Suppression of oxidative stress and 5-lipoxygenase activation by edaravone improves depressive-like behavior after concussion.

Brain concussions are a serious public concern and are associated with neuropsychiatric disorders, such as depression. Patients with concussion who suffer from depression often experience distress. Nevertheless, few pre-clinical studies have examined concussion-induced depression, and there is little information regarding its pharmacological management. Edaravone, a free radical scavenger, can exert neuroprotective effects in several animal models of neurological disorders. However, the effectiveness of edaravone in animal models of concussion-induced depression remains unclear. In this study, we examined whether edaravone could prevent concussion-induced depression. Mice were subjected to a weight-drop injury and intravenously administered edaravone (3.0 mg/kg) or vehicle immediately after impact. Serial magnetic resonance imaging showed no abnormalities of the cerebrum on diffusion T1- and T2-weighted images. We found that edaravone suppressed concussion-induced depressive-like behavior in the forced swim test, which was accompanied by inhibition of increased hippocampal and cortical oxidative stress (OS) and suppression of 5-lipoxygenase (5-LOX) translocation to the nuclear envelope in hippocampal astrocytes. Hippocampal OS in concussed mice was also prevented by the nicotinamide adenine dinucleotide phosphate oxidase inhibitor, apocynin, and administration of BWB70C, a 5-LOX inhibitor, immediately and 24 h after injury prevented depressive-like behaviors in concussed mice. Further, antidepressant effects of edaravone were observed in mice receiving 1.0 or 3.0 mg/kg of edaravone immediately after impact, but not at a lower dose of 0.1 mg/kg. This antidepressant effect persisted up to 1 h after impact, whereas edaravone treatment at 3 h after impact had no effect on concussion-induced depressive-like behavior. These results suggest that edaravone protects against concussion-induced depression, and this protection is mediated by suppression of OS and 5-LOX translocation.

Downregulation of miR-217 correlates with resistance of Ph(+) leukemia cells to ABL tyrosine kinase inhibitors.

This study found that long-term exposure of chronic myelogenous leukemia (CML) K562 cells to BCR/ABL thyrosine kinase inhibitors (TKI) caused drug-resistance in association with an increase in levels of DNA methyltransferases (DNMT) and a decrease in levels of microRNA miR-217. These observations are clinically relevant; an increase in levels of DNMT3A in association with downregulation of miR-217 were noted in leukemia cells isolated from individuals with BCR/ABL TKI-resistant Philadelphia chromosome positive acute lymphoblastic leukemia (Ph(+) ALL) and CML. Further studies with TKI-resistant K562 cells found that forced expression of miR-217 inhibited expression of DNMT3A through a miR-217-binding site within the 3'-untranslated region of DNMT3A and sensitized these cells to growth inhibition mediated by the TKI. Of note, long-term exposure of K562 cells to dasatinib (10 nM) together with 5-Aza-2'-deoxycytidine (5-AzadC) (0.1 µM) potently inhibited proliferation of these cells in association with upregulation of miR-217 and downregulation of DNMT3A in vitro. In addition, a decrease in levels of DNMT3A and an increase in levels of miR-217 were noted in K562 tumors growing in immune-deficient mice that were treated with the combination of 5-AzadC and dasatinib. Taken together, Ph(+) leukemia cells acquire TKI resistance via downregulation of miR-217 and upregulation of DNMT3A. Inhibition of DNMT3A by forced expression of miR-217 or 5-AzadC may be useful to prevent drug resistance in individuals who receive TKI.

CD82 regulates STAT5/IL-10 and supports survival of acute myelogenous leukemia cells.

We recently reported that adhesion molecule CD82 is aberrantly expressed in CD34(+) /CD38(-) leukemia stem cells (LSCs). Here, we report the results of a functional analysis of CD82 in CD34(+) /CD38(-) acute myelogenous leukemia (AML) cells. Short hairpin (sh)RNA-mediated downregulation of CD82 resulted in a decrease in the level of IL-10. In contrast, forced expression of CD82 in CD34(+)/CD38(+) AML cells by transduction with CD82-expressing lentiviral particles resulted in an increase in the levels of IL-10. Notably, exposure of CD34(+)/CD38(-) AML cells to IL-10 stimulated clonogenic growth of these cells. Moreover, downregulation of CD82 by a shRNA dephosphorylated STAT5 in CD34(+)/CD38(-) AML cells. On the other hand, forced expression of CD82 resulted in increase in the levels of p-STAT5 in CD34(+)/CD38(+) AML cells. Chromatin immunoprecipitation (ChIP) assay results indicated that STAT5A binds to the promoter region of the IL-10 gene, while reporter gene assay results indicated stimulation of IL-10 expression at the transcriptional level. These results suggest that CD82 positively regulates the STAT5/IL-10 signaling pathway. Moreover, shRNA-mediated downregulation of CD82 expression in CD34(+)/CD38(-) AML cells dephosphorylated STAT5 in immunodeficient mice. Taken together, our data suggest that the CD82/STAT5/IL-10 signaling pathway is involved in the survival of CD34(+)/CD38(-) AML cells and may thus be a promising therapeutic target for eradication of AML LSCs.

CD34⁺/CD38⁻ acute myelogenous leukemia cells aberrantly express Aurora kinase A.

We previously showed that Aurora kinase A (AURKA) is aberrantly expressed in acute myelogenous leukemia (AML) cells when compared to bone marrow mononuclear cells isolated from healthy volunteers. We have also shown that CD34(+) /CD38(-) AML cells, one of compartments enriched for leukemia stem cells in most leukemia subgroups, were relatively resistant to cytarabine-mediated growth inhibition when compared to their CD34(+) /CD38(+) counterparts. Our study attempted to identify therapeutic targets in CD34(+) /CD38(-) AML cells and found that CD34(+) /CD38(-) AML cells isolated from patients (n = 26) expressed larger amounts of AURKA than their CD34(+) /CD38(+) counterparts and CD34(+) normal hematopoietic stem/progenitor cells isolated from healthy volunteers (n = 6), as measured by real-time reverse-transcriptase polymerase chain reaction. Blockade of AURKA by the specific inhibitor MLN8237 or a short hairpin RNA (shRNA) against AURKA significantly inhibited proliferation, impaired self-renewal capability and induced apoptosis of CD34(+) /CD38(-) AML cells, in association with modulation of levels of Bcl-2 family member proteins. Importantly, inhibition of AURKA in CD34(+) /CD38(-) AML cells by MLN8237 or an shRNA significantly impaired engraftment of these cells in severely immunocompromised mice and appeared to prolong their survival. These results suggest that AURKA is a promising molecular target to eliminate chemotherapy-resistant CD34(+) /CD38(-) AML cells.

IL-1ß inhibits self-renewal capacity of dormant CD34⁺/CD38⁻ acute myelogenous leukemia cells in vitro and in vivo.

We previously showed that CD34⁺/CD38⁻ acute myelogenous leukemia (AML) cells, which contain leukemia stem cells, expressed a greater amount of the phosphorylated forms of JAK2 and STAT5 (p-JAK2 and p-STAT5) than their CD34⁺/CD38⁺ counterparts. To identify candidate cytokines that are involved in the activation of JAK2/STAT5 in CD34⁺/CD38⁻ AML cells, we compared the cytokine expression profiles of CD34⁺/CD38⁻ AML cells and their CD34⁺/CD38⁺ counterparts. Interestingly, freshly isolated CD34⁺/CD38⁻ AML cells from patients (n = 17) expressed less interleukin-1ß (IL-1ß) than their CD34⁺/CD38⁺ counterparts and CD34⁺ normal hematopoietic stem/progenitor cells from healthy volunteers (n = 6), as measured by real-time Reverse Transcription-Polymerase Chain Reaction (RT-PCR). Methylation-specific PCR found that IL-1B gene expression was silenced by methylation of the promoter region. Importantly, exposure of CD34⁺/CD38⁻ AML cells to IL-1ß (100 ng/ml) stimulated cell-cycle progression, induced apoptosis and sensitized these cells to growth inhibition by antileukemia agents. These changes occurred in conjunction with the downregulation of cyclin-dependent kinase inhibitor p21waf1, antiapoptotic proteins and p-STAT5. Forced expression of IL-1ß in CD34⁺/CD38⁻ AML cells by lentiviral transduction significantly impaired the self-renewal capacity of the cells and induced apoptosis. Additionally, when these CD34⁺/CD38⁻ AML cells with forced expression of IL-1ß were transplanted into severely immunocompromised mice, the engraftment of the cells and reconstitution of AML were significantly impaired. Taken together, our results indicate that the inhibition of STAT5 by IL-1ß may be a promising treatment strategy to eradicate leukemia stem cells in AML.

Attenuation of axonal injury and oxidative stress by edaravone protects against cognitive impairments after traumatic brain injury.

Traumatic axonal injury (TAI), a feature of traumatic brain injury (TBI), progressively evolves over hours through impaired axonal transport and is thought to be a major contributor to cognitive dysfunction. In spite of various studies suggesting that pharmacologic or physiologic interventions might reduce TAI, clinical neuroprotective treatments are still unavailable. Edaravone, a free radical scavenger, has been shown to exert neuroprotective effects in animal models of several brain disorders. In this study, to evaluate whether edaravone suppresses TAI following TBI, mice were subjected to weight drop injury and had either edaravone (3.0mg/kg) or saline administered intravenously immediately after impact. Axonal injury and oxidative stress were assessed using immunohistochemistry with antibodies against amyloid precursor protein, a marker of impaired axonal transport, and with 8-hydroxy-2'-deoxyguanosine, a marker of oxidative DNA damage. Edaravone significantly suppressed axonal injury and oxidative stress in the cortex, corpus callosum, and hippocampus 24h after injury. The neuroprotective effects of edaravone were observed in mice receiving 1.0, 3.0, or 10mg/kg of edaravone immediately after impact, but not after 0.3mg/kg of edaravone. With treatment 1h after impact, axonal injury was also significantly suppressed and this therapeutic effect persisted up to 6h after impact. Furthermore, behavioral tests performed 9 days after injury showed memory deficits in saline-treated traumatized mice, which were not evident in the edaravone-treated group. These results suggest that edaravone protects against memory deficits following TBI and that this protection is mediated by suppression of TAI and oxidative stress.

CD34⁺/CD38⁻ acute myelogenous leukemia cells aberrantly express CD82 which regulates adhesion and survival of leukemia stem cells.

To identify molecular targets in leukemia stem cells (LSCs), this study compared the protein expression profile of freshly isolated CD34(+) /CD38(-) cells with that of CD34(+) /CD38(+) counterparts from individuals with acute myelogenous leukemia (n = 2, AML) using isobaric tags for relative and absolute quantitation (iTRAQ). A total of 98 proteins were overexpressed, while six proteins were underexpressed in CD34(+) /CD38(-) AML cells compared with their CD34(+) /CD38(+) counterparts. Proteins overexpressed in CD34(+) /CD38(-) AML cells included a number of proteins involved in DNA repair, cell cycle arrest, gland differentiation, antiapoptosis, adhesion, and drug resistance. Aberrant expression of CD82, a family of adhesion molecules, in CD34(+) /CD38(-) AML cells was noted in additional clinical samples (n = 12) by flow cytometry. Importantly, down-regulation of CD82 in CD34(+) /CD38(-) AML cells by a short hairpin RNA (shRNA) inhibited adhesion to fibronectin via up-regulation of matrix metalloproteinases 9 (MMP9) and colony forming ability of these cells as assessed by transwell assay, real-time RT-PCR, and colony forming assay, respectively. Moreover, we found that down-regulation of CD82 in CD34(+) /CD38(-) AML cells by an shRNA significantly impaired engraftment of these cells in severely immunocompromised mice. Taken together, aberrant expression of CD82 might play a role in adhesion of LSCs to bone marrow microenvironment and survival of LSCs. CD82 could be an attractive molecular target to eradicate LSCs.

Involvement of galectin-9 in lung eosinophilia in patients with eosinophilic pneumonia.

BACKGROUND: Although we first found galectin-9 (Gal-9) as an eosinophil chemoattractant, its role in eosinophilic inflammation is still obscure. The purpose of the present study is to clarify the role of Gal-9 in human eosinophilic pulmonary inflammation in comparison with eotaxin (CCL11). METHODS: We measured the levels of Gal-9 and eotaxin in the bronchoalveolar lavage fluid (BALF) of patients with acute and chronic eosinophilic pneumonia (AEP and CEP). Furthermore, the biological activities (chemotaxis and apoptosis) of Gal-9 were compared with those of eotaxin using interleukin-5-primed or -unprimed eosinophils. RESULTS: The levels of Gal-9 and eotaxin in the BALF from patients with AEP and those with CEP were higher than those found in the controls. Although there was little difference in Gal-9 level between patients with AEP and patients with CEP, the eotaxin level was significantly lower in patients with CEP. In patients with AEP, the eosinophil number correlated well with both the Gal-9 and eotaxin levels. However, in patients with CEP, the eosinophil number only correlated well with the Gal-9 level. Moreover, the Gal-9 level correlated with the eotaxin level in patients with AEP, but there was no significant correlation between those levels in patients with CEP. Anti-Gal-9 antibody treatment strongly reduces eosinophil chemotactic activity in the BALF of patients with AEP and in that of patients with CEP, whereas the anti-CCR3 (receptor for eotaxin) antibody strongly reduces this activity in the BALF of patients with AEP but not in that of patients with CEP. Furthermore, Gal-9 exhibited both chemotactic and proapoptotic activities for activated eosinophils, though eotaxin only exhibited chemotactic activity. CONCLUSIONS: The present results provide two possibilities: that Gal-9 is involved in pulmonary eosinophilia in patients with AEP and CEP, and that Gal-9 exhibits regulatory functions for activated eosinophils at the site of inflammation.

Galectin-9 expands unique macrophages exhibiting plasmacytoid dendritic cell-like phenotypes that activate NK cells in tumor-bearing mice.

Galectin-9 (Gal-9) inhibits the metastasis of tumor cells by blocking their adhesion to endothelium and the extracellular matrix. In this study, we addressed the involvement of Gal-9 in anti-tumor activity. Gal-9 significantly prolonged the survival of B16F10 melanoma-bearing mice. Gal-9 increased the numbers of NK cells, CD8 T cells and macrophages in tumor-bearing mice. Gal-9-mediated anti-tumor activity was not induced in NK cell-, macrophage- and CD8 T cell-depleted mice. NK cells from Gal-9-treated mice, compared to PBS-treated mice, exhibited significantly higher cytolytic activity. Co-culture of naïve NK cells with macrophages from Gal-9-treated mice resulted in enhanced NK activity, although Gal-9 itself did not enhance the NK activity. We also found that Ly-6C(+)CD11b(+)F4/80(+) macrophages with plasmacytoid cell (pDC)-like phenotypes (PDCA-1 and B220) were responsible for the enhanced NK activity. These results provide evidence that Gal-9 promotes NK cell-mediated anti-tumor activity by expanding unique macrophages with a pDC-like phenotype.

Galectin-9 increases Tim-3+ dendritic cells and CD8+ T cells and enhances antitumor immunity via galectin-9-Tim-3 interactions.

A Tim-3 ligand, galectin-9 (Gal-9), modulates various functions of innate and adaptive immune responses. In this study, we demonstrate that Gal-9 prolongs the survival of Meth-A tumor-bearing mice in a dose- and time-dependent manner. Although Gal-9 did not prolong the survival of tumor-bearing nude mice, transfer of naive spleen cells restored a prolonged Gal-9-induced survival in nude mice, indicating possible involvement of T cell-mediated immune responses in Gal-9-mediated antitumor activity. Gal-9 administration increased the number of IFN-gamma-producing Tim-3(+) CD8(+) T cells with enhanced granzyme B and perforin expression, although it induced CD4(+) T cell apoptosis. It simultaneously increased the number of Tim-3(+)CD86(+) mature dendritic cells (DCs) in vivo and in vitro. Coculture of CD8(+) T cells with DCs from Gal-9-treated mice increased the number of IFN-gamma producing cells and IFN-gamma production. Depletion of Tim-3(+) DCs from DCs of Gal-9-treated tumor-bearing mice decreased the number of IFN-gamma-producing CD8(+) T cells. Such DC activity was significantly abrogated by Tim-3-Ig, suggesting that Gal-9 potentiates CD8(+) T cell-mediated antitumor immunity via Gal-9-Tim-3 interactions between DCs and CD8(+) T cells.

Galectin-9 suppresses tumor metastasis by blocking adhesion to endothelium and extracellular matrices.

We previously described an inverse correlation between galectin-9 (Gal-9) expression and metastasis in patients with malignant melanoma and breast cancer. This study verified the ability of Gal-9 to inhibit lung metastasis in experimental mouse models using highly metastatic B16F10 melanoma and Colon26 colon cancer cells. B16F10 cells transfected with a secreted form of Gal-9 lost their metastatic potential. Intravenous Gal-9 administration reduced the number of metastases of both B16F10 and Colon26 cells in the lung, indicating that secreted Gal-9 suppresses metastasis. Analysis of adhesive molecule expression revealed that B16F10 cells highly express CD44, integrin alpha1, alpha 4, alpha V, and beta1, and that Colon26 cells express CD44, integrin alpha2, alpha 5, alpha V, and beta1, suggesting that Gal-9 may inhibit the adhesion of tumor cells to vascular endothelium and the extracellular matrix (ECM) by binding to such adhesive molecules. Indeed, Gal-9 suppressed the binding of hyaluronic acid to CD44 on both B16F10 and Colon26 cells, and also suppressed the binding of vascular cell adhesion molecule-1 to very late antigen-4 on B16F10 cells. Furthermore, Gal-9 inhibited the binding of tumor cells to ECM components, resulting in the suppression of tumor cell migration. The present results suggest that Gal-9 suppresses both attachment and invasion of tumor cells by inhibiting the binding of adhesive molecules on tumor cells to ligands on vascular endothelium and ECM.

Beneficial effect of galectin 9 on rheumatoid arthritis by induction of apoptosis of synovial fibroblasts.

OBJECTIVE: To compare the expression of galectin 9 (Gal-9) in synovial tissue (ST) from rheumatoid arthritis (RA) patients and osteoarthritis (OA) patients and to evaluate the effects of Gal-9 on fibroblast-like synoviocytes (FLS) in these patients. METHODS: The expression of Gal-9 in ST and FLS was compared using immunohistochemical techniques. Apoptotic cells in RA and OA ST samples were detected by TUNEL assay. Apoptosis of FLS was analyzed by the sub-G(1) method in vitro. The in vivo suppressive effects of Gal-9 on collagen-induced arthritis (CIA) in a mouse model were also elucidated. RESULTS: The percentage of Gal-9-positive cells in ST samples and the amount of Gal-9 in synovial fluid samples were significantly higher in patients with RA than in patients with OA, suggesting the involvement of Gal-9 in the development of RA. Compared with the 2 wild-type Gal-9 forms, stable Gal-9, a mutant protein resistant to proteolysis, significantly induced apoptosis of FLS from RA patients. In contrast, other galectins, such as Gal-1, Gal-3, and Gal-8, did not induce apoptosis or suppress the proliferation of human RA FLS. Stable Gal-9 preferentially induced apoptosis and suppressed the proliferation of RA FLS in vitro. It also induced apoptosis of cells in RA ST implanted into SCID mice in vivo. In a mouse model of CIA, apoptotic cells were detected in the joints of stable Gal-9-treated mice, but not phosphate buffered saline-treated mice, and suppressed CIA characterized by pannus formation with inflammatory cell infiltration and bone/cartilage destruction. CONCLUSION: Gal-9-induced apoptosis of hyperproliferative RA FLS may play a critical role in the suppression of RA.

Galectin-9 inhibits CD44-hyaluronan interaction and suppresses a murine model of allergic asthma.

RATIONALE: Galectin-9 (Gal-9) belongs to the galectin family, which exhibits affinity for beta-galactosides. Gal-9 has a variety of biological activities; however, its role in allergic inflammation is unknown. OBJECTIVES: We evaluated the effect of a stable form of the human protein on allergic airway inflammation in a mite allergen-induced asthma model. METHODS: Human stable Gal-9 was given by intravenous injection to mice during antigen challenge. The effect of Gal-9 on airway inflammation and airway hyperresponsiveness (AHR) was then evaluated. MEASUREMENTS AND MAIN RESULTS: Gal-9 reduced AHR as well as Th2-associated airway inflammation. Furthermore, administration of Gal-9 as well as anti-CD44 monoclonal antibody inhibited the infiltration of peripheral blood Th2 cells into the airway. Interestingly, Gal-9 directly bound the CD44 adhesion molecule and inhibited interactions with hyaluronan (HA). Consistent with the concept that CD44-HA interactions mediate the migration of T cells into the lung, Gal-9 blocked CD44-dependent adhesion of BW5147 mouse T cells to HA. CONCLUSIONS: We conclude that Gal-9 inhibits allergic inflammation of the airway and AHR by modulating CD44-dependent leukocyte recognition of the extracellular matrix.

Development and functional analysis of eosinophils from murine embryonic stem cells.

We have established a culture system for the development of eosinophils from murine embryonic stem (ES) cells. After transferring ES cells from embryonic fibroblast cells onto macrophage colony-stimulating factor-deficient stromal cells, OP9, ES cells were cultured in the presence of interleukin (IL)-5 with either IL-3 or granulocyte-macrophage colony stimulating factor (GM-CSF) for 20 d to obtain approximately 50% eosinophils. Electron microscopy confirmed the presence of crystallized major basic protein (MBP) in the granules of some of these cells. Neither IL-5, IL-3, GM-CSF nor eotaxin alone could induce eosinophils as efficiently as the conditions described above. Eotaxin induced eosinophil development in combination with either IL-3 or IL-5. Levels of GATA-1, Friend of GATA (FOG)-1, PU.1, CCAAT/enhancer binding protein (C/EBP)alpha, C/EBPbeta, IL-3 receptor alpha (IL-3Ralpha), GM-CSF receptor alpha (GM-CSFRalpha), and MBP mRNAs were increased in ES cells 10 d after transfer onto OP9 cells. In contrast, C/EBPepsilon, IL-5Ralpha, and eosinophil peroxidase mRNAs were induced in response to IL-3 and IL-5 after transfer onto OP9 cells. Eosinophils that developed in this system expressed Gr-1, F4/80, B220, CCR3, IL-3Ralpha, IL-5Ralpha, and DX5. Finally, eosinophils developed from ES cells produced reactive oxygen species in response to Leishmania as do peripheral blood eosinophils.