RESUMEN
We previously demonstrated that interferon γ (IFN-γ) derived from donor T cells co-opts the indoleamine 2,3-dioxygenase 1 (IDO1) â aryl hydrocarbon receptor (AHR) axis to suppress idiopathic pneumonia syndrome (IPS). Here we report that the dysregulated expression of AP-1 family genes in Ahr-/- lung epithelial cells exacerbated IPS in allogeneic bone marrow transplantation settings. AHR repressed transcription of Jund by preventing STAT1 from binding to its promoter. As a consequence, decreased interleukin-6 impaired the differentiation of CD4+ T cells toward Th17 cells. IFN-γ- and IDO1-independent induction of Ahr expression indicated that the AHR agonist might be a better therapeutic target for IPS than the IDO1 activator. We developed a novel synthetic AHR agonist (referred to here as PB502) that potently inhibits Jund expression. PB502 was highly effective at inducing AHR activation and ameliorating IPS. Notably, PB502 was by far superior to the endogenous AHR ligand, L-kynurenine, in promoting the differentiation of both mouse and human FoxP3+ regulatory CD4+ T cells. Our results suggest that the IDO1-AHR axis in lung epithelial cells is associated with IPS repression. A specific AHR agonist may exhibit therapeutic activity against inflammatory and autoimmune diseases by promoting regulatory T-cell differentiation.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Neumonía , Receptores de Hidrocarburo de Aril/metabolismo , Animales , Linfocitos T CD4-Positivos/metabolismo , Indolamina-Pirrol 2,3,-Dioxigenasa/genética , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Interferón gamma/metabolismo , Ratones , Neumonía/tratamiento farmacológico , Transducción de Señal , Linfocitos T Reguladores/metabolismoRESUMEN
BACKGROUND: A wide array of histone deacetylase (HDAC) inhibitors and aryl hydrocarbon receptor (AHR) agonists commonly arrest experimental autoimmune encephalomyelitis (EAE). However, it is not known whether HDAC inhibition is linked to the AHR signaling pathway in EAE. METHODS: We investigated how the pan-HDAC inhibitor SB939 (pracinostat) exerted immunoregulatory action in the myelin oligodendrocyte glycoprotein 35-55 (MOG35-55)-induced EAE mouse model by evaluating changes in of signal transducer and activator of transcription 3 (STAT3) acetylation and the expression of indoleamine 2,3-dioxygenase 1 (IDO1) and AHR in inflamed spinal cords during EAE evolution. We proved the involvement of IDO1 and the AHR in SB939-mediated immunosuppression using Ido1-/- and Ahr-/- mice. RESULTS: Administration with SB939 halted EAE progression, which depended upon IDO1 expression in neurons of the central nervous system (CNS). Our in vitro and in vivo studies demonstrated that SB939 sustained the interleukin-6-induced acetylation of STAT3, resulting in the stable transcriptional activation of Ido1. The therapeutic effect of SB939 also required the AHR, which is expressed mainly in CD4+ T cells and macrophages in CNS disease lesions. Finally, SB939 was shown to markedly reduce the proliferation of CD4+ T cells in inflamed neuronal tissues but not in the spleen or draining lymph nodes. CONCLUSIONS: Overall, our results suggest that IDO1 tryptophan metabolites produced by neuronal cells may act on AHR in pathogenic CD4+ T cells in a paracrine fashion in the CNS and that the specific induction of IDO1 expression in neurons at disease-afflicted sites can be considered a therapeutic approach to block the progression of multiple sclerosis without affecting systemic immunity.
Asunto(s)
Encefalomielitis Autoinmune Experimental , Inhibidores de Histona Desacetilasas , Indolamina-Pirrol 2,3,-Dioxigenasa , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas , Factor de Transcripción STAT3 , Animales , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Encefalomielitis Autoinmune Experimental/inmunología , Indolamina-Pirrol 2,3,-Dioxigenasa/genética , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Indolamina-Pirrol 2,3,-Dioxigenasa/antagonistas & inhibidores , Inhibidores de Histona Desacetilasas/farmacología , Inhibidores de Histona Desacetilasas/uso terapéutico , Factor de Transcripción STAT3/metabolismo , Neuronas/efectos de los fármacos , Neuronas/patología , Neuronas/metabolismo , Ratones , Receptores de Hidrocarburo de Aril/metabolismo , Receptores de Hidrocarburo de Aril/genética , Femenino , Médula Espinal/patología , Médula Espinal/metabolismo , Médula Espinal/inmunología , Médula Espinal/efectos de los fármacos , Glicoproteína Mielina-Oligodendrócito/inmunología , Sistema Nervioso Central/inmunología , Sistema Nervioso Central/efectos de los fármacos , Sistema Nervioso Central/metabolismo , Sistema Nervioso Central/patología , Ácidos Hidroxámicos/farmacología , Ácidos Hidroxámicos/uso terapéutico , Progresión de la Enfermedad , Histona Desacetilasas/metabolismo , Histona Desacetilasas/genética , Fragmentos de Péptidos/farmacología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Interleucina-6/metabolismo , Interleucina-6/genéticaRESUMEN
The immunoregulatory effects of granulocyte colony-stimulating factor (G-CSF) on allogeneic peripheral blood cell transplantation (PBCT) have been demonstrated to reduce acute graft-versus-host disease (GVHD). However, the underlying mechanism is still not clear. In this study, we focused on the direct effects of G-CSF on donor CD4(+) T cell responses after transplantation. We observed that lethally irradiated B6D2F1 recipient mice that are transplanted with CD4(+) T cells from G-CSF-treated B6 donors showed mild attenuations in severity and mortality compared with recipients transplanted with PBS-treated CD4(+) T cells. Notably, skin GVHD was significantly reduced, but no such reduction was observed in other organs. Although there was no difference with respect to alloreactive expansion or Foxp3(+) Treg induction, the use of G-CSF-treated CD4(+) T cells significantly reduced the numbers of IL-17-producing and RORγt-expressing cells in the secondary lymphoid organs of allogeneic recipients after transplantation compared with the use of the control cells. Finally, we found that the suppressor of cytokine signaling-3 (SOCS3) expression in G-CSF-treated donor CD4(+) T cells was much higher than that in control CD4(+) T cells. Our results demonstrate that the inhibition of Th17 cell differentiation by SOCS3 induction is associated with the immunoregulatory role of G-CSF in CD4(+) T cell-mediated acute GVHD.
Asunto(s)
Linfocitos T CD4-Positivos/trasplante , Diferenciación Celular , Enfermedad Injerto contra Huésped/metabolismo , Factor Estimulante de Colonias de Granulocitos/farmacología , Células Th17/metabolismo , Enfermedad Aguda , Animales , Western Blotting , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/metabolismo , Femenino , Citometría de Flujo , Factores de Transcripción Forkhead/metabolismo , Enfermedad Injerto contra Huésped/genética , Enfermedad Injerto contra Huésped/patología , Interleucina-17/metabolismo , Ratones , Ratones Endogámicos C57BL , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/genética , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Índice de Severidad de la Enfermedad , Proteína 3 Supresora de la Señalización de Citocinas , Proteínas Supresoras de la Señalización de Citocinas/genética , Proteínas Supresoras de la Señalización de Citocinas/metabolismo , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/metabolismo , Trasplante HomólogoRESUMEN
Granulocyte colony-stimulating factor (G-CSF) mobilized peripheral blood stem cells (PBSCs) are more frequently used as the cellular source in allogeneic hematopoietic stem cell transplantation (HSCT) than bone marrow stem cells (BMSCs) because they promote more rapid engraftment and immune reconstitution. However, the underlying mechanism for this is not fully understood. Here, we investigated the role of Toll-like receptor 2 (TLR2) on PBSCs in promoting rapid engraftment after allogeneic HSCT. We found that PBSCs highly expressed TLR2 in comparison to BMSCs, and TLR2 was directly induced by G-CSF signaling. Treatment with the TLR2 ligand, Pam(3)CSK(4) (PAM), more efficiently induced myeloid differentiation of PBSCs than BMSCs. Similarly, endogenous TLR2 ligands from the serum of recipients of allogeneic transplantation more rapidly stimulated myeloid differentiation of PBSCs compared with BMSCs. PAM treatment of TLR2(-/-) syngeneic recipient mice transplanted with PBSCs resulted in significantly elevated numbers of PBSC-derived myeloid cells and spleen colony formation compared with controls. Our results demonstrate that TLR2 signaling in PBSCs correlates with their ability to rapidly differentiate into myeloid cells, resulting in improved engraftment. Thus, TLR2 may be a novel target for increasing the efficiency of allogeneic HSCT by overcoming engraftment failure or delayed engraftment.
Asunto(s)
Regulación de la Expresión Génica , Factor Estimulante de Colonias de Granulocitos/metabolismo , Trasplante de Células Madre Hematopoyéticas/métodos , Receptor Toll-Like 2/biosíntesis , Animales , Células de la Médula Ósea/citología , Diferenciación Celular , Femenino , Citometría de Flujo/métodos , Células Madre Hematopoyéticas/citología , Humanos , Leucocitos Mononucleares/citología , Ligandos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Transducción de Señal , Células Madre/citología , Receptor Toll-Like 2/genética , Trasplante HomólogoRESUMEN
Even though subclasses of macrophage have distinct roles during progression of infectious diseases, it remains poorly understood whether there is a subset-specific difference in drug responses. Here, we report that ABCG2 was expressed specifically in M2-like macrophages and that it controlled their efflux activities. Abcg2 expression is markedly induced during polarization of PMA-primed macrophages toward an M2 type. IL-4 and IL-13 induced Pparg expression through STAT6 and PPARγ in turn acted on the Abcg2 promoter for its transcription activation. Once polarized to M2-like macrophages, these cells had sustained PPARγ transcription activation of Abcg2 gene. Accordingly, interruption of this machinery by T0070907, an inverse agonist of PPARγ, was shown to be effective in Abcg2 downregulation and its efflux activity in M2-like macrophages. Taken together, our results implicate that ABCG2 of M2 macrophages may function as an important pump that plays a potential role in drug efflux and that T0070907 may be used to increase the efficacy of M2 macrophage-targeting drugs such as antibiotics.
Asunto(s)
Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/metabolismo , Benzamidas/farmacología , Activación de Macrófagos/inmunología , Macrófagos/inmunología , Proteínas de Neoplasias/metabolismo , PPAR gamma/antagonistas & inhibidores , Piridinas/farmacología , Factor de Transcripción STAT6/metabolismo , Línea Celular , Humanos , Activación de Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Macrófagos/patología , PPAR gamma/metabolismo , Fenotipo , Transducción de SeñalRESUMEN
Our previous study demonstrated that G-CSF treatment increased the expression of TLR2 in donor grafts; this contributed to rapid engraftment after allogeneic hematopoietic stem cell transplantation (HSCT) in mice. In the current study, we investigated the effects of upregulated TLR2 expression in G-CSF-mobilized donor grafts on acute graft-versus-host disease (GVHD). We found that TLR2 was highly expressed on myeloid cell populations but not T and B cells from the spleens of G-CSF-treated donor mice. After transplantation, the mortality and disease severity in recipients were not significantly different between G-CSF-treated TLR2-/- and wt donor grafts. Although endogenous TLR2 ligand was detected in the serum of both recipients, T cells from TLR2-/- and wt donors have the same ability regarding alloreactivity. Moreover, the blockade of TLR2 signaling in recipients by administering anti-TLR2 blocking antibody after BMT did not lead to a significant difference in acute GVHD compared with control IgG treatment. However, the hematopoietic ability of G-CSF-mobilized lin−c-kit+ HSCs from TLR2-/- donor grafts was lower than that from wt donor grafts. Our results demonstrate that upregulated TLR2 expression in G-CSF-mobilized donor grafts has no effect on acute GVHD, suggesting that TLR2 is a valuable target for increasing HSCT efficiency in order to enhance engraftment without exacerbating acute GVHD.
Asunto(s)
Enfermedad Injerto contra Huésped/inmunología , Factor Estimulante de Colonias de Granulocitos/farmacología , Receptor Toll-Like 4/biosíntesis , Animales , Linfocitos B/efectos de los fármacos , Linfocitos B/inmunología , Trasplante de Células Madre Hematopoyéticas/métodos , Ratones , Ratones Noqueados , Células Mieloides/efectos de los fármacos , Células Mieloides/metabolismo , Trasplante de Células Madre , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Receptor Toll-Like 4/genéticaRESUMEN
A high frequency of G-CSF-mobilized myeloid cells (gMCs) in a donor graft accelerates hematopoietic recovery after peripheral blood stem cell transplantation (PBSCT). However, because of the limited functional efficacy of gMCs, repeated transfusions of gMCs are frequently required. In this study, we investigated a strategy to improve the functional capacity of gMCs during hematopoietic engraftment after allogeneic transplantation. We found that toll-like receptor 2 (TLR2) is constitutively expressed on gMCs. Treating gMCs with the synthetic TLR2 ligand Pam(3)CSK(4) (PAM) dramatically enhanced IL-10 and TNF-α production. However, PAM treatment does not induce substantial cellular maturation. Moreover, PAM treatment significantly improved gMC survival. PAM treated gMCs significantly promoted myeloid differentiation of donor hematopoietic stem cells (HSCs), resulting in accelerated engraftment after allogeneic transplantation. Our data suggest that TLR2-stimulated gMCs may be a novel cellular therapeutic for increasing the efficiency of allogeneic hematopoietic stem cell transplantation (HSCT) by reducing infectious complications associated with delayed engraftment.
Asunto(s)
Supervivencia de Injerto , Factor Estimulante de Colonias de Granulocitos/farmacología , Trasplante de Células Madre Hematopoyéticas , Células Mieloides/efectos de los fármacos , Células Mieloides/metabolismo , Receptor Toll-Like 2/metabolismo , Animales , Diferenciación Celular , Supervivencia Celular , Citocinas/biosíntesis , Expresión Génica , Perfilación de la Expresión Génica , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Ratones , Células Mieloides/citología , Receptor Toll-Like 2/genética , Trasplante HomólogoRESUMEN
A human monoclonal antibody can be a good method for tumor diagnosis and treatment. This study is aimed at the generation of human antibody fragments against urokinase plasminogen activator (uPA) known to be related to tumor metastasis using the naive human antibody phage display library. Three clones--A2, A8, and E4--were selected from 1 x 10(10) sized human naïve antibody phage library using BIAcore rescue and screen. Clone A8 was finally selected by flow cytometry against Hep3 and HT1080, uPA overexpressing tumor cell lines. A8 clone consisted of 324 bp lambda and 402 bp heavy chains. The affinity (K(D)) of purified A8 antibody fragments was 1.44 x 10(-8) M(-1). The antibody fragment was reacted with HT1080 in a dose-dependent manner but not reacted with LS513 normal fibroblast. In this study, uPA specific human monoclonal antibody fragment A8 was made with BIAcore selection. Selected A8 was bound specifically to uPA expressed on the tumor cell surface. Further study for the application of A8 antibody clones will be needed.
Asunto(s)
Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/inmunología , Anticuerpos de Cadena Única/inmunología , Activador de Plasminógeno de Tipo Uroquinasa/inmunología , Secuencia de Aminoácidos , Anticuerpos Monoclonales/genética , Afinidad de Anticuerpos , Secuencia de Bases , Línea Celular Tumoral , Cartilla de ADN/genética , Citometría de Flujo , Humanos , Datos de Secuencia Molecular , Análisis de Secuencia de ADN , Anticuerpos de Cadena Única/genética , Resonancia por Plasmón de SuperficieRESUMEN
Tetras (tetra-arsenic oxide, As(4)O(6)) is a derivative of arsenic used in Korean traditional medicine for the treatment of cancer, but its mechanism remains largely undefined. Recently, a similar arsenic derivative, diarsenic trioxide (As(2)O(3), ATO), has been shown to mediate anti-tumor activity, therefore reigniting interest in the therapeutic effect of arsenic compounds. Here we report that Tetras can effectively mediate an anti-vascular effect on tumors, leading to delay in tumor growth and increased survival. Our study demonstrates for the first time the potential use of Tetras as a radiation therapy enhancement agent for solid tumors. These findings reveal an unappreciated role of Tetras in cancer therapy and its potential application to radiotherapy in achieving local tumor control.