Introduction

Mounting evidence suggests vitamin D as an important immune-modulator1,2. Circulating 25OHD3 is produced in the skin upon sun-exposure, dependent on e.g. season and latitude3, and circannual variation in 25OHD is well-known in Northern latitude4. 25OHD serves as substrate for conversion into the active metabolite; 1,25(OH)2D, both in the kidneys3 and locally in macrophages, dendritic cells and lymphocytes5. Immunomodulation by sufficient levels of 1,25(OH)2D includes altered differentiation and maturation of dendritic cells, macrophages and lymphocytes5, and inhibition of pro-inflammatory cytokines6. The main-effect of vitamin D on the immune system is to balance the immune-response away from the self-aggressive Th1/Th17-response and towards the tolerogenic Th2/Th-regulatory response7,8.

In Rheumatoid Arthritis (RA), a chronic inflammatory symmetric arthritis9, low levels of 25OHD are reported to be associated with markers of disease activity in cross-sectional settings10,11. 1,25(OH)2D is rarely evaluated, but seems to be inversely associated with several markers of disease activity in cross-sectional settings12.

An aggressive aiming-at-remission treatment strategy is the golden standard in RA13, and the immune-modulating activity of vitamin D and association with disease activity in cross-sectional studies suggests potential association between vitamin D and remission in RA. To the best of knowledge, only two rather small longitudinal studies evaluate associations between serum 25OHD at time of RA diagnosis and remission during follow-up14,15. Both studies report lower odds of remission in RA-patients with low 25OHD at diagnosis. Association between 1,25(OH)2D and remission was not evaluated.

The objectives of the current study were, in 158 early diagnosed, treatment-naïve Danish RA patients, to measure serum Dtotal and 1,25(OH)2D at time of diagnosis, and to analyze for associations with remission evaluated as Disease Activity Score based on 28 joints and C-reactive Protein (DAS28-CRP) ≤ 2.6 after one year. This was done to test the pre-specified hypothesis that baseline vitamin D metabolites are associated with achieving remission in early RA.

Results

At diagnosis, median Dtotal was 53.7 nmol/l, IQR 36-67. Median 1,25(OH)2D was 95.5 pmol/l, IQR 70–119. Sixty-seven patients (42%) had Dtotal below 50 nmol/L.

Obesity (BMI > 30 kg/m2) was significantly more prevalent in the low Dtotal group (30% versus 13%, p = 0.01) as a result of weight being higher (median 79 kg versus 73 kg, p = 0.03) and 1,25(OH)2D was significantly lower (median 82 pmol/L versus 110 pmol/L, p < 0.001) No other baseline variables were significantly different between Dtotal groups. (See Table 1) One-hundred-and-forty-three of the 158 patients included at baseline had disease activity evaluated one year after diagnosis, and were eligible for evaluation of remission. (See Fig. 1).

Table 1 Baseline characteristic according to Dtotal status at diagnosis.
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Figure 1
figure 1

Patient flow-diagram for year one in the original CIMESTRA study, according to Dtotal status at diagnosis - Abbreviations: Dtotal: the sum of 25OHD2 and 25OHD3.

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Results of univariate logistic regression

Diagnosis established at winter compared to summer (OR 0.4, 95% CI (0.2; 0.9) p = 0.02), Number of Tender Joints (NTJ) (OR 0.9; 95% CI (0.88; 0.98) p = 0.01) and DAS28-CRP (OR 0.7, 95% CI (0.5; 0.9) p = 0.01) at time of diagnosis were significantly inversely associated with one-year remission, independently of vitamin D metabolite levels. This indicates that it is less likely that patients diagnosed during the winter season and in patients with high NTJ or DAS28-CRP at time of diagnosis, will achieve remission. [See appendix Table 1].

Primary analysis, evaluating Dtotal groups

At year one, 96 (61%) of the 143 patients were in remission, 45 (71%) from the group with low Dtotal at diagnosis, and 51 (64%) from the group with normal Dtotal at diagnosis. (See Table 2).

Table 2 Achieving remission according to Dtotal at diagnosis.
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No significant association was found between baseline Dtotal group and remission rates in the crude model; OR 1.4, 95% CI (0.7; 2.7) p = 0.33, whereas the fully adjusted model showed statistical significantly increased odds for one year remission; OR 2.6, 95% CI (1.1; 5.9) p = 0.03, in patients with low Dtotal at time of diagnosis compared to patients with normal Dtotal. (See Table 2) For details on the definition of crude and adjusted models, see the method section. Propensity score analysis including several other factors, potentially affecting the results, did not significantly alters the estimate; OR 2.5, 95% CI (1.1; 5.8) p = 0.03, indicating that the further included factors have only negligible influence. In the fully adjusted model, remission was further inversely associated with diagnosis established in winter; OR 0.3 95% CI (0.1; 0.7) p < 0.01) and DAS28-CRP at diagnosis; OR 0.6 95% CI (0.4; 0.9) p = 0.01 and directly associated with age at diagnosis; OR 1.04, 95% CI (1.0; 1.1) p = 0.03. Stepwise inclusion of the pre-specified variables sex, age, symptom duration, DAS28-CRP at diagnosis and season at diagnosis indicates that especially adjustment for season impacts the estimates in the fully adjusted model. (See Table 3). There was no significant interaction between Dtotal levels and season of diagnosis (p = 0.7).

Table 3 Stepwise regression, including a priori chosen variables one by one.
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Model fit was tested to evaluate which model best describes the association between vitamin D metabolites, one year remission and the chosen, potential confounding variables: A likelihood-ratio test showed that fully adjusting the model improved the model fit significantly, whereas the partially adjusted model (including only sex and age at inclusion) did not improved the model fit compared to the crude model. In addition, Akaike Information criterion (AIC) decreased and Bayesian Information Criterion (BIC) increased when including all confounding variables. As we put emphasis on AIC, this is interpreted as the fully adjusted model being the model which most adequately describes the real world, resulting in the best balance between number of included variables and precision.

To estimate whether missing data for one-year remission affects the results, best- and worse case-scenarios were evaluated. These suggested potentially consequences of missing data, as estimates became insignificant in best-case scenarios. (See Table 2).

Secondary analysis, evaluating 1,25(OH)2D tertiles

Associations between the active vitamin D metabolite and one-year remission were evaluated similar to the primary analyses for Dtotal. No difference existed in odds of remission between patients with the lowest 1,25(OH)2D tertile compared to highest, neither in the univariate model; OR 1.3, 95% CI (0.5; 3.2) p = 0.59, nor in the fully adjusted model; OR 1.8, 95% CI (0.6; 4.9) p = 0.28.

Remission was negatively associated with diagnosis established at winter; OR 0.4 95% CI (0.2; 0.9) p = 0.03, and DAS28-CRP at diagnosis; OR 0.6 95% CI (0.4; 0.9) p = 0.01. [See appendix Table 2].Applying likelihood-ratio-test, both the partially and fully adjusted model showed significantly better fit than the crude model. AIC decreased with each successive adjustment, while BIC decreased from crude to fully adjusted model.

Discussion

Main finding is that the inverse association between serum Dtotal status at diagnosis and one year remission exists only in the model adjusted for season. Although largely unexplained, being diagnosed with RA in the winter is independently associated with lower odds for remission after one year of intensive treatment, suggesting that season confounds the association between vitamin D and RA disease activity.

The immunomodulatory properties of vitamin D are exerted through local conversion of 25OHD into 1,25(OH)2D1,5. Subjects with low serum Dtotal lack substrate for the local production of 1,25(OH)2D16,17, increasing the risk of an auto-aggressive Th1/Th17 response18. We surmise that our results support the potential existence of a “low vitamin D RA-subtype”, which upon anti-inflammatory treatment and relevant vitamin D supplementation is easily treated to remission. On the contrary, subjects with normal Dtotal levels and thus sufficient substrate for local 1,25(OH)2D production, who develop RA may be representative of an immune system being more imbalanced and distressed, and thus less susceptible to achieve remission, despite relevant treatment. Alternatively, our results indicate the presence of a “Winter-diagnosed RA-subtype”. Patients diagnosed at winter will inevitably have one-year remission evaluated the following winter, where the seasonal impact on disease activity will decrease probability of remission. Though, our study-design does not allow us to speculate whether “winter-diagnosed RA” is less prone to achieve remission in the summer, as we have no half-yearly evaluations.

Our results conflict with other studies evaluating vitamin D and remission in early RA: As opposed to the current study, the comparable studies did not adjusted for season in the statistical analyses, but handled potential annual impact through study design: One study is located in Columbia, a tropical country with no seasons15, while the Italian study14 only evaluated serum 25OHD in the autumn-winter-period, to minimize the seasonal impact. However, RA is diagnosed all year round, and circannual changes in light and darkness is unavoidable in Scandinavia. We find the current study to be representative of a Scandinavian, early diagnosed RA-cohort, and further sheds light on an important contextual factor in evaluation of disease activity in early RA.

The impact of season on vitamin D levels is well-known3,4 Similarly, season impacts the incidence and course of RA19,20,21, likely owing to circadian rhythms affecting pro-inflammatory cytokines22,23. This may be explained by effects of seasonal changes in vitamin D affecting the immune system24, perhaps through seasonal impact on relevant genes, independent of vitamin D metabolism25,26.

Our study design implies that one year remission is evaluated the same season as diagnosis was established, and the impact of season on disease activity at diagnosis will be mirrored in the remission-criteria applied one year after. Thus, the probability to observe one-year remission will be lower in patients diagnosed in winter. As Dtotal levels are lower in winter, we suggest this being a plausible explanation to our main finding: season is a strong confounder in the relationship between vitamin D and disease activity in RA. As season independently predicted one-year remission, we further suggest our results to be indicative of season of diagnosis being an important contextual factor in evaluation of RA disease-course. We advocate future research to take season into account when evaluating disease activity in early RA.

The primary outcome; remission, is evaluated as DAS28; a composite of subjective and objective disease-activity markers weighting pain and inflammation. Pain may persist in DAS28-remission27, whereas 1,25(OH)2D supplementation is reported to significantly relieve pain in early RA28. As the CIMESTRA strategy included increased awareness of vitamin D intake, we assume that an eventual painful proximal myopathy owing to low vitamin D was improved during follow-up. In RA, 25OHD levels may be correlated to dietary intake29, while improvement of low serum vitamin D upon supplementation in RA is associated with improved treatment response30. Moreover, RA patients with low 25OHD and no vitamin D supplementation are less frequently in remission, compared to patients receiving relevant supplementation31. Likewise, abstaining from correction of low vitamin D levels is associated with increased recurrence-rates in some32, but not all studies33. However, the current study is not an interventional study investigating effects of vitamin D supplementation on remission. Patients were supplemented after individual physicians´ evaluation of vitamin D and calcium intake, with no knowledge of actual serum Dtotal level. Suggesting that sufficient vitamin D intake, concomitant to aggressive anti-inflammatory treatment, might partly explain the results, is only hypothesis-generating, despite plausible underlying biological associations supporting the hypotheses1. Though, data concerning which patients received supplementation is not available.

If a causal association between low vitamin D levels and high RA disease activity exists, it seems reasonable that patients with more active disease shows better treatment response upon the aggressive treatment-regimen and relevant vitamin D intake, thus explaining the higher remission-rate in the group with lower Dtotal. Furthermore, if a “low vitamin D RA subtype” exists, and low vitamin D levels are associated with higher disease activity and pain11, such patients may receive higher doses of anti-inflammatory medication, and be more likely to achieve remission, partly explaining the results.

Limitations of the current study include restrictions owing to the use of post-hoc analyses in a parental RCT, designed for other purposes: The study population is rather small, supported by several estimates having large 95% CIs. However, to the best of knowledge, our study almost doubles observations upon which estimates can be made, compared to other studies evaluating vitamin D and remission in early RA14,15. The parental CIMESTRA RCT investigated the additive effect of ciclosporine or placebo to an aggressive, treat-to-target strategy, and 50% received ciclosporine. Vitamin D metabolites evaluated in the current study were obtained prior to treatment, and were not affected by ciclosporine treatment. No adjustment for ciclosporine is done, as no significant association was found between ciclosporine and one year remission34.

Possible unmeasured residual-confounders must be considered: Relevant markers of calcium metabolism, such as calcium, phosphorous and parathyroid hormone are not evaluated. Neither, information concerning sun-exposure, the main-cause to cutaneous vitamin D production, is assessed. Besides, vitamin D levels have been reported to vary according to meat consumption35,36,37. However, we are unable to account for meat-consumption in the current study.

Another potential limitation is the dichotomization of Dtotal, applied in the current study: Cutpoint defining sufficient vitamin D levels varies greatly, depending on e.g. age, race, weight, sun-exposure and diet3. The internationally accepted cut-point defining low vitamin D is 50 nmol/l38. We acknowledge that dichotomization might lead to loss of more subtle details, and that it has been argued that higher levels of vitamin D might be necessary for better health outcomes39. However, we find that the use of a well-known cut-point, and the comparability to the few similar studies increase the external validity, interpretation and clinical use of our results.

To avoid overfitting our statistical models, we evaluated model fits by AIC and concluded that the fully adjusted model was the most realistic model. As different modelling scenarios resulted in inconsistency among results, we advocate for reproduction of our results before firm conclusions are made

We believe that our data can be applied to a Scandinavian population with newly diagnosed RA. Despite data collection at the millennium, the aggressive treat-to-target strategy is still the recommendations nowadays, increasing the external validity. Our results need to be reproduced, preferably in sufficiently powered studies with vitamin D supplementation guided by serum vitamin D measurements, alongside to aggressive treatment and adjustment for season.

In conclusion, low vitamin Dtotal at time of diagnosis in early, treatment-naïve RA is associated with increased odds of achieving remission after one year, when adjusting for relevant confounding variables. Conceivably, this finding is mainly attributable to seasonal variation per se with low vitamin D levels as a potential amplifier. In order to elucidate the relationship between vitamin D status, disease activity and season of the year, follow-up visits at 6 months interval may be useful. In the meantime, clinicians and researchers should be aware of the potential impact by season on RA disease presentation.

Methods

Patients and outcomes

One-hundred-and-sixty patients were recruited from five Danish University Clinics from October 1999 to October 2002 in the CIMESTRA-study. Eligibility criteria and treatment-strategy are described elsewhere34. One-hundred-and-fifty-eight patients had Dtotal (the sum of 25OHD2 and 25OHD3) and 1,25(OH)2D) measured prior to inclusion and treatment. One-Hundred-and-forty-three patients had DAS28-CRP calculated after one year, and were included in the current study. Patients were allocated to Dtotal group at time of diagnosis, dichotomized as low: Dtotal < 50 nmol/L or normal: Dtotal ≥ 50 nmol/L. Patients who reported insufficient vitamin D and calcium intake at time of diagnosis received daily oral supplements of vitamin D3 (800 IE/10 mcg) and calcium (1500 mg), according to national guidelines. Treating physician decided eventual need for supplementation unaware of actual serum vitamin D levels, as serum levels were evaluated from biobank for this current study. NTJ, Number of Swollen Joints – 28 joint count (NSJ), Visual Analogue Scores (VAS): VASglobal-doctor, VASglobal-patient, VASpain-patient, CRP, DAS28-CRP, Health Assessment Questionnaire (HAQ), Anti Citrullinated Protein Antibodies (ACPA), Immunoglobulin M-Rheuma Factor (IgM-RF) and season of diagnosis (Winter defined as diagnosis established between November and April) were evaluated at time of diagnosis. DAS28-CRP ≤ 2.6 at one year was defined as remission. (See Fig. 1).

Measurement of the vitamin D metabolites

25OHD2 and 25OHD3 in serum were analyzed with isotope dilution liquid chromatography-mass spectrometry (LC-MS/MS) using calibrators traceable to international standard reference material NIST SRM 97240. Mean coefficients of variation (CVs) of 25OHD3 were 9.6% and 8.1% at 25 nmol/L and 48 nmol/L, respectively, and for 25OHD2, 8.5% and 8.0% at levels of 23 and 64 nmol/L, respectively41.

1,25(OH)2D was analyzed by radio Immuno Assays (RIA)42 after immune-extraction of the samples. (1,25-dihydroxy vitamin D RIA, IDS, Boldon, UK) Mean intra-assay (well-to-well) CV were 6.8% and 9.0% at levels of 90 and 220 pmol/L, respectively.

Routine laboratory assessments

IgM-RF was detected by enzyme-linked immunosorbent assay (ELISA). ACPA-IgG antibodies were determined by a second-generation ELISA (Immunoscan RA kit, Euro-diagnostica AB, Malmo, Sweden) Serum CRP was measured using standard laboratory measures.

Statistical methods

Differences between Dtotal groups for dichotomous variables at time of diagnosis were evaluated by Chi2 test. Wilcoxon-Rank-Sum test evaluated differences in continuously variables. Underlying assumptions were evaluated through study-design.

Univariate, logistic regression was performed for Dtotal, comparing levels <50 nmol/L to levels ≥50 nmol/L, in predicting DAS28-CRP ≤ 2.6 after one year. This is defined as the “crude model”. In the model defined as the “partially adjusted model”, sex and age at diagnosis were included, whereas symptom-duration prior to diagnosis, DAS28-CRP and diagnosis established at winter were further included in the fully adjusted model. These variables were chosen prior to analysis because of their previously reported ability to predict clinical remission43. or close physiological relation to vitamin D4. Despite patients originally randomized 1:1 to ciclosporine, it was decided not to adjust for ciclosporine, as no association with one-year remission was found for ciclosporine in the original CIMESTRA study34.

Further aiming at evaluating the delicate interplay between variables included in the model, a step-by-step approach was used, including sex, age, symptom duration, DAS28-CRP at diagnosis and season of diagnosis in a logistic regression one by one.

The model was subsequently evaluated for interaction between vitamin D and season of diagnosis, as a close relationship exists between season and circulating Dtotal. Likelihood ratios tests were used to evaluate model fit. AIC and BIC assesses the model fit when taking number of co-variates included into account, and thus estimates the risk of over-fitting. Emphasis is put on AIC, while BIC is also presented.

To test the robustness of the model, propensity score analysis was applied to evaluate the potential biasing impact of other variables on the outcome. The propensity score analysis included BMI, ACPA, IgM-RF, NSJ, NTJ, VASglobal-patient, HAQ and CRP at time of diagnosis to determine, if confounding by any of these factors was biasing the results.

The potential impact of the missing remission-data of the 15 patients not included in year one was evaluated using best- and worst case scenarios, coding all missing patients as being either in remission (“Best case”) or not in remission (“Worst case”).

Concerning 1,25(OH)2D, similar analyses were performed as for Dtotal, comparing lowest and middle tertile to highest tertile.

Significance is defined as the 5% level. Stata IC 1544 is used for statistical analyses.

Ethics and Consent

The study complies with the Oviedo and Helsinki declarations. The protocol was approved by Ethics Committee Region Midt (M-1959–98) and registered at www.clinicaltrials.gov (NCT00209859). All patients received verbal and written information prior to enrolment, and gave written consent at baseline.