Exosome-mediated delivery of super-repressor IκBα alleviates inflammation and joint damages in rheumatoid arthritis.

BACKGROUND: This study aims to investigate the potential anti-inflammatory effects of exosomes engineered to carry super-repressor IκB (Exo-srIκB), an exosome-based NF-κB inhibitor, in the context of RA. METHODS: Peripheral blood mononuclear cells (PBMCs) and synovial fluid mononuclear cells (SFMCs) were collected from patients diagnosed with RA and treated with Exo-srIκB to test the therapeutic potential. Flow cytometry analysis was performed to assess the production of inflammatory cytokines (IL-17A and GM-CSF) by the cells. ELISA was utilized to measure the levels of TNF-α, IL-17A, IL-6, and GM-CSF. Arthritis was induced in SKG mice by intraperitoneal injection of curdlan. DBA/1 J mice were used in collagen-induced arthritis (CIA) experiments. After the development of arthritis, mice were injected with either Exo-Naïve (control exosome) or Exo-srIκB. Arthritis scores were recorded biweekly, and histological observations of the ankle joint were conducted using H&E and safranin-O staining. Additionally, bone erosion was evaluated using micro-CT imaging. RESULTS: In the ex vivo study involving human PBMCs and SFMCs, treatment with Exo-srIκB demonstrated a notable reduction in inflammatory cytokines. Furthermore, in both the SKG and CIA models, Exo-srIκB treatment exhibited significant reductions in inflammation, cartilage destruction, and bone erosion within the joint tissues when compared to the Exo-Naive control group. Additionally, the radiographic score assessed through microCT showed a significant decrease compared to the Exo-Naive control group. CONCLUSION: Overall, these findings suggest that Exo-srIκB possesses anti-inflammatory properties in human RA cells and animal models, making it a promising therapeutic candidate for the treatment of RA.

The Effect of Super-Repressor IkB-Loaded Exosomes (Exo-srIκBs) in Chronic Post-Ischemia Pain (CPIP) Models.

Complex regional pain syndrome (CRPS) is a condition associated with neuropathic pain that causes significant impairment of daily activities and functioning. Nuclear factor kappa B (NFκB) is thought to play an important role in the mechanism of CRPS. Recently, exosomes loaded with super-repressor inhibitory kappa B (Exo-srIκB, IκB; inhibitor of NFκB) have been shown to have potential anti-inflammatory effects in various inflammatory disease models. We investigated the therapeutic effect of Exo-srIκB on a rodent model with chronic post-ischemia pain (CPIP), a representative animal model of Type I CRPS. After intraperitoneal injection of a vehicle, Exo-srIκB, and pregabalin, the paw withdrawal threshold (PWT) was evaluated up to 48 h. Administration of Exo-srIκB increased PWT compared to the vehicle and pregabalin, and the relative densities of p-IκB and IκB showed significant changes compared to the vehicle 24 h after Exo-srIκB injection. The levels of several cytokines and chemokines were reduced by the administration of Exo-srIκB in mice with CPIP. In conclusion, our results showed more specifically the role of NFκB in the pathogenesis of CRPS and provided a theoretical background for novel treatment options for CRPS.

Exosome-Based Delivery of Super-Repressor IκBα Alleviates Alcohol-Associated Liver Injury in Mice.

Activation of Kupffer cells (KCs) by gut-derived lipopolysaccharide (LPS) instigates nuclear factor-κB (NF-κB)-mediated inflammatory responses in alcohol-associated liver diseases (ALD). Here, we utilized a novel optogenetically engineered exosome technology called 'exosomes for protein loading via optically reversible protein-protein interactions (EXPLOR)' to efficiently deliver the super-repressor IκB-loaded exosomes (Exo-srIκB) to the liver and examined its therapeutic potential in acute-on-chronic alcohol-associated liver injury. We detected enhanced uptake of DiI-labeled Exo-srIκB by LPS-treated inflammatory KCs, which suppressed LPS-induced inflammatory gene expression levels. In animal experiments, a single intravenous injection of Exo-srIκB prior to alcohol binge drinking significantly attenuated alcohol-associated hepatic steatosis and infiltration of neutrophils and macrophages but not a liver injury. Notably, three consecutive days of Exo-srIκB injection remarkably reduced alcohol-associated liver injury, steatosis, apoptosis of hepatocytes, fibrosis-related gene expression levels in hepatic stellate cells, infiltration of neutrophils and macrophages, and inflammatory gene expression levels in hepatocytes and KCs. In particular, the above effects occurred with inhibition of nuclear translocation of NF-κB in liver tissues, and these beneficial effects of Exo-srIκB on ALD were shown regardless of doses. Our results suggest an exosome-based modulation of NF-κB activity in KCs by Exo-srIκB as a novel and efficient therapeutic approach in ALD.

Manufacturing Therapeutic Exosomes: from Bench to Industry.

Process of manufacturing therapeutics exosome development for commercialization. The development of exosome treatment starts at the bench, and in order to be commercialized, it goes through the manufacturing, characterization, and formulation stages, production under Good Manufacturing Practice (GMP) conditions for clinical use, and close consultation with regulatory authorities. Exosome, a type of nanoparticles also known as small extracellular vesicles are gaining attention as novel therapeutics for various diseases because of their ability to deliver genetic or bioactive molecules to recipient cells. Although many pharmaceutical companies are gradually developing exosome therapeutics, numerous hurdles remain regarding manufacture of clinical-grade exosomes for therapeutic use. In this mini-review, we will discuss the manufacturing challenges of therapeutic exosomes, including cell line development, upstream cell culture, and downstream purification process. In addition, developing proper formulations for exosome storage and, establishing good manufacturing practice facility for producing therapeutic exosomes remains as challenges for developing clinicalgrade exosomes. However, owing to the lack of consensus regarding the guidelines for manufacturing therapeutic exosomes, close communication between regulators and companies is required for the successful development of exosome therapeutics. This review shares the challenges and perspectives regarding the manufacture and quality control of clinical grade exosomes.

Targeted Delivery of Exosomes Armed with Anti-Cancer Therapeutics.

Among extracellular vesicles, exosomes have gained great attention for their role as therapeutic vehicles for delivering various active pharmaceutical ingredients (APIs). Exosomes "armed" with anti-cancer therapeutics possess great potential for an efficient intracellular delivery of anti-cancer APIs and enhanced targetability to tumor cells. Various technologies are being developed to efficiently incorporate anti-cancer APIs such as genetic materials (miRNA, siRNA, mRNA), chemotherapeutics, and proteins into exosomes and to induce targeted delivery to tumor burden by exosomal surface modification. Exosomes can incorporate the desired therapeutic molecules via direct exogenous methods (e.g., electroporation and sonication) or indirect methods by modifying cells to produce "armed" exosomes. The targeted delivery of "armed" exosomes to tumor burden could be accomplished either by "passive" targeting using the natural tropism of exosomes or by "active" targeting via the surface engineering of exosomal membranes. Although anti-cancer exosome therapeutics demonstrated promising results in preclinical studies, success in clinical trials requires thorough validation in terms of chemistry, manufacturing, and control techniques. While exosomes possess multiple advantages over synthetic nanoparticles, challenges remain in increasing the loading efficiency of anti-cancer agents into exosomes, as well as establishing quantitative and qualitative analytical methods for monitoring the delivery of in vivo administered exosomes and exosome-incorporated anti-cancer agents to the tumor parenchyma.

Exosome-based delivery of super-repressor IκBα ameliorates kidney ischemia-reperfusion injury.

Ischemia-reperfusion injury is a major cause of acute kidney injury. Recent studies on the pathophysiology of ischemia-reperfusion-induced acute kidney injury showed that immunologic responses significantly affect kidney ischemia-reperfusion injury and repair. Nuclear factor (NF)-ĸB signaling, which controls cytokine production and cell survival, is significantly involved in ischemia-reperfusion-induced acute kidney injury, and its inhibition can ameliorate ischemic acute kidney injury. Using EXPLOR, a novel, optogenetically engineered exosome technology, we successfully delivered the exosomal super-repressor inhibitor of NF-ĸB (Exo-srIĸB) into B6 wild type mice before/after kidney ischemia-reperfusion surgery, and compared outcomes with those of a control exosome (Exo-Naïve)-injected group. Exo-srIĸB treatment resulted in lower levels of serum blood urea nitrogen, creatinine, and neutrophil gelatinase-associated lipocalin in post-ischemic mice than in the Exo-Naïve treatment group. Systemic delivery of Exo-srIĸB decreased NF-ĸB activity in post-ischemic kidneys and reduced apoptosis. Post-ischemic kidneys showed decreased gene expression of pro-inflammatory cytokines and adhesion molecules with Exo-srIĸB treatment as compared with the control. Intravital imaging confirmed the uptake of exosomes in neutrophils and macrophages. Exo-srIĸB treatment also significantly affected post-ischemic kidney immune cell populations, lowering neutrophil, monocyte/macrophage, and T cell frequencies than those in the control. Thus, modulation of NF-ĸB signaling through exosomal delivery can be used as a novel therapeutic method for ischemia-reperfusion-induced acute kidney injury.

Biodistribution and Pharmacokinectics of Liposomes and Exosomes in a Mouse Model of Sepsis.

Exosomes have attracted considerable attention as drug delivery vehicles because their biological properties can be utilized for selective delivery of therapeutic cargoes to disease sites. In this context, analysis of the in vivo behaviors of exosomes in a diseased state is required to maximize their therapeutic potential as drug delivery vehicles. In this study, we investigated biodistribution and pharmacokinetics of HEK293T cell-derived exosomes and PEGylated liposomes, their synthetic counterparts, into healthy and sepsis mice. We found that biodistribution and pharmacokinetics of exosomes were significantly affected by pathophysiological conditions of sepsis compared to those of liposomes. In the sepsis mice, a substantial number of exosomes were found in the lung after intravenous injection, and their prolonged blood residence was observed due to the liver dysfunction. However, liposomes did not show such sepsis-specific effects significantly. These results demonstrate that exosome-based therapeutics can be developed to manage sepsis and septic shock by virtue of their sepsis-specific in vivo behaviors.

Cell-specific activation of RIPK1 and MLKL after intracerebral hemorrhage in mice.

Receptor-interacting protein kinase-1 (RIPK1) is a master regulator of cell death and inflammation, and mediates programmed necrosis (necroptosis) via mixed-lineage kinase like (MLKL) protein. Prior studies in experimental intracerebral hemorrhage (ICH) implicated RIPK1 in the pathogenesis of neuronal death and cognitive outcome, but the relevant cell types involved and potential role of necroptosis remain unexplored. In mice subjected to autologous blood ICH, early RIPK1 activation was observed in neurons, endothelium and pericytes, but not in astrocytes. MLKL activation was detected in astrocytes and neurons but not endothelium or pericytes. Compared with WT controls, RIPK1 kinase-dead (RIPK1D138N/D138N) mice had reduced brain edema (24 h) and blood-brain barrier (BBB) permeability (24 h, 30 d), and improved postinjury rotarod performance. Mice deficient in MLKL (Mlkl-/-) had reduced neuronal death (24 h) and BBB permeability at 24 h but not 30d, and improved post-injury rotarod performance vs. WT. The data support a central role for RIPK1 in the pathogenesis of ICH, including cell death, edema, BBB permeability, and motor deficits. These effects may be mediated in part through the activation of MLKL-dependent necroptosis in neurons. The data support development of RIPK1 kinase inhibitors as therapeutic agents for human ICH.

MCP-1 and MIP-3α Secreted from Necrotic Cell-Treated Glioblastoma Cells Promote Migration/Infiltration of Microglia.

BACKGROUND/AIMS: Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults. The defining characteristics of GBM are diffuse infiltration of tumor cells into normal brain parenchyma, rapid growth, a high degree of infiltration of microglia and macrophages, and the presence of necrosis. Microglia/macrophages are frequently found in gliomas and they extensively infiltrate GBM tissue, up to 30% of total tumor mass. However, little is known about the effect of necrotic cells (NCs) on microglia infiltration in GBM and the tumor-infiltrating microglia-induced factors in GBMs. METHODS: In this study, to address whether necrosis or necrosis-exposed GBM cells affect the degree of microglia/macrophage infiltration, migration and invasion/infiltration assays were performed. Culture supernatants and nuclear extracts of CRT-MG cells treated or untreated with necrotic cells were analyzed using a chemokine array and electrophoretic mobility shift assay, respectively. RESULTS: The presence of NCs promoted the migration/infiltration of microglia, and GBM cell line CRT-MG cells exposed to NCs further enhanced the migration and infiltration of HMO6 microglial cells. Treatment with NCs induced mRNA and protein expression of chemokines such as Monocyte Chemoattractant Protein-1 (CCL2/MCP-1) and Macrophage Inflammatory Protein-3α (CCL20/MIP-3α) in CRT-MG cells. In particular, CCL2/MCP-1 and CCL20/MIP-3α were significantly increased in NC-treated CRT-MG cells. NCs induced DNA binding of the transcription factors Nuclear Factor (NF)-κB and Activator Protein 1 (AP-1) to the CCL2/MCP-1 and CCL20/MIP-3α promoters, leading to increased CCL2/MCP-1 and CCL20/MIP-3α mRNA and protein expression in CRT-MG cells. CONCLUSION: These results provide evidence that NCs induce the expression of CCL2/MCP-1 and CCL20/MIP-3α in glioblastoma cells through activation of NF-κB and AP-1 and facilitate the infiltration of microglia into tumor tissues.

3-Oxoacid CoA transferase 1 as a therapeutic target gene for cisplatin-resistant ovarian cancer.

Ovarian cancer (OC) is the second leading cause of mortality from gynecological malignancies and has the highest mortality rate worldwide. As it is commonly asymptomatic during the early stages of the disease, >70% of patients with OC are diagnosed at advanced stages with metastasis. Despite treatment methods, including optimal debulking surgery and chemotherapy with the platinum-based drug cisplatin, OC recurrence is often inevitable, with an overall 5-year survival rate of 45%, mostly due to the steady development of cisplatin resistance. To identify genes involved in cisplatin resistance, the present study determined the half-maximal inhibitory concentrations of eight different OC cell lines and classified them into two groups (sensitive and resistant). mRNA expression was analyzed with GeneChip Human Gene 1.0 ST Arrays, and DNA methylation profiles were evaluated with the HumanMethylation450 BeadChip. Using an integrated approach of analyzing gene expression levels and DNA methylation profiles simultaneously, 26 genes were selected that were differentially expressed and methylated between the resistant and sensitive groups. Among these 26 genes, 3-oxoacid CoA transferase 1 (OXCT1), which was demonstrated to be downregulated and hypermethylated at promoter CpGs in the cisplatin-resistant group compared with the cisplatin-sensitive group, was selected for further investigation. Treatment with a DNA methyltransferase inhibitor restored hypermethylation-mediated gene silencing of OXCT1 in the cisplatin-resistant group, but not in the cisplatin-sensitive group. Furthermore, overexpression of OXCT1 conferred sensitivity to cisplatin in OC cells. The results of the present study suggest that OXCT1 serves an important role in conferring cisplatin sensitivity, and may provide a potential therapeutic target for cisplatin chemotherapy in patients with recurrent OC.

Effect of Necrosis on the miRNA-mRNA Regulatory Network in CRT-MG Human Astroglioma Cells.

PURPOSE: Glioblastoma multiforme (GBM) is the most common adult primary intracranial tumor. The remarkable features of GBM include central necrosis. MicroRNAs (miRNAs) have been considered as diagnostic/prognostic biomarkers for many cancers, including glioblastoma. However, the effect of necrosis on the miRNA expression profile and predicted miRNA-mRNA regulatory information remain unclear. The purpose of this study is to examine the effect of necrotic cells on the modulation of miRNA and mRNA expression profiles and miRNA-mRNA network in CRT-MG cells. MATERIALS AND METHODS: We used human astroglioma cells, CRT-MG, treated with necrotic CRT-MG cells to examine the effect of necrosis on the modulation of miRNA and mRNA by next-generation sequencing. For preparation of necrotic cells, CRT-MGcellswere frozen and thawed through cycle of liquid nitrogen-water bath. The putative miRNA-mRNA regulatory relationshipwas inferred through target information, using miRDB. RESULTS: The necrotic cells induced dysregulation of 106 miRNAs and 887 mRNAs. Among them, 11 miRNAs that had a negative correlation value of p < 0.05 by the hypergeometric test were screened, and their target mRNAs were analyzed by Gene Ontology enrichment analysis. Using the Kyoto Encyclopedia of Genes and Genomes database, we also found several necrotic cell treatment-activated pathways that were modulated by relevant gene targets of differentially expressed miRNAs. CONCLUSION: Our result demonstrated that dysregulation of miRNA and mRNA expression profiles occurs when GBM cells are exposed to necrotic cells, suggesting that several miRNAs may have the potential to be used as biomarkers for predicting GBM progression and pathogenesis.

Leptin Suppresses Glutamate-Induced Apoptosis Through Regulation of ERK1/2 Signaling Pathways in Rat Primary Astrocytes.

BACKGROUND/AIMS: Leptin is a hormone expressed by adipose tissue that regulates body energy homeostasis and weight loss by activating leptin receptors in the hypothalamus. Leptin receptors are also expressed in astrocytes. An anti-apoptosis effect of leptin in brain has recently been reported. However, the anti-apoptosis mechanism of leptin in the brain is unknown. METHODS: To investigate whether leptin exerts protective effects against glutamate-induced apoptosis in astrocytes, we performed cell viability assays and apoptosis assays using rat primary astrocytes. Intracellular signaling pathways involved in anti-apoptosis effects of leptin were analyzed by immunoblotting together with a leptin mutant (S120A/T121A) with antagonist function and pharmacological inhibitors. RESULTS: We found that glutamate-induced apoptosis in rat primary astrocytes was significantly decreased by treatment with leptin. Leptin inhibited glutamate-induced phosphorylation of ERK1/2 in astrocytes. The leptin S120A/T121A mutant did not inhibit glutamate-induced ERK1/2 phosphorylation and ERK1/2-mediated apoptosis. CONCLUSIONS: Collectively, our results provide initial evidence that leptin exerts an anti-apoptotic effect against glutamate toxicity through activation of intracellular signaling pathways which reverse glutamate-induced ERK1/2 phosphorylation in primary astrocytes. Therefore, our findings suggest that leptin might be considered a candidate for potential therapeutic applications in glutamate-induced brain excitotoxicity.

Effect of glucose level on chemical hypoxia- and hydrogen peroxide-induced chemokine expression in human glioblastoma cell lines.

Glioblastoma multiforme (GBM) is the most common primary intracranial tumor in adults and has poor prognosis. The GBM-specific tumor microenvironment (TME) plays a crucial role in tumor progression, immune escape, local invasion, and metastasis of GBM. Here, we demonstrate that hypoxia, reactive oxygen species (ROS), and differential concentration of glucose influence the expression of cytokines and chemokines, such as IL-6, IL-8, and IP-10, in human glial cell lines. Treatment with cobalt chloride (CoCl2) and hydrogen peroxide (H2O2) significantly increased the expression levels of IL-6, IL-8, and IP-10 in a dose-dependent manner in CRT-MG and U251-MG astroglioma cells, but not in microglia cells. However, we found strikingly different patterns of expression of cytokines and chemokines between H2O2-treated CRT-MG cells cultured in low- and high-glucose medium. These results suggest that astroglioma and microglia cells exhibit distinct patterns of cytokine and chemokine expression in response to CoCl2 and H2O2 treatment, and different concentrations of glucose influence this expression under either hypoxic or oxidant-enriched conditions.

Necrotic cells influence migration and invasion of glioblastoma via NF-κB/AP-1-mediated IL-8 regulation.

Glioblastoma multiforme (GBM) is the most common primary intracranial tumor in adults and has poor prognosis. Diffuse infiltration into normal brain parenchyma, rapid growth, and the presence of necrosis are remarkable hallmarks of GBM. However, the effect of necrotic cells on GBM growth and metastasis is poorly understood at present. In this study, we examined the biological significance of necrotic tissues by exploring the molecular mechanisms underlying the signaling network between necrotic tissues and GBM cells. The migration and invasion of the GBM cell line CRT-MG was significantly enhanced by treatment with necrotic cells, as shown by assays for scratch wound healing and spheroid invasion. Incubation with necrotic cells induced IL-8 secretion in CRT-MG cells in a dose-dependent manner. In human GBM tissues, IL-8 positive cells were mainly distributed in the perinecrotic region, as seen in immunohistochemistry and immunofluorescence analysis. Necrotic cells induced NF-κB and AP-1 activation and their binding to the IL-8 promoter, leading to enhanced IL-8 production and secretion in GBM cells. Our data demonstrate that when GBM cells are exposed to and stimulated by necrotic cells, the migration and invasion of GBM cells are enhanced and facilitated via NF-κB/AP-1 mediated IL-8 upregulation.

The epidemiology and economic burden of Clostridium difficile infection in Korea.

The prevalence of Clostridium difficile infection and the associated burden have recently increased in many countries. While the main risk factors for C. difficile infection include old age and antibiotic use, the prevalence of this infection is increasing in low-risk groups. These trends highlight the need for research on C. difficile infection. This study pointed out the prevalence and economic burden of C. difficile infection and uses the representative national data which is primarily from the database of the Korean Health Insurance Review and Assessment Service, for 2008-2011. The annual economic cost was measured using a prevalence approach, which sums the costs incurred to treat C. difficile infection. C. difficile infection prevalence was estimated to have increased from 1.43 per 100,000 in 2008 to 5.06 per 100,000 in 2011. Moreover, mortality increased from 69 cases in 2008 to 172 in 2011. The economic cost increased concurrently, from $2.4 million in 2008 to $7.6 million, $10.5 million, and $15.8 million in 2009, 2010, and 2011, respectively. The increasing economic burden of C. difficile infection over the course of the study period emphasizes the need for intervention to minimize the burden of a preventable illness like C. difficile infection.

Transcriptional regulation of adrenomedullin by oncostatin M in human astroglioma cells: implications for tumor invasion and migration.

Adrenomedullin (ADM), a secretory peptide with multiple functions in physiological to pathological conditions, is upregulated in several human cancers, including brain, breast, colon, prostate, and lung cancer. However, the molecular mechanisms underlying the regulation of ADM expression in cancerous cells are not fully understood. Here, we report that oncostatin M (OSM), a cytokine belonging to the interleukin-6 family, induces ADM expression in astroglioma cells through induction of signal transducer and activator of transcription-3 (STAT-3) phosphorylation, nuclear translocation, and subsequent DNA binding to the ADM promoter. STAT-3 knockdown decreased OSM-mediated expression of ADM, indicating that ADM expression is regulated by STAT-3 in astroglioma cells. Lastly, scratch wound healing assay showed that astroglioma cell migration was significantly enhanced by ADM peptides. These data suggest that aberrant activation of STAT-3, which is observed in malignant brain tumors, may function as one of the key regulators for ADM expression and glioma invasion.

SHP-2 binds to caveolin-1 and regulates Src activity via competitive inhibition of CSK in response to H2O2 in astrocytes.

Reactive oxygen species (ROS) regulate diverse cellular functions by triggering signal transduction events, such as Src and mitogen-activated protein (MAP) kinases. Here, we report the role of caveolin-1 and Src homology 2 domain-containing protein tyrosine phosphatase 2 (SHP-2) in H2O2-induced signaling pathway in brain astrocytes. H2O2-mediated oxidative stress induced phosphorylation of caveolin-1 and association between p-caveolin-1 and SHP-2. SHP-2 specifically bound to wild-type caveolin-1 similarly to c-Src tyrosine kinase (CSK), but not to phosphorylation-deficient mutant of caveolin-1 (Y14A), and interfered with complex formation between caveolin-1 and CSK. In the presence of CSK siRNA, binding between caveolin-1 and SHP-2 was enhanced by H2O2 treatment, which led to reduced Src phosphorylation at tyrosine (Tyr) 530 and enhanced Src phosphorylation at Tyr 419. In contrast, siRNA targeting of SHP-2 facilitated H2O2-mediated interaction between caveolin-1 and CSK and enhanced Src phosphorylation at Tyr 530, leading to subsequent decrease in Src downstream signaling, such as focal adhesion kinase (FAK) and extracellular signal-related kinase (ERK). Our results collectively indicate that SHP-2 alters Src kinase activity by interfering with the complex formation between CSK and phosphotyrosine caveolin-1 in the presence of H2O2, thus functions as a positive regulator in Src signaling under oxidative stress in brain astrocytes.