Hongmin Zhou

The Tim-3 ligand galectin-9 negatively regulates CD8+ alloreactive T cell and prolongs survival of skin graft.

CD8+ alloreactive T cells are the key mediators of accelerated rejection. Vigorous CD8+ alloreactive T cells responses against alloantigens, which is the main effector mechanism in acute allograft rejection, has been well described. But the molecular mechanisms to dampen activated CD8+ T cells are largely unknown. On the other hand, Tim-3 is a molecule expressed on terminally differentiated CD4+ Th1 cells. Engaging Tim-3 with its ligand galectin-9 causes an inhibitory signal, resulting in apoptosis of Th1 cells and negatively regulates Th1 type immunity. However, the question whether CD8+ T cells express surface molecular Tim-3 has not been fully elucidated. In this study, we have investigated which CD8+ subset express molecular Tim-3 by flow cytometric assay. In addition, cytotoxic assay was applied to analyze whether CD8+ alloreactive T cells were sensitive to galectin-9 induced apoptosis. Here, our results demonstrated that Tim-3 was expressed on activated CD8+ alloreactive T cells (CD8+CD44highCD62Llow), but not expressed on naïve CD8+ T cells. Furthermore, alloreactive CD8+ cytotoxic T cells were sensitive to galectin-9 induced apoptosis both in vitro and vivo, resulting in attenuation of CD8+ alloreactive T cells mediated cytotoxicity and prolonged survival of skin graft.

Activation of Tim-3–Galectin-9 pathway improves survival of fully allogeneic skin grafts

T cell immunoglobulin and mucin domain (Tim)-3 is a molecule expressed on terminally differentiated murine Th1 cells but not on Th2 cells. Identification of Galectin-9 as a ligand for Tim-3 has now firmly established the Tim-3-Galectin-9 pathway as an important regulator of Th1 immunity, which results in apoptosis of Th1 cells. Here, we demonstrate that engagement of Tim-3 by mouse recombinant Galectin-9 remarkably suppresses allograft rejection and improves survival of allogeneic skin grafts. Furthermore, administration of recombinant Galectin-9 decreases Tim-3 positive cells in draining lymph node and selectively inhibits production of IFN-gamma after skin transplantation. At last, even low dose of Galectin-9 (1 microg/ml) can obviously inhibit TCR crosslinking-induced primary T cell proliferation in vitro. These observations suggest that Tim-3-Galectin-9 pathway plays an important role in the termination of productive Th1-immune response and could lead to developing novel therapies in transplant medicine.

Galectin-9 Significantly Prolongs the Survival of Fully Mismatched Cardiac Allografts in Mice

Background. The involvement of T-cell immunoglobulin mucin-3 (Tim-3) in the regulation of solid organ transplantation rejection is largely unknown. We used galectin-9 (Tim-3 ligand) to evaluate the effect and mechanisms of Tim-3/galectin-9 pathway in an allogeneic heart transplant model. Methods. The murine cardiac transplant model from BALB/c (H-2d) to C57BL/6 (H-2b) was built. The recipients were administered with galectin-9 for 7 days from day 1 or day 3 posttransplant. The complete cessation of cardiac contractility was defined as the observation endpoint. The effect of galectin-9 on cell proliferation was assessed by mixed lymphocyte reaction. Histology and immunohistochemistry were performed to estimate the severity of rejection. The phenotype and cytokine profile of lymphocytes were analyzed by flow cytometry. Results. In vitro, galectin-9 suppressed the proliferation of lymphocytes mixed lymphocyte reactions in a dose- and &bgr;-galactoside-dependent manner. In vivo, galectin-9 treatment from day 1 to day 3 posttransplant achieved similar survival time of grafts (median survival time, 22.7±1.2 vs. 23.0±1.0 days). The prolonged survival time was associated with reduced infiltration of CD4 and CD8 lymphocytes in allografts. Furthermore, the intragraft transcriptional profiling in galectin-9-treated group showed reduction of IFN-&ggr; and IL-17 mRNA but elevation of Ebi-3 and galectin-9 mRNA. Flow cytometry analysis indicated that galectin-9 treatment reduced the ratio of T helper (Th) 1 and Th17 cells in the draining lymph nodes and peripheral blood. Conclusions. A short-course administration of galectin-9 significantly prolonged the survival of fully allogeneic cardiac allografts, which was associated with the suppression of Th1 and Th17 immune responses.

Transforming growth factor beta 1 plays an important role in inducing CD4+CD25+forhead box P3+ regulatory T cells by mast cells

The role of mast cells (MCs) in the generation of adaptive immune responses especially in the transplant immune responses is far from being resolved. It is reported that mast cells are essential intermediaries in regulatory T cell (Treg) transplant tolerance, but the mechanism has not been clarified. To investigate whether bone marrow‐derived mast cells (BMMCs) can induce Tregs by expressing transforming growth factor beta 1 (TGF‐β1) in vitro, bone marrow cells obtained from C57BL/6 (H‐2b) mice were cultured with interleukin (IL)‐3 (10 ng/ml) and stem cell factor (SCF) (10 ng/ml) for 4 weeks. The purity of BMMCs was measured by flow cytometry. The BMMCs were then co‐cultured with C57BL/6 T cells at ratios of 1:2, 1:1 and 2:1. Anti‐CD3, anti‐CD28 and IL‐2 were administered into the co‐culture system with (experiment groups) or without (control groups) TGF‐β1 neutralizing antibody. The percentages of CD4+CD25+forkhead box P3 (FoxP3)+ Tregs in the co‐cultured system were analysed by flow cytometry on day 5. The Treg percentages were significantly higher in all the experiment groups compared to the control groups. These changes were deduced by applying TGF‐β1 neutralizing antibody into the co‐culture system. Our results indicated that the CD4+ T cells can be induced into CD4+CD25+FoxP3+ T cells by BMMCs via TGF‐β1.

Characterization of acute renal allograft rejection by human serum proteomic analysis

SummaryTo identify acute renal allograft rejection biomarkers in human serum, two-dimensional differential in-gel electrophoresis (2-D DIGE) and reversed phase high-performance liquid chromatography (RP-HPLC) followed by electrospray ionization mass spectrometry (ESI-MS) were used. Serum samples from renal allograft patients and normal volunteers were divided into three groups: acute rejection (AR), stable renal function (SRF) and normal volunteer (N). Serum samples were firstly processed using Multiple Affinity Removal Column to selectively remove the highest abundance proteins. Differentially expressed proteins were analyzed using 2-D DIGE. These differential protein spots were excised, digested by trypsin, and identified by RP-HPLC-ESI/MS. Twenty-two differentially expressed proteins were identified in serum from AR group. These proteins included complement C9 precursor, apolipoprotein A-IV precursor, vitamin D-binding protein precursor, beta-2-glycoprotein 1 precursor, etc. Vitamin D-binding protein, one of these proteins, was confirmed by ELISA in the independent set of serum samples. In conclusion, the differentially expressed proteins as serum biomarker candidates may provide the basis of acute rejection noninvasive diagnosis. Confirmed vitamin D-binding protein may be one of serum biomarkers of acute rejection. Furthermore, it may provide great insights into understanding the mechanisms and potential treatment strategy of acute rejection.To identify acute renal allograft rejection biomarkers in human serum, two-dimensional differential in-gel electrophoresis (2-D DIGE) and reversed phase high-performance liquid chromatography (RP-HPLC) followed by electrospray ionization mass spectrometry (ESI-MS) were used. Serum samples from renal allograft patients and normal volunteers were divided into three groups: acute rejection (AR), stable renal function (SRF) and normal volunteer (N). Serum samples were firstly processed using Multiple Affinity Removal Column to selectively remove the highest abundance proteins. Differentially expressed proteins were analyzed using 2-D DIGE. These differential protein spots were excised, digested by trypsin, and identified by RP-HPLC-ESI/MS. Twenty-two differentially expressed proteins were identified in serum from AR group. These proteins included complement C9 precursor, apolipoprotein A-IV precursor, vitamin D-binding protein precursor, beta-2-glycoprotein 1 precursor, etc. Vitamin D-binding protein, one of these proteins, was confirmed by ELISA in the independent set of serum samples. In conclusion, the differentially expressed proteins as serum biomarker candidates may provide the basis of acute rejection noninvasive diagnosis. Confirmed vitamin D-binding protein may be one of serum biomarkers of acute rejection. Furthermore, it may provide great insights into understanding the mechanisms and potential treatment strategy of acute rejection.

Role of Wnt/β-catenin signaling pathway in the mechanism of calcification of aortic valve

SummaryAortic valve calcification is a common disease in the elderly, but its cellular and molecular mechanisms are not clear. In order to verify the hypothesis that Wnt/β-catenin signaling pathway is involved in the process of calcification of aortic valve, porcine aortic valve interstitial cells (VICs) were isolated, cultured and stimulated with oxidized low density lipoprotein (ox-LDL) for 48 h to induce the differentiation of VICs into osteoblast-like cells. The key proteins and genes of Wnt/β-catenin signaling pathway, such as glycogen synthase kinase 3β (GSK-3β) and β-catenin, were detected by using Western blotting and real-time polymerase chain reaction (PCR). The results showed that the VICs managed to differentiate into osteoblast-like cells after the stimulation with ox-LDL and the levels of proteins and genes of GSK-3β and β-catenin were increased significantly in VICs after stimulation for 48 h (P<0.05). It is suggested that Wnt/β-catenin signaling pathway may play a key role in the differentiation of VICs into osteoblast-like cells and make great contribution to aortic valve calcification.Aortic valve calcification is a common disease in the elderly, but its cellular and molecular mechanisms are not clear. In order to verify the hypothesis that Wnt/β-catenin signaling pathway is involved in the process of calcification of aortic valve, porcine aortic valve interstitial cells (VICs) were isolated, cultured and stimulated with oxidized low density lipoprotein (ox-LDL) for 48 h to induce the differentiation of VICs into osteoblast-like cells. The key proteins and genes of Wnt/β-catenin signaling pathway, such as glycogen synthase kinase 3β (GSK-3β) and β-catenin, were detected by using Western blotting and real-time polymerase chain reaction (PCR). The results showed that the VICs managed to differentiate into osteoblast-like cells after the stimulation with ox-LDL and the levels of proteins and genes of GSK-3β and β-catenin were increased significantly in VICs after stimulation for 48 h (P<0.05). It is suggested that Wnt/β-catenin signaling pathway may play a key role in the differentiation of VICs into osteoblast-like cells and make great contribution to aortic valve calcification.

Galectin-7 in cardiac allografts in mice: increased expression compared with isografts and localization in infiltrating lymphocytes and vascular endothelial cells.

We sought to identify elevated expression of galectin-7, a T-cell-binding protein, in renal allograft recipients undergoing acute rejection episodes. Allografts and isografts were examined immunohistologically and using quantitative Western blot analysis for galectin-7 protein. The expression of galectin-7 in T lymphocytes from draining lymph nodes in the recipient mice was examined using flow cytometry. We observed galectin-7 to gradually increase with time in the allografts to be significantly higher than that in isografts at days 3, 5, and 7 postoperative: 0.85 ± 0.03 versus 0.69 ± 0.05; 1.15 ± 0.11 versus 0.81 ± 0.02; and 2.02 ± 0.12 versus 0.81 ± 0.05 (P < .05). The majority of galectin-7 was located in the infiltrating lymphocytes and vascular endothelial cells. Furthermore, the percentage of CD4+galectin-7+ and CD8+galectin-7+ T cells from draining lymph nodes in the allograft group was higher than that in the isograft group: 28.0 ± 1.0% versus 1.2 ± 0.2%; and 12.4 ± 0.8% versus 0.4 ± 0.1% (P < .01). In conclusion, galectin-7 relates to acute allograft rejection and T-cell responses possibly as an accelerant.

Galectin-9 in combination with rapamycin induces cardiac allograft tolerance in mice.

Background Galectin-9 serves opposing roles in the innate and adaptive immune systems. Galectin-9 triggers T-cell immunoglobulin mucin-3 (Tim-3) on T helper type 1 (Th1) cells, thereby terminating Th1 immunity and protecting allografts from host immune attacks. Meanwhile, galectin-9 promotes the maturation of dendritic cells (DCs) that deliver proinflammatory signals. We previously showed that galectin-9 significantly prolongs cardiac allograft survival in mice but failed to induce tolerance. This study aimed at improving the administration protocol to induce allograft tolerance. We examined whether rapamycin can reverse the proinflammatory effects of galectin-9 on DCs and whether rapamycin synergizes with galectin-9 to induce cardiac allograft tolerance. Methods Monocytes/DCs from cardiac allografts were assessed for Tim-3 expression by flow cytometry. Costimulatory molecules CD80/CD86 were measured on galectin-9/rapamycin–treated bone marrow–derived DCs by flow cytometry. We performed heterotopic cervical cardiac transplantation using BALB/c donors and C57BL/6 recipients and assessed graft survival time. T cells of long-term surviving recipients were immunoassayed for interferon-&ggr; and interleukin-4 secretion. Results Allograft-infiltrating monocytes/DCs expressed high Tim-3 levels (47.3%±5.6%). Expression of CD80/CD86 was up-regulated on galectin-9–treated bone marrow–derived DCs, which was reversed by rapamycin. Combined treatment with galectin-9 and rapamycin promoted the permanent acceptance of fully mismatched grafts (survival time >180 days; n=6). However, treatment with galectin-9 or rapamycin alone was not sufficient to induce tolerance. Galectin-9/rapamycin–induced tolerance was associated with low donor-specific interferon-&ggr; and interleukin-4 secretion. Conclusions Rapamycin inhibits proinflammatory effects of galectin-9 on DCs. Combined treatment of galectin-9 and rapamycin promotes allografts tolerance, which is associated with reduced Th1 and Th2 responses.

Quantitative analysis of cell tracing by in vivo imaging system

SummaryIn vivo imaging system (IVIS) is a new and rapidly expanding technology, which has a wide range of applications in life science such as cell tracing. By counting the number of photons emitted from a specimen, IVIS can quantify biological events such as tumor growth. We used B16F10-luc-G5 tumor cells and 20 Babl/C mice injected subcutaneously with B16F10-luc-G5 tumor cells (1×106 in 100 μL) to develop a method to quantitatively analyze cells traced by IVIS in vitro and in vivo, respectively. The results showed a strong correlation between the number of tumor cells and the intensity of bioluminescence signal (R2=0.99) under different exposure conditions in in vitro assay. The results derived from the in vivo experiments showed that tumor luminescence was observed in all mice by IVIS at all days, and there was significant difference (P<0.01) between every two days from day 3 to day 14. Moreover, tumor dynamic morphology could be monitored by IVIS when it was invisible. There was a strong correlation between tumor volume and bioluminescence signal (R2=0.97) by IVIS. In summary, we demonstrated a way to accurately carry out the quantitative analysis of cells using IVIS both in vitro and in vivo. The data indicate that IVIS can be used as an effective and quantitative method for cell tracing both in vitro and in vivo.In vivo imaging system (IVIS) is a new and rapidly expanding technology, which has a wide range of applications in life science such as cell tracing. By counting the number of photons emitted from a specimen, IVIS can quantify biological events such as tumor growth. We used B16F10-luc-G5 tumor cells and 20 Babl/C mice injected subcutaneously with B16F10-luc-G5 tumor cells (1×106 in 100 μL) to develop a method to quantitatively analyze cells traced by IVIS in vitro and in vivo, respectively. The results showed a strong correlation between the number of tumor cells and the intensity of bioluminescence signal (R2=0.99) under different exposure conditions in in vitro assay. The results derived from the in vivo experiments showed that tumor luminescence was observed in all mice by IVIS at all days, and there was significant difference (P<0.01) between every two days from day 3 to day 14. Moreover, tumor dynamic morphology could be monitored by IVIS when it was invisible. There was a strong correlation between tumor volume and bioluminescence signal (R2=0.97) by IVIS. In summary, we demonstrated a way to accurately carry out the quantitative analysis of cells using IVIS both in vitro and in vivo. The data indicate that IVIS can be used as an effective and quantitative method for cell tracing both in vitro and in vivo.

Donor pretreatment with adenosine monophosphate-activated protein kinase activator protects cardiac grafts from cold ischaemia/reperfusion injury

OBJECTIVES Adenosine monophosphate-activated protein kinase (AMPK) is a master regulator of energy metabolism and has been shown to be protective in ischaemia/reperfusion injury (IRI). We hypothesized that preactivation of AMPK with an activator before donor heart procurement could protect heart grafts from cold IRI. METHODS Donor Sprague-Dawley rats were injected intravenously with AMPK activator 5-amino-imidazole-4-carboxamide ribonucleotide (AICAR) or vehicle 30 min before heart procurement. Heart grafts were then preserved in histidine-tryptophan-ketoglutarate (HTK) solution at 4°C for 8 h. After preservation, grafts were immediately mounted on the Langendorff perfusion system and perfused with Krebs-Henseleit buffer at 37°C for 1 h. Adenosine triphosphate (ATP) and malondialdehyde (MDA) content in graft tissue were quantified post-preservation and post-reperfusion. After reperfusion, isolated heart function was assessed using a pressure transducer; cumulative release of creatine kinase (CK) and lactate dehydrogenase (LDH) into the perfusate was measured to assess cardiomyocyte necrosis; ultrastructural changes in the mitochondria of the grafts were examined using transmission electron microscopy (TEM). RESULTS After preservation, myocardial ATP content in the pretreated hearts was significantly higher than in the control hearts (3.247 ± 0.3034 vs 1.817 ± 0.2533 µmol/g protein; P < 0.05). AICAR-pretreated heart grafts exhibited significantly higher coronary flow (9.667 ± 0.3159 vs 8.033 ± 0.2459 ml/min; P < 0.05) and left ventricular developing pressure (58.67 ± 2.894 vs 42.67 ± 3.333 mmHg; P < 0.05) than the vehicle treated after reperfusion. Cumulative release of CK (300.0 ± 25.30 vs 431.7 ± 42.39 U/l; P < 0.05) and LDH (228.0 ± 16.68 vs 366.8 ± 57.41 U/l; P < 0.05) in the perfusate was significantly lower in the AICAR-pretreated group than that in the control group. Myocardial MDA content was also reduced in the pretreated group (0.5167 ± 0.1046 vs 0.9333 ± 0.1333 nmol/mg protein; P < 0.05). TEM suggested that the mitochondrial structure of AICAR-pretreated hearts was much better preserved. Moreover, AICAR-pretreated hearts significantly diminished cytosolic cytochrome c release after reperfusion. CONCLUSIONS This study demonstrates that pretreatment with AMPK activator AICAR significantly protects heart grafts from extended cold IRI. This novel protocol may be useful and feasible in clinical heart transplantation.