| BackgroundPrimary immune thrombocytopenia (ITP) is currently defined as an acquired autoimmune disorder in which platelets are prematurely destroyed in the reticuloendothelial system by platelet autoantibodies, charaeterized by a low peripheral blood platelet count and inereased number of megakaryocytes in bone marrow. Classically, ITP is primarily due to various platelet membrane autoantibodies, including platelet glyeoproteins such as glycoprotein Ⅱb/Ⅲa (GP Ⅱb/Ⅲa) or GPIb/Ⅸ complexes autoantibodies opsonizing the individual’s platelets, resulting in markedly enhanced Fc receptor (FcR)-mediated phagocytosis and destruction by macrophages in the reticuloendothelial system within the spleen.But the pathogenesis of ITP has obvious heterogeneity. With the in-depth study, abnormal humoral immunity plays a decisive role in the pathogenesis of ITP point of view has been challenged. Since platelet autoantibodies can be detected in only50-70%of ITP patients and remission can occur despite the presence of platelet autoantibodies. Most studies suggest that ITP patients showed Thl response mode, and Thl response can promote antibody production, but also directly promote lymphocyte cytotoxicity. Therefore, in recent years the cellular immune dysfunction in the pathogenesis of ITP is more and more attention. The study found the abnormal cellular immunity plays an important role in the pathogenesis of ITP, the balance between Th1/Th2cell subsets imbalance, quantity of CTL and Th17cells increased, the number of Treg cells decreased and abnormal function is a key factor to abnormal ITP cell immune responses.Interleukin (IL)-27, a novel member of the IL-12family, play an important role in the regulation of T cell differentiation. Research confirms, IL-27can promote Thl differentiation from naive T cells through signal transducer and activator of transcription (STAT)1and STAT3-dependent pathways, inducing the production of IFN-γ. However, more recent studies revealed s that IL-27could inhibit the expression of retinoid related orphan receptor gamma (RORyt), one of the key lineage-specific transcription factors that direct Th17development. Therefore, IL-27is a pleiotropic immunomodulatory cytokines play a dual role in the process of immune response.ITP is an autoimmune disorder that has also shown a Thl dominant profile and up-regulated Th17expression. The roles of IL-27in ITP remain unresolved and the conclusions of the study are controversial. We hypothesized that IL-27may participate in ITP pathogenesis by differentiation of Th cells. In the present study, we will combine IL-27plasma levels and messenger RNA (mRNA) expression, analyze the relationship between IL-27with Th cell subsets, cytokines secretion and laboratory parameters to explore IL-27in the pathogenesis of ITP.ObjectiveWe researched the expression level of IL-27in order to evaluate the role of IL-27in the pathogenesis of ITPMethods1.31cases of cITP patients. Normal controls come from36healthy volunteers. 2. Thl, Th2and Th17cells were evaluated by flow cytometry.3. The plasma levels of IL-27, IFN-y, IL-4and IL-17A in peripheral blood of cITP patients and healthy controls were determined by FlowCytomix Technology.4. The mRNA levels of IL-27, T-bet, GATA-3and RORyt were detected and measured by real-time reverse transcriptase polymerase chain reaction (RT-PCR).5. Separate mononuclear cells from peripheral blood of cITP patients and controls, use exogenous rhIL-27to culture2d. Th1, Th2and Th17cells were evaluated by flow cytometry.Results1. The plasma concentrations of IL-27in cITP patients were found to be significantly increased compared with healthy controls (60.42±43.68vs32.91±20.37pg/ml, P=0.001), plasma IFN-y levels of cITP patients were significantly increased compared with healthy controls (40.12±19.14vs23.21±10.87pg/ml, P<0.001), plasma IL-4levels were decreased considerably compared with healthy controls (57.69±17.71vs78.7±29.82pg/ml, P=0.001), while IL-17A levels were significantly increased compared with healthy controls (29.6±28.08vs9.21±7.9pg/ml, P<0.001).2. The percentage of Thl cells was significantly higher in the cITP patients than in the healthy controls (19.38±9.18%vs13.19±5.33%, P=0.004). In addition, the percentage of Th17cells was also significantly higher in the cITP patients than in the healthy controls (2.39±1.32%vs1.07±0.51%, P<0.001). However, the percentage of Th2cells was significantly lower in the cITP patients than in the healthy controls (1.15±0.52%vs1.9±0.64%, P<0.001).3. The plasma level of IL-27were positively correlated with the percentage of Thl cells in the PBMCs population in cITP patients (r=0.733, P<0.001). In this study, we did not find any correlation between the plasma levels of IL-27and the percentage of Th2(r=-0.166, P=0.371), Th17(r=0.229, P=0.215) cells in the PBMCs of cITP patients. Because an insufficient platelet count is a main bleeding cause of cITP, we analyzed the association between IL-27and platelet counts in cITP patients. There was no significant correlation between IL-27levels and the platelet counts (r=0.194, P=0.296).4. The mRNA levels of IL-27, T-bet and RORyt in cITP patients were significantly higher than those in healthy controls (IL-27:cITP3.46fold vs control, P<0.05; T-bet:cITP7.37fold vs control, P<0.05; RORyt:cITP11.1fold vs control, P<0.05). There was no significant difference in GATA-3mRNA level between cITP and healthy controls (P>0.05).5. Exogenous rhIL-27significantly up-regulated the Thl cells in cITP patients and normal controls (cITP patients:19.63±5.88%vs31.7±10.71%, P=0.007; normal controls:9.93±2.66%vs18.46±4.84%, P=0.002). In contrast, the Th2cells down-regulated in the presence of rhIL-27(cITP patients:1.15±0.21%vs0.96±0.18%, P=0.031; normal controls:2.15±0.49%vs1.68±0.40%, P=0.035). In normal controls, rhIL-27could down-regulate Th17cells significantly, but had no effect in cITP patients (cITP patients:2.41±0.57%vs2.33±0.4%, P=0.62; normal controls:1.38±0.45%vs0.81±0.35%, P=0.016).Conclusions1. The mRNA levels of IL-27and the plasma concentrations of IL-27in cITP patients were significantly elevated, suggesting that IL-27might be involved in the pathogenesis of cITP.2. The percentage of Thl and Th17cells accompanied by the mRNA expression of T-bet and RORyt were significantly higher in cITP patients than in healthy controls, indicating that Thl and Th17dominant disease in cITP, and cellular immune abnormalities is important in the pathogenesis of cITP. 3. The plasma level of IL-27were positively correlated with the percentage of Th1cells in the PBMCs population in cITP patients, and exogenous rhIL-27could significantly up-regulate the percentage of Thl cells in vitro, indicating that IL-27is by promoting Thl cell differentiation involved in the pathogenesis of cITP. BackgroundPrimary immune thrombocytopenia (ITP) is currently defined as an acquired autoimmune disorder in which platelets are prematurely destroyed in the reticuloendothelial system by platelet autoantibodies. Although auto-reactive B lymphocytes secreting anti-platelet antibodies are considered as the primary immunologic defect in ITP, T-lymphocyte abnormalities are considered important in the pathogenesis of ITP. It has become reported that the high Th1/Th2ratio was closely related to the etiology and disease status of ITP, and described a higher Thl response, and quantity of Thl7cells increased, the number of Treg cells decreased and abnormal function is a key factor to abnormal ITP cell immune responses. However, the mechanism of cellular immune abnormalities in patients with ITP remains indecipherable.Interleukin (IL)-12family cytokines is composed of heterodimeric cytokines, including IL-12, IL-23, IL-27and IL-35, they are produced by antigen-presenting cells (APC) such as macrophages and dendritic cells and play critical roles in the differentiation of helper T (Th) cells. IL-12induces IFN-Y production through activating signal transducer and activator of transcription (STAT)4signaling pathway and enhances cellular immune response, IL-23induces the production of IL-17by activating STAT3, promoting development of Th17cells, IL-27has a close relationship with the occurrence, development, and prognosis of many immune inflammatory diseases related to Thl, and mediates immune homeostasis. IL-27exerts a negative effect on immunomodulation by inhibiting the differentiation of Th2and Thl7cells, and IL-35may be a potential immunosuppressive cytokine. IL-12family cytokines play an important role in regulating the differentiation of Th cells. However, the exact roles of IL-12family members in ITP and their correlation to disease progression remain unknown.In recent years, some reports have shown that high-dose dexamethasone (HD-DXM) might be a promising alternative to prednisone as the first-line treatment in ITP patients. HD-DXM could correct Thl polarization by altering the Th1/Th2cytokine profile, and increased expression of CD4+CD25+Treg and FoxP3. In the present study, we investigate the roles of IL-12family cytokines in ITP and the possible effects of HD-DXM on IL-12family members in ITP patients. All of these will provide reference data for further study of the role of IL-12family cytokines in the pathogenesis of ITP.ObjectiveThis study aims to investigate expression of IL-12family cytokines IL-12, IL-23, IL-27and IL-35in chronic immune thrombocytopenia (cITP) patients and the effect of HD-DXM treatment on their expression.Methods1.31cases of cITP patients. Normal controls come from36healthy volunteers.2. DXM was administered orally or intravenous drip at a dose of40mg per day for4consecutive days.3. The plasma levels of IL-12p70, IL-23, IL-27, IFN-γ, IL-4and IL-17A in peripheral blood of cITP patients and healthy controls were determined by FlowCytomix Technology.4. The plasma levels of IL-35in peripheral blood of cITP patients and healthy controls were determined by enzyme-linked immunosorbent assay (ELISA).Results1. Complete response was observed in19(61.29%), response in6(19.35%), and no response in6(19.35%) of the31patients. The platelet counts of cITP patients after HD-DXM treatment were significantly increased compared to that of patients before treatment (P<0.01). No bleeding or other obvious complications were observed throughout the treatment.2. Compared with the normal controls, in cITP patients before HD-DXM treatment, the plasma levels of IL-12p70, IL-23, IL-27, IFN-y and IL-17A were increased significantly (IL-12p70:27.32±8.19vs14.21±6.23pg/ml, P<0.01; IL-23:789.25±340.52vs477.42±141.65pg/ml, P<0.01; IL-27:60.42±43.68vs32.91±20.37pg/ml, P=0.001; IFN-y:40.12±19.14vs23.21±10.87pg/ml, P<0.001; IL-17A:29.6±28.08vs9.21±7.9pg/ml, P<0.001). On the contrary, IL-35and IL-4levels were considerably decreased (IL-4:57.69±17.71vs78.7±29.82pg/ml, P=0.001; IL-35:7.82±2.88vs11.89±5.11pg/ml, P<0.01).3. The plasma levels of IL-12p70, IL-23, IL-27, IFN-y and IL-17A were decreased significantly (IL-12p70:16.08±5.48vs27.32±8.19pg/ml, P<0.01; IL-23:517.54±166.67vs789.25±340.52pg/ml, P<0.01; IL-27:32.58±20.21vs60.42±43.68pg/ml, P=0.001; IFN-γ:23.84±11.13vs40.12±19.14pg/ml, P<0.001; IL-17A:11.44±8.48vs29.6±28.08pg/ml, P<0.001) and normalized after HD-DXM treatment. The plasma levels of IL-35and IL-4were increased significantly (IL-4:77.74±20.70vs57.69±17.71pg/ml, P=0.001; IL-35:11.75±6.76vs7.82±2.88pg/ml, P<0.01) after the treatment. There were no significant plasma levels of all of cytokines differences between the post-treatment patients and the normal controls (P>0.05). 4. The plasma levels of IL-12p70, IL-23were negative correlated with platelet counts in pre-treatment (IL-12p70:r=-0.561, P<0.01; IL-23:r=-0.475, P<0.01)as well as post-treatment (IL-12p70:r=-0.489, P<0.01; IL-23:r=-0.570, P<0.01) cITP patients. In this study, we did not find any correlation between the plasma levels of IL-27, IL-35with platelet counts in cITP patients before and after HD-DXM treatment.Conclusions1. HD-DXM treatment is safe and effective, can be used as an alternative initial therapy for cITP.2. The plasma levels of IFN-y, IL-4, and IL-17A in the patients included in the present study further implied a Th1and Th17-dominated cytokine profile in cITP. More interestingly, we found that HD-DXM therapy for cITP could cause a shift in the Thl/Th2/Thl7cytokine balance to the same levels as normal controls, leading to a more balanced Th1/Th2/Thl7cytokine profile response in vivo.3. CITP patients have increased plasma levels of IL-12p70, IL-23and IL-27and decreased plasma levels of IL-35, HD-DXM can correct IL-12family cytokines levels in chronic ITP patients. As immunosuppressant, DXM may play its role in cITP treatment partly through regulating expression of the IL-12family cytokines levels.4. IL-12p70and IL-23are found to be correlated with disease progression in c ITP. |
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