Increased serum soluble interleukin-2 receptor levels in dermatomyositis are associated with Th17/Treg immune imbalance

Dermatomyositis (DM) represents a multifaceted chronic inflammatory myopathy, primarily manifesting as progressive deterioration of muscular and cutaneous tissues. Despite an incomplete comprehension of DM's etiology and pathogenesis, current evidence implicates the involvement of T lymphocyte infiltration, extensive cytokine release, myositis-specific antibodies, and myositis-associated antibodies in disease development. Serum soluble interleukin-2 receptor (sIL-2R) frequently serves as a marker for T cell activation; however, its role remains elusive. Consequently, this investigation sought to elucidate the association between sIL-2R levels, peripheral blood lymphocyte subset counts, and related cytokines in DM patients, with the aim of uncovering the intricate mechanisms underlying DM and establishing a theoretical foundation for the implementation of precise, targeted, individualized immunomodulatory therapy. In this study, a cohort of 60 dermatomyositis (DM) patients, comprising 32 with inactive DM and 28 with active DM, was enrolled and stratified into inactive and active groups based on the Myositis Disease Activity Visual Analogue Scale (MYOACT). Flow cytometry was employed to quantify the absolute counts of peripheral lymphocyte subsets and CD4+T cell subsets in each group, while a flow cytometry bead array was utilized to measure serum cytokine levels. In a comparative analysis between healthy individuals and patients diagnosed with DM, we observed a marked elevation in serum sIL-2R concentrations (P < 0.001) and T-helper 17 cell/regulatory T cell (Th17/Treg) ratios (P < 0.01) within the latter group. A positive correlation was identified between serum sIL-2R levels and various parameters, including ESR, CRP, VAS, AST, CKMB, LDH, HBDH, PT, APTT, DDi, IL-6, IL-10, and IFN-γlevels (P < 0.05). In contrast, serum sIL-2R levels demonstrated a negative correlation with LY, HGB, ALB, Th17 cell populations, and Th17/Treg cell ratios (P < 0.05). Employing multivariate logistic regression, we identified serum sIL-2R concentrations as an independent risk factor for both disease activity and hepatic involvement in DM patients. Moreover, receiver operating characteristic (ROC) curve analyses revealed that serum sIL-2R levels significantly contributed to the differentiation of disease activity and the detection of liver involvement in DM patients, with areas under the ROC curve (AUC) of 0.757 (95% CI 0.630–0.884, P = 0.001) and 0.826 (95% CI 0.717–0.935, P < 0.001), respectively. This study highlights the potential utility of serum sIL-2R levels as a valuable biomarker for assessing disease activity and liver involvement in dermatomyositis. Elevated serum concentrations of sIL-2R were observed in patients with DM, exhibiting significant associations with Th17 cell populations and Th17/ Treg ratios. These findings indicate that sIL-2R may be implicated in the immunopathogenesis of DM, thereby warranting further investigation to elucidate its role in the disease process.


Introduction
Dermatomyositis (DM) represents an autoimmune disorder characterized by the presence of a distinct rash and symmetrical proximal muscle weakness.Notable cutaneous manifestations encompass Gottron's papules, heliotrope rash, V-sign, and mechanic's hands.Laboratory findings frequently reveal elevated muscle enzyme levels and inflammatory markers.
Importantly, DM extends beyond skin and muscle involvement, potentially affecting critical organs such as the lungs, heart, kidneys, liver, and gastrointestinal tract, thereby necessitating comprehensive management and monitoring of the disease [1].
The etiology and pathogenesis of DM remain incompletely understood, with current evidence pointing towards a multifactorial origin involving genetic, environmental, and immunological factors.Certain individuals present with specific human leukocyte antigen (HLA) molecules, suggesting a genetic predisposition to the disease [2].Environmental triggers, such as viral infections and ultraviolet (UV) exposure, have the potential to initiate or exacerbate DM, resulting in aberrant immune system responses [3].From an immunological perspective, research has primarily focused on T lymphocyte infiltration, cytokine release, and the involvement of myositis-specific antibodies (MSAs) and myositis-associated antibodies (MAAs) in the disease process.During the pathogenesis of DM, T lymphocytes accumulate and release pro-inflammatory cytokines, such as tumor necrosis factor (TNF) and interleukins (ILs), in the vicinity of damaged muscle fibers, thereby exacerbating muscle injury and inflammation [4,5].Moreover, MSAs and MAAs produced by the immune system of DM patients may exert direct or indirect pathogenic effects on myocytes and other tissues, further propelling disease progression [6].
Recent investigations have highlighted the pivotal role of interleukin-2 (IL-2) in modulating immune cell differentiation, as it exerts pleiotropic effects on various immunological processes.IL-2 not only activates effector T cells, promoting immune responses, but also fosters regulatory T cell (Treg) proliferation, thereby maintaining immune tolerance.Consequently, IL-2 plays a significant role in infectious diseases, autoimmune disorders, and malignancies.IL-2 mediates its effects through the IL-2 receptor (IL-2R), a complex composed of three chains: a specific α chain (CD25) and β (CD122) and γ (CD132) chains shared with other cytokine receptors [7].Following T cell activation, IL-2Rα is rapidly expressed on the cell surface, where it binds IL-2, consequently inducing T cell proliferation via autocrine and paracrine mechanisms [8].Low-dose IL-2 has been employed as a therapeutic approach in the management of systemic lupus erythematosus (SLE), type 1 diabetes mellitus (T1D), Sjogren's syndrome, and other autoimmune diseases, with some studies reporting promising efficacy [9][10][11] Soluble interleukin-2 receptor (sIL-2R), a component of the IL-2Rα chain found on the membrane of activated lymphocytes, enters the circulatory system upon shedding from the cell membrane and is regarded as a crucial biological marker of T cell activation [12].Elevated levels of sIL-2R can be detected in healthy individuals, with further increases observed in patients suffering from infections, inflammatory conditions, and autoimmune diseases.In typical immune responses, sIL-2R interacts with IL-2 to modulate the proliferation, differentiation, and cytokine production of T cells, facilitating the regulation of immune system balance and preventing excessive immune responses.However, sIL-2R can also contribute to pathological processes by disrupting immune homeostasis in autoimmune diseases [13].Previous investigations have demonstrated marked elevations of sIL-2R in various systemic conditions, including Sjögren's syndrome, systemic lupus erythematosus, sarcoidosis, and IgG4-related diseases [14][15][16][17].Comparable results have been reported in DM, with He et al. noting increased sIL-2R levels in DM patients, displaying significant correlations with disease severity and acute phase reactants such as erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP), thus serving as disease activity markers [18].Nonetheless, the relationship between sIL-2R concentrations and the absolute counts of peripheral blood lymphocyte subsets, as well as associated cytokines, remains elusive.Therefore, this study aims to analyze the correlations between serum sIL-2R levels and clinical parameters, autoantibodies, peripheral blood lymphocyte counts, and related cytokines in DM patients, with the goal of elucidating the significance of sIL-2R in DM pathogenesis and providing insights for further investigation into its underlying mechanisms.

Patients
A cohort of 60 DM patients, comprising 14 males and 46 females with a mean age of 51.93 ± 14.56 years, was enrolled from the Rheumatology Department of the Second Affiliated Hospital of Shanxi Medical University between October 2020 and October 2022.All patients were diagnosed based on the established criteria by Bohan and Peter [19,20].Exclusion criteria encompassed acute and chronic infections, hematologic disorders, severe liver and kidney dysfunction, malignant tumors, and concurrent autoimmune diseases such as systemic lupus erythematosus, ankylosing spondylitis, or rheumatoid arthritis.Additionally, 30 age and sex-matched healthy controls (HCs), comprising individuals undergoing physical examinations during the same period, were included for comparative analysis.
Disease activity was assessed based on clinical symptoms and laboratory test results, incorporating MYOACTmucocutaneous score, MYOACT-muscle score, and MYOACT-total disease activity score.Inactive dermatomyositis was defined by a score of 0 across all three categories, with this status persisting for one month or longer.Active dermatomyositis was characterized by one or more scores exceeding 0, or all three scores registering 0 but lasting for less than one month.Physician's evaluation of total disease activity was measured using an independent visual analog scale (VAS) derived from the Myositis Disease Activity Assessment Tool (MDAAT) score.Disease activity was appraised using the VAS tool in accordance with the patient's corresponding clinical symptoms, signs, or laboratory abnormalities, with scores ranging from 0 to 10.
Organ involvement was characterized by the presence of additional organ dysfunction at the time of DM diagnosis.Lung involvement encompassed symptoms such as dyspnea, dysphonia, and interstitial lung disease, while heart involvement included arrhythmias and heart failure.Gastrointestinal involvement manifested as dysphagia and gastroesophageal reflux, whereas liver involvement was identified by elevated liver enzymes.Kidney involvement comprised elevated creatinine levels, proteinuria, microscopic hematuria, and renal failure, among other manifestations.

Demographic and disease characteristics
Data collected from all patients included age, sex, DM duration, clinical presentation, and information regarding the presence or absence of treatment.

Laboratory data
Laboratory data were gathered, encompassing white blood cell count (WBC), platelet (PLT) count, erythrocyte sedimentation rate (ESR), C-reactive protein (CRP) levels, coagulation function, muscle enzymes, absolute counts of lymphocytes and CD4+T cell subsets in peripheral blood, as well as peripheral blood cytokines.All laboratory measurements were performed using fresh blood samples collected following a morning fast.

Statistical analysis
For quantitative data, continuous variables with normal distribution were examined and presented as mean ± standard deviation, while non-normally distributed variables were expressed as median (interquartile range).Differences in variables were compared between groups using the independent samples t-test for normally distributed data and the Mann-Whitney U test for non-normally distributed data.Categorical variables were presented as frequencies and compared using the Chi-square test.Correlations between the two clinical measures were determined employing Spearman's rank correlation.Logistic regression analysis was utilized to identify risk factors associated with disease activity in DM patients.Receiver operating characteristic (ROC) curves were applied to detect optimal cutoff values and validity for specific variables, with P values (two-tailed) less than or equal to 0.05 considered statistically significant.Statistical calculations were conducted using SPSS 26.0 and GraphPad Prism 8.0.

Clinical and demographic characteristics
The primary demographic characteristics, disease features, and laboratory data of the 60 DM patients (32 inactive DM patients and 28 active DM patients) are presented in Table 1.The mean ages of the inactive and active groups were 53.72 ± 12.15 and 49.89 ± 16.89, respectively.The active group exhibited a higher frequency of clinical manifestations, such as fever (p = 0.002) and positive rash (p = 0.013), compared to the inactive group.

Peripheral blood lymphocyte subset and cytokine levels in DM Patients Across Varying Disease Activity Groups
Compared to healthy controls, T cells, CD4 + cells, CD8 + cells, NK cells, and total lymphocytes exhibited a significant decrease in DM patients belonging to the active group.T cells, CD8 + cells, NK cells, and total lymphocytes were also reduced in DM patients in the inactive group compared to healthy controls, although no significant difference was observed between DM patients in the inactive and active groups (Fig. 2a).
Upon further analysis of CD4 + cell subsets for differences between the three groups, the results revealed that the number of Th1 cells and Treg cells in DM patients within the active group was significantly lower than that in the healthy group (P < 0.001).In immune responses, Th17 cells and Treg cells counterbalance each other and maintain a dynamic equilibrium, and their ratio can reflect the presence of immune imbalances in the body to a certain extent.Our study found that the Th17/Treg cell ratio was significantly increased in DM patients (P < 0.01), suggesting that the reduced number of Treg cells and the imbalance of the Th17/Treg cell ratio may be associated with disease progression (Fig. 2b).
By comparing cytokine levels in DM patients across different disease activity groups and healthy controls, we observed that the levels of sIL-2R, IL-6, IL-10, IL-17, IFN-γ, and TNF-α were significantly elevated in DM patients within the inactive and active groups, with results being statistically significant.In addition to sIL-2R (p = 0.018), IL-6 and IFN-γ levels also displayed a significant upward trend in the active group compared to the inactive group (p = 0.011 and p = 0.034, respectively) (Fig. 2c).3a).Upon analyzing the association with peripheral blood lymphocyte counts, serum sIL-2R was found to negatively correlate with total lymphocytes, total T lymphocytes, CD4 + cells, CD8 + cells, Th2 cells, Th17 cells, and Th17/Treg cell ratio while positively correlating with the percentage of Treg cells (Fig. 3b).Moreover, a significant positive correlation was observed between serum sIL-2R levels and interleukin-6 (IL-6), interleukin-10 (IL-10), and interferon-gamma (IFN-γ), with correlation coefficients of 0.443, 0.416, and 0.273, respectively, which were statistically significant (Fig. 4).

Association between Serum sIL-2R Levels and Autoantibodies in DM Patients
By comparing serum sIL-2R levels between DM patients with positive and negative antibodies, our results revealed no significant differences in serum sIL-2R levels among patients with positive and negative antibodies for MDA5, anti-Ro52, and anti-Jo-1 (Table 2).In comparison to DM patients with negative anti-Ro52 antibody, serum CK and TNF-α were significantly elevated (P < 0.05).The neutrophil-to-lymphocyte ratio (NLR) serves as a biomarker for assessing human health status, inflammation, immune system response, and disease severity.DM patients positive for anti-Jo-1 antibodies exhibited lower NLRs compared to those negative for anti-Jo-1 antibodies.Furthermore, serum APTT, IL-17, and TNF-α levels were significantly higher in DM patients with positive anti-MDA5 antibodies (Fig. 5).

Clinical value of sIL-2R in DM disease
As serum sIL-2R levels increased, DM patients showed an increased rate of liver involvement, and the difference was statistically significant (p = 0.006) (Table 3).Logistic regression was used to analyze the correlation between cytokines and disease activity and liver involvement in DM .092,95% CI 1.000-1.002,p = 0.009) was associated with liver involvement.All cytokines with p-values less than 0.05 in univariate analysis were considered in multivariate analysis and levels of sIL-2R were found to remain independent factors associated with disease activity and liver involvement in DM patients (Tables 4, 5).
In addition to correlation analysis, ROC analysis was performed to evaluate the clinical utility of sIL-2R in identifying disease activity and liver involvement in DM patients.The results demonstrated that the optimal cut-off value of sIL-2R for distinguishing between inactive and active DM patients was 910.5, and the optimal cut-off value for determining liver involvement in DM patients was 1118.5.The area under the ROC curve (AUC) for disease activity was 0.757 (95% CI 0.630-0.884,P = 0.001) and 0.826 (95% CI 0.717-0.935,P < 0.001) for liver involvement.The sensitivity and specificity for assessing disease activity were 78.6% and 71.9%, respectively, while those for liver involvement were 85.7% and 78.3%, respectively.All DM patients were further stratified into two groups according to the cut-off values: the elevated sIL-2R group and the non-elevated sIL-2R group.The elevated sIL-2R group exhibited a significantly higher proportion of active disease compared to the non-elevated sIL-2R group (71.00% vs 20.70%, P < 0.001).Moreover, the elevated sIL-2R group displayed a significantly higher proportion of liver involvement than the nonelevated sIL-2R group (54.50% vs 5.30%, P < 0.001).These findings suggest that sIL-2R may serve as a risk predictor for disease activity and liver involvement in DM patients (Fig. 6).

Discussion
The pathological alterations in DM are diverse, and its pathogenesis is intricate.Identifying rapid and accurate clinical indicators related to DM is crucial for patient treatment and prognostic evaluation.In line with previous research, we observed elevated serum sIL-2R levels in DM patients compared to healthy controls [21,22].Furthermore, we detected an increased Th17/Treg cell ratio in DM patients.Multivariate logistic regression analysis indicates that elevated serum sIL-2R is associated with DM disease activity and liver involvement, further suggesting that serum sIL-2R may participate in the inflammatory immunoregulatory process and play a pivotal role in the etiological mechanism.This finding offers novel insights for further investigation into     DM, and may have predictive value for disease development and severity.Interestingly, apart from traditional relevant markers, we observed a significant positive correlation between sIL-2R and coagulation parameters (PT, APTT, and DDi), which could imply coagulation abnormalities in DM patients.sIL-2R might be involved in regulating coagulation in DM patients, thereby exacerbating disease development and progression.Previous studies have revealed a close interaction between the immune and coagulation systems, with inflammation and coagulation disorders considered common pathophysiological processes in numerous autoimmune diseases [23].In DM, inflammation and immune abnormalities may lead to vascular endothelial cell injury, hemodynamic alterations, and activation of coagulation factors, which induce thrombosis and microcirculatory disturbances.Myocardial involvement in DM can result in sudden death, suggesting that immune microthrombi and a hypercoagulable state in vivo may represent crucial new mechanisms in DM, in addition to electrophysiological disorders and other related factors.These findings provide a novel therapeutic strategy to improve DM patient clinical outcomes by intervening in the interaction between the immune and coagulation systems.Moreover, our results align with those of other autoimmune diseases: D-Di, a response indicator of hypercoagulability and hyperfibrinolysis in the body, has been shown to increase to some extent in many autoimmune diseases, such as systemic lupus erythematosus and rheumatoid arthritis [24,25].This observation suggests that sIL-2R may play a similar role in various autoimmune diseases, providing new insights for diagnosis and treatment in these conditions.
In numerous preceding investigations, a substantial decrease in the quantity of Treg cells within the peripheral blood of DM patients has been consistently observed [26,27].This finding aligns with our conclusions and further substantiates the notion that the diminution of Treg cells and their principal effector cytokines in the skin and serum of DM patients may play a pivotal role in the disease's pathogenesis.Furthermore, our research has unveiled a negative correlation between serum sIL-2R concentrations and Th17 cell counts, as well as a positive correlation between sIL-2R levels and Treg cell proportions.This implies that serum sIL-2R may exert immunosuppressive effects by attenuating Th17 cell activity or enhancing Treg cell activity.Nevertheless, the precise mechanism through which serum sIL-2R inhibits Th17 cell activity and fosters Treg cell activity remains elusive.Potential explanations include sIL-2R's capacity to impact Th17 cell proliferation and differentiation by competing with IL-2R binding sites on the cellular surface, consequently reducing IL-2's affinity for IL-2R [28].Interestingly, it has been reported that sIL-2R, upon forming a complex with IL-2, can amplify the biological activity of IL-2, thereby promoting Treg cell growth and functionality more effectively [29].Additionally, sIL-2R might modulate Th17 cell function by influencing signaling pathways associated with the differentiation and activity of Th17 and Treg cells [30].Such pathways include STAT3, RORγt, and TGF-β, as well as STAT5, FoxP3, and TGF-β.Moreover, sIL-2R could potentially affect the function and homeostasis of Th17 and Treg cells by orchestrating other signaling pathways [31], such as the Akt/mTOR, PI3K/Akt, and NF-κB pathways.In our cross-sectional analysis, we observed a counterintuitive negative correlation between IL-17 and Th17 cells, as well as the Th17/ Treg ratio.Th17 cells are a class of IL-17-producing T cells known for their pathogenic roles in various autoimmune diseases.This finding diverges from established knowledge, prompting an exploration of potential explanations.Firstly, Th17 cells are not a homogenous population but comprise diverse subsets with potentially distinct functions.Secondly, IL-17 is not exclusively secreted by Th17 cells but also by other immune and nonimmune cells, including γδ T cells, natural killer (NK) cells, and monocytes [32,33].These cells may contribute to the pathogenesis and progression of autoimmune diseases such as DM.Furthermore, other immune cells and cytokines, including Treg cells, IL-10 and TGF-β, may modulate the differentiation of Th17 cells and IL-17 production.Immune cells coordinate the intensity and duration of immune responses through intricate feedback mechanisms.A similar feedback loop may exist in DM, whereby elevated IL-17 levels trigger a decrease in Th17 cell numbers to mitigate excessive inflammation.Notably, DM represents a heterogeneous group of diseases, and pathophysiological distinctions may underlie the contrasting correlation patterns between IL-17 and Th17 cells.Further investigation into the relationship between IL-17 and Th17 cells in DM patients and the mechanisms governing their interplay will enhance our understanding of the pathophysiological processes underlying dermatomyositis.
In conclusion, an imbalance between Th17 and Treg cells constitutes a critical mechanism underpinning the pathogenesis of DM. sIL-2R may modulate the Th17/Treg balance by suppressing Th17 cell proliferation or enhancing Treg cell proliferation, thereby ameliorating DM symptoms.A significant correlation was observed between serum sIL-2R levels and the Th17/Treg ratio, suggesting that sIL-2R serves as a valuable indicator for monitoring Th17/Treg immune imbalances in patients with DM.Elucidating the mechanisms by which sIL-2R regulates the equilibrium between Th17 and Treg cells will facilitate the identification of novel therapeutic targets and strategies for treating autoimmune diseases such as DM.
In conjunction with sIL-2R, our study revealed notable elevations in IL-6 and INF-γlevels within the active DM cohort, indicating an intensified inflammatory state and immune response in DM patients.IL-6, a pleiotropic cytokine possessing both pro-and anti-inflammatory properties, has been shown to facilitate muscle cell depletion and may serve as a predictive biomarker for disease activity in adult and adolescent DM populations [34].INF-γ, a multifaceted lymphokine with extensive immunomodulatory effects, assumes a crucial role in DM pathogenesis.Elevated INF-γ levels have been observed in DM patients presenting with rapidly progressive interstitial lung disease and can be detected in muscle tissue samples [34,35].Furthermore, our investigation uncovered a significant positive correlation between serum sIL-2R concentrations and those of IL-6, IL-10, and IFN-γ.This suggests a potential synergistic influence on the pathological processes underlying DM, thereby impacting the disease's progression and course.In conclusion, the intricate interplay between sIL-2R and other cytokines embodies the complex interactions of the immune system in DM, furnishing a theoretical foundation for the development of targeted therapeutic interventions.
DM is frequently complicated by pulmonary interstitial disease (ILD), particularly in patients positive for the MDA5 antibody [36].These individuals are predisposed to acute progressive pulmonary interstitial disease and face poor prognoses, resulting in increased morbidity and mortality among myositis patients.In this study, we investigated the association between serum biomarkers and myositis-specific and related autoantibodies by analyzing DM patients with and without MDA5 antibodies.Our results demonstrated that IL-17 levels were significantly higher in MDA5 antibody-positive patients compared to those negative for the antibody.This finding is consistent with previous reports indicating that IL-17 promotes pulmonary fibrosis in multiple studies [37].Nevertheless, we observed no significant differences in serum sIL-2R levels between patients positive and negative for MDA5, anti-Ro52, and anti-Jo-1 antibodies.
DM is known to affect multiple organ systems, including the liver.Upon conducting a multiple logistic regression analysis, we found that serum sIL-2R levels constitute an independent factor influencing disease activity and liver involvement.Moreover, ROC analysis supports the utility of serum sIL-2R levels in detecting DM disease activity and liver involvement, suggesting that these levels serve as potential biomarkers for assessing both aspects.Our findings indicate that serum sIL-2R levels may hold diagnostic value in evaluating disease activity and liver involvement in DM patients, and could potentially be employed as an indicator for clinical assessment of activity and risk of liver involvement in future studies.
Although our study presents noteworthy and clinically pertinent findings, certain limitations warrant consideration.Firstly, this retrospective investigation encompasses clinical and serological data collected from patients at a single medical center.Secondly, the relatively small patient sample size necessitates further enlargement to enable a more comprehensive examination of DM pathogenesis and the identification of more robust indicators for evaluating disease-related outcomes.

Conclusion
In this comprehensive analysis, we observed markedly reduced serum sIL-2R levels in DM patients compared to those in a healthy control cohort.Concurrently, significant correlations were identified between serum sIL-2R concentrations in DM patients and liver enzymes, muscle enzymes, peripheral blood Treg cells, Th17 cells, as well as the Th17/ Treg balance.The elevated serum sIL-2R levels are associated with heightened risk for DM activity and liver involvement, implying a potential connection between sIL-2R and the DM disease course.Consequently, sIL-2R may represent a promising target for the prevention and treatment of DM, warranting further investigation to elucidate its mechanistic role and therapeutic potential in this context.

Fig. 3 3
Fig. 3 Heat map of correlation of serum sIL-2R levels.a Heat map of correlation between serum sIL-2R levels and clinical activity indicators.b Heat map of correlation between serum sIL-2R level and peripheral blood lymphocytes (*P < 0.05, **P < 0.01, ***P < 0.001)

Fig. 6
Fig. 6 Clinical value of sIL-2R in DM disease.a ROC curve for sIL-2R evaluation of DM disease activity.b ROC curve of sIL-2R for evaluation of liver involvement in DM

Table 1
Clinical characteristics of inactive and active groups *

Table 4
Logistic regression analysis of disease activity and cytokines in patients with DM *

Table 5
Logistic regression analysis of hepatic involvement and cytokines in patients with DM *