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Phosphatase and tensin homolog (PTEN) is thought to mediate B cell activation by negatively regulating the phosphoinositide 3-kinase (PI3K) signaling pathway. This pathway is important for activation, growth, and proliferation. Although enhanced B cell receptor (BCR) signaling contributes to increased B cell activity in immune thrombocytopenia (ITP), the role of PTEN is unclear. In this study, we analyzed B cells of ITP patients using flow cytometry and found that all B cell subsets, excluding memory B cells, showed lower PTEN expression than cells from healthy controls (HCs). PTEN expression was also positively-correlated with blood platelet count, although levels were lower in patients who were platelet autoantibody-positive compared with those who were negative. We next evaluated the effects of IL-21, anti-IgM, and CD40L on PTEN expression, demonstrating that they were potent inducers of PTEN expression in normal B cells. Induction of PTEN expression was lower in B cells of ITP patients. We also found that IL-21 increased the proportion of plasma cells in peripheral blood mononuclear cells (PBMCs) of ITP patients, independent of BCR signaling. This effect was reproducible using PTEN inhibitors with cells from HCs. In summary, defective PTEN expression, regulation, and function all contribute to the B cell hyper-responsiveness that associates with ITP., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Primary immune thrombocytopenia (ITP) is a disease of autoimmunity in which there are Th1/Th2 imbalance and disordered cytokine profiles. CXC chemokine ligand 16 (CXCL16) was proved to implicate in some autoimmune diseases. Our research aimed to determine plasma soluble CXCL16 (sCXCL16) levels and its effects in ITP. We used ELISA to measure plasma sCXCL16, IFN-γ and IL-4 and flow cytometry to determine expression of CXCR6 on lymphocyte subsets. We used real-time PCR to detect the CXCL16 and CXCR6 mRNA expression. Additionally, plasma sCXCL16, CXCL16 and CXCR6 mRNA levels of 8 patients were monitored before and after treatment. We found that patients with active ITP had higher circulating sCXCL16 in plasma than healthy controls and patients in remission. Meanwhile, negative relationships between sCXCL16 and platelet count, IL-4 and positive relationships between sCXCL16 and IFN-γ, IFN-γ/IL-4 ratio were observed. Besides, expression of CXCR6 on lymphocyte subsets and mRNA levels of CXCL16 and CXCR6 were all increased in active ITP. Additionally, plasma sCXCL16 and IFN-γ levels and CXCR6 mRNA expression were down-regulated after effective treatment compared with those before treatment. Thus, increased plasma sCXCL16 might be implicated in the pathogenesis of ITP and have a relationship with Th1/Th2 imbalance., (Copyright © 2017. Published by Elsevier Ltd.)

Primary immune thrombocytopenia (ITP) is an autoimmune disease with many immune dysfunctions including T helper type 1 cell (Th1) polarization and regulatory T cells (Tregs) deficiency. This study aimed to determine the effects of TLR4 on Treg differentiation and the cytokine production of peripheral blood mononuclear cells (PBMCs) from patients with ITP. We found that expression of TLR4 on monocytes was significantly decreased in patients with active ITP than that in healthy controls and it had positive correlation with platelet count. However, there was no expression of TLR4 on CD4 +  T cells. The result of further experiments in vitro showed that lipopolysaccharide (LPS) stimulation could enhance TLR4 expression on monocytes. Additionally, activation of TLR4 with LPS could promote differentiation of Treg cells and anti-TLR4 attenuated this effect. There was no significant difference about Th17 cells among three subgroups. However, the Th17/Treg cell ratio was decreased after stimulation with LPS and increased with anti-TLR4. Moreover, activation of TLR4 with LPS could significantly promote the secretion of interleukin-10 (IL-10) and transforming growth factor-β1 (TGFB1), while anti-TLR4 significantly suppressed the secretion of them. Nevertheless, the secretion of IL-17A did not reach the statistical difference among three subgroups. In summary, decreased TLR4 appears to cause Tregs abnormality in ITP by modulating Tregs differentiation and immunoregulatory cytokines.

Primary immune thrombocytopenia (ITP) is an acquired autoimmune disorder, and loss of immune tolerance has been implicated in ITP pathogenesis. CD8 + CD28 - suppressor (Ts) cells have an immunosuppression function and are involved in several autoimmune disorders. However, the role of Ts cells in ITP is currently not clear. Here, flow cytometry was used to detect the CD8 + CD28 - CD127 - proportion, which was decreased in active ITP patients compared with that of controls. Function analysis showed that immunosuppression of CD8 + CD28 - Ts cells in ITP patients was impaired. Mechanistic studies have shown that CD8 + CD28 - Ts cells from controls can downregulate CD80 and upregulate LILRB4 (ITL3) and LILRB2 (ILT4) expression on CD14 + monocytes, whereas these abilities were not found in Ts cells from ITP patients. Furthermore, Inducible T-cell costimulatory (ICOS) expression on the Ts cell surface after activation was decreased whereas programmed death 1 and interleukin 10 expression was not changed in ITP patients compared with those of controls. In summary, the down-regulated quantity and function of Ts cells in active patients indicated that a Ts defect was involved in ITP. Moreover, decreased ICOS expression and the loss of the ability to regulate co-stimulator expression on antigen-presenting cells partly explained the defective Ts-mediated suppression., (© 2017 John Wiley & Sons Ltd.)

CD72 is a co-receptor of B cells and regulates B cell activation. Although aberrant expression of CD72 has been reported in primary immune thrombocytopenia (ITP), it is uncertain whether this aberrant expression is restricted to specific B cell subsets. Furthermore, the mechanisms that regulate CD72 expression are unknown. In this study, we found higher frequency of CD19 + B cells, CD19 + CD27 + memory B cells and lower frequency of CD19 + CD27 - naive B cells in active ITP patients compared with controls and patients in remission. CD72 expression on CD19 + CD27 + cells was upregulated in active ITP patients and correlated with platelet count and anti-platelet autoantibodies. In vitro, CD40L could specifically induce CD72 upregulation on CD19 + CD27 + B cells. In combination with CD40L, interleukin (IL) 10 and BAFF (also termed TNFSF13B) further enhanced CD72 expression on CD19 + CD27 + B cells, whereas IL21 reduced CD72 upregulation. CD72mRNA expression after CD40L stimulation was increased in ITP patients and controls. Significant increase of CD40L on CD4 + T cells was correlated with CD72 expression on CD19 + CD27 + B cells in ITP patients. In conclusion, upregulation of CD72 expression on CD27 + memory B cells might take part in the pathogenesis of ITP. Elevated CD40L on CD4 + cells combined with cytokines might contribute to the upregulation of CD72 expression on CD27 + memory B cells., (© 2017 John Wiley & Sons Ltd.)

TMEVPG1 is a long noncoding RNA, which can promote Interferon gamma (IFNG) transcript as an enhancer. It has been reported that plasma IFNG concentration was elevated and partly due to dysregulation of micro RNAs in patients with primary immune thrombocytopenia (ITP). However, the effect of long noncoding RNA on IFNG expression in ITP is currently not well understood. To explore whether TMEVPG1 is involved in ITP, this study examined the TMEVPG1 expression and analyzed the association between TMEVPG1 transcript and IFNG expression in patients with ITP and healthy controls. The result showed that TMEVPG1 expression in peripheral blood mononuclear cells (PBMCs) from active ITP patients were lower than in that of healthy controls. Further activation for 24 h in vitro resulted in decreased TMEVPG1 while elevated IFNG mRNA and protein expressions in activated PBMCs compared with un-activated PBMCs both in ITP patients and healthy controls. However, TMEVPG1 and IFNG mRNA had same up-regulated tendency after short-time activation (1, 2 and 4 h) in healthy controls. In addition, we detected T helper 1 (Th1) cell associated transcription factors T-bet, STAT1 and STAT4 mRNA levels, and found T-bet mRNA expression was lower in PBMCs from ITP patient in remission compared with healthy controls. In conclusion, we speculated that TMEVPG1 could promote IFNG transcription, and IFNG over-expression negative feedback regulated on TMEVPG1 expression, which resulted in decreased TMEVPG1 in ITP patients.

Primary immune thrombocytopenia (ITP) is an autoimmune disorder that is characterized by low platelet count. Glucocorticoids (GCs) resistance is a great challenge in the treatment of ITP. P-glycoprotein (P-gp) is a widely studied protein, which is associated with drug resistance. However, in ITP, the functional activity and immune regulation mechanism of P-gp remain uncertain. In this study, we evaluated the expression and functional activity of P-gp in different lymphocyte subsets, explored the correlation between P-gp function and GCs resistance and investigated the role of P-gp in ITP pathogenesis. Results indicated that the functional activity and mRNA level of P-gp were significantly higher in GCs-nonresponsive patients than in GCs-responsive patients with ITP. However, these differences in P-gp were only significant in CD8 + T cells. P-gp function was related to disease activity rather than GCs therapy. P-gp was involved in secreting granzyme B and perforin, maintaining autoreactive lymphocytes survival and enhancing autologous platelets lysis in ITP. In conclusion, over-functional P-gp might play an important role in the pathogenesis of ITP and induce GCs resistance in nonresponsive ITP patients. The blockage of P-gp could be a promising therapeutic approach for GCs-resistant patients with ITP.

Primary immune thrombocytopenia (ITP) is an acquired autoimmune disease. 1α, 25-dihydroxyvitamin D 3 [1,25(OH) 2 D 3 ] and vitamin D receptor (VDR) play important immune-suppressive roles in immune system. It has been reported that serum 1,25(OH) 2 D 3 were lower in ITP patients. In this study, we evaluated local 1,25(OH) 2 D 3 level and VDR mRNA expression further, and determined whether 1,25(OH) 2 D 3 /VDR were correlated with T cell dysfunction in ITP patients. We found that 1,25(OH) 2 D 3 /VDR levels were decreased in active ITP patients, and 1,25(OH) 2 D 3 had significant anti-inflammatory effects on ITP patients, including both anti-proliferation of peripheral blood mononuclear cells (PBMCs) and reversing the abnormal T cells polarization. 1,25(OH) 2 D 3 inhibited the differentiation of T helper (Th)1 and Tc1 cells but induced the differentiation of Th2, Tc2 and T regulatory (Treg) cells in ITP patients. However, the percentage of Th17 cells were not affected obviously with 1,25(OH) 2 D 3 . In addition, 1,25(OH) 2 D 3 also suppressed pro-inflammatory cytokines (INF-γ and IL-17A) but promoted anti-inflammatory cytokine (IL-10) secretion in ITP patients. In conclusion, decreased 1,25(OH) 2 D 3 /VDR might participate in the pathogenesis of ITP, and appropriate supplement of 1,25(OH) 2 D 3 may be a promising treatment., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Stromal cell-derived factor-1 (SDF-1), signaling through CXCR4, is implicated in megakaryopoiesis and platelet production. SDF-1 rs2297630 is a functional polymorphism in linkage disequilibrium with other functional variants in SDF-1. This study aimed to investigate the role of SDF-1 rs2297630 in chronic ITP. The genotypes were determined by polymerase chain reaction-restriction fragment length polymorphism and confirmed by direct sequencing. Immature platelet fraction (IPF) was performed using Sysmex XE-2100. Anti-platelet autoantibodies were assayed by enzyme-linked immunosorbent assay. The main characteristics at diagnosis and the outcome of chronic ITP in 201 Chinese patients were retrospectively reviewed. There was no significant difference in either genotype or allelic distribution between ITP patients and the controls (p = 0.114; p = 0.787). However, both heterozygote (GA) and homozygote minor allele (AA) patients had significantly increased megakaryocyte quantity compared to homozygote genotype (GG) patients at diagnosis (p = 0.011). The mean IPF values of GA and AA genotype patients were higher than those observed in the GG genotype patients when platelet counts ≤50 × 10(9)/L at diagnosis (p = 0.007). Patients with GA and AA genotype showed a higher response rate to standard treatments than patients with GG genotype (p < 0.001). In particular, GA and AA genotype patients had a significantly increased chance of responding to steroids, intravenous immunoglobulin (IVIG), and thrombopoietin analogs (p = 0.007; p = 0.029; p = 0.034, respectively). No significant difference was found between anti-platelet antibodies and genotypes (p = 0.296). In summary, the SDF-1 rs2297630 was associated with platelet production and treatment response in Chinese patients with chronic ITP.

Background: Primary immune thrombocytopenia (ITP) is an immune-mediated disorder in which cellular immunity deficiency and disturbed cytokine profiles have been found. Semaphorin 5A (Sema5A) has been showed to be implicated in cellular immune response. We aimed to evaluate the role of Sema5A in patients with chronic ITP., Methods: Plasma levels of Sema5A, T helper (Th) cytokines (interferon [IFN] -γ,interleukin [IL]-4,IL-17A) were determined by enzyme-linked immunosorbent assay (ELISA) in ITP patients and healthy controls. Using real-time quantitative polymerase chain reaction (RT-PCR), mRNA levels of Sema5A and its receptor plexin-B3, plexin-A1 in peripheral blood mononuclear cells(PBMCs)were studied in all subjects. Specific anti-platelet autoantibodies were measured by the Pak Auto method. The dynamic change of plasma Sema5A and mRNA levels of its receptors was measured in 9 patients after effective therapy., Results: Plasma Sema5A levels were significantly increased in active patients with chronic ITP compared to patients in remission and healthy controls. Elevated levels of Sema5A were found positively correlated with higher levels of plasma IFN-γ, IFN-γ/IL-4 ratio and negatively correlated with lower levels of plasma IL-4, platelet counts in ITP patients. The mRNA plexin-B3 was decreased in active ITP patients and inversely correlated with plasma Sema5A levels. Additionally, plasma levels of Sema5A and IFN-γ were reduced with up-regulation of plexin-B3 expression after effective treatment., Conclusions: Our data demonstrated elevated plasma Sema5A in chronic ITP patients might be involved in Th1 polarization by down-regulating receptor plexin-B3 expression and correlated with disease activity., (Copyright © 2015 Elsevier Ltd. All rights reserved.)