Vitamin D levels in Indian systemic lupus erythematosus patients: association with disease activity index and interferon alpha

Introduction Low levels of vitamin D have been associated with several autoimmune disorders including multiple sclerosis, rheumatoid arthritis, type 1 diabetes and systemic lupus erythematosus (SLE). The major source of vitamin D is sunlight but exposure of SLE patients to UV rays has been shown to exacerbate disease pathology. Studies in various populations have shown an association between low vitamin D levels and higher SLE disease activity. Methods We enrolled 129 patients who fulfilled American College of Rheumatology criteria in the study. There were 79 treatment-naïve cases and 50 patients who were under treatment for underlying SLE. There were 100 healthy subjects from similar geographical areas included as controls. Plasma 25-OH vitamin D3 and interferon (IFN)-α levels were quantified by enzyme-linked immunosorbent assay (ELISA). The gene expression level of IFN-α was determined by quantitative real-time reverse transcriptase polymerase chain reaction (RT-PCR). Results Plasma 25-OH vitamin D3 significantly correlated in an inverse manner with systemic lupus erythematosus disease activity index (SLEDAI) scores (P <0.0001, r = -0.42), anti-dsDNA (P <0.0001, r = -0.39), plasma IFN-α (P <0.0001, r = -0.43) and levels of IFN-α gene expression (P = 0.0009, r = -0.45). Further, plasma levels of IFN-α positively correlated with gene expression of IFN-α (P <0.0001, r = 0.84). Treatment-naïve SLE patients displayed significantly higher plasma levels of IFN-α compared to patients under treatment (P <0.001) and controls (P <0.001). Conclusions These results suggest an important role of vitamin D in regulating disease activity in SLE patients and the need to supplement vitamin D in their treatment.


Introduction
Systemic lupus erythematosus (SLE) is an autoimmune disorder which appears in a group of individuals and which is related to several factors, including environmental and host genetics that contribute to the development of the disease [1]. Patients with SLE develop an immune response against numerous, mostly intracellular self-antigens. This results in formation of immune complexes that get deposited in vascular beds in most organs of the body. Immune complex deposition causes local inflammation and tissue damage that probably amplify the autoimmune response [2]. This has serious consequences on the outcome of the disease.
The importance of vitamin D in various autoimmune disorders has been reported. Vitamin D deficiency has been associated with multiple sclerosis (MS), rheumatoid arthritis (RA), type 1 diabetes mellitus, inflammatory bowel disease (IBD), mixed connective tissue disease, autoimmune thyroid disease, scleroderma and SLE [3][4][5]. Vitamin D supplementation improves disease outcome in various animal models of MS [6], RA [7], type 1 diabetes mellitus [8], IBD [9], autoimmune encephalomyelitis [10] and SLE [11]. The role of vitamin D in murine models of SLE has been investigated to a limited degree. Administration of vitamin D and its synthetic analogs to murine models has resulted in improved dermatological manifestations [11], reduced proteinuria [12] and increased survival [12,13]. An earlier report highlighted vitamin D 3 insufficiency in two-thirds, and deficiency (<10 ng/ml) in approximately one-fifth of SLE patients [14]. In addition, serum vitamin D 3 (25-OH) levels have been found to correlate inversely with SLE disease activity index (SLEDAI) scores [15][16][17].
The major source of vitamin D is the conversion of 7dehydrocholesterol to previtamin D 3 in the skin when exposed to solar ultraviolet radiation [18]. Previtamin D 3 then gets converted to vitamin D 3 (cholecalciferol) through a heat-mediated process in the skin [18]. A lesser amount of vitamin D 3 (25-OH) is obtained from foods that supply less than 20% of the body's requirements. Vitamin D 3 undergoes two hydroxylations to achieve its functional form. The first hydroxylation occurs in the liver resulting in 25-hydroxyvitamin D (25(OH)D 3 ) or calcidiol, which is normally quantified for evaluating vitamin D status, and the second hydroxylation takes place in the kidney to its active form 1,25-dihydroxyvitamin D3 (1, 25(OH) 2 D) [18]. In addition to the liver and kidney, hydroxylation of vitamin D 3 also occurs in the lymph nodes and skin [19].
Several studies worldwide have investigated the role of vitamin D 3 in the pathogenesis of SLE. However, to date, there have been no reports from an Indian population. Although the prevalence of SLE in India is rare (3 per 100,000) [20], the survival rates of these patients (5-year: 70%; 10-year: 50%) are low compared to Western cohorts [21,22]. Interestingly, vitamin D 3 insufficiency or deficiency appears to be widespread in the Indian subcontinent [23], which makes it important to analyze its role in the background of SLE from an Indian cohort. We have addressed this issue in a tertiary-care, hospitalbased, case-control study, to assess the role of vitamin D 3 in SLE in a cohort from eastern India.

Subjects
The patients recruited for the study were all inpatients, admitted to the Department of Medicine, under the Clinical Immunology and Rheumatology unit of SCB Medical College, Cuttack, Odisha. As described earlier [24][25][26], diagnosis of SLE was based on the revised American College of Rheumatology (ACR) classification criteria [27]. After a detailed clinical examination and laboratory investigation, the clinical manifestations were categorized. The clinical profiles of 129 SLE patients are summarized in Table 1. Since, SLE affects women primarily [28], 50 agematched healthy females (medical students: HCA) and 50 healthy subjects from similar geographical areas (HCB) were included as healthy controls (HC). None of the controls reported any history of autoimmune disorder. About 5 ml blood in EDTA was collected from each participant. The study was approved by the institutional ethics committee of SCB Medical College, Cuttack. Informed consent was obtained from each patient and healthy control.

25-OH vitamin D quantification in plasma
The plasma levels of 25-OH Vitamin D were quantified by enzyme-linked immunosorbent assay (ELISA) kit (CPC, Euroimmun, Lübeck, Germany) according to the manufacturer's instructions. Vitamin D deficiency was defined as plasma levels of 25-OH vitamin D <10 ng/ml and insufficiency as 10 to 30 ng/ml [18].

Quantification of plasma interferon alpha
Plasma levels of interferon (IFN)-α were measured by ELISA kit (Bender MedSystems Inc., Burlingame, CA, USA) according to the manufacturer's protocol.

RNA extraction and reverse transcription
According to the manufacturer's instructions, total RNA was isolated from 250 μl of whole blood by TRIzol LS reagent (Invitrogen, Carlsbad, CA, USA). RNA concentration was determined by spectrophotometry using an Implen NanoPhotometer (Implen, Munich, Germany). To remove any traces of genomic DNA, 1 μg of total RNA was then treated with 2U DNase (Sigma-Aldrich, St Louis, MO, USA) for 30 min at 37°C. DNase-treated RNA was reverse transcribed with a hexamer primer using a First Strand cDNA Synthesis kit (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer's instructions. Once the cDNA was synthesized, its fidelity was tested by PCR and stored at −70°C.

Real-time PCR assay
Real-time PCR assay of IFN-α was carried out as described earlier [29]. Briefly, reactions were set up in a total volume of 20 μl using 2 μl of cDNA, 10 μl of MESA GREEN qPCR MasterMix Plus (Eurogentec, Seraing, Belgium) and 10 picomole each of gene-specific primer (IFN-α (sense: 5′-TTCCTCCTGYYTGAWGGACAGA-3; antisense: 5′-GATCTCATGATTTCTGCTCTGACA-3′), glyceraldehyde-3 phosphate dehydrogenase (G3PDH) was taken as control (sense: 5′-GGTATCGTGGAAGGACT CATGAC-3′; antisense: 5′-ATGCCAGTGAGCTTCCC GTTCAGC-3′)) and performed in the MJ Research DNA Engine Opticon Real-Time Thermal Cycler (MJ Research, Waltham, MA, USA).The cycling conditions were: 95°C for 4 min; 35 cycles of 95°C for 30 s, 55°C for 30 s and 72°C for 30 s with a single fluorescence measurement; a final elongation step was carried out at 72°C for 10 min. Specificity of the PCR products was confirmed by analysis of the dissociation curve. The melting curve program consisted of temperatures between 55 and 95°C with a heating rate of 0.1°C/s and a continuous fluorescence measurement.
Additionally, the amplicons' expected size and the absence of nonspecific products were confirmed by analysis of the real-time PCR products in 1% agarose gel in 1 × TBE, stained with ethidium bromide and visualized under ultraviolet light (expected product size of IFN-α: 375 bp and G3PDH: 187 bp). IFN-α gene expression in each sample was calculated by the 2 -ΔCt method (ΔCt = Ct of IFN-α -Ct of GAPDH) [30].

Statistical analysis
All statistical analysis was performed by GraphPad prism 5.01 (GraphPad Software, San Diego, CA, USA). Distribution of plasma 25-OH vitamin D 3 and IFN-α in treatment-naïve SLE patients, controls and treated patients were assessed by D' Agostino-Pearson omnibus normality test. Based on the results of the normality test, the association of 25-OH vitamin D 3 and IFN-α with clinical disease was analyzed by analysis of variance (ANOVA) or Kruskal-Wallis test followed by an appropriate post test. Correlation of 25-OH vitamin D 3 with double-stranded (ds)DNA, SLEDAI scores and IFN-α was analyzed by Spearman's correlation test. Further correlation of IFN-α gene expression with plasma IFN-α and 25-OH vitamin D 3 levels was analyzed by Spearman's correlation test. A P value <0.05 was considered as significant.

Clinical characteristics of SLE patients
One hundred and twenty-nine patients were enrolled in the current study. Baseline characteristics are shown in Table 1. There were 125 (97%) females and 4 (3%) males with a mean age (standard deviation) of 28.14 (8.43) years. The mean duration of disease (standard deviation) was 2.90 years (2.66). Out of the 129 SLE patients, 50 patients included in the study were already on treatment for SLE and were also receiving supplements of oral calcium and vitamin D 3 at the time of blood collection ( Table 1). The other 79 patients were treatment-naive cases, undiagnosed earlier and the details of the treatment received for their complaints before hospitalization were not known since the patients had not maintained any records. The clinical profiles of patients were as follows: photosensitivity rash (26%), malar rash (57%), discoid rash (11%), oral ulcer (59%), arthritis (60%), neuropsychiatric disease (9%), myocarditis (2%), serositis (5%), nephritis (37%) and vasculitis (13%) ( Table 1).

Plasma 25-OH vitamin D 3 levels in SLE patients and healthy controls
Plasma levels of 25-OH vitamin D 3 in SLE patients and healthy controls were quantified by ELISA and the results are shown in Figure 1. Patients under vitamin D 3 supplementation (treated cases) displayed significantly higher levels of 25-OH vitamin D 3 compared to treatment-naïve patients (P <0.001) and healthy controls (P <0.001). The levels of plasma 25-OH vitamin D 3 in treatment-naïve SLE patients and healthy controls were comparable.
Vitamin D 3 levels negatively correlated with SLEDAI scores and anti-dsDNA Analysis of data in SLE patients revealed a significant negative correlation between plasma 25-OH vitamin D 3 levels with SLEDAI scores (P <0.0001, r = −0.42) (Figure 2A) and anti-dsDNA (P <0.0001, r = −0.39) ( Figure 2B). Further, SLE patients were categorized into two groups: treatmentnaïve and those under treatment. As shown in Figure 2C and D, irrespective of treatment status, the plasma levels of 25-OH vitamin D 3 negatively correlated with SLEDAI scores. In addition, a further analysis of the relationship between 25-OH vitamin D 3 and modified SLEDAI scores (eliminating the anti-dsDNA positive score of 2 from SLEDAI), revealed identical results in both the groups ( Figures 2E and F). These findings indicate a significant association between 25-OH vitamin D 3 and disease activity in SLE.

Correlation between 25-OH vitamin D 3 and IFN-α
The role of IFN-α in SLE has been clearly documented and its significant correlation with SLEDAI scores has been demonstrated in patients from different populations [31,32]. As shown in Figure 3A, a significant negative correlation was observed between 25-OH vitamin D 3 levels and plasma IFN-α (P <0.0001, r = −0.43). Treatment-naïve patients displayed higher levels of plasma IFN-α compared to SLE patients on treatment (P <0.001) and healthy controls (P <0.001) ( Figure 3B) suggesting an important role of IFN-α in modulating disease activity.

Correlation of IFN-α gene expression with plasma IFN-α and 25-OH vitamin D 3 levels
To validate the robustness of IFN-α data, considering that it is an evanescent cytokine, we quantified IFN-α gene expression by RT-PCR in SLE patients (n = 49) and correlated the values with plasma levels of IFN-α and 25-OH vitamin D 3 . As shown in Figure 4A, a strong positive correlation was observed between IFN-α plasma levels and its gene expression (P <0.0001, r = 84). In addition, IFN-α gene expression negatively correlated with plasma 25-OH vitamin D 3 (P = 0.0009, r = −45) ( Figure 4B).

Association of plasma IFN-α with SLE disease severity
Role of IFN-α in the pathogenesis of SLE is an important issue that is being investigated [32]. We analyzed the association of IFN-α with disease severity. As shown in Figure 5A, plasma levels of IFN-α positively correlated with SLEDAI scores (r = 0.26, P = 0.002) and patients with severe phenotype displayed significantly higher levels of IFN-α compared to those with mild disease manifestations (P = 0.01) ( Figure 5B). However, duration of disease did not correlate with plasma IFN-α levels (data not shown). We also observed a significant inverse correlation between plasma 25-OH vitamin D 3 and IFNα levels. This correlation held true while analyzing the association between 25-OH vitamin D 3 levels and IFN-α gene expression.

Discussion
The role of vitamin D 3 in autoimmune disorders has been the subject of several studies with regard to its importance as an immune regulator [33]. This is the first study from India to demonstrate an association between vitamin D 3 and SLE, highlighting its significant inverse correlation with SLEDAI scores, anti-dsDNA and IFN-α. These are markers of disease activity and IFN-α is closely associated with disease pathogenesis. Low levels of vitamin D 3 in SLE patients have been reported compared to healthy controls in different populations [34]. Interestingly, mean plasma levels of 25-OH vitamin D3 were not significantly different among treatment-naïve SLE cases (11.61 ng/ml), healthy medical students (9.55 ng/ml) and other healthy controls from same locality (13.36 ng/ml). Vitamin D 3 insufficiency has been   reported to be widely prevalent in the Indian subcontinent irrespective of the social class [23]. Two groups of healthy controls were analyzed, which included medical students (HCA), who led a lifestyle marked by poor exposure to sunlight and irregular dietary habits, and a group of healthy subjects from the same locality (HCB). Interestingly, 63% of healthy medical students were deficient and 37% were insufficient of vitamin D 3 . Furthermore, 94% of the other groups of healthy controls were either deficient or insufficient of vitamin D 3 . This was an important observation considering India being a tropical country with lots of sunshine. However, the facts were contrary and several hypotheses have been discussed to explain the discrepancy. Higher melanin concentration in the skin [35], current lifestyle changes, avoidance of sunlight and poor food habits are some of the causes attributed to the widespread prevalence of low vitamin D 3 among Indians. Low vitamin D 3 may not be cause for development of SLE but persons with low serum levels are likely to suffer from severe disease. The current cross-sectional study does not address the issue of cause and effect relationship between vitamin D 3 and SLE.
There are several interesting observations in the current study that points to an important role for vitamin D 3 in disease modulation. One of them being a significant inverse correlation between vitamin D 3 and SLEDAI scores and the other association is between vitamin D 3 and anti-dsDNA. Association between plasma vitamin D 3 and SLEDAI scores has not been uniform across observations: several studies have reported a negative correlation [15][16][17], while others have found none [36][37][38][39].
One of the important functions of vitamin D 3 is maintenance of homeostasis of B cells [40]. Low levels of vitamin D 3 contribute to hyperactivity of B cells and enhanced production of autoantibodies [41]. Furthermore,  vitamin D 3 is known to modulate various immunological pathways [33] and thus could have a defining role in the development, progression and pathogenesis of SLE. Vitamin D 3 also inhibits differentiation of dendritic cells (DCs) and T-helper cells (CD4+) [42], enhances T regulatory cell proliferation and suppresses release of inflammatory mediators [43], which collectively help in control of autoimmune disorders.
In recent years, the role of interferon in the pathogenesis of lupus has been widely investigated. Higher levels of IFN-α were observed in our SLE patients compared to healthy controls, corroborating earlier observations [32,[44][45][46]. The interferon levels were significantly low in patients under treatment compared to treatmentnaïve cases, supporting its possible role in disease modulation. Furthermore, IFN-α could be a marker of disease activity and low levels in treated patients could indicate response to therapy.
Interestingly, our study revealed a strong negative correlation of vitamin D 3 with IFN-α (P <0.0001, r = −0.52). The robustness of the assay was validated by assessment of IFN-α gene expression, which corroborated with the earlier observations on the association between plasma IFN-α and vitamin D 3 . There are no reports in the literature assessing the association between IFN-α and vitamin D 3 .
In active SLE overexpression of interferon-inducible genes (IFN signature) has been reported [47]. The major source of IFN-α in SLE patients are activated DCs. Maturation/activation of DCs and production of IFN-α has been observed to be inhibited by vitamin D in in vitro studies [48,49]. A direct role for vitamin D 3 in modulating lupus activity has been demonstrated in animal models [11][12][13]. Our observations, although cross-sectional, and studies on experimental models, provide evidence for a disease-modulating role for vitamin D 3 , which could be a promising therapeutic adjunct in the treatment of SLE. In view of the limited number of drugs available for the treatment of lupus and the low cost of vitamin D 3 therapy, there is a strong case for its use routinely.

Conclusions
To conclude, vitamin D deficiency is prevalent among healthy Indians as well as among SLE patients. The significant inverse correlation of vitamin D 3 with SLEDAI scores, anti-dsDNA and IFN-α highlights its immunemodulatory role contributing to disease outcome. Although the present study indicates a necessity for vitamin D 3 supplementation in the management of SLE patients, larger randomized controlled trials would be necessary to define the daily requirement and optimum blood levels of vitamin D 3 that are effective in influencing disease outcome.

Competing interests
The authors declare that they have no competing interests.
Authors' contributions MM, SD and SSP were involved in samples collection, data management and clinical categorization of samples. AdKP carried out quantification of IFN-α by ELISA, real-time PCR, data interpretation and wrote the first draft of the manuscript. AnKP and SC quantified IFN-α gene expression and interpreted the results. RT performed all routine tests including measurement of vitamin D. RT, BR and BKD made a contribution in the design, data interpretation, work supervision and critically revising the manuscript. All authors read and approved the manuscript.