Background Accumulating data has showed that Glucocorticoid-induced Tumor Necrosis Factor Receptor Family-related Protein (GITR), with its ligand (GITRL), plays an important role in promoting T-cell-mediated immunity and exacerbating autoimmunity in animal models, including transient inhibition of Tregs and enhancement of Th17 cells. But its pathogenesis role in primary Sjögren syndrome (pSS) remains unclear.
Objectives Our study aimed to evaluate whether GITRL is related to disease severity and multi-organ involvement in pSS patients and explore the possible mechanisms.
Methods 78 pSS patients and 44 healthy controls were recruited, and the serum level of GITRL was measured by ELISA, and the serum levels of interleukin (IL) -17A, IL-17E, IL-17F, IL-1β, IL-22 and IL-23 were tested by Luminex. CD4+ T lymphocytes were isolated from PBMCs of 5 healthy donors by CD4+ T Cell isolation kit and cultured in TexMACS™ GMP medium with anti-CD3 monoantibody (mAb), anti-CD28 mAb and rhIL-2. For Th17 cell differentiation and the pathway exploration of pAkt, pS6, pSTAT3 and pSTAT5, CD4+T cells were treated either with recombinant human GITRL protein or without the protein for 5 days. Surface marker, intracellular cytokine and signal proteins were detected by flow cytometry. Clinical and laboratory data were also collected.
Results Serum level of GITRL was significantly higher in pSS patients than in HC (Figure 1). There is a negative correlation between elevated levels of serum GITRL and WBC, Neutrophils%, PLT, C3 and C4, and positive correlation with Lymphocytes%, IgG and RF (Table 1). Interestingly, pSS patients with overt hypothyroidism showed higher level of serum GITRL comparing to those with subclinical hypothyroidism and normal thyroid function (Figure 2). Patients with pulmonary involvement had higher serum GITRL level (Figure 3), and patients of moderate to high disease activity (ESSDAI≥5) also showed higher serum level of GITRL (Figure 4). Moreover, Serum GITRL level was positively correlated with IL-17A, IL-17E, IL-17F, IL-1β, IL-22 and IL-23 (Table 2). After GITRL treatment, we found an obvious expansion of CD4+IL-17A+T (Th17) cells and there might be a dose-dependent effect (Figure 5). We also found the mean fluorescence intensity (MFI) of pAkt, pS6, pSTAT3 and pSTAT5 increased after GITRL stimulation (Figure 6).
Conclusions Our results identified the function of GITRL in exacerbating disease activity in pSS patients, and enhancing the differentiation of Th17 cells and the secretion of Th17-related cytokines, and the possible mechanisms might be through the pathways of pAkt, pS6, pSTAT3 and pSTAT5. This finding might provide the theoretical basis of anti-GITRL mAb as a novel therapeutic tageting on Th17 cells in the treatment for pSS patients.
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Disclosure of Interest Y. Gan: None declared, X. Sun Grant/research support from: National Science Foundation of China, L. Li: None declared, J. He: None declared, Z. Li: None declared
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