Vitamin D as a Key Player in Modulating Rheumatoid Arthritis-derived Immune Response

Rheumatoid arthritis (RA) is a systemic inflammatory disease with chronic nature of joints related to autoimmunity. Vitamin D was found to modulate cell growth, function of immune cells and antiinflammatory action. The aims of that study were to investigate serum level of vitamin D and some cytokines and to identify the correlation between vitamin D and these cytokines in RA. Totally 40 RA patients without vitamin D supplement were involved in this study. Serum level of vitamin D, interleukin-6 (IL-6), IL-10, IL-35, C-reactive protein (CRP) and tumor necrosis factor α (TNF-α), all of them were measure in all patients by enzyme-linked immunosorbent assay (ELISA). Patients were classified according to Vitamin D levels into two groups; RA patients with Vit. D deficiency (n=25) and RA patients with Vit. D sufficiency (n=15). IL-6 was lower significantly (P = 0.03) in RA patients with Vit. D sufficiency than RA patients with Vit. D deficiency. IL-10 and IL-35 were higher significantly (P = 0.0234, P = 0.0356 respectively) in RA patients with Vit. D sufficiency than RA patients with Vit. D deficiency. Vit. D was significantly positively correlated with both IL-10 (r = 0.4516, P = 0.0034) and IL-35 (r = 0.3424, P = 0.0329) and negatively correlated with IL-6 (r = -0.3188, P = 0.0479). Sufficient serum level of Vit. D is correlated with higher level of anti-inflammatory cytokines (IL-10 and IL-35) and lower level of IL-6. This support the immunomodulatory effect of Vit. D in RA.

TNF-a is a prevalent pro-inflammatory cytokine in RA pathogenesis and is highly expressed the synovial fluid. Selective blockage of TNF-a leads to relieve of RA progression 24,25 .
IL-6 plays a crucial role in RA pathogenesis, thus it promotes autoantibodies production and regulation of TH-cells differentiation 26 . Also its signaling cascades trigger bone erosion, joint inflammation 27 , acute phase response, synovial fibroblasts proliferation and promote hematopoietic cells 28 .
Vitamin D sufficient level have been related to reduced serum IL-6 29 by the mastery of vitamin D to suppress the synthesis of IL-6 transcription thru the stimulation of MAPK phosphatase-1 (MKP1) which leads to p38 inhibition by its dephosphorelation. The dephosphorelated p38 resulting in down regulation of IL-6 gene transcription 30 .
On the other side, vitamin D could up-regulate the anti-inflammatory cytokines as IL-10 31 and IL-4 32 and. This was proven by in vitro study that reported vitamin D3 therapy could enhance the production of Th2 lymphocytes which in turn amplified the manufacture of IL-4, IL-5, and IL-10 33 . IL-10 is a sturdy anti-inflammatory cytokine and plays a vital role in relieving RA signs 34 through inhibiting both the infiltration and activation of neutrophils in the synovial fluids 35 , constraining pro-inflammatory cytokines expression as TNF-a 36 , pointing macrophages to polarize into an M2 phenotype. In vitro study reported that, nullification of IL-10 activity in RA synovial membrane cultures resulted in augmented level of pro-inflammatory cytokines like IL-1β and TNF 36 .
IL-35 is a heterodimeric cytokine and it is one of IL-12 family 37 . IL-35 could down regulation T-cell proliferation and induce regulatory T cells (iTr35) as a result of the creation of pSTAT1:pSTAT4 heterodimers 38 . IL-35 chiefly released by Treg cells 39 , however a recent study reported that, IL-35 is also released by regulatory B cells (Breg) 40,41 .
Several studies have revealed the antiinflammatory properties of IL-35, in which it could promote Treg-cells and deter Th17 cells in many experimental inflammatory diseases models such as collagen induced arthritis (CIA) 42 , also in vitro treatment with IL-35 inhibited fibroblast-like synoviocytes (FLS) proliferation and promoted its apoptosis in a dose-dependent manner cultured from CIA mice 43 . Furthermore, IL-35 could suppress the expression of vascular endothelial growth factor (VEGF) and repressed angiogenesis in CIA mice model thus made the severe synovitis much better 44 .
The aims of that study were to investigate the serum level of vitamin D and some cytokines and identify the correlation between vitamin D and these cytokines and the correlation among the cytokines.

Patients
That study was conducted during 2019 after approval of the ethical committee of the university hospital (Ref number 17100135). A written informed consent was obtained after discussing a detailed description of the study with the patient. Patients were informed that they can withdraw their consent to participate at any time. The work was done in agreement with the Declaration of Helsinki.
According to ACR/EULAR criteria for classification of RA disease (45), Forty patients attending routinely outpatient clinic of Rheumatology, Department of Rehabilitation and Physical Medicine, Assiut University Hospital, Egypt diagnosed with RA were selected in this study. All the recruited patients underwent a thorough clinical assessment to detect the clinical activity of the disease using Rheumatoid disease activity score DAS28, patients should have a score less than 2.6 for at least 3 months prior enrollment to the study 46 .
In addition, all patients were receiving Methotrexate and Leflunomide. The exclusion criteria included: active RA (DAS>2.6), patients receive vitamin D supplement, patients receive corticosteroids, Patients with inflammatory bowel disease, patients with any other autoimmune diseases or patients aged younger than 15 years. Patients are classified into 2 groups according vitamin D level; RA patients 25-OH Vit. D deficient and RA patients 25-OH Vit. D sufficient.

Healthy volunteers
Forty healthy volunteers without rheumatoid arthritis, inflammatory bowel disease or any other autoimmune disease have been recruited in this study to measure their serum 25-OH Vitamin D only.

serum samples
Five millimeters of fasting venous blood sample were collected from the selected RA patients and healthy volunteers in a sterile plain tube and left to clot. Then, centrifuged. Each serum sample was distributed in 5 cryogenic vials and deposited at -80°C for extra analysis.

Determination of total 25-OH Vitamin D (25-OH Vit. D)
Serum concentration (ng/mL) of total 25-OH Vit. D was determined for both RA patients and healthy volunteers by enzyme immunoassay by following the manufacturer protocol of Epitope Diagnostic kit (San Diego, CA: 92121, USA). According to manufacturer guidelines vitamin D deficiency will be fewer than 20 ng/mL and optimal vitamin D level will be 20-70 ng/mL.

Measurement of C-reactive protein (CRP)
Serum concentration (ng/mL) was measured for RA patients according to manufacturer protocol. HIGH SENSITIVITY C-REACTIVE PROTEIN

Statistical analysis
All data were documented on Microsoft Excel. All statistical analyses were carried out by means of the program GraphPad Prism version 8.0.1 (244) (San Diego, CA, USA). All results are informed as mean together with standard deviation (SD).

Demographic data
Totally 80 persons, of them 40 patients (38 females and 2 males) from 2 governorates; Assiut and Sohage, upper Egypt, Egypt. The mean age for RA patients was 43.23 ± 9.037 (Table 2). There was no statistically significant difference among the two RA groups within age. All patients with active RA according to diagnosis based on ACR/EULAR criteria. It was obvious from the rate   Table 2). Thus patients are classified into 2 groups; RA patients 25-OH Vit. D deficient and RA patients 25-OH Vit. D sufficient. The variance between the two groups were statistically significant with P value < 0.0001 (Table 2) (Fig. 1). Also for healthy volunteers 18 persons were found to be sufficient in 25-OH Vit. D level with mean ± SD 27.12 ± 3.705, however 22persons were found to be deficient in 25-OH Vit. D level with mean ± SD 18.50 ± 1.017. the difference in total serum 25-OH Vit. D level between RA patients and healthy volunteers were significant with P value 0.0193 (Table 1). Serum 25-OH Vit. D level correlated significantly with the serum level of both IL-10 (r = 0.4516, P = 0.0034) and IL-35 (r = 0.3424, P = 0.0329) ( Table 3) (Fig.  2). Moreover, serum 25-OH Vit. D level negatively correlated significantly with the serum level of IL-6 (r = -0.3188, P = 0.0479). While, 25-OH Vit. D did not correlate with both TNF-a (r = -0.1141, P = 0.4834) and c-reactive protein (CRP) (r = 0.009931, P = 0.9515) ( Table 3) (Fig. 2).

Serum IL-35 cytokine level and correlation with other parameters
Serum IL-35 in all patients was 4340 ± 1058. The serum IL-35 in RA patients 25-OH Vit. D deficient group was 4022 ± 716.3, and in RA patients 25-OH Vit. D sufficient group was 4729 ± 1309 (Table 2) (Fig. 1). Moreover, the variance among the 2 groups in IL-35 was statistically significant with P value 0.0356 (Table 2). Serum, IL-35 level correlated significantly with serum 25-OH Vit. D level, serum IL-6 and serum IL-10 ( Table  3) (Fig. 2), but in the same time neither TNF-a nor CRP correlated with serum IL-35 level (data not shown).

disCussiON
Many types of inflammatory diseases as RA have been linked to low vitamin D serum level [47][48][49][50] , although many studies reported that serum vitamin D level was not necessary to be lower than in healthy controls 49  Many recent studies reported that, decreased serum level of vitamin D is associated with many types of inflammatory diseases as rheumatoid arthritis (RA) [47][48][49][50] . However other studies indicated that, serum level of vitamin D in rheumatic patients is not certainly lower than in healthy volunteers 49 . A microarray dissection reported that, 5% of the human genome and the physiological attitude of not less than 36 different types of cells are under the regulation of vitamin D either by indirect or direct action 51 . Furthermore, a recent study reported that, there were no statistically significant variance in vitamin D level between persistent RA or reactive arthritis and healthy control present in that study 6 , which also support the believe that, vitamin D level is not necessarily correlated with inflammatory disease. Another recent study reported that, 84% of RA patients present in in that study were deficient in serum vitamin D level, while only 34%  47 . This meta-analysis research also, reported that, the level of vitamin D was inversely correlated with RA disease activity 47 . In the light of our results and other studies results, we think that, vitamin D deficiency is highly prevailing in RA patients. TNF-%, IL-17, IL-6 and IL-1 are predominant pro-inflammatory cytokines in RA chronic inflammation of joints by both cartilage and bone distortion 53 . From recent researches it has been apparent, the value of vitamin D on the health and the activity of many types of diseases, thus beside its well-known role on bone metabolism 8,54,55 , vitamin D possesses a leading role on the cardiovascular and immune system. It had been approved that, normal vitamin D level is essential for normal physiological function of both innate and adaptive immune response and also for self-antigen tolerance 8,[56][57][58][59] . This role is imputed to the ability of vitamin D to increase the expression of the anti-inflammatory cytokines like IL-10 and decrease the expression of proinflammatory cytokines such as IL-6 and TNF-a, accordingly vitamin D deficiency is proved to be associated with loss of immune tolerance to self-antigen and thus to autoimmune diseases like RA disease 60 . In the present study, the serum IL-6 level in all RA patients was 5.383 ± 1.731, moreover, IL-6 was higher in RA patients 25-OH Vit. D deficient group (5.775 ± 1.822) than in RA patients 25-OH Vit. D sufficient group (4.539 ± 1.218) with statistically significant variance (P = 0.0294). In the same time, 25-OH Vit. D level negatively correlated significantly with IL-6 level (r = -0.3188, P = 0.0479). Also, Caraba, Crisan 61 revealed that vitamin D had an inverse significant correlation with both IL-6 and TNF-a. An earlier study indicated that, the intensity of systematic inflammation is inversely associated with vitamin D serum level 62 . Another study reported that, they did not catch any correlation among serum vitamin D level and serum IL-6, moreover they reported that, neither DAS28 score (Score of 28 joints) nor IL-6 differ significantly amid vitamin D diminish and replete groups 63 . In the light of most previous studies and our result that, IL-6 was higher in RA patients 25-OH Vit. D deficient group (5.775 ± 1.822) than in RA patients 25-OH Vit. D sufficient group (4.539 ± 1.218) with statistically significant difference (P = 0.0294), we support the hypothesis that normal vitamin D level is a vital and has an inhibitory effect on IL-6 cytokine.
TNF promote synovial-fibroblast, endothelial cell and leukocyte activation, pain receptor sensitization and angiogenesis which all together are main characteristics of RA 64 . Activation of synovial-fibroblast by TNF-a results in the production of CTGF (connective tissue growth factor) causes hyper activation of osteoclasts, leading to joint deterioration 65 . In our study, the serum TNF-a in all patients was 255.5 ± 58.24. Furthermore, in RA patients 25-OH Vit. D deficient group TNF-a serum level was 255.6 ± 50.03 and in RA patients 25-OH Vit. D sufficient group was 250.1 ± 61.01 without significant variance (P = 0.7316) amid the two groups. Likewise, 25-OH Vit. D level did not correlate with TNF-a level (r = -0.1141, P = 0.4834) and this is in agreement with previous study 66 . Another study reported that, there were no significant variance in serum TNF-a level amid vitamin D sufficiency group and vitamin D deficiency group 67 . Also they reported that, increased sTNFr1 (soluble tumor necrosis factor-receptor-1) and decreased sTNFr2 (soluble tumor necrosis factor-receptor-2) significantly in vitamin D sufficiency group than in vitamin D deficiency group 67 . Thus, based on the results of our study and the mentioned previous studies, serum TNF-a is not affected by serum vitamin D status without vitamin D supplement.
Our study revealed that, the serum CRP in all patients was 9007 ± 2860. The CRP level in RA patients 25-OH Vit. D deficient group was 9105 ± 2713, and in RA patients 25-OH Vit. D sufficient group was 8843 ± 3181 without statistically significant variance amid the two groups (P = 0.7828). Also, serum vitamin D level did not correlate with serum CRP level (r = 0.009931, P = 0.9515). These findings agree with previous study (63). The pro-inflammatory cytokine IL-6 is linked with CRP release 68 , thus, based on this finding and our results, we believe that, vitamin D status without supplement has no effect on neither IL-6 nor its role in releasing acute phase proteins.
Our study revealed that, serum level of IL-35 in all patients was 4340 ± 1058. Interestingly, IL-35 was higher in RA patients 25-OH Vit. D sufficient group (4729 ± 1309) than RA patients 25-OH Vit. D deficient group (4022 ± 716.3) with significant difference (P = 0.0356). Furthermore, serum 25-OH Vit. D level correlated significantly with serum IL-35 (r = 0.3424, P = 0.0329). the data about IL-35 is controversial, that early study reported that an early study reported that, invitro TNF a stimulation resulted in enhancing the expression of both p35 and EBI3 subunits of IL-35 69 . Not only this but also IL-35 itself stimulated the expression of many pro-inflammatory particles in mononuclear cells 69 . In collagen induced arthritis (CIA) mice model, IL-35 was used as remedy for CIA mice orally by using the engineered Lactococcus lactis strain as vector 70 . IL-35 exhibited anti-inflammatory properties by significantly decreasing IFN-γ and IL-17 levels and increasing the level of both IL-10 and CCR6+ Tregs (regulatory T cells) 70 , thus, this supporting the anti-inflammatory features of IL-35. Another study reported that, IL-35 possesses a role in Treg-mediated repression of autoimmunity in RA 71 . Another studies confirmed the role of IL-35 in hindering the processes linked to (vascular endothelial growth factor) VEGF/ Ang2/Tie2 pathway 72,73 , also they indicated that IL-35 activity stimulated neovascularization, and promoted inflammatory process during the acute phase of RA 72 . Moreover, 74 reported that IL-35 is positively correlated significantly with Treg percentage, and in the same time Trge percentage negatively correlated with DAS28 in RA patients, which confirm the anti-inflammatory effect of IL-35 in RA.
Interestingly, in the current study, IL-35 also, correlated significantly with both IL-6 (r = 0.3829, P = 0.0147) and IL-10 (r = 0.6005, P = <0.0001). The immunomodulatory effect of IL-35 as anti-inflammatory may be due to its ability to upregulate the expansion of both regulatory T cells and IL-10 and in the same time inhibit the differentiation of Th 17 cells and suppress the proliferation of Teff cells 39,42 . However, another study reported that, IL-35 inversely correlated significantly with 28-joint DAS and in the same time in contrast to our results, IL-35 did not correlate with IL-10 71 . Furthermore, another study reported that, Tregs are divided into two distinct subsets; one subsets produce the suppressive cytokine IL-35 and CCR7, located in the T cell zone, while the other subset producing high level of the suppressive cytokine IL-10,granzymes, ICOS and multiple chemokine receptors responsible for migration to peripheral non-lymphoid tissues 75 .
Also, we found that IL-6 correlated significantly with IL-10 (r = 0.5065, P = 0.0009). it was reported that; higher level of IL-10 is produced in response to higher inflammatory state in patients with RA 66 .
Finally, our results revealed that, IL-6 correlated significantly with TNF-a (r = 0.3841, P = 0.0144) which is in agreement with other studies (76). It was reported that inhibition of TNF-a in a synovial cell culture resulted in down regulation of the production of other pro-inflammatory cytokines 77 . It was reported that TNF-a promote the production of IL-6 from synoviocytes in RA patients 78 .

CONClusiON
This study demonstrated that, serum level of IL-10 and IL-35 were higher in 25-OH Vit. D sufficient group than 25-OH Vit. D deficient group with significant difference. also IL-6 serum level was lower in in 25-OH Vit. D sufficient group than 25-OH Vit. D deficient group with significant difference. the levels of TNF-a and CRP also were lower in 25-OH Vit. D sufficient group than 25-OH Vit. D deficient group without significant difference. also 25-OH Vit. D correlated significantly with both IL-10 and IL-35, and in the same time 25-OH Vit. D negatively correlated significantly with IL-6. From our study the sufficient 25-OH Vit. D serum level could potentiate the production of antiinflammatory cytokines (IL-10 and IL-35) and also could repress the release of pro-inflammatory IL-6. Thus we support the hypothesis that 25-OH Vit. D act as immunomodulatory agent in RA diseases and the serum level of 25-OH Vit. D is important and must be assessed for all patients with not only RA disease but also for all inflammatory autoimmune diseases.