1,25-(OH)₂D3 was shown to act synergistically with the biologic drug abatacept to inhibit T cell activation driven by anti-CD3 cross-linking, and promote a pro-regulatory CD28 phenotype [136]. The potential for enhancing the effects of biologics with simple, low-risk addition of 1,25-(OH)₂D3 is interesting, and further work is required to validate this initial in vitro finding.

Cellular Targets for Vitamin D in RA

The pathogenesis of RA involves both innate and adaptive immune activities. Adaptive CD4+ T cells are critical in the pathogenesis of RA. For example, T cells are a source of RANKL, leading to osteoclast activation and subsequent joint destruction in RA [137]. However, antigen-presenting cells (APCs) such as DC also contribute to RA by providing the necessary co-stimulatory signals required for CD4+ T cell activation [138, 139]. Not only do APCs activate T cell proliferation, but they also influence T cell phenotype (Th1/Th2/Th17/Treg) and subsequent cytokine profile, and Th1/17 are both known to be relevant to the pathogenesis of RA [140, 141]. With this in mind, it is clear that the immunomodulatory activities of 1,25-(OH)₂D3 described earlier in the review (see Fig. 1) have the potential to influence both the innate and adaptive immune cell types that contribute to the dysregulated immunity associated with RA. In a murine model of RA, tolerogenic DCs (tolDCs) were observed to reduce severity and progression of RA disease by increasing IL-10-producing T cell numbers and reducing Th17 cell counts [142]. It is therefore interesting to note that 1,25-(OH)₂D3 can induce tolDC [143], and tolDCs, generated ex vivo using 1,25-(OH)₂D3 have been proposed as a potential strategy for RA therapy [144]. In this instance, 1,25-(OH)₂D3 was used in combination with the glucocorticoid dexamethasone which is known to promote a tolDC phenotype [58].

In addition to enhanced activity of Th1 and Th17 cells, RA disease is also characterised by reduced Treg activity, including decreased Treg numbers [145], a reduction in Treg: Th1/Th17 ratio [146], altered Treg function [147] and differences in Treg number and function in the peripheral blood compared with the synovium [148]. However, in some studies, numbers of circulating Treg in RA were similar to those found in healthy controls [149] and osteoarthritis patients [150]. Tregs are likely to be a key target for vitamin D in RA. In animal models of experimental autoimmune encephalitis (the most widely used mouse model of MS), IBD and T1D, 1,25-(OH)₂D3 promotes a Treg phenotype and augment IL-10 production, thus inhibiting Th17 responses and ameliorating disease [54]. In studies using human Tregs, the 1,25-(OH)₂D3 analogue TX527 skewed the Treg cell phenotype in favour of migration to sites of inflammation [151], whilst also promoting a stable Treg phenotype [152]. The growing pool of ex vivo and in vitro evidence linking vitamin D and Treg function now requires replication in vivo, particularly in diseases such as RA.

To date, most studies of the T cell actions of 1,25-(OH)₂D3 have been based on the analysis of mixed populations of circulating T cells from healthy donors. However, in recent studies, we have shown that T cells from synovial fluid of RA patients’ inflamed joints are relatively insensitive to 1,25-(OH)₂D3, despite expressing the VDR machinery required for 1,25-(OH)₂D3 signalling [153]. This is due, in part, to decreased 1,25-(OH)₂D3 responsiveness in the memory T cells that predominate in RA synovial fluid, but also involves tissue-specific effects, with synovial fluid memory T cells showing decreased responses to 1,25-(OH)₂D3 relative to peripheral blood memory T cells [153]. Based on these observations, we have proposed that immunomodulatory responses to vitamin D at tissue sites of inflammation are impaired by target cell insensitivity to 1,25-(OH)₂D3. If this is the case, then conventional analysis of the effects of vitamin D using circulating blood immune cells can only provide a limited picture of immunomodulation by vitamin D in diseases such as RA. Likewise, to overcome the vitamin D-insensitivity observed in RA patient, inflamed joints may require alternative strategies. This could include the use of higher doses of vitamin D supplements to enhance localised tissue levels of 1,25-(OH)₂D3, or the use of vitamin D as an adjunct to other RA therapies. With respect to the latter, we have recently shown that 1,25-(OH)₂D3 can more potently inhibit T cell activation when used in combination with the CD28 co-stimulatory blocker abatacept [154].

Beyond the actions of innate and adaptive immune cells, synovial fibroblasts (synoviocytes) also play an important role in the pathogenesis of RA. In studies using the immortalised synoviocyte cell line, MH7A, 1,25-(OH)₂D3 has been shown to promote synoviocyte apoptosis, which might protect against RA, but only when cells were treated with both tumor necrosis factor α (TNFα) and 1,25-(OH)₂D3 [155]. These observations suggest that TNFα is required for 1,25-(OH)₂D3 to have anti-inflammatory effects in RA, which is a paradoxical observation given the use of TNFα inhibitors as biological treatment in RA. Conversely, other studies have reported synergistic effects of TNFα inhibition and 1,25-(OH)₂D3 in suppressing Th17-mediated inflammation [156], suggesting a complex interaction between TNFα and 1,25-(OH)₂D3. Other studies using MH7A cells have shown synergistic effects of interleukin 1β (IL-1β) and 1,25-(OH)₂D3 in suppressing the production of IL-6 and TNFβ levels, and Th17-inducing cytokines (IL-1β, IL-6 and IL-23) synergistically enhanced the pro-regulatory effect of 1,25-(OH)₂D3 on T cell phenotype [136], further emphasising important interactions between 1,25-(OH)₂D3 and pro-inflammatory cytokines in RA [157]. Collectively, these observations suggest that the beneficial effects of 1,25-(OH)₂D3 are most potent when the threshold for activation of the immune response is breached, leading to concomitant production of inflammatory cytokines. Therefore, in the setting of inflammation, vitamin D appears to function as a negative-feedback regulator, attenuating the inflammatory immune responses. Vitamin D may also influence the synovial microenvironment by modulating factors that influence joint bone and cartilage damage. In studies using synoviocytes and articular chondrocytes from RA patients, 1,25-(OH)₂D3 was shown to regulate matric metalloproteinases and prostaglandins, but only in the presence of IL-1β [158], suggesting, as outlined earlier, that 1,25-(OH)₂D3 is only effective as a regulator of synoviocytes in the setting of RA disease inflammation.

Below is the link to the electronic supplementary material.

Conflict of interest

Stephanie R. Harrison, Danyang Li, Louisa E. Jefery, Karim Raza, and Martin Hewison declare that they have no conflict of interest.