So-Youn Woo

CD40-Mediated Transcriptional Regulation of the IL-6 Gene in B Lymphocytes: Involvement of NF-κB, AP-1, and C/EBP

Engagement of CD40 by its ligand CD154 induces IL-6 production by B lymphocytes. We previously reported that this IL-6 production is dependent upon binding of the adapter protein TNF receptor-associated factor 6 to the cytoplasmic domain of CD40, while binding of TNF receptor-associated factors 2 and 3 is dispensable, as is the activation-induced nuclear translocation of NF-κB. The present study was designed to characterize CD40-mediated transcriptional control of the IL-6 gene in B cells. CD40 engagement on B lymphocytes activated the IL-6 promoter, and mutations in the putative binding sites for AP-1 and C/EBP transcription factors reduced this activation. Interestingly, a mutation in the putative NF-κB binding site completely abrogated the basal promoter activity, thus also rendering the promoter unresponsive to CD40 stimulation, suggesting that this site is required for binding of NF-κB constitutively present in the nucleus of mature B cells. The expression of dominant negative Fos or C/EBPα proteins, which prevent binding of AP-1 or C/EBP complexes to DNA, also reduced CD40-mediated IL-6 gene expression. Furthermore, CD40 stimulation led to phosphorylation of c-Jun on its activation domain, implicating CD40-mediated Jun kinase activation in the transcriptional regulation of IL-6 production.

Mesenchymal stem cells showed the highest potential for the regeneration of injured liver tissue compared with other subpopulations of the bone marrow

We have previously reported that bone marrow cells (BMCs) participate in the regeneration after liver injury. However, it is not established that this is the result of differentiation of hematopoietic stem cells (HSCs), mesenchymal stem cells (MSCs) or the combination of both. We investigated the contribution of each cell fraction to the regenerative process. First, we confirmed that transplanted stem cells migrate directly to injured liver tissue without dispersing to other organs. Next, we divided green fluorescent protein (GFP)‐expressing BMCs into three populations as mononuclear cells, MSCs and HSCs. We then compared the engraftment capacity after transplantation of each fraction of cells into liver‐injured mice. Of these, the MSCs transplanted group showed the highest GFP fluorescence intensities in liver tissue by flow cytometry analysis and confocal microscopic observation. Furthermore, MSCs showed differentiation potential into hepatocytes when co‐cultured with injured liver cells, which suggests that MSCs showed highest potential for the regeneration of injured liver tissue compared with those of the other two cell refractions.

IL-17 and IL-22 enhance skin inflammation by stimulating the secretion of IL-1β by keratinocytes via the ROS-NLRP3-caspase-1 pathway

BACKGROUND The pathogenesis of inflammatory skin disease involves the release of cytokines from keratinocytes, and one of these, IL-1β, has been previously implicated in inflammatory skin disease. T(h)17 cells, a subset of T(h) cells involved in autoimmunity and inflammation, possess IL-1β receptors and secrete cytokines such as IL-17 and IL-22 in response to IL-1β stimulation. A mutation in the inflammasome protein NLRP3 (NACHT, LRR and PYD domains-containing protein 3) causes excess production of IL-1β, resulting in an augmentation of T(h)17-dominant pathology. METHODS To determine the feedback effect, if any, of IL-17 and/or IL-22 on the secretion of IL-1β from keratinocytes, we stimulated the human keratinocyte cell line HaCaT, as well as caspase-1-deficient mice, with IL-17 or IL-22. RESULTS We found that treatment with IL-17 and IL-22 causes an increase in IL-1β via the activation of NLRP3 by a process that involves the generation of reactive oxygen species. Moreover, skin inflammation induced by IL-17 and IL-22 was lower in caspase-1 knockout (KO) mice relative to that induced by IL-1β treatment. Additionally, skin inflammation induced by the drug imiquimod was lower in caspase-1 KO mice than in wild-type mice. CONCLUSION These results indicate that cytokines from T(h)17 cells may potentiate IL-1β-mediated skin inflammation and result in phenotypic alterations of keratinocytes via a feedback mechanism.

Tonsil-derived mesenchymal stromal cells: evaluation of biologic, immunologic and genetic factors for successful banking

BACKGROUND AIMS Although mesenchymal stromal cells (MSC) from human palatine tonsils (tonsillar MSC, T-MSC) have been isolated, whether T-MSC isolated from multiple donors are feasible for cell banking has not been studied. METHODS T-MSC before and after a standard protocol of cryopreservation and thawing were assessed regarding several basic characteristics, including colony-forming unit-fibroblast features, MSC-specific surface antigen profiles, and inhibition of alloreactive T-cell proliferation. In vitro mesodermal differentiation potentials to adipocytes, osteocytes and chondrocytes were detected by staining with either cell-specific dyes or antibody after incubation with each appropriate differentiation medium. Expression of mesoderm-specific genes was also quantified by real-time polymerase chain reaction (PCR) assay. Expression profiles of endoderm-specific genes were identified by reverse transcription PCR assay. The feasibility of T-MSC in future engraftment was tested by short tandem repeat (STR) analysis using genomic DNA isolated randomly from three independent subjects. RESULTS Both fresh and cryopreserved-thawed T-MSC showed a similar high proliferation capacity and expressed primitive cell-surface markers. Hematopoietic cell markers, HLA-DR, co-stimulatory molecules and follicular dendritic cell markers were not detected. In addition to mesodermal differentiation, fresh and cryopreserved-thawed cells also underwent endodermal differentiation, as evidenced by the expression of endoderm-specific genes including forkhead box A2 (FoxA2), SIX homeobox 1 (Six1) and chemokine (C-C motif) ligand 21 (CCL21). Both cells significantly decreased phorbol 12- myristate 13-acetate (PMA)-induced T-cell proliferation. T-MSC from three independent donors formed chimerism in STR analysis. CONCLUSIONS Our results demonstrate for the first time that T-MSC are a potentially good source for MSC banking.

Filaggrin knockdown and Toll‐like receptor 3 (TLR3) stimulation enhanced the production of thymic stromal lymphopoietin (TSLP) from epidermal layers

Abstract:  Keratinocytes constitute the first‐line barrier against exogenous antigens and contain Toll‐like receptors (TLRs), which function as pattern‐recognition molecules to activate antimicrobial innate immune responses. In an effort to ascertain whether or not filaggrin (filament‐aggregating protein) expression affected the TLR‐mediated responses of keratinocytes, we transfected filaggrin siRNA into HaCaT human keratinocyte cells and determined that thymic stromal lymphopoietin (TSLP) and IL‐6 secretion were increased by poly(I:C) stimulus. Additionally, TSLP expression is increased in filaggrin knockdown as well as TLR3 stimulation in reconstituted human epidermal layers. Therefore, the findings of this study show that reduced filaggrin levels may influence innate immune responses via TLR stimuli and may contribute to the pathogenesis of inflammatory skin disease via TSLP expression.

Mesenchymal stromal cells inhibit graft-versus-host disease of mice in a dose-dependent manner

BACKGROUND AIMS Graft-versus-host disease (GvHD) remains a major complication after allogeneic hematopoietic cell transplantation (HCT). Recent literature demonstrates a potential benefit of human mesenchymal stromal cells (MSC) for the treatment of refractory GvHD; however, the optimal dose remains uncertain. We set out to develop an animal model that can be used to study the effect of MSC on GvHD. METHODS A GvHD mouse model was established by transplanting C3H/he donor bone marrow (BM) cells and spleen cells into lethally irradiated BALB/c recipient mice. MSC were obtained from C3H/he mice and the C3H/10T1/2 murine MSC line. RESULTS The mRNA expression of Foxp3 in regional lymph nodes (LN) localized with T cells was markedly increased by the addition of C3H10T1/2 cells in a real-time polymerase chain reaction (PCR). Using a mixed lymphocyte reaction, we determined the optimal splenocyte proliferation inhibition dose (MSC:splenocyte ratios 1:2 and 1:1). Three different C3H10T1/2 cell doses (low, 0.5 x 10(6), intermediate, 1 x 10(6), and high, 2 x 10(6)) with a consistent splenocyte dose (1 x 10(6)) were evaluated for their therapeutic potential in an in vivo GvHD model. The clinical and histologic GvHD score and Kaplan-Meier survival rate were improved after MSC transplantation, and these results demonstrated a dose-dependent inhibition. CONCLUSIONS We conclude that MSC inhibit GvHD in a dose-dependent manner in this mouse model and this model can be used to study the effects of MSC on GvHD.

Mesenchymal stem cells restore CCl4-induced liver injury by an antioxidative process.

We have investigated BM (bone marrow)‐derived MSCs (mesenchymal stem cells) for the treatment of liver injury. It was hypothesized that MSC‐mediated resolution of liver injury could occur through an antioxidative process. After being injected with CCl4 (carbon tetrachloride), mice were injected with syngenic BM‐derived MSCs or normal saline. Oxidative stress activity of the MSCs was determined by the analysis of ROS (reactive oxygen species) and SOD (superoxide dismutase) activity. In addition, cytoprotective genes of the liver tissue were assessed by real‐time PCR and ARE (antioxidant‐response element) reporter assay. Up‐regulated ROS of CCl4‐treated liver cells was attenuated by co‐culturing with MSCs. Suppression of SOD by adding an SOD inhibitor decreased the effect of MSCs on injured liver cells. MSCs significantly increased SOD activity and inhibited ROS production in the injured liver. The gene expression levels of Hmox‐1 (haem oxygenase‐1), BI‐1 (Bax inhibitor‐1), HGF (hepatocyte growth factor), GST (glutathione transferase) and Nrf2 (nuclear factor‐erythoid 2 p45 subunit‐related factor 20), attenuated by CCl4, were increased up to basal levels after MSC transplantation. In addition, MSCs induced an ARE, shown by luciferase activity, which represented a cytoprotective response in the injured liver. Evidence of a new cytoprotective effect is shown in which MSCs promote an antioxidant response and supports the potential of using MSC transplantation as an effective treatment modality for liver disease.

Tonsil-derived Mesenchymal Stem Cells Ameliorate CCl4–induced Liver Fibrosis in Mice via Autophagy Activation

Liver transplantation is the treatment of choice for chronic liver failure, although it is complicated by donor shortage, surgery-related complications, and immunological rejection. Cell transplantation is an alternative, minimally invasive treatment option with potentially fewer complications. We used human palatine tonsil as a novel source of mesenchymal stem cells (T-MSCs) and examined their ability to differentiate into hepatocyte-like cells in vivo and in vitro. Carbon tetrachloride (CCl4) mouse model was used to investigate the ability of T-MSCs to home to the site of liver injury. T-MSCs were only detected in the damaged liver, suggesting that they are disease-responsive. Differentiation of T-MSCs into hepatocyte-like cells was confirmed in vitro as determined by expression of hepatocyte markers. Next, we showed resolution of liver fibrosis by T-MSCs via reduction of TGF-β expression and collagen deposition in the liver. We hypothesized that autophagy activation was a possible mechanism for T-MSC-mediated liver recovery. In this report, we demonstrate for the first time that T-MSCs can differentiate into hepatocyte-like cells and ameliorate liver fibrosis via autophagy activation and down-regulation of TGF-β. These findings suggest that T-MSCs could be used as a novel source for stem cell therapy targeting liver diseases.

Gene Expression Profile of Megakaryocytes from Human Cord Blood CD34+ Cells Ex Vivo Expanded by Thrombopoietin

Previously, we investigated the process of megakaryocytopoiesis during ex vivo expansion of human cord blood (CB) CD34+ cells using thrombopoietin (TPO) and found that megakaryocytopoiesis was closely associated with apoptosis. To understand megakaryocytopoiesis at the molecular level, we performed a microserial analysis of gene expression (microSAGE) in megakaryocytes (MKs) and nonmegakaryocytes (non‐MKs) derived from human CB CD34+ cells by ex vivo expansion using TPO, and a total of 38,909 tags, representing 8,976 unique genes, were identified. In MKs, many of the known genes, including coagulation factor VII, P‐selectin (CD62P), pim‐1, azurocidin, defensin, and CD48 were highly expressed; meanwhile, those genes encoding some small G proteins of the Ras family (Rab 7 and Rab 11A) and glutathione S transferase family (1, 4, A2, omega, and pi) showed lower expression levels in MKs. These gene expression profiles will be useful to understand megakaryocytopoiesis at the molecular level, including apoptosis and related signal transduction pathways.

Transplantation of bone marrow cells reduces CCl4 -induced liver fibrosis in mice.

Background: We investigated the reversibility of liver fibrosis induced with a CCl4 injection and the role of stem cells in reversing the hepatic injury. Furthermore, the most effective cell fraction among bone marrow cells (BMCs) in the repair process was analysed.