Serum Levels of IL-12, IL-6, IL-10, IL-17 and IFN-γ  in Female Students with Vitamin D Deficiency

doi:10.21203/rs.3.rs-832276/v1

Background: Several studies demonstrated the regulatory effects of vitamin D₃ on the immune system. The aim of this study was to investigate the serum levels of IL-12, IL-6, IL-10, IL-17 and IFN-g cytokines in 11-17 year-old female students with 25-hydroxyvitamin D₃ (25-(OH) D₃) (VitD3) deficiency.

Methods: 100 subjects with VitD3deficiency were selected as the case group and an equal number of individuals with sufficient levels of VitD3 were selected as a control group. The serum levels of IL-10, IL-6, IL-12, IFN-g and IL-17 were measured by ELISA method.

Results: The results showed that the serum concentrations of IL-12 (P= 0.005) and IFN-g (P= 0.019) were significantly lower in VitD3-deficient group compared to control group. There were no significant differences in serum levels of IL-6 (P= 0.66), IL-10 (P= 0.647) and IL-17 (P= 0.159) between cases and controls.

Conclusion: These results suggest that VitD3deficiency may result in decreased inflammatory cytokines production, which are required for proper immune functions. However, the effects of VitD3 on IL-6 and IL-17A, as the pro-inflammatory and IL-10, as anti-inflammatory cytokines needs more investigations.

Immunology

25-hydroxyvitamin D3

Vitamin D deficiency

Cytokines

The body receives its own vitamin D₃ ((3β,5Z,7E)-9,10-secocholesta- 5,7,10(19)-trien-3-ol) through synthesis in skin utilizing UV radiation and foods [1]. In some countries, the women are at lower skin utilizing UV radiation, so the population is at risk of VitD3 deficiency [2–4].

Recently scientists have found out the effects of VitD3 and its receptor on the immune system, as it is shown, immune cells like macrophages, T and B lymphocytes and natural killer (NK) cells have receptors for VitD3 [5]. Furthermore, several studies have proposed that VitD3 can be considered as immunoregulator [6, 7]. It has been demonstrated that immune cells utilize cytokines to perform their functions [8].As it is clear, cytokines are the regulator proteins of immune responses for regulation of target cells, including immune cells [9, 10]. For instance, interferon-gamma (IFN-γ) is a cytokine produced and released by several cells, including T CD₄⁺ and T CD₈⁺ lymphocytes as well as NK cells [11]. This cytokine is able to affect antigen processing cells such as dendritic cells and macrophages by increasing their ability to present antigens via MHC class II and by inducing the cells to produce immune irritant substances like IL-12 [12]. IL-12 is capable of activating T CD₄⁺ and T CD₈⁺ lymphocytes and also NK cells to fulfill their main functions and produce inflammatory cytokines such as IFN-γ in a positive feedback manner [13].

Th17 lymphocytes contribute to the primary defense against infections by releasing IL-17 [14, 15]. IL-6 is also an innate immune cytokine that can increase production of inflammatory cytokines and also inhibit the production and function of T regulatory cells [8, 9].

Decrease in secretion of inflammatory cytokines (IL-12, IFN-γ, and IL-17) may lead to disorders in immune system response such as susceptibility to infectious diseases and cancers. On the other hand, elevated levels of these cytokines could lead to inflammatory and autoimmune diseases, including hypersensitivities [8], through which, the immune cells produce IL-10 by regulatory T lymphocytes, macrophages and other cells to modulate immune response [16]. Now a day, there are some evidences demonstrate that VitD3 regulates many aspects of our life, such as bone marrow cells, which are influenced of immune system, through innate immunity receptors, such as toll like receptors (TLRs) [17]. Interestingly, our previous study revealed that there are significant correlations between Ca, P, PTH, ALP and serum levels of IL-10, IL-17 and IFN-γ in the female student with VitD3 deficiency [18]. Thus, VitD3 can also affect the immune system via indirect mechanisms, like alteration in blood mineral factors.

Regarding the effects of VitD3 on the immune system and immune cells, and the importance of the cytokines in this scenario, and based on the fact that in the many countries, the women are at lower skin utilizing UV radiation, and the possibility of the VitD3 deficiency risk in the population [2], we aimed to assess the serum levels of produced cytokines by principal cells of the immune system, including Th1 lymphocyte and NK cells (IFN-γ), regulatory T lymphocyte (IL-10), Th17 lymphocytes (IL-17) and macrophages (IL-6 and IL-12) in the female students with VitD3 deficiency and the control group.

Subjects

This descriptive and cross-sectional study was carried out in Rafsanjan, a city in southeast Iran (30°24′24″N 55°59′38″E). The students were selected by random sampling method from all high school children residing in Rafsanjan. Exclusion criteria included a chronic illness, rheumatoid arthritis, thyroid, parathyroid and adrenal diseases, diabetes, cancer, renal and liver failure, Cushing’s syndrome, taking calcium, multivitamin, or VitD3 supplements during 2 weeks prior to sampling, injection of VitD3 during the previous 6 months, use of medication known to affect bone metabolism and anticonvulsant drugs. Of the 260 girls who accepted participation in our study, 10 girls were excluded from the study and at last, 250 healthy subjects participated in the study.

Participants completed questionnaires regarding age, education, residency (urban or rural), type of accommodation (apartment or house with a yard), skin color (white or dark), menstruation status (regular or irregular), type of coverage (wearing veil or not), exercise (total hours per week spent doing sports or other physical activities), diet (e.g., fish and egg consumption), exposure to sunlight (minutes per day), and body mass index (BMI).

VitD3₃-deficient students were selected based on the serum concentration of 25-(OH) D₃ ≤ 20 ng/ml [19], and individuals with 25-(OH) D₃ serum concentration more than 20 ng/ml and less than or equal to 36 ng/ml were considered as normal subjects. Cases were matched to controls regarding age, education, residency, type of accommodation, skin color, menstruation status, type of coverage, exercise, diet, exposure to sunlight, and BMI. Finally, 100 subjects with 25-(OH) D₃ deficiency were selected as cases and an equal number of individuals with sufficient levels of 25-(OH) D₃ were selected as controls.

With the approval of our University Ethic Committee (IR.RUMS.REC.5) and taking written informed consent forms from parents and participants, blood sample was collected in fasting conditions. Sera were stored at − 20 ºC for future analysis.

Evaluation of VitD3 serum levels

Serum levels of 25-(OH) D₃ of 250 female students aged 11–17 years [20], were evaluated using commercial Radioimmunoassay (RIA) kit (DIA source kit, Belgium) according to the manufacturer`s instruction. Briefly, 25-(OH) D₃ in the samples and standards were extracted using acetonitrile. Then the samples and standards combination with I¹²⁵25-(OH) D₃ have been added to the anti-25-(OH) D₃ pre-coated tubes. 25-(OH) D₃ competes with I¹²⁵25-(OH) D₃ to interact with pre-coated anti-25-(OH) D₃. Finally, the radiation by I¹²⁵25-(OH) D₃ was evaluated using 1275 MINIGAMA Counter (LKB Company, Finland).

Cytokine assay

The serum levels of IL-10, IL-6, IL-12, IFN-γ and IL-17 were measured using commercial ELISA Kit (eBioscience, ESP) according to the manufacturer’s guidelines. The sensitivities of the kits were 2 pg/mL and were able to detect up to 400 pg/mL.

Statistical analysis

Statistical analysis was performed using SPSS version 16. The normal distribution of the data was calculated using One-Sample Kolmogorov-Smirnov test. The analysis revealed normal distribution of the data, hence the parametric tests were used to analyze the data. Accordingly, the difference between VitD3-deficient and normal (control) groups regarding age, Length, Weight, BMI, Waist, Hip, VitD3, Sun exposure, IL-10, IL-6, IL-12, IFN-γ and IL-17 was analyzed using Student’s t test and the relationship between variables was assessed by Pearson correlation coefficient. Data were reported as Mean ± SEM. The P values of less than 0.05 were considered statistically significant.

Table 1 indicates demographic characteristics of participants. Our results showed that serum concentrations of IL-12 were 2.34 ± 4.98 in cases and 4.79 ± 8.28 in controls. The statistical analysis revealed that the difference was significant (P = 0.005). The results also demonstrated that serum levels of IFN-γ were significantly (P = 0.019) decreased in the case group (19.47 ± 10.52) when compared to healthy controls (22.88 ± 10.41). There were no significant differences regarding IL-6 (P = 0.66), IL-10 (P = 0.647) and IL-17 (P = 0.159) between two groups (Fig. 1).

Table 1

indicates demographic characteristics of vit D deficiency and control groups. Results are shown as mean ± SD.
Variable	Control	VitD3 deficient female
Age (year)	2.07 ± 13.86	1.75 ± 14.06
(cm) Length	8.73 ± 157	7.2 ± 157.93
(kg) Weight	12.73 ± 49	10.06 ± 50.68
(kg/m2) BMI	4.16 ± 19.61	3.7 ± 20.26
(cm)Waist	9.58 ± 62.1	7.88 ± 63.05
(cm)Hip	10.81 ± 84.48	8.97 ± 86.03

The results revealed that there are no correlations among age, weight, BMI, waist, hip, sun exposure and length with serum levels of IL-6, IL-10, IL-12, IL-17, and IFN-γ in the participants (Table 2), while there was a weak positive correlation between serum levels of VitD3 and IFN-γ (r = 0.151, P = 0.032). There were not significant correlations between serum levels of VitD3 and other cytokines (Table 2).

Table 2

The correlation among weight, BMI, waist, hip, length, sun exposure and vitamin D with serum levels of IL-6, IL-10, IL-12, IL-17, and IFN-γ in the participants.
Variables	Test	IL-6	IL-10	IL-12	IL-17	IFN-γ	Vitamin D3
Weight	Pearson correlation P value	0.001 0.986	0.061 0.417	− 0.033 0.648	− 0.100 0.236	− 0.053 0.454	-0.068 0.283
BMI	Pearson correlation P value	-0.021 0.800	-0.069 0.357	0.027 0.716	-0.023 0.788	-0.038 0.595	-0.029 0.645
Waist	Pearson correlation P value	-0.004 0.958	0.032 0.675	0.023 0.753	-0.087 0.301	-0.079 0.267	-0.033 0.599
Hip	Pearson correlation P value	0.052 0.527	0.064 0.398	-0.020 0.787	-0.082 0.332	-0.076 0.286	-0.021 0.871
Length	Pearson correlation P value	-0.021 0.800	-0.069 0.357	0.027 0.716	-0.023 0.788	-0.038 0.595	-0.091 0.154
Vitamin D3	Pearson correlation P value	0.021 0.795	0.019 0.803	0.070 0.342	-0.067 0.431	0.151 0.032	1 -
Sun exposure	Pearson correlation P value	0.102 0.216	0.080 0.289	0.032 0.661	0.016 0.850	0.000 0.999	0.027 0.671

Vitamin D can modulate the innate and adaptive immune responses through a complex network. Our results revealed that serum levels of IL-12 and IFN-γ were significantly lower in the VitD3-deficient group in comparison to the control group, but no significant differences were observed in other cytokines, including IL-6, IL-10, and IL-17. Our results suggest that VitD3 deficiency in female students may result in defective immune system function and inability to produce adequate rates of important cytokines such as IL-12 and IFN-γ, the main cellular immunity cytokines [8, 21]. Therefore, it could be concluded that, VitD3-deficient individuals may be at risk of infection and many other disorders, due to defect in immune function following decline in the inflammatory cytokines.

Obviously, IL-12 and IFN-γ are important cytokines, which connect innate and acquired immunity [22]. Hence, it may be concluded that decreased IL-12 and IFN-γ cytokines in female students may result in inadequate cellular immune responses.

Adorini et al., showed that the administration of 1,25-(OH)₂ D₃ in patients with multiple sclerosis (MS) and controls significantly inhibited IL-17 production in both groups, but had no effect on IL-4 production. Moreover, VitD3 receptor agonists directly inhibit Th1 cytokines such as IL-2 and IFN-γ [23]. Deluca et al., [24] determined the mRNA levels that encode cytokines in the lymph node lymphocytes of control mice and those treated with 1,25-(OH)₂D₃. They found that VitD3 administration markedly increased TGF-ß1 and IL-4 mRNA levels, while there was a decrease in IFN-γ and TNF-α expression.

Correale et al., evaluated the effect of VitD₃ on cytokine productions by T cells using purified CD4⁺ T cells [25]. The results demonstrated that VitD₃ administration resulted in high numbers of IL-10 producing T cells and low numbers of IL-6 and IL-17 producing cells, whereas no changes were found in the number of T cells producing IL-4 or IFN-γ. They also showed that the combination of VitD₃ and IL-10 resulted in elevated numbers of human IL-10 producing T cells compared to the impacts of VitD₃. However, the study by Correale et al., were performed on the cells separated from MS patients in the in vitro condition, while in the present study, the correlations between VitD3 and serum levels of the cytokines were performed in the in vivo condition. Therefore, no significant difference in the amount of IL-10 between the VitD3-deficient and control groups may be related to its in vivo conditions, which needs more investigations. The reason for this discrepancy remains to be explained.

VitD3 inhibits the differentiation of monocytes into dendritic cells, and suppresses IL-12 secretion in vitro. Treatment of dendritic cells with VitD3 down-regulates the expression of co-stimulatory molecules and reduces IL-12 production [26]. Our result showed that the serum levels of IL-12 were significantly lower in the VitD3-deficient group compared to control group. Therefore, the decreased in the levels of IL-12 and IFN-γ in the VitD3-deficient group supports the importance of the regulatory role of VitD3 in the immune system.

As mentioned previously, the relation between VitD3 serum levels and cytokine production is controversial in several investigations. For instance, it has been reported that supplementation of VitD3 or 25(OH)D₃ decreases IL-12 [27]. In agreement with the study, Barker and colleagues demonstrated that the serum concentrations of IFN-γ are significantly higher in vitamin D-deficient group compared to vitamin D-sufficient group [28]. The results are in contrast with our results which showed that serum levels of IL-12 and IFN-γ decreased in the VitD3-deficient group. It may be hypothesized that gender could be considered as a main interference factor in the investigations regarding the roles of VitD3 on the cytokine production because all of the subjects in our study were female, while the investigations by Barker et al., and Bischoff-Ferrari et al., were performed on both male and female subjects. Additionally, various ethnics and genetics could be considered as interference factors, which may affect cytokine productions [29]. However, there are some investigations which are in agreement with our results. For example, Barker et al., reported that supplementation of the vitamin D increased IFN-γ in vitamin D deficient individuals [30]. Interestingly, several studies demonstrated that vitamin D has immunomodulatory effects on the expression of pro-inflammatory cytokines such as IL-12, IL-18, TNF-α and IFN-γ during human pro-inflammatory based diseases [31–33]. Based on the fact that our participants were healthy female students, it seems that vitamin D has controversial functions due to the in vivo condition.

Additionally, although IL-17A has antagonist effects with IFN-γ and IL-12 [34], it is produced in response to fungi and bacterial infections and also participates in the pathophysiology of autoimmune diseases [35]. Therefore, it may be hypothesized that IL-17A have not been down-regulated in the VitD3-deficient group because of antagonist effects with IFN-γ and IL-12, as down-regulation of IFN-γ and IL-12 leads to up-regulation of IL-17A. Furthermore, due to the fact that cytokines/chemokines show their functions in a networked manner [36], IL-17A may try to retrieve the immunodeficiency following decreased expression of IFN-γ and IL-12 in the VitD3-deficient group.

Our results also demonstrated that there is no relation between age, weight, BMI, waist, hip, sun exposure and length with serum levels of IL-6, IL-10, IL-12, IL-17, and IFN-γ in the participants. Thus, it may be concluded that production of the cytokines is not associated with secretion of the pro/anti-inflammatory cytokines in our population (female students). Our previous investigations also confirmed the results and demonstrated that there is no association between the parameters like age, diet and so on with serum levels of IL-6, IL-10, IL-12, IL-17, and IFN-γ in the female students [18].

These data indicated that VitD3 plays important roles in immune processes and acts as a potential modulator of the immune system, and those female students with hypovitaminosis D₃ may be at increased risk of many disorders.

25-(OH) D3: 25-hydroxyvitamin vitamin D3

IFN-g: interferon-gamma

CD: Cluster of differentiation

TLR: Toll-like receptor

NK: natural killer

MHC: Major histocompatibility complex

Th17: T-helper 17

Ca: Calcium

P: Phosphorus

PTH: Parathyroid hormone

ALP: Alkaline phosphatase

Ethics approval and consent to participate

The ethics committee of Rafsanjan University of Medical Sciences approved this study (Ethical codes: ID: (IR.RUMS.REC.5). Written informed consent was obtained from the parents and participants. The data of Participants kept confidential and was only accessible to the study investigators

Consent for publication:

Not applicable.

Availability of data and material

The datasets in the current study are available from the corresponding author on reasonable request.

Competing interests

Authors have no conflict of interest to declare.

Funding

Rafsanjan University of Medical Sciences has funded the current project.

Authors' contributions

GA has prepared the idea, proposal and examinations. ZJ has collected the samples and performed the experiments. SI and SN help the ZJ to collect the samples. VB and MNK help ZJ to perform the experiments. MNK participate in preparation of manuscript. MKA was the supervisor and wrote the manuscript.

Acknowledgements

This work was supported by grants from Physiology Research Center, Kerman University of Medical Sciences. The authors thankfully acknowledge the support of the dissertation grant. The Authors also acknowledge Professor Hamid Najafipour for his critical comments on the paper.

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No competing interests reported.

Serum Levels of IL-12, IL-6, IL-10, IL-17 and IFN-γ in Female Students with Vitamin D Deficiency

Status:

Version 1

Abstract

Figures

Introduction

Material And Methods

Subjects

Evaluation of VitD3 serum levels

Cytokine assay

Statistical analysis

Results

Discussion

Conclusion

Abbreviations

Declarations