Enhancing autophagy protects platelets in immune thrombocytopenia patients.

BACKGROUND: Immune thrombocytopenia (ITP) is an autoimmune bleeding disorder and involves increased apoptosis of platelets. Autophagy is an essential process for platelets to maintain their life and physiological functions. However, the role of autophagy in ITP platelets was previously unclear. METHODS: In the present study, the expression of autophagy-related protein and autophagy flux were detected in platelets from ITP patients and healthy controls by immunofluorescence staining and immunoblotting, and the influence of autophagy on the viability and apoptosis of ITP platelets was further explored. RESULTS: We found that platelet autophagy was diminished in ITP patients. Platelet autophagy in ITP was regulated by the PI3K/AKT/mTOR pathway, with mTOR (mammalian target of rapamycin) as a negative regulator and class III PtdIns3K playing a crucial role in the process. Importantly, the small-molecule compound ABO (6-amino-2,3-dihydro-3-hydroxymethyl-1,4-benzoxazine) enhanced autophagy in ITP platelets. Enhancing platelet autophagy alleviated platelet destruction by inhibiting apoptosis and improving platelet viability. CONCLUSIONS: These results suggest a role for autophagy regulation in the pathogenesis of ITP, and offer a novel treatment for these patients.

Novel Murine Model of Immune Thrombocytopaenia through Immunized CD41 Knockout Mice.

Immune thrombocytopaenia (ITP) is the most common autoimmune bleeding disorder, where platelets are destroyed by auto-antibodies and/or cell-mediated mechanisms. To understand the pathogenesis of ITP and explore novel therapeutics, three types of animal models have been used: passive ITP, secondary ITP and platelet-induced ITP. However, the first two are not ideal for chronic ITP pathophysiology where both T cell and B cell play important roles in platelet destruction. The most efficient model to mimic chronic ITP is developed by Chow et al through transferring splenocytes from platelet-immune CD61-knockout (KO) mice into mice with severe combined immunodeficiency (SCID). However, placental defects are evident in 25% of CD61-KO females and post-natal haemorrhage does occur, reducing the survival rate of embryos and pups. Compared with CD61-KO mice, CD41-KO ones do not present such problems. In our study, we employ CD41-KO mice as another source of immunized spleen cells. We evaluated our model with existing standards. Transferred SCID mice presented typical features of ITP, such as reduced platelet counts in the peripheral blood, increased anti-platelet antibody levels in the serum and reduced mature megakaryocytes in the bone marrow. What is more, lymphocyte-depletion experiments showed the role of CD8+ T cells in mature megakaryocyte decrease and thrombocytopaenia. And we confirmed the antibody-mediated platelet destruction by phagocytosis in the spleen. Our study develops another efficient murine ITP model through immunized CD41-KO mice.

Dysregulated miR34a/diacylglycerol kinase ζ interaction enhances T-cell activation in acquired aplastic anemia.

Acquired aplastic anemia is an idiopathic paradigm of human bone marrow failure syndrome, which involves active destruction of hematopoietic stem cells and progenitors by cytotoxic T cells in the bone marrow. Aberrant expression of microRNAs in T cells has been shown to lead to development of certain autoimmune diseases. In the present study, we performed a microarray analysis of miRNA expression in bone marrow CD3+ T cells from patients with aplastic anemia and healthy controls. Overexpression of miR34a and underexpression of its target gene diacylglycerol kinase (DGK) ζ in bone marrow mononuclear cells were validated in 41 patients and associated with the severity of aplastic anemia. Further, the level of miR34a was higher in naïve T cells from patients than from controls. The role of miR34a and DGKζ in aplastic anemia was investigated in a murine model of immune-mediated bone marrow failure using miR34a-/- mice. After T-cell receptor stimulation in vitro, lymph node T cells from miR34a-/- mice demonstrated reduced activation and proliferation accompanied with a less profound down-regulation of DGKζ expression and decreased ERK phosphorylation compared to those from wild-type C57BL6 control mice. Infusion of 5 × 106 miR34a-/- lymph node T cells into sublethally irradiated CB6F1 recipients led to increased Lin-Sca1+CD117+ cells and less vigorous expansion of CD8+ T cells than injection of same number of wild-type lymph node cells. Our study demonstrates that the miR34a/DGKζ dysregulation enhances T-cell activation in aplastic anemia and targeting miR34a may represent a novel molecular therapeutic approach for patients with aplastic anemia.

The catalytic region and PEST domain of PTPN18 distinctly regulate the HER2 phosphorylation and ubiquitination barcodes.

The tyrosine phosphorylation barcode encoded in C-terminus of HER2 and its ubiquitination regulate diverse HER2 functions. PTPN18 was reported as a HER2 phosphatase; however, the exact mechanism by which it defines HER2 signaling is not fully understood. Here, we demonstrate that PTPN18 regulates HER2-mediated cellular functions through defining both its phosphorylation and ubiquitination barcodes. Enzymologic characterization and three crystal structures of PTPN18 in complex with HER2 phospho-peptides revealed the molecular basis for the recognition between PTPN18 and specific HER2 phosphorylation sites, which assumes two distinct conformations. Unique structural properties of PTPN18 contribute to the regulation of sub-cellular phosphorylation networks downstream of HER2, which are required for inhibition of HER2-mediated cell growth and migration. Whereas the catalytic domain of PTPN18 blocks lysosomal routing and delays the degradation of HER2 by dephosphorylation of HER2 on pY(1112), the PEST domain of PTPN18 promotes K48-linked HER2 ubiquitination and its rapid destruction via the proteasome pathway and an HER2 negative feedback loop. In agreement with the negative regulatory role of PTPN18 in HER2 signaling, the HER2/PTPN18 ratio was correlated with breast cancer stage. Taken together, our study presents a structural basis for selective HER2 dephosphorylation, a previously uncharacterized mechanism for HER2 degradation and a novel function for the PTPN18 PEST domain. The new regulatory role of the PEST domain in the ubiquitination pathway will broaden our understanding of the functions of other important PEST domain-containing phosphatases, such as LYP and PTPN12.

Blockade of Tim-3 pathway ameliorates interferon-gamma production from hepatic CD8+ T cells in a mouse model of hepatitis B virus infection.

T cell immunoglobulin- and mucin-domain-containing molecule-3 (Tim-3) has been reported to participate in the pathogenesis of inflammatory diseases. However, whether Tim-3 is involved in hepatitis B virus (HBV) infection remains unknown. Here, we studied the expression and function of Tim-3 in a hydrodynamics-based mouse model of HBV infection. A significant increase of Tim-3 expression on hepatic T lymphocytes, especially on CD8+ T cells, was demonstrated in HBV model mice from day 7 to day 18. After Tim-3 knockdown by specific shRNAs, significantly increased IFN-gamma production from hepatic CD8+ T cells in HBV model mice was observed. Very interestingly, we found Tim-3 expression on CD8+ T cells was higher in HBV model mice with higher serum anti-HBs production. Moreover, Tim-3 knockdown influenced anti-HBs production in vivo. Collectively, our data suggested that Tim-3 might act as a potent regulator of antiviral T-cell responses in HBV infection.