Knockdown Of TSG-6 Research Articles

A novel therapeutic approach for inflammatory bowel disease by exosomes derived from human umbilical cord mesenchymal stem cells to repair intestinal barrier via TSG-6

BackgroundExosomes as the main therapeutic vectors of mesenchymal stem cells (MSC) for inflammatory bowel disease (IBD) treatment and its mechanism remain unexplored. Tumor necrosis factor-α stimulated gene 6 (TSG-6) is a glycoprotein secreted by MSC with the capacities of tissue repair and immune regulation. This study aimed to explore whether TSG-6 is a potential molecular target of exosomes derived from MSCs (MSCs-Exo) exerting its therapeutic effect against colon inflammation and repairing mucosal tissue.MethodsTwo separate dextran sulfate sodium (DSS) and 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced IBD mouse models were intraperitoneally administered MSCs-Exo extracted from human umbilical cord MSC (hUC-MSC) culture supernatant. Effects of MSCs-Exo on intestinal inflammation, colon barrier function, and proportion of T cells were investigated. We explored the effects of MSCs-Exo on the intestinal barrier and immune response with TSG-6 knockdown. Moreover, recombinant human TSG-6 (rhTSG-6) was administered exogenously and colon inflammation severity in mice was evaluated.ResultsIntraperitoneal injection of MSCs-Exo significantly ameliorated IBD symptoms and reduced mortality rate. The protective effect of MSCs-Exo on intestinal barrier was demonstrated evidenced by the loss of goblet cells and intestinal mucosa permeability, thereby improving the destruction of tight junctions (TJ) structures and microvilli, as well as increasing the expression of TJ proteins. Microarray analysis revealed that MSCs-Exo administration downregulated the level of pro-inflammatory cytokines and upregulated the anti-inflammatory cytokine in colon tissue. MSCs-Exo also modulated the response of Th2 and Th17 cells in the mesenteric lymph nodes (MLN). Reversely, knockdown of TSG-6 abrogated the therapeutic effect of MSCs-Exo on mucosal barrier maintenance and immune regulation, whereas rhTSG-6 administration showed similar efficacy to that of MSCs-Exo.ConclusionsOur findings suggested that MSCs-Exo protected against IBD through restoring mucosal barrier repair and intestinal immune homeostasis via TSG-6 in mice.

Therapeutic effect of secretome from TNF-α stimulated mesenchymal stem cells in an experimental model of corneal limbal stem cell deficiency.

To explore the secretome efficacy in tumor necrosis factor (TNF)-α stimulated mouse mesenchymal stem cells (MSCs) in a murine model of corneal limbal alkali injury. Corneal limbal stem cell deficiency (LSCD) was created in the eyes of male C57 mice. Concentrated conditioned medium from TNF-α stimulated MSCs (MSC-CMT) was applied topically for 4wk, with basal medium and conditioned medium from MSCs as controls. Corneal opacification, corneal inflammatory response, and corneal neovascularization (NV) were evaluated. Corneal epithelial cell apoptosis, corneal conjunctivation, and inflammatory cell infiltration were assessed with TUNEL staining, CK3 and Muc-5AC immunostaining, and CD11b immunofluorescence staining, respectively. The effect of TSG-6 was further evaluated by knockdown with short hairpin RNA (shRNA). Compared to the controls, topical administration of MSC-CMT significantly ameliorated the clinical symptoms of alkali-induced LSCD, with restrained corneal NV, reduced corneal epithelial cell apoptosis, and inhibition of corneal conjunctivation. In addition, MSC-CMT treatment significantly reduced CD11b+ inflammatory cell infiltration, and inhibited the expression of pro-inflammatory cytokines (IL-1β, TNF-α and IL-6). Furthermore, the promotion of corneal epithelial reconstruction by MSC-CMT was largely abolished by TSG-6 knockdown. Our study provides evidence that MSC-CMT enhances the alleviation of corneal alkali injuries, partially through TSG-6-mediated anti-inflammatory protective mechanisms. MSC-CMT may serve as a potential strategy for treating corneal disorders.

Impact of Marine-Based Biomaterials on the Immunoregulatory Properties of Bone Marrow-Derived Mesenchymal Stem Cells: Potential Use of Fish Collagen in Bone Tissue Engineering.

A key issue in the field of tissue engineering and stem cell therapy is immunological rejection after the implantation of allogeneic bone marrow-derived mesenchymal stem cells (BMSCs). In addition, maintaining the immunoregulatory function of BMSCs is critical to achieving tissue repair. In recent years, scientists have become interested in fish collagen because of its unique osteoinductive activity. However, it is still unclear whether osteogenically differentiated BMSCs induced by fish collagen maintain their immunoregulatory functions. To address this question, BMSCs were isolated from 8-week-old male BALB/c mice, and a noncontact coculture model was established consisting of macrophages and BMSCs treated with hydrolyzed fish collagen (HFC). Cell proliferation of the macrophages was determined by MTT. The gene and protein expression levels of the M1 and M2 macrophage markers were measured by real-time PCR and enzyme-linked immunosorbent assay (ELISA). To study the role of TNF-α-induced gene/protein 6 (TSG-6), TSG-6 was targeted by short interfering RNA (siRNA) in BMSCs, then the osteogenic differentiation ability of the BMSCs was examined by western blotting. The mRNA expression levels of interleukin-10 (IL-10), CCL22 (a macrophage-derived chemokine), tumor necrosis factor α (TNF-α), and interleukin-12 (IL-12), and the protein expression levels of arginase-1 (Arg-1) and inducible nitric oxide synthase (iNOS) of macrophages cocultured with TSG-6-siRNA–BMSCs+HFC were detected by real-time PCR and western blotting, respectively. The results showed that the osteogenically differentiated BMSCs induced by HFC did not affect the proliferation of macrophages. Osteogenically differentiated BMSCs induced by HFC promoted the expression of M2 macrophage markers IL-10 and CCL22, while HFC inhibited the expression of M1 macrophage markers, including TNF-α and IL-12. The TSG-6 knockdown led to a decrease in the production of TSG-6 without impairing the expression of bone sialoprotein (BSP), osteocalcin (OCN), and Runt-related transcription factor 2 (RUNX2) by BMSCs. TSG-6 silencing significantly counteracted the effect of HFC, and the expression of IL-10, CCL22, and Arg-1 were all decreased in the macrophages cocultured with TSG-6-siRNA–BMSCs+HFC, while that of TNF-α, IL-12, and iNOS were increased relative to the BMSCs+HFC group. The data demonstrated that osteogenically differentiated BMSCs induced by fish collagen retained their immunomodulatory functions. This study provides an additional scientific basis for future applications of fish collagen as an osteogenic component in the fields of tissue engineering and stem cell therapy.

Exosomes derived from TSG-6 modified mesenchymal stromal cells attenuate scar formation during wound healing

Mesenchymal stromal cell (MSC)-derived exosome therapy has emerged as an effective therapy strategy for the pathological scar formation. However, the underlying mechanisms have not been completely understood. In the current study, we investigate the therapeutic effect of TSG-6 modified MSC-derived exosomes on a mouse full-thickness wound model and provide evidence of a possible mechanism for MSC-derived exosomes to prevent from scar formation. Overexpression and knockdown of TSG-6 were conducted by lentivirus infection into hBMSCs. Exosomes were isolated from cell culture and identified by transmission electron microscopy and Western blot. C57BL/6J mice were performed of full-thickness skin wounds and treated with exosomal suspension or TSG-6-neutralizing antibody. H&E staining was subjected to observe the pathological changes of scar tissues. Immunohistochemistry, ELISA, real time-PCR and Western blot were applied to detect the expressions of relevant molecules. The results showed that subcutaneous injection of TSG-6 overexpressed MSC-derived exosomes effectively ameliorated scar pathological injury, decreased inflammatory molecular secretion and attenuated collagen deposition in a mouse skin wound model. Reversely, knockdown of TSG-6 abrogated the therapeutic effect of MSC-derived exosomes on scarring. Moreover, TSG-6-neutralizing antibody counteracted the effect of TSG-6 overexpressed MSC-derived exosomes in preventing scar formation. In conclusion, we demonstrated that exosomes derived from TSG-6 modified MSCs suppressed scar formation via reducing inflammation and inhibiting collagen deposition.

Mesenchymal stem cells alleviate liver injury induced by chronic-binge ethanol feeding in mice via release of TSG6 and suppression of STAT3 activation

BackgroundMesenchymal stem cells (MSCs) are a population of pluripotent cells that might be used for treatment of liver disease. However, the efficacy of MSCs for mice with alcoholic hepatitis (AH) and its underlying mechanism remains unclear.MethodsMSCs were isolated from the bone marrow (BM) of 4–6-week-old male C57BL/6 N mice. AH was induced in female mice by chronic-binge ethanol feeding for 10 days. The mice were given intraperitoneal injections of MSCs with or without transfection or AG490, recombinant mouse tumor necrosis factor (TNF)-α-stimulated gene/protein 6 (rmTSG-6), or saline at day 10. Blood samples and hepatic tissues were collected at day 11. Various assays such as biochemistry, histology, and flow cytometry were performed.ResultsMSCs reduced AH in mice, decreasing liver/body weight ratio, liver injury, blood and hepatic lipids, malondialdehyde, interleukin (IL)-6, and TNF-ɑ, but increasing glutathione, IL-10, and TSG-6, compared to control mice. Few MSCs engrafted into the inflamed liver. Knockdown of TSG-6 in MSCs significantly attenuated their effects, and injection of rmTSG-6 achieved similar effects to MSCs. The signal transducer and activator of transcription 3 (STAT3) was activated in mice with AH, and MSCs and rmTSG-6 inhibited the STAT3 activation. Injection of MSCs plus AG490 obtained more alleviation of liver injury than MSCs alone.ConclusionsBM-MSCs injected into mice with AH do not engraft the liver, but they secrete TSG-6 to reduce liver injury and to inhibit STAT3 activation.

The Potassium SK Channel Activator NS309 Protects Against Experimental Traumatic Brain Injury Through Anti-Inflammatory and Immunomodulatory Mechanisms.

Neuroinflammation plays important roles in neuronal cell death and functional deficits after TBI. Small conductance Ca2+-activated K+ channels (SK) have been shown to be potential therapeutic targets for treatment of neurological disorders, such as stroke and Parkinson’s disease (PD). The aim of the present study was to investigate the role of SK channels in an animal model of TBI induced by controlled cortical impact (CCI). The SK channels activator NS309 at a concentration of 2 mg/kg was administered by intraperitoneal injection, and no obviously organ-related toxicity of NS309 was found in Sprague-Dawley (SD) rats. Treatment with NS309 significantly reduced brain edema after TBI, but had no effect on contusion volume. This protection can be observed even when the administration was delayed by 4 h after injury. NS309 attenuated the TBI-induced deficits in neurological function, which was accompanied by the reduced neuronal apoptosis. The results of immunohistochemistry showed that NS309 decreased the number of neutrophils, lymphocytes, and microglia cells, with no effect on astrocytes. In addition, NS309 markedly decreased the levels of pro-inflammatory cytokines (IL-1β, IL-6 and TNF-α) and chemokines (MCP-1, MIP-2, and RANTES), but increased the levels of anti-inflammatory cytokines (IL-4, IL-10, and TGF-β1) after TBI. The results of RT-PCR and western blot showed that NS309 increased TSG-6 expression and inhibited NF-κB activation. Furthermore, knockdown of TSG-6 using in vivo transfection with TSG-6 specific shRNA partially reversed the protective and anti-inflammatory effects of NS309 against TBI. In summary, our results indicate that the SK channel activator NS309 could modulate inflammation-associated immune cells and cytokines via regulating the TSG-6/NF-κB pathway after TBI. The present study offers a new sight into the mechanisms responsible for SK channels activation with implications for the treatment of TBI.

Serum-Free Medium Enhances the Immunosuppressive and Antifibrotic Abilities of Mesenchymal Stem Cells Utilized in Experimental Renal Fibrosis.

Serum used in culture medium brings risks of immune reactions or infections and thus may hinder using ex vivo expanded mesenchymal stem cells (MSCs) for medical treatment. Here, we cultured MSCs in a serum‐free medium (SF‐MSCs) and in a medium containing 10% fetal bovine serum (10%MSCs) and investigated their effects on inflammation and fibrosis. MSC‐conditioned medium suppressed transforming growth factor‐β1–induced phosphorylation of Smad2 in HK‐2 cells, with no significant difference between the two MSCs. This finding suggests that the direct antifibrotic effect of SF‐MSCs is similar to that of 10%MSCs. However, immunohistochemistry revealed that renal fibrosis induced by unilateral ureteral obstruction in rats was more significantly ameliorated by the administration of SF‐MSCs than by that of 10%MSCs. Coculture of MSCs and monocytic THP‐1 cell‐derived macrophages using a Transwell system showed that SF‐MSCs significantly induced polarization from the proinflammatory M1 to the immunosuppressive M2 phenotype macrophages, suggesting that SF‐MSCs strongly suppress the persistence of inflammation. Furthermore, the gene expression of tumor necrosis factor‐α–induced protein 6 (TSG‐6), which inhibits the recruitment of inflammatory cells, was higher in SF‐MSCs than in 10%MSCs, and TSG‐6 knockdown in SF‐MSCs attenuated the anti‐inflammatory responses in unilateral ureteral obstruction rats. These findings imply that SF culture conditions can enhance the immunosuppressive and antifibrotic abilities of MSCs and the administration of ex vivo expanded SF‐MSCs has the potential to be a useful therapy for preventing the progression of renal fibrosis. Stem Cells Translational Medicine 2018;7:893–905

TSG-6 attenuates inflammation-induced brain injury via modulation of microglial polarization in SAH rats through the SOCS3/STAT3 pathway

BackgroundAn acute and drastic inflammatory response characterized by the production of inflammatory mediators is followed by stroke, including SAH. Overactivation of microglia parallels an excessive inflammatory response and worsened brain damage. Previous studies indicate that TSG-6 has potent immunomodulatory and anti-inflammatory properties. This study aimed to evaluate the effects of TSG-6 in modulating immune reaction and microglial phenotype shift after experimental SAH.MethodsThe SAH model was established by endovascular puncture method for Sprague–Dawley rats (weighing 280–320 g). Recombinant human protein and specific siRNAs for TSG-6 were exploited in vivo. Brain injury was assessed by neurologic scores, brain water content, and Fluoro-Jade C (FJC) staining. Microglia phenotypic status was evaluated and determined by Western immunoblotting, quantitative real-time polymerase chain reaction (qPCR) analyses, flow cytometry, and immunofluorescence labeling.ResultsSAH induced significant inflammation, and M1-dominated microglia polarization increased expression of TSG-6 and neurological dysfunction in rats. rh-TSG-6 significantly ameliorated brain injury, decreased proinflammatory mediators, and skewed microglia towards a more anti-inflammatory property 24-h after SAH. While knockdown of TSG-6 further induced detrimental effects of microglia accompanied with more neurological deficits, the anti-inflammation effects of rh-TSG-6 were associated with microglia phenotypic shift by regulating the level of SOCS3/STAT3 axis.ConclusionsTSG-6 exerted neuroprotection against SAH-induced EBI in rats, mediated in part by skewing the balance of microglial response towards a protective phenotype, thereby preventing excessive tissue damage and improving functional outcomes. Our findings revealed the role of TSG-6 in modulating microglial response partially involved in the SOCS3/STAT3 pathway and TSG-6 may be a promising therapeutic target for the treatment of brain injury following SAH.

Mesenchymal Stem Cells Ameliorated Glucolipotoxicity in HUVECs through TSG-6.

Glucolipotoxicity is one of the critical causal factors of diabetic complications. Whether mesenchymal stem cells (MSCs) have effects on glucolipotoxicity in human umbilical vein endothelial cells (HUVECs) and mechanisms involved are unclear. Thirty mM glucose plus 100 μM palmitic acid was used to induce glucolipotoxicity in HUVECs. MSCs and HUVECs were co-cultured at the ratio of 1:5 via Transwell system. The mRNA expressions of inflammatory factors were detected by RT-qPCR. The productions of reactive oxygen species (ROS), cell cycle and apoptosis were analyzed by flow cytometry. The tumor necrosis factor-α stimulated protein 6 (TSG-6) was knockdown in MSCs by RNA interference. High glucose and palmitic acid remarkably impaired cell viability and tube formation capacity, as well as increased the mRNA expression of inflammatory factors, ROS levels, and cell apoptosis in HUVECs. MSC co-cultivation ameliorated these detrimental effects in HUVECs, but no effect on ROS production. Moreover, TSG-6 was dramatically up-regulated by high glucose and fatty acid stimulation in both MSCs and HUVECs. TSG-6 knockdown partially abolished the protection mediated by MSCs. MSCs had protective effects on high glucose and palmitic acid induced glucolipotoxicity in HUVECs, and TSG-6 secreted by MSCs was likely to play an important role in this process.

Mesenchymal stem/stromal cells precondition lung monocytes/macrophages to produce tolerance against allo- and autoimmunity in the eye

Intravenously administered mesenchymal stem/stromal cells (MSCs) engraft only transiently in recipients, but confer long-term therapeutic benefits in patients with immune disorders. This suggests that MSCs induce immune tolerance by long-lasting effects on the recipient immune regulatory system. Here, we demonstrate that i.v. infusion of MSCs preconditioned lung monocytes/macrophages toward an immune regulatory phenotype in a TNF-α-stimulated gene/protein (TSG)-6-dependent manner. As a result, mice were protected against subsequent immune challenge in two models of allo- and autoimmune ocular inflammation: corneal allotransplantation and experimental autoimmune uveitis (EAU). The monocytes/macrophages primed by MSCs expressed high levels of MHC class II, B220, CD11b, and IL-10, and exhibited T-cell-suppressive activities independently of FoxP3(+) regulatory T cells. Adoptive transfer of MSC-induced B220(+)CD11b(+) monocytes/macrophages prevented corneal allograft rejection and EAU. Deletion of monocytes/macrophages abrogated the MSC-induced tolerance. However, MSCs with TSG-6 knockdown did not induce MHC II(+)B220(+)CD11b(+) cells, and failed to attenuate EAU. Therefore, the results demonstrate a mechanism of the MSC-mediated immune modulation through induction of innate immune tolerance that involves monocytes/macrophages.

MSCs inhibit bone marrow-derived DC maturation and function through the release of TSG-6

Dendritic cells (DCs) are potent antigen-presenting cells (APCs) that are characterized by the ability to take up and process antigens and prime T cell responses. Mesenchymal stem cells (MSCs) are multipotent cells that have been shown to have immunomodulatory abilities, including inhibition of DC maturation and function in vivo and in vitro; however, the underlying mechanism is far from clear. In this study we found that MSCs can inhibit the maturation and function of bone marrow-derived DCs by releasing TSG-6. In the presence of MSCs, lower expression of mature DC surface phenotype (CD80, CD86, MHC-II, and CD11c) was observed. In addition, typical DC functions, such as the production of IL-12 and the ability to prime T cells, were decreased when co-cultured with MSCs. In contrast, knockdown of TSG-6 reduced the inhibitory effect of MSCs on DC. Moreover, we found that TSG-6 can suppress the activation of MAPKs, and NF-κB signaling pathways within DCs during Lipopolysaccharides (LPS) stimulation. In conclusion, we suggest that TSG-6 plays an important role in MSCs-mediated immunosuppressive effect on DC.

TSG-6 Produced by hMSCs Delays the Onset of Autoimmune Diabetes by Suppressing Th1 Development and Enhancing Tolerogenicity

Genetic and immunological screening for type 1 diabetes has led to the possibility of preventing disease in susceptible individuals. Here, we show that human mesenchymal stem/stromal cells (hMSCs) and tumor necrosis factor-α–stimulated gene 6 (TSG-6), a protein produced by hMSCs in response to signals from injured tissues, delayed the onset of spontaneous autoimmune diabetes in NOD mice by inhibiting insulitis and augmenting regulatory T cells (Tregs) within the pancreas. Importantly, hMSCs with a knockdown of tsg-6 were ineffective at delaying insulitis and the onset of diabetes in mice. TSG-6 inhibited the activation of both T cells and antigen-presenting cells (APCs) in a CD44-dependent manner. Moreover, multiple treatments of TSG-6 rendered APCs more tolerogenic, capable of enhancing Treg generation and delaying diabetes in an adoptive transfer model. Therefore, these results could provide the basis for a novel therapy for the prevention of type 1 diabetes.

Intravenous Mesenchymal Stem Cells Prevented Rejection of Allogeneic Corneal Transplants by Aborting the Early Inflammatory Response

Mesenchymal stem/progenitor cells (MSCs) were reported to enhance the survival of cellular and organ transplants. However, their mode of action was not established. We here used a mouse model of corneal allotransplantation and demonstrated that peri-transplant intravenous (i.v.) infusion of human MSCs (hMSCs) decreased the early surgically induced inflammation and reduced the activation of antigen-presenting cells (APCs) in the cornea and draining lymph nodes (DLNs). Subsequently, immune rejection was decreased, and allograft survival was prolonged. Quantitative assays for human GAPDH revealed that <10 hMSCs out of 1 × 10(6) injected cells were recovered in the cornea 10 hours to 28 days after i.v. infusion. Most of hMSCs were trapped in lungs where they were activated to increase expression of the gene for a multifunctional anti-inflammatory protein tumor necrosis factor-α stimulated gene/protein 6 (TSG-6). i.v. hMSCs with a knockdown of TSG-6 did not suppress the early inflammation and failed to prolong the allograft survival. Also, i.v. infusion of recombinant TSG-6 reproduced the effects of hMSCs. Results suggest that hMSCs improve the survival of corneal allografts without engraftment and primarily by secreting TSG-6 that acts by aborting early inflammatory responses. The same mechanism may explain previous reports that MSCs decrease rejection of other organ transplants.

Tumour necrosis factor-stimulated gene (TSG)-6 controls epithelial–mesenchymal transition of proximal tubular epithelial cells

Progressive renal disease is characterized by accumulation of extracellular matrix in the renal cortex. Proximal tubular cells (PTC) may contribute to disease through a process of epithelial–mesenchymal-transition (EMT): phenotypic change, disruption of the tubular basement membrane and migration into the interstitium. Hyaluronan (HA) synthesis and its extracellular organization by hyaladherins affect cell fate in other systems: this study investigated the role of the hyaladherin, tumour necrosis factor-stimulated gene (TSG)-6, in PTC EMT triggered in vitro by transforming growth factor (TGF)β1. TGFβ1 triggered the loss of PTC epithelial phenotype with 60% decreased expression of E-cadherin and 2–3-fold induction of alpha-smooth muscle actin (α-sma). It also increased the expression of TSG-6, HA-synthase-(HAS)2 and the HA-receptor, CD44, to a peak at 8–12 h, remaining elevated thereafter. Immuno-localization of HA demonstrated that unstimulated PTC assembled HA in cables and that treatment with TGFβ1 initiated cable disassembly with formation of dense HA-pericellular coats. Stable knockdown of TSG-6 with short-hairpin-RNA increased E-cadherin and HAS2 expression, produced loose HA-pericellular coats, HA cables were absent and cell migration was slowed. Treatment of transfectants with TGFβ1 did not induce α-sma, alter E-cadherin, pericellular-HA or migration but did induce HAS2. This was dependent on the expression of CD44 and was inhibited by CD44-specific siRNA. In summary, TSG-6 was central to EMT through effects on HA macromolecular structure and through CD44-dependent triggering of cell responses. These findings suggest that controlling the assembly of HA by proximal tubular cells may be a novel approach towards intervention in renal disease.