The changes of Treg and Th17 cells relate to serum 25(OH)D in patients with initial-onset childhood systemic lupus erythematosus

Background T helper 17 (Th17) cells and regulatory T cells (Treg) are known to play a crucial role in the pathogenesis of systemic lupus erythematosus (SLE). Improving the balance between Treg and Th17 cells can be a promising new therapeutic target in SLE patients. Vitamin D has a significant impact on the immune inflammatory process and the immune cells involved in this process. The purpose of this study is to investigate the relationship between Th17, Treg, cytokines, and serum 25 hydroxyvitamin D [25(OH)D] in patients with initial-onset childhood SLE. Methods A total of 82 children aged <18 years with initial-onset SLE were included, as well as 60 healthy subjects during the same period at the Pediatrics Department of the Second Hospital of Hebei Medical University. The chemiluminescence method was performed to detect serum 25(OH)D levels. Flow cytometry was used to evaluate Treg and Th17 cells. An enzyme-linked immunosorbent assay kit was used to evaluate plasma interleukin (IL)-23, IL-17, IL-10, IL-6, and tumor necrosis factor alpha (TNF-α) concentrations. Result The serum 25(OH)D levels in patients with initial-onset childhood SLE were significantly lower than those in the healthy controls. The proportion of lupus nephritis (LN) was higher in the vitamin D insufficiency group (71.4%) compared with the vitamin D sufficiency group (30.3%) (p < 0.05). The SLE disease activity index (SLEDAI) was higher in the vitamin D insufficiency group (median = 14) than that in the vitamin D sufficiency group (median = 9) (p < 0.05).The 25(OH)D level was positively correlated with the Treg ratio (r = 0.337, p = 0.002), and it was negatively correlated with the Th17 cell ratio (r = −0.370, p = 0.001). The serum 25(OH)D level had a negative correlation with IL-23 (r = −0.589, p < 0.001), IL-17(r = −0.351, p = 0.001), TNF-α (r = −0.283, p = 0.01), IL-6 (r = −0.392, p < 0.001), and IL-10 (r = −0.313, p = 0.004) levels. Conclusion The serum 25(OH)D levels decreased in patients with initial-onset childhood SLE. There was a negative correlation between the serum 25(OH)D levels and SLEDAI. The serum 25(OH)D levels in patients with initial-onset childhood SLE were negatively correlated with the Th17 ratio and related cytokines, while positively correlated with the Treg ratio.


Introduction
Systemic lupus erythematosus (SLE) is a chronic systemic autoimmune disease that causes chronic inflammation and damages multiple tissues and organs, including the central nervous system, skin mucosa, cardiovascular system, kidneys, and joints.The pathogenesis of SLE is not fully understood.SLE is characterized by polyclonal activation of T and B lymphocytes.In addition to an imbalance of T helper 1 (Th1) and T helper 2 (Th2) cells, the regulatory T cells (Treg) and T helper 17 (Th17) cells are known to play a crucial role in the pathogenesis of SLE (1).Studies have found that quantity anomalies or/and functional defects of Treg and Th17 cells were associated with flares and organ damages in SLE patients (2).Th17 cells secrete a profile of potent pro-inflammatory cytokines, including interleukin-17 , and potent tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) upon certain stimulation (3).Interleukin-23 (IL-23) is an important cytokine that promotes the secretion of interleukin-17 by Th17 cells to maintain pathological status by combining with IL-23 receptor (4).Although it is widely believed that Treg cells play a preventive role in autoimmunity, the data on SLE are inconsistent (5).Interleukin-10 (IL-10) is secreted not only by Th2 cells but also by Treg cells.The differentiation and proliferation of Treg and Th17 cells are regulated by multiple cytokines including IL-10, IL-23, IL-17, and IL-6 (6).Regulating the balance between Treg and Th17 cells will be a promising new therapeutic target in SLE patients.
Vitamin D is an important steroid hormone that has significant effects on bone health and the cardiovascular system (7).Vitamin D also has some non-classical effects, such as immune modulatory effects (8).Many studies have found that most patients with autoimmune diseases worldwide suffer from vitamin D deficiency.These studies have also emphasized the relationship between decreased serum vitamin D levels and disease activity in SLE and rheumatoid arthritis (9)(10)(11).Vitamin D has great impact on immune cells as well as the inflammatory cascade.The receptors of Vitamin D are commonly accessible for many adaptive immune cells including T cells, B cells, macrophages, and dendritic cells (12).
Whether vitamin D can act on Treg and Th17 cells remains largely unexplored.Therefore, the purpose of this study is to investigate the relationship between Th17, Treg, cytokines, and serum 25 hydroxyvitamin D [25(OH)D] in patients with initialonset childhood SLE.

Study subjects
A total of 82 children aged <18 years with initial-onset SLE who were admitted to the Pediatrics Department of the Second Hospital of Hebei Medical University between April 2020 and February 2023 were included in this study.All patients met the 1997 American College of Rheumatology (ACR) classification criteria for SLE (13) or the 2012 Systemic Lupus Erythematosus International Collaborating Clinics (SLICC) classification criteria for SLE (14).The disease activity was assessed using the SLE Disease Activity Index-2000 (SLEDAI-2K).The exclusion criteria were as follows: certain diseases that affect vitamin D metabolism (gastrointestinal surgery, liver metabolic diseases, tumors, etc.); and vitamin D supplementation by oral medication within the past 3 months.This study involved 60 healthy subjects during the same period as healthy controls (HC).This study was approved by the ethics committee of the Second Hospital of Hebei Medical University (protocol number 2021-R307).

Determination of serum 25(OH)D level
Blood was collected between 6:00 and 7:00 in the morning, and the children were fasted from food and water overnight before the blood samples were collected.The chemiluminescence method was performed for the detection of serum 25(OH)D levels, the kit was provided by Siemens Healthcare Diagnostics Inc. (USA), and the analysis was done using an ADVIA Centaur XP automatic chemiluminescence immunoassay analyzer.A vitamin D insufficiency was defined as serum 25(OH)D level of < 20 ng/ml, and a vitamin D sufficiency was defined as serum 25(OH)D level of ≥ 20 ng/ml.

Flow cytometry 2.4.1. Sample and cell preparations
All participants fasted from water after 12 p.m. the previous day, and peripheral venous blood samples of approximately 5 ml were collected between 6:00 and 7:00 in the morning.Blood samples were anticoagulated with ethylenediaminetetraacetic acid dipotassium (EDTA-K2), which was used to isolate and identify Treg and Th17 cell subsets.Peripheral blood mononuclear cells (PBMCs) were obtained through Ficoll density gradient.PBMCs were suspended at a density of 2 × 10 6 cells/ml on a complete culture medium (RPMI 1640 supplemented with 100 µg/ml streptomycin, 100 U/ml penicillin, 2 mM glutamine, and 10% heat-inactivated fetal calf serum) to obtain and analyze Th17 cell subset.The cell suspension was transferred to 24-well culture plates with a concentration of 25 ng/ml of phorbol ethyl ester (PMA), 1.7 ml of moneomycin (MN), and 1 ml of ionomycin (LC), and then incubated at 37°C under a 5% CO 2 environment for 4 h.For Treg cells analysis, PBMCs were suspended at a density of 2 × 10 7 cells/ml.

Surface and intracellular staining
To analyze Th17 cell subset, the cells were fixed and permeabilized according to the manufacturer's instructions, and then intracellularly stained with PE-conjugated anti-IL-17 monoclonal antibodies.Th17 cells were labeled as CD4 + IL-17A+.For Treg analysis, the cells were surface-stained, and then fixed permeabilized, then stained with PE anti-human Foxp3 according to the manufacturer's instructions.Treg cells were labeled as CD4 + CD25 + FoxP3+.Homotypic controls were used to verify specificity and perform compensation correction.All antibodies were provided by eBioscience.Stained cells were analyzed by flow cytometry analysis using a FACSCalibur flow cytometer (BD biosciences) with FlowJo software (Tree Star, San Carlos, CA, USA).

Statistical analysis
The data were statistically analyzed by using the SPSS version23.0program.The data were presented as the mean ± standard deviation (SD) or median, and the categorical variables were expressed as frequencies and percentages.The rates were compared between two or more groups using chi-square test or Fisher's exact test.A non-parametric Mann-Whitney U test was used to compare the data between groups.Pearson correlation analysis was used for variables that conformed to a normal distribution, and Spearman correlation analysis was used for variables that did not conform to a normal distribution.Statistically significant was defined as p-value less than 0.05.

Serum 25(OH)D level
As shown in Figure 1, the serum 25(OH)D levels in patients with initial-onset childhood SLE were significantly lower than those in the healthy control group (25.60 ± 6.87 ng/ml for HC, 18.86 ± 5.18 ng/ml for SLE).

Comparison of clinical and laboratory findings in pediatric SLE patients with different 25(OH)D levels
In this study, the patients with initial-onset childhood SLE were divided into two groups based on their serum 25(OH)D levels.The clinical manifestations and laboratory parameters were compared between the two groups.The proportion of lupus nephritis (LN) was higher in the vitamin D insufficiency group (71.4%) compared with the vitamin D sufficiency group (30.3%) (p < 0.05).SLEDAI was higher in the vitamin D insufficiency group (median = 14) than that in the vitamin D sufficiency group (median = 9) (p < 0.05).The probability of pulmonary involvement and anemia was higher in the vitamin D insufficiency group.The SLEDAI-2K score was higher in the vitamin D insufficiency group (median = 14) than that in the vitamin D sufficiency group (median = 9) (p < 0.05), indicating higher disease activity in SLE.Compared with the vitamin D sufficiency group (0.54 ± .024g/L), SLE patients in the vitamin D insufficiency group had lower levels of C3 (0.39 ± 0.27 g/L) (p < 0.05).It was worth noting that SLE children with insufficient vitamin D had lower serum calcium levels (Table 1).

Discussion
SLE is a chronic autoimmune disease distinguished by autoantibodies development and persistent inflammation that damages multiple organs.The clinical manifestations and severity of pediatric SLE are not completely the same as those of adult SLE, with childhood SLE having more severe clinical manifestations and being more prone to involving important organs compared with adult SLE.However, research on childhood SLE has not been widely reported (12).
The increase of Th17 and the decrease of Treg subsets were reported to be the main factors related to organ damages and auto-antibodies production in SLE patients (15).The elevation of the proportion of Th17 cells with pro-inflammatory effects was reported to be positively related to the disease activity of SLE (15).Treg cells have immunosuppressive function and can induce and maintain the self-immune tolerance of the body.The    Reports confirmed that the mTOR signaling pathway regulates the proliferation, differentiation, and functions of Treg cells (16).Specifically, mTORC1 promotes the expansion of proinflammatory lymphocyte subsets such as Th17; mTORC2 drives the proliferation of T follicle helper cells, promoting the activation of B cells and generation of auto-antibodies (17).Both mTORC1 and mTORC2 can control the differentiation and maturation of CD4 + CD25 + Foxp3 + Treg cells (17).In SLE patients, the abnormal metabolism of T cells, including high mTOR activation, increased glutaminolysis, active lipid synthesis, and enhanced glycolysis, all contribute to the differentiation and function of Th17.The metabolic disorder of T cell is a potential mechanism for Th17/Treg imbalance in SLE patients (1).
The serum 25(OH)D levels for children with SLE were obviously lower than those for healthy control children.Consistent with our findings, multiple studies worldwide have found lower levels of 25(OH)D in adults with SLE (10).Although insufficiency and/or deficiency of vitamin D have been reported in children with SLE (11,12), there is relatively few studies on vitamin D levels in pediatric SLE.There are various reasons for the decrease of serum vitamin D levels in patients   (30,31).IL-6 inhibits the expression of Foxp3 during Treg differentiation (32).Relative studies showed that the levels of inflammatory cytokines, such as IL-6, IL-1, IL-18, and TNF-α, were significantly reduced with the vitamin D treatment group in SLE patients.On the other hand, vitamin D treatment upregulated IL-10 expression (33,34).A study found a positive correlation between elevated serum 25 (OH)D and elevated IL-10.The author showed that after 8 weeks of vitamin D treatment, the levels of IL-10 significantly increased, while there was no significant change in the level of TGF-β1 in multiple sclerosis patients (35).However, our study showed that the levels of IL-10 increased in patients with initialonset childhood SLE, and a negative correlation can be observed between serum vitamin D levels and blood IL-10 levels, which was inconsistent with other studies.
Vitamin D, as an immune regulatory factor, participates in innate and adaptive immunity (36).The immune regulatory role of vitamin D in autoimmune diseases has always been a focus of research (37).Multiple epidemiological studies worldwide have found vitamin D deficiency or insufficiency in various autoimmune diseases (37).Vitamin D not only regulates Th17 and Treg cell differentiation, but also acts on other T lymphocyte subsets, B cells, dendritic cells, etc.Both T cells and B cells express VDR, which is an important target for vitamin D to exert immune regulation.Vitamin D induces tolerance phenotype by acting on antigen-presenting cell, monocyte, natural killer cell, and dendritic cells, enhance chemotaxis of neutrophil (38).
Osteoporosis can occur in patients with SLE, including juvenile patients, possibly due to chronic inflammation affecting bone metabolism and the use of glucocorticoids and other drugs (39).It is recommended to monitor the calcium and phosphorus metabolism as well as vitamin D levels in pediatric SLE patients.
Our research has limitations.The sample size included in this study is not large enough and cannot be subjected to a stratified analysis.We only studied Treg and Th17 cells and related cytokines, but did not include other lymphocyte subpopulations.We studied the relation between the serum 25 (OH)D levels and the ratio of Treg and Th17 cells in peripheral blood, but did not conduct a double-blind controlled randomized study to observe the changes in Treg and Th17 cells after vitamin D treatment.The molecular mechanism by which vitamin D acts on Treg and Th17 cells in SLE patients is not yet well understood.These will be explored in our future research.

Conclusion
The imbalance of Treg and Th17 cell differentiation leads to the suppression of immune function and promotes the development of SLE.In patients with initial-onset childhood SLE, the changes of serum vitamin D levels can affect the proportion of Treg cell subset and TH17 cell subset and can also affect the levels of cytokines related to these T cell subpopulations.The molecular mechanism of action of vitamin D and lymphocyte subpopulations in SLE is complex.Further exploration should be conducted on the role and mechanism of vitamin D in regulating Th17 and Treg subsets, providing a basis for immunotherapy in pediatric SLE.

FIGURE 1
FIGURE 1 Serum 25 hydroxyvitamin D [25(OH)D] levels were decreased in patients with initial-onset childhood SLE, n = 60 for HC and n = 82 for SLE.
decrease of Treg and its dysfunction play a very important role in the pathogenesis of SLE(1).Injecting Treg into SLE mice could alleviate inflammation and reduce tissue damage (1).Our study found that the ratio of Th17 cells significantly elevated in initialonset childhood SLE, while the proportion of Treg significantly decreased compared with healthy controls.However, there have been reports of an increase of the percentage of Treg and Th17 cells rather than a decrease of the number of Treg in SLE patients(2).The research results on the ratio of Treg in SLE patients are inconsistent.Therefore, it is currently believed that not only abnormal proportions but, more importantly, abnormal functions of Treg are involved in the pathogenesis of SLE.

FIGURE 2 Flow
FIGURE 2 Flow cytometry analysis of Treg subset (A) and Th17 cell subset (C) in peripheral blood, n = 60 for HC and n = 82 for SLE.The correlation analysis of 25 (OH)D levels and Treg subset (B) and Th17 cell subset (D) in peripheral blood in patients with initial-onset childhood SLE (n = 82).

TABLE 2
Statistically significant results are highlighted in bold.

TABLE 3
Correlations of the Treg ratio with the clinical and laboratory parameters.
Statistically significant results are highlighted in bold.

TABLE 1
Demographic, clinical, and laboratory characteristics of the studied groups.

TABLE 4
Correlations of the Th17 cell ratio with the clinical and laboratory parameters.