Lingyu Zeng

Endothelial Injury, an Intriguing Effect of Methotrexate and Cyclophosphamide During Hematopoietic Stem Cell Transplantation in Mice

OBJECTIVE Elevated circulating endothelial cells (EC) in peripheral blood are an important indicator of endothelial damage and graft-versus-host disease (GVHD). However, the injured endothelial vasculature may in turn promote GVHD. In this study, we investigated whether methotrexate or cyclophosphamide, two conventional chemotherapeutic agents used in hematopoietic stem cell transplantation, caused endothelial injury and what were the functional consequences of this injury on GVHD. METHODS Six to 8-week-old female mice were randomly separated into three groups, including methotrexate (15 mg/m2 on day 1 and 10 mg/m2 on days 3, 6, and 11), cyclophosphamide (60 mg/kg; days 1, 2), and PBS saline alone (control). Circulating EC (CD31+CD133(-)CD45low) and the CD4+/CD8+ T lymphocytes in the peripheral blood were estimated by flow cytometry on days 1, 3, 5, 7, 9, 11, 16, 21, and 26. The morphologic changes of the endothelium were examined by phase contrast light microscopy to determine the integrity of the endothelial vasculature. RESULTS Elevated EC were detected at day 1 or 3 in mice receiving cyclophosphamide or methotrexate, respectively, with a peak increase at day 5 or day 3, respectively. The ratio of CD4+/CD8+ T lymphocytes showed a delayed increase in the peak to day 11 for both groups. In the cyclophosphamide group, there was significant apomorphosis with necrosis/thrombosis under light microscopy, whereas only apomorphosis was noticed in the methotrexate group. In both groups, EC showed hydropsia and cytomembrane damage. CONCLUSIONS Circulating EC increase during the early phases of cyclophosphamide or methotrexate conditioning, suggesting that both chemotherapeutic drugs induce endothelial damage, which occurs a little earlier than suppression of the immune system.

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.

Irradiation is an early determinant of endothelial injury during hematopoietic stem cell transplantation.

OBJECTIVE We investigated the degree and the time course of endothelial injury in mice pretreated with lethal or reduced-intensity irradiation administered before transplantation. MATERIALS AND METHODS Six- to eight-week-old female mice were randomly allocated into three groups: lethal-intensity irradiation (8.5 Gy, group 1), reduced-intensity irradiation (5.0 Gy, group 2), or nonirradiated controls (group 3). After conditioning, circulating endothelial cells (CD31+, CD133(-), and CD45low) and peripheral blood CD4+ or CD8+ T-lymphocyte subpopulations were enumerated using flow cytometry at various times. The morphologic changes in endothelium were examined at phase-contrast light microscopy. RESULTS Circulating endothelial cells showed an earlier and higher peak in the lethal irradiation group compared with the reduced-intensity irradiation group, which exhibited a protean elevation in cell numbers. There were no visible histopathologic changes during the early stage of endothelial damage. CONCLUSIONS Lethal and reduced doses of irradiation induced endothelial injury in a dose-dependent manner. Endothelial damage may occur before graft-vs-host disease and its related complications.

MiR-150 impairs inflammatory cytokine production by targeting ARRB-2 after blocking CD28/B7 costimulatory pathway

MiR-150, a major modulator negatively regulating the development and differentiation of various immune cells, is widely involved in orchestrating inflammation. In transplantation immunity, miR-150 can effectively induce immune tolerance, although the underlying mechanisms have not been fully elucidated. In the current study, we found that miR-150 is elevated after blocking CD28/B7 co-stimulatory signaling pathway and impaired IL-2 production by targeting ARRB2. Further investigation suggested that miR-150 not only repressed the level of ARRB2/PDE4 directly but also prevented AKT/ARRB2/PDE4 trimer recruitment into the lipid raft by inhibiting the activities of PI3K and AKT through the cAMP-PKA-Csk signaling pathway. This leads to the interruption of cAMP degradation and subsequently results in inhibition of the NF-kB pathway and reduced production of both IL-2 and TNF. In conclusion, our study demonstrated that miR-150 can effectively prevent CD28/B7 co-stimulatory signaling transduction, decrease production of inflammatory cytokines, such as IL-2 and TNF, and elicit the induction of immune tolerance. Therefore, miR-150 could become a novel potential therapeutic target in transplantation immunology.

Engineered regulatory T cells prevent graft-versus-host disease while sparing the graft-versus-leukemia effect after bone marrow transplantation

Regulatory T cells (Tregs) can prevent graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation (BMT). Here we developed a lentivirus-based strategy to ectopically express Foxp3 in mouse CD4(+)CD25(-) T cells. These cells shared similar immunophenotypes and biological features of natural Tregs. Co-injection of engineered Tregs with donor bone marrow cells and splenocytes prevented recipients from lethal GVHD. Furthermore, we showed that graft-versus-leukemia (GVL) effect against EL4/DsRed leukemic cells was maximally preserved while GVHD was minimized during exposure to engineered Tregs in a mouse leukemia model. These findings provide a novel approach to preventing GVHD while maintaining GVL effect during BMT.

Imbalanced expression of Bcl-xL and Bax in platelets treated with plasma from immune thrombocytopenia.

Immune thrombocytopenia is a heterogeneous autoimmune disease, characterized by accelerated platelet destruction and impaired platelet production. Bcl-xL and Bax play an opposite role in the regulation of apoptotic process with Bcl-xL for cell survival and Bax for cell apoptosis. Given the critical roles in the regulation of platelet apoptosis, whether Bcl-xL or Bax was involved in the pathogenesis of ITP remains unknown. The aim of this study is to evaluate the expression profile of Bcl-xL and Bax in platelets treated with ITP plasma. Normal washed platelets were treated with plasma from 20 active ITP patients or 10 age and gender-matched control to mimic the ITP in vivo environment. Mitochondrial depolarization, platelet apoptosis and activation were measured by flow cytometry. Expression of Bcl-xL, Bax and caspase-3 were also measured by quantitative real-time PCR and western blot. Our results demonstrated increased mitochondrial depolarization, platelet apoptosis and activation in platelets after treated with ITP plasma in comparison to control. In addition, decreased expression of Bcl-xL, increased expression of Bax and activity of caspase-3 were also observed. Furthermore, a negative correlation of Bcl-xL with Bax was found in platelets treated with ITP plasma. In conclusion, imbalanced expression of Bcl-xL and Bax might be associated with platelet apoptosis in ITP and therapeutically targeting them might be a novel approach in the treatment of ITP.

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.

Control of mouse graft-versus-host disease following allogeneic bone marrow transplantation by blocking the CD28/B7 signaling pathway with lentiviral vector-mediated RNA interference.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is an effective way to cure hematological malignancies. However, graft-versus-host disease (GVHD) following transplantation limits the clinical application to some extent. The donor T lymphocytes play a central role in the occurrence and development of GVHD. Control of GVHD by inhibition of T cell proliferation by blocking the CD28/B7 signaling pathway with RNA interference has not been examined. In this study, we constructed a lentiviral vector carrying CD28 shRNA and generated genetically engineered splenocytes through transduction in a murine allogeneic bone marrow transplantation model. The survival and the occurrence of GVHD in transplanted mice were monitored every day. Liver, intestine, skin, and other tissues from the mice in each group were used for histological examination. We also determined plasma concentrations of interleukin (IL)-2, IL-4, IL-5, IL-10, IL-13, and interferon gamma (IFN-γ). Recipient bone marrow from mice that had survived for an extended period was examined to detect chimerism. We succeeded in suppressing the expression of CD28 gene and controlling mouse GVHD following allogeneic bone marrow transplantation in the engineered spleen cell group. These suggest that blocking the CD28/B7 signal transduction pathway with lentiviral vector-mediated RNA interference effectively controlled the occurrence of mouse GVHD following allogeneic bone marrow transplantation. Its mechanism could be due to the inhibition of T cell proliferation and, simultaneously, the promotion of the differentiation of TH0 to TH2 cells, thereby reducing GVHD in the mouse transplantation model.