Hai Cheng

Altered balance between Th1 and Th17 cells in circulation is an indicator for the severity of murine acute GVHD.

Disbalance of Th1 and Th17 can lead to inflammatory diseases and autoimmunity. Acute graft-versus-host disease (GVHD) is now considered as a compound and dysregulated immune response. Both Th1 and Th17 have been implicated in the pathophysiology of acute GVHD. Disbalance of Th1 and Th17 may also play a critical role in mediating acute GVHD. In this study, we investigated the Th1/Th17 imbalance in peripheral blood through out the pathological process of acute GVHD, using a GVHD model of C57BL/6 (H-2(b)) donor to BALB/c (H-2(d)) recipient. We also analyzed the correlation between Th1/Th17 ratio and severity of acute GVHD, and explored the potential function of Th1/Th17 imbalance in acute GVHD. Recipients received 5×10(6) spleen cells (SP) underwent more severe acute GVHD, compared with the ones received 5×10(5) SP. Severe GVHD bearing recipients had much higher proportion of Th1 cells but lower proportion of Th17 cells, compared with mild GVHD bearing ones. The Th1/Th17 ratios in both groups underwent relative changes according to the changing proportions of Th1 and Th17 cells and showed positive correlation with clinical scores of acute GVHD. We describe the changing proportions of Th1 and Th17 cells in acute GVHD, and we find that Th1/Th17 ratio is an accurate indicator for predicting the severity of acute GVHD.

CXCR4-transduced mesenchymal stem cells protect mice against graft-versus-host disease

Mesenchymal stem cells (MSCs) possessing immunoregulatory activities have been evaluated in the treatment of graft-versus-host disease (GVHD). However, the immunomodulatory effects of MSCs are not always successfully achieved in some animal models, and this deficiency may be caused in part by poor homing of these cells to hematopoietic tissues. In this study, we assessed the immunsuppressive capacity of lentiviral vector transduced MSCs expressing CXCR4 in a major histocompatibility complex (MHC)-mismatched mouse model of bone marrow (BM) transplantation from C57BL/6 donors to BALB/c recipients. The survival, body weight and clinical score of GVHD in transplanted mice were monitored. Liver, intestine and skin from mice in each group were obtained for histological examination. Plasma concentrations of interleukin (IL)-2, IL-4, IL-6, IL-10, IFN-γ, TNF-α and IL-17A were also determined using a Cytometric Bead Array. CXCR4 over-expressing MSCs maintained their immunsuppressive capacity and showed enhanced migration capacity in vitro. In the mouse GVHD model, treatment with CXCR4 over-expressing MSCs decreased the mortality rate and attenuated clinical and pathological GVHD scores. Moreover, compared with control groups, the plasma IL-2, IL-6, IFN-γ and TNF-α levels in recipients infused with CXCR4 over-expressing MSCs were significantly decreased, while those of IL-4 and IL-10 were increased. In conclusion, our report reveals that CXCR4-transduced MSCs effectively controlled the occurrence of mouse GVHD following allogeneic BM transplantation.

Deviated balance between Th1 and Th17 cells exacerbates acute graft-versus-host disease in mice

BACKGROUND Th1/Th17 imbalance had been indicated to mediate several kinds of inflammatory diseases. We deduce that Th1/Th17 imbalance might also contribute to the pathogenesis of acute graft-versus-host disease (GVHD). This study is to investigate the relation between Th1/Th17 imbalance and acute GVHD. METHODS We applied a murine GVHD model of C57BL/6 (H-2(b)) donor to BALB/c (H-2(d)) recipient by treating the recipients with low dose of halofuginone (HF), which is competent in selectively inhibiting Th17 differentiation and facilitating Th1 differentiation. Recipient mice were monitored for survival rate, body weight change, clinical symptoms and pathological evidence of acute GVHD. We also measured the proportions of Th1 and Th17 cells in circulation and expression levels of IFN-γ and IL-17A in tissues involved in GVHD. RESULTS Firstly, we confirm the existence of Th1/Th17 imbalance in acute GVHD and Th1/Th17 imbalance positively correlates with severity of acute GVHD. Secondly, low dose of HF augments Th1/Th17 imbalance by driving the Th1/Th17 balance to a Th1-dominant reaction. Finally, augmented Th1/Th17 imbalance leads to aggravated systemic GVHD. An increased Th1-type reaction results in aggravated hepatic and intestinal GVHD, and inhibiting Th17 differentiation is sufficient to alleviate pulmonic impairment. CONCLUSION Our study is indicative for a critical role of Th1/Th17 imbalance in the pathogenesis of murine GVHD.

RNA interference-mediated silencing of NANOG leads to reduced proliferation and self-renewal, cell cycle arrest and apoptosis in T-cell acute lymphoblastic leukemia cells via the p53 signaling pathway.

NANOG is critical for maintaining the self-renewal and proliferative properties of embryonic stem cells. Here we found that cultured T-cell acute lymphoblastic leukemia (T-ALL) cells, as well as human primary T-ALL cells, express a functional variant of NANOG. NANOG mRNA is derived predominantly from a retrogene locus termed NANOGP8. Furthermore, we showed that RNA interference-mediated NANOG knockdown inhibited cell proliferation, reduced self-renewal, promoted apoptosis and arrested the cell cycle through a p53-mediated pathway in leukemic cells. These findings demonstrate the oncogenic potential of this pluripotent gene in human T-ALL cells.

Bone Marrow–derived Endothelial Progenitor Cells Promote Hematopoietic Reconstitution After Hematopoietic Stem Cell Transplantation

OBJECTIVE A hematopoietic deficit is a serious complication after hematopoietic stem cell transplantation. It has been shown that fetal blood-derived endothelial progenitor cells (EPCs) can promote hematopoietic reconstitution after transplantation. This study investigated whether EPCs from bone marrow (BM) of adult mice could promote hematopoietic reconstitution. METHODS Lethally irradiated BALB/c mice were administered BM cells or BM cells plus EPCs. RESULTS The results showed that EPC-treated mice displayed accelerated recovery of peripheral blood white blood cells and reticulocytes. But the platelets were not significantly different with versus without EPCs. Accelerated recovery of BM sinusoidal vessels, promotion of stem cell implantation, and decreased adipocyte formation were associated with the mechanism. Systemic administration of anti-vascular endothelial cadherin antibody neutralized these effects significantly. CONCLUSION These data showed that BM-derived EPC infusions augmented hematopoiesis suggesting a new approach to promote hematopoiesis.

TH17 cells are critical for skin-specific pathological injury in acute graft-versus-host disease.

Interleukin-17 (IL-17), which is important for host defens, has been implicated in autoimmune and chronic inflammatory diseases. As knockout mice lack IL-17 expression in δγT, NKT-like cells, studies investigating the association between TH17 cells and cutaneous graft-versus-host disease (GVHD) in animal models have reported conflicting results. To determine the role of TH17 cells in cutaneous GVHD, we developed an acute GVHD model using C57BL/6(H-2(b)) donors to BABL/c (H-2(d)) recipients. Blood samples and skin were examined for inflammation and infiltrating cells using histology and fluorescence-activated cell sorter (FACS) on days 6 and 15 after bone marrow transplantation. We found donor T cells to mediate severe cutaneous inflammation, which was ameliorater by administration of halofuginone (HF) to the recipients. Mechanistically, we demonstrate the severe tissue damage during this disorder to be associated with the production of IL-17 and the expansion of IL-17-producing CD4(+) cells. Specific inhibition of TH17 differentiation and function by HF reduced disease severity. Thus, TH17 cells are sufficient to induce acute cutaneous GVHD.

Halofugine prevents cutaneous graft versus host disease by suppression of Th17 differentiation

Abstract Halofuginone, isolated from Dichroa febreifuga, is a potent inhibitor of skin collagen in chronic graft-versus-host disease (GVHD). To evaluate the effect of halofuginone on the development of cutaneous GVHD, we developed a murine model based on BALB/c (H-2d) as recipients with transplantation of C57BL/6(H-2b) bone marrow plus splenocytes. Halofuginone or its vehicle dimethyl sulfoxide (DMSO) was given introperitoneally at a dose of 5 ug/mouse daily from one day before transplantation until 20 days post-transplantation. Halofuginone-treated recipients showed only very mild appearance of cutaneous GVHD, whereas DMSO-treated recipients rapidly showed manifestation of severe cutaneous GVHD, indicating a protective effect of halofuginone in cutaneous GVHD. After injected with halofuginone, we observed a decrease in the number of CD4+ interleukin (IL)-17+ cells and a parallel increase in that of CD4+ interferon (IFN)-gamma+ cells in peripheral blood. This shift between CD4+ IL-17+ cells and CD4+ IFN-gamma+ cells developed through modulation of cytokine profile indicated by a marked increase in the levels of IFN-gamma, tumor necrosis factor (TNF)-alpha, and IL-6. The level of IL-10 was not changed obviously. Mechanistically, we demonstrate that severe tissue damage was associated with the production of IL-17 and expansion of CD4+IL-17+ cells during this disorder. Specific inhibition of Th17 differentiation by halofuginone reduced disease severity. Our results indicate a significant role of halofuginone in suppressing cutaneous GVHD, apparently through effect on inhibition of Th17 cells differentiation.

Busulfan Triggers Intrinsic Mitochondrial-Dependent Platelet Apoptosis Independent of Platelet Activation

As a nonspecific alkylating antineoplastic agent, busulfan has been widely used in the treatment of patients with chronic myeloid leukemia. In vitro and in vivo studies demonstrated busulfan-induced cell apoptosis. Whether busulfan triggers platelet apoptosis remains unclear. This study aimed to evaluate the role of busulfan in platelet apoptosis. Isolated human platelets were incubated with busulfan followed by analysis of platelet apoptosis by flow cytometry or western blot, including mitochondrial depolarization, expression of Bcl-2, and Bax and caspase 3 activation. Meanwhile, platelet activation, expression of glycoprotein Ibα (GPIbα), glycoprotein VI (GPVI), and IIb3 and platelet aggregation in response to collagen and adenosine diphosphate (ADP) were measured. Additionally, busulfan was injected into mice with or without administration of caspase inhibitor QVD-Oph to investigate its effect on platelet lifespan. Our results showed that busulfan-treated platelets displayed increased mitochondrial membrane depolarization, decreased expression of Bcl-2, increased expression of Bax and caspase 3 activation in dose-dependent manner, which were inhibited by QVD-Oph. Platelet activation was not observed in busulfan-treated platelets as showed by no increased P-selectin expression and PAC-1 binding. However, busulfan reduced collagen- or ADP-induced platelet aggregation without affecting expression of GPIbα, GPVI, and IIb3. Furthermore, busulfan reduced circulating platelet lifespan which was ameliorated by QVD-Oph in mice. In conclusion, busulfan triggers mitochondrial-dependent platelet apoptosis and reduces platelet lifespan in mice. These data suggest targeting caspase activation might be beneficial in the prophylaxis of platelet apoptosis-associated thrombocytopenia after administration of busulfan.

Co-transplantation of Hematopoietic Stem Cells and Cxcr4 Gene-Transduced Mesenchymal Stem Cells Promotes Hematopoiesis

Mesenchymal stem cells (MSCs) are a promising candidate for cellular therapies. Co-transplantation of MSCs and hematopoietic stem cells (HSCs) promotes successful engraftment and improves hematopoietic recovery. In this study, the effects of co-transplantation of HSCs and mouse bone marrow (BM)-derived MSCs overexpressing CXCR4 (CXCR4-MSC) on CXCR4-MSC homing capacity and the reconstitution potential in lethally irradiated mice were evaluated. Recovery of donor-derived peripheral blood leukocytes and platelets was accelerated when CXCR4-MSCs were co-transplanted with BM cells. The frequency of c-kit+Sca+Lin− HSCs was higher in recipient BM following co-transplantation of CXCR4-MSCs compared with the EGFP-MSC control and the BMT only groups. Surprisingly, the rate of early engraftment of donor-derived BM cells in recipients co-transplanted with CXCR4-MSCs was slightly lower than in the absence of MSCs on day 7. Moreover, co-transplantation of CXCR4-MSCs regulated the balance of T helper cells subsets. Hematopoietic tissue reconstitution was evaluated by histopathological analysis of BM and spleen. Co-transplantation of CXCR4-MSCs was shown to promote the recovery of hematopoietic organs. These findings indicate that co-transplantation of CXCR4-MSCs promotes the early phase of hematopoietic recovery and sustained hematopoiesis.

An increased expression profile of Th9/IL-9 correlated with Th17/IL-17 in patients with immune thrombocytopenia

Abstract Immune thrombocytopenia (ITP) is a heterogeneous autoimmune disease, characterized by dysregulation of cellular immunity. Th9 cells were recently identified as a new subtype of Th cells, characterized by preferential production of IL-9. Given the pleiotropic function of IL-9, Th9 cells are demonstrated to be involved in various autoimmune diseases. However, whether Th9 cells are involved in the pathogenesis of ITP remains unclear. In this study, 49 active ITP patients, 39 ITP with remission and 20 healthy controls were included. Peripheral blood mononuclear cells (PBMCs) were isolated from ITP and controls for measuring Th9 and Th17 cells by flow cytometry. Meanwhile, RNA was isolated from PBMCs for the measurement of the mRNA level of PU.1, IRF4, BATF, and RORγt by quantitative real-time PCR. Plasma levels of IL-9 and IL-17 were detected by ELISA. Our results showed that higher expressions of Th9, IL-9, and associated transcription factors (PU.1, IRF4, and BATF) were found in active ITP patients and restored to the normal level (except IL-9) in patients in remission. Meanwhile, Th9 cells and the IL-9 plasma level were positively correlated with Th17 cells and the IL-17 level in ITP patients, respectively. Moreover, a positive correlation of IRF4 or BATF with RORγt was found. In conclusion, an aberrant expression profile of Th9/IL-9 was associated with pathogenesis of ITP possibly through cooperatively working with Th17/IL-17 and therapeutically targeting Th9/IL-9 might be a novel approach in the treatment of ITP.