METHODS
Subjects
In this retrospective study, peripheral blood samples were obtained from 45 psoriatic patients (24 men and 21 women; 21–58 years of age; 17 psoriasis vulgaris (PV), 18 psoriasis arthritis (PA), 5 erythrodermic psoriasis (EP), and 5 generalized pustular psoriasis (GPP)) and 40 healthy controls (19 men and 21 women; 23–53 years of age) from February 2020 to November 2021. The inclusion criteria were a documented diagnosis of psoriasis and a Psoriasis Area and Severity Index (PASI) ≥ 10. The exclusion criteria were another severe or progressive autoimmune/inflammatory disease; haematological disorders; vital organ failure; cancer; and active HIV, hepatitis B virus, or Epstein–Barr virus infections.
Study Design and Treatment
The 45 psoriatic patients received methotrexate at 7.5 mg per week for 12 weeks to control disease activity. From 13 to 24 weeks, the patients received conventional therapy combined with three cycles of low-dose IL-2 therapy using recombinant human IL-2 (rhIL-2, Beijing, China). Each of the three cycles consisted of daily subcutaneous injections of IL-2 for 2 consecutive weeks, followed by a 2-week break. The daily dose in every cycle was 0.5 million international units (MIU) of IL-2 for all patients. This study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of the Second Hospital of Shanxi Medical University (No. 2021-YX-010). Written informed consent was obtained from all participates prior to their enrolment in this study.
Measurement of Inflammatory Indices
The peripheral neutrophil count, leukocyte count, lymphocyte count, platelet count, neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), erythrocyte sedimentation rate (ESR), and c-reactive protein (CRP) concentration in serum were recorded.
Blood Sample Collection and Preparation
Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized peripheral blood by density gradient centrifugation with Ficoll–Paque PREMIUM 1.077 (GE Healthcare Life Sciences, Pittsburgh, PA). The cells were then resuspended in RPMI 1640 medium containing phorbol 12-myristate13-acetate (PMA) (50 ng/ml), GolgiStop (1 µl), and ionomycin (1 µg/ml) for 5 h in an incubator at 37 °C with 5% CO2.
Cell Staining
The stimulated cells were stained with fluorescein isothiocyanate (FITC)–labelled CD4 antibody for 30 min at 4 °C. After surface staining, the cells were fixed, permeabilized, and stained with interferon-γ (IFN-γ)-phycoerythrin (PE) (Th1), IL-4-PE (Th2), and IL-17-PE (Th17). For Treg cells, the PBMCs were incubated with FITC–labelled CD4 and allophycocyanin (APC)-labelled CD25 antibodies at 4 °C for 30 min. The cells were then treated with 1 ml of freshly prepared fixation/permeabilization solution followed by staining with PE-labelled Foxp3 antibody.
Flow Cytometric Analysis
The percentages of Th1, Th2, Th17, and Treg cells were detected by flow cytometry (Calibur, BD, USA) and analysed by fluorescence-activated cell sorting (FACS) (Canto II, BD Biosciences, San Jose, CA) followed by data analysis using FlowJo 7.6 software (TreeStar, San Carlos, CA).
Cytometric Bead Array
The cytokines in serum from peripheral blood were analysed using a cytometric bead array human Th1, Th2, Th17, and Treg cytokine kit (Becton Dickinson, USA) along with a FACS Calibur flow cytometer. The beads were coated with antibodies that specifically reacted with each of the cytokines to be detected in the multiplex system. The beads could be differentiated by their sizes and their distinct spectral addresses. The serum concentrations of IL-2, IL-4, IL-6, IL-10, IL-17, IFN-γ, and tumour necrosis factor (TNF)-α were determined using the cytometric bead array technique.
Evaluation of Therapeutic Efficiency
Therapeutic efficiency was primarily evaluated using the quantitative PASI score, which measures the severity of psoriatic lesions based on area coverage and plaque appearance, including scaling, infiltration, and erythema. PASI 50 and PASI 75 indicated 50% and 75% improvements in PASI, respectively. PA patients were identified according to the criteria of the American College of Rheumatology (ACR20 response). At weeks 0 and 24 of treatment, all observation indices were reviewed, and the adverse reactions of patients were recorded in detail.
Statistical Analysis
SPSS version 22.0 was used for all data analyses (SPSS Inc., Chicago, IL, USA), and P < 0.05 was considered statistically significant. Continuous variables were expressed as mean ± standard deviation, and the two groups were compared using Student’s t-test. One-way analysis of variance and least significant difference (LSD) test were used to analyse multiple groups. Median (interquartile range) and Wilcoxon test were used for abnormally distributed data when there were two groups. Kruskal–Wallis and LSD tests were used for multiple groups. For categorical variables, counts (percentages) were described and compared using the χ2 test. Relationships between two variables were evaluated by Spearman correlation analysis.
DISCUSSION
Th17/Treg immune imbalance has been reported to contribute to the pathogenesis of psoriasis [
1‐
3]. Normally, naïve CD4
+ T cells differentiate into Th17 and Treg cells. The Th17 cells produce IL-6, IL-17, and TNF-α to mediate the inflammatory reaction in psoriasis. Meanwhile, Treg cells produce suppressive cytokines such as IL-10, transforming growth factor-β, and cell–cell interaction, thereby playing an important role in immunosuppression. Th17 and Treg cells regulate each other to maintain immune balance [
6‐
8].
In this study, the proportion of Th17 cells in peripheral blood was significantly increased in the patients with psoriasis compared with the healthy controls, and the Th17 cell proportion positively correlated with NLR, ESR, CRP, and PASI. Moreover, the Treg cell percentage was dramatically decreased and negatively correlated with NLR, ESR, CRP, and PASI. These changes resulted in a dramatic increase in the Th17/Treg ratio, which increased in the following order: PV < PA < EP < GPP. The Th17/Treg ratio positively correlated with NLR, ESR, CRP, and PASI. However, no significant differences in Th1, Th2, or the Th1/Th2 ratio were observed between the psoriasis and healthy groups. These results suggest that the percentages of Th17 and Treg cells are closely related to psoriasis disease severity.
Our findings agree with Ma
et al. who reported Th17/Treg immune imbalance in PV patients [
9]. However, Zhang
et al. found that Th17 and Treg cells were increased in psoriatic patients in the peripheral circulation, both of which positively correlated with PASI score [
10,
11]. These distinct results may stem from differences in disease states, Treg definition, the time frame from which the PASI scores were derived, and the investigated psoriasis subtypes [
12,
13]. Overall, these studies suggest that Th17/Treg immune imbalance potentially contributes to psoriasis pathogenesis.
The immune imbalance between Th17 and Treg cells is associated with the disorder of pro-inflammatory and anti-inflammatory cytokines in psoriasis. In this study, the serum concentrations of IL-10 in the psoriasis group were decreased compared to the healthy group, and the IL-10 concentrations negatively correlated with NLR, ESR, CRP, and PASI. Reduction in IL-10 concentrations indirectly showed that Treg cells’ suppressive function was impaired, and this could not effectively resist the pro-inflammatory effect of increased Th17 cells secretion of IL-17. The serum concentrations of IL-17 were higher in the psoriatic patients compared to the healthy controls. As a result, the IL-17/IL-10 ratio was increased in the psoriasis group compared to the healthy group, and it positively correlated with NLR, PLR, ESR, CRP, and PASI. The increased IL-17/IL-10 ratio further confirms the imbalance of Th17/Treg cells in psoriasis.
IL-6 is a critical factor in the homeostatic balance between Th17 and Treg cells, and high concentrations of IL-6 increase Th17 cell production in cooperation with transforming growth factor-β and decrease Treg cell activity [
14,
15]. In this study, the concentrations of IL-6, IFN-γ, and TNF-α were significantly elevated in the psoriasis group compared with the healthy group. The
serum concentrations of IL-6 positively correlated with ESR and PASI; the
serum concentrations of IFN-γ positively correlated with PASI, and the TNF-α
serum concentrations positively correlated with NLR, ESR, CRP, and PASI. Yang
et al. reported that the secretion of IFN-γ and TNF-α by Tregs was increased in psoriatic patients, suggesting that the conversion of Tregs to Th1 cells may impair Treg cell function, leading to the excessive activation of conventional T cells in autoimmune diseases [
16]. Josefowicz
et al. also reported the impaired function of Treg cells, leading to Th2 cell overactivation [
17]. These Treg defects were associated with disease severity, and the recovery of Treg may be an effective method for the treatment of psoriasis.
Low-dose IL-2 therapy aims to compensate for the shortage of Treg cells and expand their population. Treg cells are more sensitive to IL-2 and require by far much lower doses of IL-2 for their stimulation because Treg cells expressing CD25 on the surface can form high-affinity IL-2 receptor complex, which is composed of α (CD25), β (CD122), and γ chain (CD132). In contrast, CD4
+ conventional T cells, CD8
+ T cells, or natural killer cells do not express CD25 and need high-dose IL-2 for stimulation [
5,
18,
19]. IL-2 directly activates and phosphorylates the transcription factor STAT5, which upregulates the key transcription factor Forkhead helix protein 3 (Foxp3) of Treg cells, thereby inducing the differentiation of Treg cells and maintaining their activity [
20,
21]. However, IL-2 activation of STAT5 can inhibit the expression of retinoid-related orphan receptor γt and then inhibit the differentiation of Th17 cells [
22,
23]. As mentioned above, IL-2 is a crucial factor in the production and maintenance of Treg cells and reverses the imbalance of Th17/Treg cells.
Low-dose IL-2 has attracted attention in the treatment of autoimmune diseases. Clinical studies have shown that many immune-related diseases, such as systemic lupus erythematosus (SLE), rheumatoid arthritis, polymyositis/dermatomyositis, Sjögren’s syndrome, Treg cell defects, and Th17/Treg cell imbalance, may benefit from low-dose IL-2 treatment [
24‐
28]. Humrich
et al. used low-dose IL-2 to treat SLE with moderate-to-severe disease activity, supporting that Treg defects were associated with disease severity and could be corrected using low-dose IL-2, especially for active and refractory SLE [
24]. More recently, in an open-label, multicentre phase I/IIa clinical trial that used low-dose IL-2 across 11 individual diseases suffering from autoimmune inflammatory conditions, the level of Treg cells in patients significantly increased (2.0 ± 0.6) fold, without effector T cell activation, and the clinical symptoms improved significantly [
29].
In this study, 45 psoriatic patients were treated with low-dose IL-2 combined with methotrexate, which greatly amplified the percentage of Treg cells. The proportion of Th17 cells and the Th17/Treg ratio decreased significantly. No significant differences in Th1, Th2, or the Th1/Th2 ratio were observed after treatment. The percentage of Treg cells was fivefold more than that of Th17 cells, which means anti-inflammatory Tregs play important roles in restoring the Th17 and Treg cell balance in psoriasis. Wang
et al. reported that in PA patients that exhibited low Treg numbers, low-dose IL-2 combination with conventional therapy not only increased the absolute numbers of Tregs but also rapidly alleviated PA disease activity as indicated by the disease activity score without any apparent side effects [
30]. Therefore, these findings strengthen the concept that low-dose IL-2 corrects Treg cell defects, promotes Treg cell expansion, and restores immune balance.
At the same time, low-dose IL-2 treatment also significantly increased the concentrations of anti-inflammatory cytokines (IL-4 and IL-10) but decreased the IFN-γ/IL-4 and IL-17/IL-10 ratios and regulated the production of pro-inflammatory factors (IFN-γ, IL-6, IL-17, and TNF-α), leading to significant decreases in the IFN-γ/IL-4 and IL-17/IL-10 ratios. These results indicate that low-dose IL-2 can not only restore the activity of Treg cells but also improve the pro-inflammatory environment in psoriasis and rebuild the immune balance.
Low-dose IL-2 in psoriatic patients can reduce disease activity by promoting the expansion of circulating Tregs and restoring immune balance. After low-dose IL-2 treatment, the clinical manifestations of psoriasis improved, and the PASI 50 and PASI 75 were 68.89% and 22.22% at week 24, respectively. The ACR20 percentages were 35% after treatment in PA. Low-dose IL-2 was well tolerated, and most adverse events were mild and of a transient nature, suggesting a favourable safety profile. He
et al. reported low-dose IL-2 therapy in a randomised, double-blind, placebo-controlled clinical trial in 60 patients with active SLE. At week 12, the SLE responder index-4 response rates were 55.17% and 30.00% for the IL-2 and placebo groups, respectively. The prednisone dose was reduced by more than 50% at week 24. The levels of anti-dsDNA antibodies were decreased in the IL-2 group compared with the placebo group, whereas the complement C3 and C4 levels were increased. An expansion of the Treg cells was observed in this study. The number of Treg in peripheral blood was increased, the Tfh and Th17 cells were decreased, and the (Tfh+Th17)/Treg ratio was decreased [
31,
32].
After administering low-dose IL-2 for psoriasis, it also decreased the inflammatory indicators of disease activity, including the leukocyte count, neutrophil count, ESR, CRP, and NLR in
serum after treatment. NLR is a new inflammatory index calculated by dividing the number of neutrophils by the number of lymphocytes. Neutrophils reflect systemic inflammation, and lymphocytes reflect the immune status in psoriatic patients [
33,
34]. We found that circulating NLR was significantly higher in the psoriasis group compared to the healthy group. NLR positively correlated with the levels of Th17, IL-17, and TNF-α along with the Th17/Treg and IL-17/IL-10 ratios. NLR negatively correlated with Treg cells and IL-10 concentrations. After low-dose IL-2 treatment, NLR levels decreased in the
serum of psoriatic patients. Kim
et al. reported that NLR was increased in psoriatic patients compared to the control group and that NLR positively correlated with PASI [
35]. Altogether, these results indicate that NLR combined with conventional inflammatory indicators is useful markers for evaluating systemic inflammation in psoriatic patients. Thus, NLR may serve as simple, convenient, and cost-effective biomarkers to monitor the course of psoriasis before and after systemic therapy [
33].
In conclusion, inflammatory diseases are associated with insufficient Tregs in psoriasis, leading to Th17/Treg immune imbalance, which contributes to psoriasis pathogenesis, supporting the regulation of Th17/Treg imbalance as a potential psoriasis treatment. Notably, low-dose IL-2 not only selectively activated and expanded Treg cells but also, with conventional therapies, may improve the anti-inflammatory environment and rebuild immune balance in psoriatic patients. Low-dose IL-2 can rapidly alleviate disease activity and improve clinical symptoms significantly. Low-dose IL-2 showed good safety and was well tolerated. As a new immunomodulatory therapy, low-dose IL-2 may be an effective treatment option for psoriatic patients.