Synthetic TGF-ß Signaling Agonist-Treated Dendritic Cells Induce Tolerogenicity and Antirheumatic Effects.

The newly synthesized compound TGF-ß signaling agonist (T74) is a small molecule associated with the TGF-ß receptor signaling pathway. Tolerogenic dendritic cells (tDCs) have been used to examine immunosuppressive and anti-inflammatory effects in multiple autoimmune disease models. The aim of this study was to investigate whether treatment of DCs with T74 has an antirheumatic effect in a mouse model of collagen-induced arthritis (CIA). Bone marrow-derived cells were obtained from DBA/1J mice and differentiated into DCs. T74-treated DCs (T74-DCs) were generated by treating bone marrow-derived DCs with LPS, type II collagen, and T74. T74-DCs expressed lower levels of surface molecules and inflammatory cytokines associated with antigen presentation and T cell stimulation. The ability of T74-DCs to differentiate effector T cells was lower than that of T74-untreated DCs (NT-DCs), but T74-DCs increased the regulatory T (Treg) cell differentiation in vitro. DBA/1J mice received two subcutaneous (s.c.) injections of type II collagen to establish CIA. Mice then received two s.c. injections of T74-DCs or NT-DCs. Joint inflammation was ameliorated in the paws of T74-DC-treated mice. Additionally, Treg populations in T74-DC-treated mice were higher than in NT-DC-treated or PBS-treated CIA mice. Taken together, these results demonstrate that T74 induces tolerance in DCs, and that T74-mediated DCs exert antirheumatic effects via induction of Tregs.

TNF-α Induces Mitophagy in Rheumatoid Arthritis Synovial Fibroblasts, and Mitophagy Inhibition Alleviates Synovitis in Collagen Antibody-Induced Arthritis.

Mitophagy is a selective form of autophagy that removes damaged mitochondria. Increasing evidence indicates that dysregulated mitophagy is implicated in numerous autoimmune diseases, but the role of mitophagy in rheumatoid arthritis (RA) has not yet been reported. The aim of the present study was to determine the roles of mitophagy in patient-derived RA synovial fibroblasts (RASFs) and in the collagen antibody-induced arthritis mouse model. We measured the mitophagy marker PTEN-induced putative kinase 1 (PINK1) in RASFs treated with tumor necrosis factor-α (TNF-α) using Western blotting and immunofluorescence. Arthritis was induced in PINK1-/- mice by intraperitoneal injection of an anti-type II collagen antibody cocktail and lipopolysaccharide. RA severity was assessed by histopathology. PINK1 expression and damaged mitochondria increased in TNF-α treated RASFs via increased intracellular levels of reactive oxygen species. PINK1 knockdown RASFs decreased cellular migration and invasion functions. In addition, PINK1-/- mice with arthritis exhibited markedly reduced swelling and inflammation relative to wild-type mice with arthritis. Taken together, these findings suggest that regulation of PINK1 expression in RA could represent a potential therapeutic and diagnostic target for RA.

Functional Ambivalence of Dendritic Cells: Tolerogenicity and Immunogenicity.

Dendritic cells (DCs) are the most potent professional antigen-presenting cells (APCs) and inducers of T cell-mediated immunity. Although DCs play a central role in promoting adaptive immune responses against growing tumors, they also establish and maintain peripheral tolerance. DC activity depends on the method of induction and/or the presence of immunosuppressive agents. Tolerogenic dendritic cells (tDCs) induce immune tolerance by activating CD4+CD25+Foxp3+ regulatory T (Treg) cells and/or by producing cytokines that inhibit T cell activation. These findings suggest that tDCs may be an effective treatment for autoimmune diseases, inflammatory diseases, and infertility.

TGF-ß/IL-7 Chimeric Switch Receptor-Expressing CAR-T Cells Inhibit Recurrence of CD19-Positive B Cell Lymphoma.

Chimeric antigen receptor (CAR)-T cells are effective in the treatment of hematologic malignancies but have shown limited efficacy against solid tumors. Here, we demonstrated an approach to inhibit recurrence of B cell lymphoma by co-expressing both a human anti-CD19-specific single-chain variable fragment (scFv) CAR (CD19 CAR) and a TGF-ß/IL-7 chimeric switch receptor (tTRII-I7R) in T cells (CD19 CAR-tTRII-I7R-T cells). The tTRII-I7R was designed to convert immunosuppressive TGF-ß signaling into immune-activating IL-7 signaling. The effect of TGF-ß on CD19 CAR-tTRII-I7R-T cells was assessed by western blotting. Target-specific killing by CD19 CAR-tTRII-I7R-T cells was evaluated by Eu-TDA assay. Daudi tumor-bearing NSG (NOD/SCID/IL2Rγ-/-) mice were treated with CD19 CAR-tTRII-I7R-T cells to analyze the in vivo anti-tumor effect. In vitro, CD19 CAR-tTRII-I7R-T cells had a lower level of phosphorylated SMAD2 and a higher level of target-specific cytotoxicity than controls in the presence of rhTGF-ß1. In the animal model, the overall survival and recurrence-free survival of mice that received CD19 CAR-tTRII-I7R-T cells were significantly longer than in control mice. These findings strongly suggest that CD19 CAR-tTRII-I7R-T cell therapy provides a new strategy for long-lasting, TGF-ß-resistant anti-tumor effects against B cell lymphoma, which may lead ultimately to increased clinical efficacy.

Rosiglitazone-dependent dissociation of HuR from PPAR-γ regulates adiponectin expression at the posttranscriptional level.

Peroxisome proliferator-activated receptor (PPAR)-γ has been implicated as a key player in the regulation of adiponectin levels via both transcriptional and posttranscriptional mechanisms. Herein, we show that PPAR-γ interacts with human antigen R (HuR) and that the PPAR-γ-HuR complex dissociates following activation of PPAR-γ by rosiglitazone, a specific ligand of PPAR-γ. This rosiglitazone-dependent dissociation of HuR from PPAR-γ leads to nucleocytoplasmic shuttling of HuR and its binding to the 3'-UTR of adiponectin mRNA. PPAR-γ with H321A and H447A double mutation (PPAR-γH321/447A), a mutant lacking ligand-binding activity, impaired HuR dissociation from the PPAR-γ-HuR complex, resulting in reduced nucleocytoplasmic shuttling, even in the presence of rosiglitazone. Consequently, rosiglitazone up-regulated adiponectin levels by modulating the stability of adiponectin mRNA, whereas these effects were abolished by HuR ablation or blocked in cells expressing the PPAR-γH321/447A mutant, indicating that the interaction of PPAR-γ and HuR is a critical event during adiponectin expression. Taken together, the findings demonstrate a novel mechanism for regulating adiponectin expression at the posttranscriptional level and suggest that ligand-mediated activation of PPAR-γ to interfere with interaction of HuR could offer a therapeutic strategy for inflammation-associated diseases that involve decreased adiponectin mRNA stability.-Hwang, J. S., Lee, W. J., Hur, J., Lee, H. G., Kim, E., Lee, G. H., Choi, M.-J., Lim, D.-S., Paek, K. S., Seo, H. G. Rosiglitazone-dependent dissociation of HuR from PPAR-γ regulates adiponectin expression at the posttranscriptional level.

Pdlim4 is essential for CCR7-JNK-mediated dendritic cell migration and F-actin-related dendrite formation.

Dendritic cells (DCs) are the most potent professional antigen (Ag)-presenting cells and inducers of T cell-mediated immunity. A previous microarray analysis identified PDZ and LIM domain protein 4 (Pdlim4) as a candidate marker for DC maturation. The aim of this study was to investigate whether Pdlim4 influences DC migration and maturation. Mouse bone marrow-derived DCs were transduced lentivirally with Pdlim4 short hairpin RNA and examined by confocal microscopy, flow cytometry, ELISA, and Western blotting. Pdlim4 was highly induced in LPS-stimulated mature DCs (mDCs). Pdlim4-knockdown mDCs showed reduced expression of molecules associated with Ag presentation and T-cell costimulation, reduced cytokine production, and functional defects in their ability to activate T cells. Moreover, Pdlim4 was necessary for mDC migration via C-C chemokine receptor type 7 (CCR7)-JNK in in vitro Transwell assays. The importance of Pdlim4 in DC migration was confirmed with an in vivo migration model in which C57BL/6 mice were injected with fluorescently labeled DCs in the footpad and migration to the popliteal lymph nodes was assessed by flow cytometry. Moreover, dendrite formation in mDCs was remarkably attenuated under Pdlim4 knockdown. Taken together, these results demonstrate that Pdlim4 is necessary for DC migration via CCR7-JNK, dendrite formation, and subsequent development of functional T-cell responses.-Yoo, J.-Y., Jung, N.-C., Lee, J.-H., Choi, S.-Y., Choi, H.-J., Park, S.-Y., Jang, J.-S., Byun, S.-H., Hwang, S.-U., Noh, K.-E., Park, Y., Lee, J., Song, J.-Y., Seo, H. G., Lee, H. S., Lim, D.-S. Pdlim4 is essential for CCR7-JNK-mediated dendritic cell migration and F-actin-related dendrite formation.

Asymptomatic Clostridium perfringens Inhabitation in Intestine Can Cause Inflammation, Apoptosis, and Disorders in Brain.

Clostridium perfringens (CP) is a foodborne pathogen. The bacterium can also inhabit human gut without symptoms of foodborne illness. However, the clinical symptoms of long-term inhabitation have not been known yet. Therefore, the objective of this study was to elucidate the relationship between intestinal CP and other internal organs. Phosphate-buffered saline (PBS) and CP were orally injected into 5-week-old (YOUNG) and 12-month-old C57BL6/J (ADULT) mice. Gene expression levels related to inflammation (tumor necrosis factor-α [TNF-α], interleukin [IL]-1ß, and IL-6) and oxidative stress (superoxide dismutase [SOD]1, SOD2, SOD3, glutathione reductase [GSR], glutathione peroxidase [GPx]3, and catalase [CAT]) responses were evaluated in the brain, small intestine, and liver. In addition, apoptosis-related (BCL2-associated X [BAX]1 and high-mobility group box-1 [HMGB1]) and brain disorder-related genes (CCAAT-enhancer-binding protein [C/EBP]-ß, C/EBPδ, C/EBP homologous protein [CHOP], and amyloid precursor protein [APP]) as brain damage markers were examined. The protein expressions in the brain were also measured. Gene expression levels of inflammation and oxidative stress responses were higher (p < 0.05) in brains of CP-YOUNG and CP-ADULT mice, compared with PBS-YOUNG and PBS-ADULT, and the gene expression levels were higher (p < 0.05) in brains of CP-ADULT mice than CP-YOUNG mice. Apoptosis-related (BAX1 and HMGB1) and brain disorder-related genes (C/EBPß, C/EBPδ, CHOP, and APP) were higher (p < 0.05) in brains of CP-challenged mice, compared with PBS-challenged mice. Even oxidative stress response (GPx and SOD2), cell damage-related (HMGB1), and ß-amyloid proteins were higher (p < 0.05) in brains of CP- than in PBS-challenged mice. C/EBP protein was higher (p < 0.05) in CP-YOUNG, compared with PBS-YOUNG mice. However, these clinical symptoms were not observed in small intestine and liver. These results indicate that although asymptomatic intestinal CP do not cause foodborne illness, their inhabitation may cause brain inflammation, oxidative stress, apoptosis, and cell damage, which may induce disorders, especially for the aged group.

Activation of peroxisome proliferator-activated receptor delta suppresses BACE1 expression by up-regulating SOCS1 in a JAK2/STAT1-dependent manner.

Neuronal expression of beta-secretase 1 (BACE1) has been implicated in the progression of Alzheimer's disease. However, the mechanisms that regulate BACE1 expression are unclear. Here, we show that peroxisome proliferator-activated receptor delta (PPARδ) decreases BACE1 expression by up-regulating suppressor of cytokine signaling 1 (SOCS1) in SH-SY5Y neuroblastoma cells. The activation of PPARδ by GW501516, a specific PPARδ agonist, inhibited expression of BACE1. This effect was abrogated by shRNA-mediated knockdown of PPARδ and by treatment with the PPARδ antagonist GSK0660, indicating that PPARδ is involved in GW501516-mediated suppression of BACE1 expression. On the other hand, GW501516-activated PPARδ induced expression of SOCS1, which is a negative regulator of cytokine signal transduction, at the transcriptional level by binding to a PPAR response element in its promoter. This GW501516-mediated induction of SOCS1 expression led to down-regulation of BACE1 expression via inactivation of signal transducer and activator of transcription 1. GW501516-activated PPARδ suppressed the generation of neurotoxic amyloid beta (Aß) in accordance with the decrease in BACE1 expression. Taken together, these results indicate that PPARδ attenuates BACE1 expression via SOCS1-mediated inhibition of signal transducer and activator of transcription 1 signaling, thereby suppressing BACE1-associated generation of neurotoxic Aß.

Infarcted Myocardium-Primed Dendritic Cells Improve Remodeling and Cardiac Function After Myocardial Infarction by Modulating the Regulatory T Cell and Macrophage Polarization.

BACKGROUND: Inflammatory responses play a critical role in left ventricular remodeling after myocardial infarction (MI). Tolerogenic dendritic cells (tDCs) can modulate immune responses, inducing regulatory T cells in a number of inflammatory diseases. METHODS: We generated tDCs by treating bone marrow-derived dendritic cells with tumor necrosis factor-α and cardiac lysate from MI mice. We injected MI mice, induced by a ligation of the left anterior descending coronary artery in C57BL/6 mice, twice with tDCs within 24 hours and at 7 days after the ligation. RESULTS: In vivo cardiac magnetic resonance imaging and ex vivo histology confirmed the beneficial effect on postinfarct left ventricular remodeling in MI mice treated with tDCs. Subcutaneously administered infarct lysate-primed tDCs near the inguinal lymph node migrated to the regional lymph node and induced infarct tissue-specific regulatory T-cell populations in the inguinal and mediastinal lymph nodes, spleen, and infarcted myocardium, indicating that a local injection of tDCs induces a systemic activation of MI-specific regulatory T cells. These events elicited an inflammatory-to-reparative macrophage shift. The altered immune environment in the infarcted heart resulted in a better wound remodeling, preserved left ventricular systolic function after myocardial tissue damage, and improved survival. CONCLUSIONS: This study showed that tDC therapy in a preclinical model of MI was potentially translatable into an antiremodeling therapy for ischemic tissue repair.

Activation of PPARδ attenuates neurotoxicity by inhibiting lipopolysaccharide-triggered glutamate release in BV-2 microglial cells.

Neuroinflammation-associated release of glutamate from activated microglia has been implicated in the progression of neurodegenerative diseases. However, the regulatory mechanisms underlying this glutamate release are poorly understood. Here, we show that peroxisome proliferator-activated receptor delta (PPARδ) modulates neurotoxicity by inhibiting glutamate release in lipopolysaccharide (LPS)-activated BV-2 microglial cells. Activation of PPARδ by GW501516, a specific PPARδ agonist, inhibited glutamate release in BV-2 cells. This effect of GW501516 was significantly blocked by shRNA-mediated knockdown of PPARδ and by treatment with GSK0660, a specific PPARδ antagonist, indicating that PPARδ is associated with blockade of glutamate release. Additionally, GW501516-activated PPARδ suppressed generation of reactive oxygen species and expression of gp91phox, a functional subunit of NADPH oxidase 2, in BV-2 cells stimulated with LPS. The inhibitory effect of GW501516 on gp91phox expression and glutamate release was further potentiated in the presence of AG490, a specific inhibitor of janus kinase 2 (JAK2), leading to the inhibition of signal transducer and activator of transcription 1 (STAT1). By contrast, GW501516 upregulated the expression of suppressor of cytokine signaling 1 (SOCS1), an endogenous inhibitor of JAK2. Furthermore, neurotoxicity induced by conditioned media from LPS-stimulated BV-2 cells was significantly reduced when conditioned media from BV-2 cells treated with both LPS and GW501516 were used. These results indicate that PPARδ attenuates LPS-triggered neuroinflammation by enhancing SOCS1-mediated inhibition of JAK2/STAT1 signaling, thereby inhibiting neurotoxicity associated with glutamate release.

Ligand-Activated Peroxisome Proliferator-Activated Receptor δ Attenuates Vascular Oxidative Stress by Inhibiting Thrombospondin-1 Expression.

Thrombospondin-1 (TSP-1) is implicated in vascular diseases associated with oxidative stress, such as abdominal aortic aneurysms, ischemia-reperfusion injury, and atherosclerosis. However, the regulatory mechanisms underlying TSP-1 expression are not fully elucidated. In this study, we found that peroxisome proliferator-activated receptor δ (PPARδ) inhibited oxidative stress-induced TSP-1 expression and migration in vascular smooth muscle cells (VSMCs). Activation of PPARδ by GW501516, a specific ligand for PPARδ, significantly attenuated hydrogen peroxide (H2O2)-induced expression of TSP-1 in VSMCs. Small interfering RNA-mediated knockdown of PPARδ and treatment with GSK0660, a selective PPARδ antagonist, reversed the effect of GW501516 on H2O2-induced expression of TSP-1, suggesting that PPARδ is associated with GW501516 activity. Furthermore, JNK (c-Jun N-terminal kinase), but not p38 and ERK (extracellular signal-regulated kinase), mediated PPARδ-dependent inhibition of TSP-1 expression in VSMCs exposed to H2O2. GW501516- activated PPARδ also reduced the H2O2-induced generation of reactive oxygen species, concomitant with inhibition of VSMC migration. In particular, TSP-1 contributed to the action of PPARδ in the regulation of H2O2-induced interleukin-1ß expression. These results suggest that PPARδ-modulated downregulation of TSP-1 is associated with reduced cellular oxidative stress, thereby inhibiting H2O2-induced pheno-typic changes in vascular cells.

Dendritic Cell Immunotherapy Combined with Cytokine-Induced Killer Cells Effectively Suppresses Established Hepatocellular Carcinomas in Mice.

BACKGROUND: The response of hepatocellular carcinoma (HCC) to immunotherapy is often disappointing and new strategies are clearly needed. The aim of the present study was to investigate whether cytokine-induced killer (CIK) cells combined with a dendritic cell vaccination enhanced cytotoxicity against hepatocarcinoma tumor cells in an in vivo animal model. METHODS: CIKs and DCs were prepared from C3H/HeJ mice by conventional methods, the dendritic cell (DC) pulsed with a MH134 cell lysate, DC or CIK alone were used as controls. Cell phenotypes were analyzed by flow cytometry, cytokine secretion levels were determined by enzyme-linked immunosorbent assay (ELISA), and cytotoxicity was assessed by means of an in vitro lactate dehydrogenase (LDH) release assay. A mouse hepatocarcinoma cell MH134-bearing mice model was established to test the in vivo anti-tumor efficacy of the system. RESULTS: CIK cells combined with DC therapy resulted in significant inhibition of tumor growth compared with the control group, whereas the decrease in tumor growth in mice that had been treated with CIK or DC alone did not reach the level of statistical significance. The combination therapy led to a further increase in the population of cytotoxic T cells (CTLs) in vivo, compared to the CIK or DC alone therapy. In addition, the combination therapy significantly enhanced cytotoxic activity against MH134 cells. CONCLUSION: Taken together, these results show that a DC + CIK vaccination is more effective than DC or CIK alone therapy for the treatment of hepatocarcinoma cancer.

Induction of galectin-1 by TGF-ß1 accelerates fibrosis through enhancing nuclear retention of Smad2.

Fibrosis is one of the most serious side effects in cancer patients undergoing radio-/ chemo-therapy, especially of the lung, pancreas or kidney. Based on our previous finding that galectin-1 (Gal-1) was significantly increased during radiation-induced lung fibrosis in areas of pulmonary fibrosis, we herein clarified the roles and action mechanisms of Gal-1 during fibrosis. Our results revealed that treatment with TGF-ß1 induced the differentiation of fibroblast cell lines (NIH3T3 and IMR-90) to myofibroblasts, as evidenced by increased expression of the fibrotic markers smooth muscle actin-alpha (α-SMA), fibronectin, and collagen (Col-1). We also observed marked and time-dependent increases in the expression level and nuclear accumulation of Gal-1. The TGF-ß1-induced increases in Gal-1, α-SMA and Col-1 were decreased by inhibitors of PI3-kinase and p38 MAPK, but not ERK. Gal-1 knockdown using shRNA decreased the phosphorylation and nuclear retention of Smad2, preventing the differentiation of fibroblasts. Gal-1 interacted with Smad2 and phosphorylated Smad2, which may accelerate fibrotic processes. In addition, up-regulation of Gal-1 expression was demonstrated in a bleomycin (BLM)-induced mouse model of lung fibrosis in vivo. Together, our results indicate that Gal-1 may promote the TGF-ß1-induced differentiation of fibroblasts by sustaining nuclear localization of Smad2, and could be a potential target for the treatment of pulmonary fibrotic diseases.

Dendritic Cell-Based Immunotherapy: The Importance of Dendritic Cell Migration.

Dendritic cells (DCs) are specialized antigen-presenting cells that are crucial for maintaining self-tolerance, initiating immune responses against pathogens, and patrolling body compartments. Despite promising aspects, DC-based immunotherapy faces challenges that include limited availability, immune escape in tumors, immunosuppression in the tumor microenvironment, and the need for effective combination therapies. A further limitation in DC-based immunotherapy is the low population of migratory DC (around 5%-10%) that migrate to lymph nodes (LNs) through afferent lymphatics depending on the LN draining site. By increasing the population of migratory DCs, DC-based immunotherapy could enhance immunotherapeutic effects on target diseases. This paper reviews the importance of DC migration and current research progress in the context of DC-based immunotherapy.

Predictive DNA damage signaling for low­dose ionizing radiation.

Numerous studies have attempted to develop biological markers for the response to radiation for broad and straightforward application in the field of radiation. Based on a public database, the present study selected several molecules involved in the DNA damage repair response, cell cycle regulation and cytokine signaling as promising candidates for low­dose radiation­sensitive markers. The HuT 78 and IM­9 cell lines were irradiated in a concentration­dependent manner, and the expression of these molecules was analyzed using western blot analysis. Notably, the activation of ataxia telangiectasia mutated (ATM), checkpoint kinase 2 (CHK2), p53 and H2A histone family member X (H2AX) significantly increased in a concentration­dependent manner, which was also observed in human peripheral blood mononuclear cells. To determine the radioprotective effects of cinobufagin, as an ATM and CHK2 activator, an in vivo model was employed using sub­lethal and lethal doses in irradiated mice. Treatment with cinobufagin increased the number of bone marrow cells in sub­lethal irradiated mice, and slightly elongated the survival of lethally irradiated mice, although the difference was not statistically significant. Therefore, KU60019, BML­277, pifithrin­α, and nutlin­3a were evaluated for their ability to modulate radiation­induced cell death. The use of BML­277 led to a decrease in radiation­induced p­CHK2 and γH2AX levels and mitigated radiation­induced apoptosis. On the whole, the present study provides a novel approach for developing drug candidates based on the profiling of biological radiation­sensitive markers. These markers hold promise for predicting radiation exposure and assessing the associated human risk.

Poliovirus receptor inhibition in breast cancer cells induces antitumor immunity via T cell activation.

Radiotherapy (RT) is a commonly used treatment option for patients with cancer because it can effectively control tumor growth and kill tumor cells. However, the impact of RT goes beyond direct tumor cell killing because it can change the tumor microenvironment by altering surrounding tissues and infiltrating cells and modulating the expression of immune checkpoints. Poliovirus receptor (PVR, cluster of differentiation (CD)155), a member of the nectin-like molecule family, is overexpressed in many human cancers. However, its role in the tumor growth and T-cell immune responses of triple-negative breast cancer (TNBC) remains unclear. In the present study, we observe that radiation exposure increases PVR expression in MDA-MB-231 and BT549 cells. Silencing PVR not only inhibited the proliferation of breast cancer cells but also significantly enhanced the cytotoxicity of cytotoxic T lymphocytes (CTLs) compared with the control or RT groups. Treatment of T cells with PVR decreased CD8+ T cells, increased CD4+ T cells, and induced PVR ligands such as T cell immunoreceptor with immunoglobulin and ITIM domain, CD226, and CD96. However, after treatment with PVR, CTL responses decreased and secretion of interferon-γ, tumor necrosis factor-α, interleukin (IL)-2, IL-6, and IL-10 was significantly inhibited. In contrast, PVR knockdown increased the production of these cytokines, illustrating the immunosuppressive function of PVR. Suppression of PVR using an anti-PVR antibody inhibited 4T1 tumor growth by increasing immune cell infiltration. These results provide new insights into the role of PVR in TNBC and highlight its potential as a target for T cell-mediated immunotherapy in breast cancer.

Activation of peroxisome proliferator-activated receptor-δ attenuates glutamate-induced neurotoxicity in HT22 mouse hippocampal cells.

Glutamate-induced neurotoxicity has been implicated in the pathogenesis of neurodegenerative disorders; however, little is known about the cellular events that underlie neurotoxicity or how to impede these events. This study demonstrates that peroxisome proliferator-activated receptor (PPAR)-δ regulates glutamate-induced neurotoxicity in HT22 mouse hippocampal cells. Activation of PPARδ by GW501516, a specific ligand, significantly inhibited glutamate-induced cell death and reactive oxygen species (ROS) production in HT22 cells. The siRNA-mediated knockdown of PPARδ abrogated the effects of GW501516 in neuronal toxicity and ROS production induced by glutamate. In addition, ligand-activated PPARδ reduced the glutamate-induced level of intracellular calcium ions (Ca(2+)) by modulating the influx of Ca(2+) from the extracellular space. Similarly, glutamate-induced cell death and intracellular Ca(2+) levels were attenuated in the presence of LY83583, an inhibitor of soluble guanylyl cyclase. Taken together, these results suggest that PPARδ plays an important role in glutamate-induced neurotoxicity by modulating oxidative stress and Ca(2+) influx.

Mesenchymal stem cell-mediated immature dendritic cells induce regulatory T cell-based immunosuppressive effect.

Immature dendritic cells (imDCs) are increasingly viewed as mediators of T-cell tolerance. We investigated factors enabling induction of regulatory T (Treg) cells through syngeneic imDC/mesenchymal stem cell (MSC) co-cultures in vitro and immunosuppressive effects of MSC-mediated imDCs (MSCs were excluded after 72 h co-culture) in vivo. In these experiments, we found that Foxp3(+) Treg cell population remarkably increased after the T cell priming phase when splenocytes were co-cultured with both imDCs and MSCs, presumably inducing naïve T cells into Treg cells by MSCs and imDCs. In parallel, TGF-ß secretion was markedly induced from the imDC+MSC+splenocyte culture supernatant to a significant level at 72-h co-culture, compared to the MSC or imDC+splenocyte co-culture. Based on these results, using a murine melanoma tumor model, we confirmed that the subcutaneous injection of B16 cells induced a perfect tumor incidence in allogeneic recipients when MSC-mediated DCs were coinjected. Consequently, these results suggested that immune tolerance with MSC-mediated DCs leads to immunosuppression induced by at least Foxp3-specific Treg cells. This tool may be useful in clinical trials due to the yet unknown side effects of stem cell therapy.

Activation of peroxisome proliferator-activated receptor γ by rosiglitazone inhibits lipopolysaccharide-induced release of high mobility group box 1.

Peroxisome proliferator-activated receptors (PPARs) are shown to modulate the pathological status of sepsis by regulating the release of high mobility group box 1 (HMGB1), a well-known late proinflammatory mediator of sepsis. Ligand-activated PPARs markedly inhibited lipopolysaccharide- (LPS) induced release of HMGB1 in RAW 264.7 cells. Among the ligands of PPAR, the effect of rosiglitazone, a specific ligand for PPARγ, was superior in the inhibition of HMGB1 release induced by LPS. This effect was observed in cells that received rosiglitazone before LPS or after LPS treatment, indicating that rosiglitazone is effective in both treatment and prevention. Ablation of PPARγ with small interfering RNA or GW9662-mediated inhibition of PPARγ abolished the effect of rosiglitazone on HMGB1 release. Furthermore, the overexpression of PPARγ markedly potentiated the inhibitory effect of rosiglitazone on HMGB1 release. In addition, rosiglitazone inhibited LPS-induced expression of Toll-like receptor 4 signal molecules, suggesting a possible mechanism by which rosiglitazone modulates HMGB1 release. Notably, the administration of rosiglitazone to mice improved survival rates in an LPS-induced animal model of endotoxemia, where reduced levels of circulating HMGB1 were demonstrated. Taken together, these results suggest that PPARs play an important role in the cellular response to inflammation by inhibiting HMGB1 release.

PKR, a p53 target gene, plays a crucial role in the tumor-suppressor function of p53.

Type I IFN-induced expression of dsRNA-activated protein kinase (PKR) during viral infection is a well-established antiviral mechanism. However, little is known about the expression of PKR in the context of p53 and about PKR involvement in p53-mediated tumor suppression. Here, we report that PKR is a p53 target gene and plays an important role in the tumor-suppressor function of p53. Activation of p53 by genotoxic stress induces a significant level of PKR expression by acting on the newly identified cis-acting element (ISRE), which is separated from the IFN-stimulated responsive element on the PKR promoter, resulting in translational inhibition and cell apoptosis. The genotoxin-mediated inhibition of translation is associated with the p53/PKR/elF2a (eukaryotic initiation factor-2alpha) pathway. To some extent, p53 activation induced by DNA damage facilitates cell apoptosis by activating PKR. PKR-knockdown human colon cancer cells grew rapidly in nude mice and proved resistant to anti-cancer drugs. These data indicate that p53-mediated tumor suppression can be attributed at least in part to the biological functions of PKR induced by p53 in genotoxic conditions.