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Migration and characteristics of thrombocytes during cold-exposure in xenopus laevis
S82
Poster Presentations/Experimental Hematology 43 (2015) S51–S106
3124 - ALLOGENEIC T CELLS DISRUPT MEDULLARY THYMIC EPITHELIAL CELL FORMATION AND INDIRECTLY LEAD TO CHRONIC GRAFT-VS-HOST DISEASE Antonia M. Mueller1,2, Dullei Min2, Casey Burnett2, Mareike Florek2, Kenneth Weinberg2, and Judith A. Shizuru2 1 University Hospital Zurich, Zurich, Switzerland; 2Stanford University, Stanford, California, USA
3126 - MIGRATION AND CHARACTERISTICS OF THROMBOCYTES DURING COLD-EXPOSURE IN XENOPUS LAEVIS Ayaka Murase1, Nao Kashiwase1, Yuta Tanizaki2, Takato Otani1, Yoko Mochizuki1, and Takashi Kato1,2 1 Waseda University, Graduate School of Advanced Science and Engineering, Shinjuku, Tokyo, Japan; 2Waseda University, Department of Biology, School of Education, Shinjuku, Tokyo, Japan
Graft-vs-host disease (GVHD) remains a major complication after allogeneic hematopoietic cell transplantation (HCT). Acute (a) GVHD results from direct donor T cell (TC) damage of organs. In contrast, the biology of chronic (c) GVHD with its various autoimmune-like manifestations remains poorly understood. We studied the pathophysiology of cGVHD in an MHC-matched mouse model (C57BL/6 into BALB.B) using lethal irradiation and HCT of pure hematopoietic stem cells (HSC; cKIT+Thy1.1loSca1+Lin-) or HSC + TC. Recipients of HSC remained healthy; mice given HSC+TC developed aGVHD with a mortality of w30%. Survivors stabilized around d+45, but developed cGVHD after 6-12m with sclerodermatous skin excoriations, cataracts, and liver fibrosis/cirrhosis. Thymuses were a major target of aGVHD resulting in severe hypocellularity and disruption of the organ architecture. During the months of lymphoid reconstitution thymuses of HSC+TC-recipients showed markedly lower expression of cytokeratin 5 (CK5) than HSC recipients. CK5 marks medullary thymic epithelial cells (mTEC) that provide a specialized microenvironment for survival, proliferation, and differentiation of immature TC. Expression of AIRE, a transcription factor in the thymic medulla that controls negative selection during TC maturation was low in all transplanted groups. During aGVHD only TH1-donor TC, but not CD4+IL17+ (TH17)-cells were detectable. Starting at 2 months TH17 cells emerged, first in intestines then liver and skin (all typical GVHD target organs) increasing until 6-12 months post-HCT. Of note, TH17 cells originated from donor-HSC, not adaptively transferred mature TC. Even recipients of pure HSC showed increasing proportions of TH17 cells over time, and could manifest discrete signs of cGVHD. From our model we hypothesize the thymus is damaged by conditioning regimen and alloreactive donor TC. Disruption of mTEC may compromise negative selection of nascent HSC-derived donor TC, thereby misguide TC maturation and ultimately result in the emergence of TH17 donor-TC in target organs of chronic GVHD.
There are several studies on hypothermia-induced thrombocytopenia in various animals. Although it has been considered that the cause of hypothermia-induced thrombocytopenia is the sequestration of platelets to portal system, the detailed mechanism remains to be understood. In this study, we characterized the thrombocytes (TBCs) during cold-exposure in African clawed frogs. We counted the number of TBCs in the peripheral blood and the spleen after exposing frogs to 5 C by flow cytometry by using anti TBC antibody. After 6 hours later, TBCs decreased in the peripheral blood, while increased in the spleen. Moreover, in order to examine the kinetics of peripheral TBCs, we counted TBCs after returning frogs to 22 C after exposing them to 5 C for 6 hours. The number of TBCs recovered to the equivalent level as normal frogs in 2 hours after rewarming, indicating that TBCs were stored in the spleen or kept in the blood vessel during cold-exposure. Since we first hypothesized that the cause of cold-induced thrombocytopenia was due to the structural change of sugar chain on TBCs, we analyzed the structural change of N-linked glycan on the surface of TBCs by flow cytometry with biotinylated lectins. However, there was no singnificant indication of the change between normal frogs and those exposed to low temperature. Secondly, we hypothesized that cold-induced thrombocytopenia was caused by TBC activation; therefore we tested cellular morphological changes of TBCs under the low temperature condition. TBCs were exposed to cold temperature in vivo and in vitro for 2 hours, and observed the shape of TBCs by imaging with a scanning electron microscope. TBCs transformed from spindle-shape to sphereshape, and TBCs membrane surface got wrinkled. Furthermore, the shape of TBCs returned to former states when rewarmed. These results suggested that TBCs were aggregated in the blood vessel. We discuss the characteristics of TBCs exposed to low temperature, which might be the novel model to elucidate the mechanism of hypothermia-induced thrombocytopenia.
3125 - INHIBITION OF GPR56 BY PI-POLYAMIDE TARGETING THE EVI1-BINDING SITE WITH SUPPRESSION OF LEUKEMIA Kazuko N. Kaneda, Yusuke Saito, Akira Suekane, Shunsuke Shimosaki, and Kazuhiro Morishita Faculty of Medicine, University of Miyazaki, Miyazaki, Japan
3127 - A STRESS-RESPONSIVE TRANSCRIPTIONAL FACTOR NRF2 ACTIVATES HEMATOPOIETIC STEM CELLS Shohei Murakami1, Masayuki Yamamoto2, and Hozumi Motohashi1 1 IDAC, Tohoku Univ, Sendai, Japan; 2Grad Schl Med, Tohoku Univ, Sendai, Japan
Pyrrole-imidazole (PI) polyamides, are nuclease-resistant novel compounds that inhibit transcription factors by binding to the minor groove of DNA. We constructed a PI polyamide compound for the specific EVI1-binding site of human GPR56 promoter region (PI/GPR56-1), which is one of important EVI1-target genes in Gcoupled receptor (GPCR) for maintenance of HSC or LSC. In AML cells with EVI1-high expression, PI/GPR56-1 bound to the EVI1-binding sequence at the GPR56 promoter region and specifically reduced GPR56 expression with induction of growth suppression and apoptosis. In vivo effect of the PI/GPR56-1 treatment was examined by subcutaneous xenograft tumors in Balb/c-RJ mice. After transplantation of UCSD/AML1 cells, optimal PI/GPR56-1 or PI/AP-2 as a control, was injected via their tail veins once a week for a month, and their tumor sizes were evaluated. Tumor sizes in PI/GPR56-1-treated mice were significantly reduced as compared with control PI/AP-2-treated mice and the treatment of PI/GPR56-1 did not affect to murine bone marrow cells and various blood cells. Moreover, we injected leukemia cells to tail vein and determined the effect of PI/GPR56-1 to the leukemia model in immunodeficient NOG mice. The survival time of the PI/GPR56-1treated mice was significantly extended as compared with PI/AP-2-treated mice as a control without any side effects. Therefore, the treatment of PI polyamide to AML with EVI-1 high expression is one of useful therapeutic compounds as an anticancer drug without serious side effects.
KEAP1-NRF2 system is a major regulator of cellular redox balance. NRF2 is an inducible transcription factor, and KEAP1 is its negative regulator. When KEAP1 is inactivated by oxidative stress, NRF2 exerts its activity and induces cytoprotective genes. Accumulating evidence shows that KEAP1-NRF2 system acts as a regulator of cell proliferation and differentiation beyond the canonical role for the cellular protection. Under normal conditions, hematopoietic stem cells (HSCs) are maintained quiescent, but begin to proliferate and differentiate in response to environmental alterations. However, how HSCs are driven into proliferation is still elusive. Here, we examined the contribution of KEAP1-NRF2 system to the stress-responsive proliferation of HSCs. To this end, we investigated Keap1 conditional knockout mice, Keap1F/F::Mx1-Cre. Whereas the number of long-term HSCs was not different between the Keap1-deficient mice and controls, Keap1-deficient HSCs showed low bone marrow reconstitution after competitive transplantation, which can be canceled by Nrf2 deletion. Importantly, Keap1-deficient HSCs enhanced exit from quiescence, which is recapitulated in another Keap1 conditional knockout mice, Keap1F/F::Vav1Cre. Because the homing capacity and the cell death were not changed regardless of Keap1 genotype, the attenuated reconstitution capacity of Keap1-deficient HSCs is likely attributable to the exit from quiescence. To elucidate whether the NRF2-mediated cell cycle entry of HSCs is induced by exogenous stimuli, we treated Nrf2-null mice with an Nrf2 inducer, CDDO-Im, and found that CDDO-Im treatment induced cell cycle entry of HSCs, which was not observed on the Nrf2 knockout background. These results show that NRF2 activation enhances exit of HSCs from quiescent state, and thereby contributes to the initiation of proliferation and differentiation of HSCs.
Poster Presentations/Experimental Hematology 43 (2015) S51–S106
3124 - ALLOGENEIC T CELLS DISRUPT MEDULLARY THYMIC EPITHELIAL CELL FORMATION AND INDIRECTLY LEAD TO CHRONIC GRAFT-VS-HOST DISEASE Antonia M. Mueller1,2, Dullei Min2, Casey Burnett2, Mareike Florek2, Kenneth Weinberg2, and Judith A. Shizuru2 1 University Hospital Zurich, Zurich, Switzerland; 2Stanford University, Stanford, California, USA
3126 - MIGRATION AND CHARACTERISTICS OF THROMBOCYTES DURING COLD-EXPOSURE IN XENOPUS LAEVIS Ayaka Murase1, Nao Kashiwase1, Yuta Tanizaki2, Takato Otani1, Yoko Mochizuki1, and Takashi Kato1,2 1 Waseda University, Graduate School of Advanced Science and Engineering, Shinjuku, Tokyo, Japan; 2Waseda University, Department of Biology, School of Education, Shinjuku, Tokyo, Japan
Graft-vs-host disease (GVHD) remains a major complication after allogeneic hematopoietic cell transplantation (HCT). Acute (a) GVHD results from direct donor T cell (TC) damage of organs. In contrast, the biology of chronic (c) GVHD with its various autoimmune-like manifestations remains poorly understood. We studied the pathophysiology of cGVHD in an MHC-matched mouse model (C57BL/6 into BALB.B) using lethal irradiation and HCT of pure hematopoietic stem cells (HSC; cKIT+Thy1.1loSca1+Lin-) or HSC + TC. Recipients of HSC remained healthy; mice given HSC+TC developed aGVHD with a mortality of w30%. Survivors stabilized around d+45, but developed cGVHD after 6-12m with sclerodermatous skin excoriations, cataracts, and liver fibrosis/cirrhosis. Thymuses were a major target of aGVHD resulting in severe hypocellularity and disruption of the organ architecture. During the months of lymphoid reconstitution thymuses of HSC+TC-recipients showed markedly lower expression of cytokeratin 5 (CK5) than HSC recipients. CK5 marks medullary thymic epithelial cells (mTEC) that provide a specialized microenvironment for survival, proliferation, and differentiation of immature TC. Expression of AIRE, a transcription factor in the thymic medulla that controls negative selection during TC maturation was low in all transplanted groups. During aGVHD only TH1-donor TC, but not CD4+IL17+ (TH17)-cells were detectable. Starting at 2 months TH17 cells emerged, first in intestines then liver and skin (all typical GVHD target organs) increasing until 6-12 months post-HCT. Of note, TH17 cells originated from donor-HSC, not adaptively transferred mature TC. Even recipients of pure HSC showed increasing proportions of TH17 cells over time, and could manifest discrete signs of cGVHD. From our model we hypothesize the thymus is damaged by conditioning regimen and alloreactive donor TC. Disruption of mTEC may compromise negative selection of nascent HSC-derived donor TC, thereby misguide TC maturation and ultimately result in the emergence of TH17 donor-TC in target organs of chronic GVHD.
There are several studies on hypothermia-induced thrombocytopenia in various animals. Although it has been considered that the cause of hypothermia-induced thrombocytopenia is the sequestration of platelets to portal system, the detailed mechanism remains to be understood. In this study, we characterized the thrombocytes (TBCs) during cold-exposure in African clawed frogs. We counted the number of TBCs in the peripheral blood and the spleen after exposing frogs to 5 C by flow cytometry by using anti TBC antibody. After 6 hours later, TBCs decreased in the peripheral blood, while increased in the spleen. Moreover, in order to examine the kinetics of peripheral TBCs, we counted TBCs after returning frogs to 22 C after exposing them to 5 C for 6 hours. The number of TBCs recovered to the equivalent level as normal frogs in 2 hours after rewarming, indicating that TBCs were stored in the spleen or kept in the blood vessel during cold-exposure. Since we first hypothesized that the cause of cold-induced thrombocytopenia was due to the structural change of sugar chain on TBCs, we analyzed the structural change of N-linked glycan on the surface of TBCs by flow cytometry with biotinylated lectins. However, there was no singnificant indication of the change between normal frogs and those exposed to low temperature. Secondly, we hypothesized that cold-induced thrombocytopenia was caused by TBC activation; therefore we tested cellular morphological changes of TBCs under the low temperature condition. TBCs were exposed to cold temperature in vivo and in vitro for 2 hours, and observed the shape of TBCs by imaging with a scanning electron microscope. TBCs transformed from spindle-shape to sphereshape, and TBCs membrane surface got wrinkled. Furthermore, the shape of TBCs returned to former states when rewarmed. These results suggested that TBCs were aggregated in the blood vessel. We discuss the characteristics of TBCs exposed to low temperature, which might be the novel model to elucidate the mechanism of hypothermia-induced thrombocytopenia.
3125 - INHIBITION OF GPR56 BY PI-POLYAMIDE TARGETING THE EVI1-BINDING SITE WITH SUPPRESSION OF LEUKEMIA Kazuko N. Kaneda, Yusuke Saito, Akira Suekane, Shunsuke Shimosaki, and Kazuhiro Morishita Faculty of Medicine, University of Miyazaki, Miyazaki, Japan
3127 - A STRESS-RESPONSIVE TRANSCRIPTIONAL FACTOR NRF2 ACTIVATES HEMATOPOIETIC STEM CELLS Shohei Murakami1, Masayuki Yamamoto2, and Hozumi Motohashi1 1 IDAC, Tohoku Univ, Sendai, Japan; 2Grad Schl Med, Tohoku Univ, Sendai, Japan
Pyrrole-imidazole (PI) polyamides, are nuclease-resistant novel compounds that inhibit transcription factors by binding to the minor groove of DNA. We constructed a PI polyamide compound for the specific EVI1-binding site of human GPR56 promoter region (PI/GPR56-1), which is one of important EVI1-target genes in Gcoupled receptor (GPCR) for maintenance of HSC or LSC. In AML cells with EVI1-high expression, PI/GPR56-1 bound to the EVI1-binding sequence at the GPR56 promoter region and specifically reduced GPR56 expression with induction of growth suppression and apoptosis. In vivo effect of the PI/GPR56-1 treatment was examined by subcutaneous xenograft tumors in Balb/c-RJ mice. After transplantation of UCSD/AML1 cells, optimal PI/GPR56-1 or PI/AP-2 as a control, was injected via their tail veins once a week for a month, and their tumor sizes were evaluated. Tumor sizes in PI/GPR56-1-treated mice were significantly reduced as compared with control PI/AP-2-treated mice and the treatment of PI/GPR56-1 did not affect to murine bone marrow cells and various blood cells. Moreover, we injected leukemia cells to tail vein and determined the effect of PI/GPR56-1 to the leukemia model in immunodeficient NOG mice. The survival time of the PI/GPR56-1treated mice was significantly extended as compared with PI/AP-2-treated mice as a control without any side effects. Therefore, the treatment of PI polyamide to AML with EVI-1 high expression is one of useful therapeutic compounds as an anticancer drug without serious side effects.
KEAP1-NRF2 system is a major regulator of cellular redox balance. NRF2 is an inducible transcription factor, and KEAP1 is its negative regulator. When KEAP1 is inactivated by oxidative stress, NRF2 exerts its activity and induces cytoprotective genes. Accumulating evidence shows that KEAP1-NRF2 system acts as a regulator of cell proliferation and differentiation beyond the canonical role for the cellular protection. Under normal conditions, hematopoietic stem cells (HSCs) are maintained quiescent, but begin to proliferate and differentiate in response to environmental alterations. However, how HSCs are driven into proliferation is still elusive. Here, we examined the contribution of KEAP1-NRF2 system to the stress-responsive proliferation of HSCs. To this end, we investigated Keap1 conditional knockout mice, Keap1F/F::Mx1-Cre. Whereas the number of long-term HSCs was not different between the Keap1-deficient mice and controls, Keap1-deficient HSCs showed low bone marrow reconstitution after competitive transplantation, which can be canceled by Nrf2 deletion. Importantly, Keap1-deficient HSCs enhanced exit from quiescence, which is recapitulated in another Keap1 conditional knockout mice, Keap1F/F::Vav1Cre. Because the homing capacity and the cell death were not changed regardless of Keap1 genotype, the attenuated reconstitution capacity of Keap1-deficient HSCs is likely attributable to the exit from quiescence. To elucidate whether the NRF2-mediated cell cycle entry of HSCs is induced by exogenous stimuli, we treated Nrf2-null mice with an Nrf2 inducer, CDDO-Im, and found that CDDO-Im treatment induced cell cycle entry of HSCs, which was not observed on the Nrf2 knockout background. These results show that NRF2 activation enhances exit of HSCs from quiescent state, and thereby contributes to the initiation of proliferation and differentiation of HSCs.