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Abstract
Background Systemic sclerosis (SSc) is a chronic inflammatory disease with complex pathogenesis. The role of regulatory T cells (Tregs) in the development of SSc has started being studied during the last decade with new aspects being disclosed continuously. Although there is a general agreement in the medical literature regarding the decreased functional capacity of circulating Tregs in SSc, the alteration of absolute number of Treg cells as well as orther CD4 +T lymphocyte subsets in SSc is still unclear.
Objectives The aim of the present study was to explore the absolute number status of CD4 +T subsets in peripheral blood of patients with SSc using our modified flow cryometric method and investigate whether the imbalance of Th17 and Treg cells can be corrected by supplementing low dose interleukin −2 (IL-2).
Methods The peripheral CD4 +T subsets from 54 patients with SSc and 30 healthy control subjects were analysed. The patients were divided into the untreated group (n=29) and treated group (n=25). The patients were also divided into group A (n=28) who only used low-dose glucocorticoids (GCS), and group B (n=26) who also used immunosuppressant cyclophosphamide (CYC), methotrexate (MTX), leflunomide (LEF) or mycophenolate mofetil (MMF). In addition, 21 patients from 54 patients, given a small dose of IL-2 (50WIU) treatment for a 5 day course, divided into pre-treatment group and post-treatment group. Directly using the results from flow cytometry combined with internal standard beads, absolute number of peripheral CD4 +T subsets from the subjects in each group were calculated.
Results Although there were some changes among CD4 +T cell subsets in peripheral blood from these SSc patients, the major alteration was the reductions of Treg cell absolute number. Compared with the normal controls, the number of CD4+CD25+FOXP3+Treg cells were significantly decreased in all patients (p=0.001), in untreated group (p=0.029), in treated group (p=0.029), in group A (p=0.006) and in group B (p=0.004). The ratio of Th17/Treg in total patients (p=0.008) and group B (p=0.001) were significantly higher than that in normal control group, and the group B was significantly higher than the group A (p=0.032). Moreover, the number of Th17 cells in group B was significantly higher than that in group A (p=0.023). After IL-2 treatment, the absolute number of CD4+CD25+FOXP3+Treg cells (p<0.001) were significantly increased, while the number of Th1, Th2 and Th17 cells were slightly higher than those before treatment with IL-2 (p>0.05). Since Th17 cells increases significantly lower than Treg cells, so their ratio decreases significantly (p=0.001) to get re-balance.
Comparison of the levels of CD4+T lymphocyte subgroups among different groups. (A and D) The percentage of Th2 cells were significantly increased in untreated group and Group A compared with healthy controls. (B and E) The number of CD4+CD25+FOXP3+Treg cells decreased in patients with SSc, whether or not they use immunosuppressive agents. (C and F) The ratio of Th17/Treg cells was found to be significantly increased in Group A, as well as between Group B and Group A. *P<0.05; **P<0.01; ***P<0.001.
Comparison of the levels of CD4+ T lymphocyte subgroups among different groups as well as before and after IL-2 treatments. (A) The percentage of Th2 cell were significantly increased before and after IL-2 treatment compared with healthy controls, and the percentage of Treg cells significantly increased after IL-2 treatment. (B) The number of CD4+CD25+FOXP3+Treg cells decreased in patients before IL-2 treatment, but increased significantly after IL-2 treatment (p<0.05); while Th1, Th2 and Th17 cells after IL-2 treatment were slightly higher than those before treatment (p>0.05). (C) The ratio of Th17/Treg cells were found to be significantly increased after IL-2 treatment. *P<0.05, **P<0.01, ***P< 0.001.
Conclusions The absolute number of peripheral CD4 +CD25+FOXP3+Treg cells decreased in untreated patients, indicating that this reduction arising from the disease itself. Our findings suggest that SSc progression is associated with the absolute decrease of peripheral Treg cells. Although there was no statistically significant different, absolute number of the Treg cells trended to decrease in patients treated with immunosuppressive agents. The use of IL-2 can effectively up-regulate Treg cells as well as increase Th17 to a certain extent, which restores the balance of Th17/Treg cells, but the long-term effect needs further investigation.
Disclosure of Interest None declared