Chemical modification of hyaluronan oligosaccharides differentially modulates hyaluronan-hyaladherin interactions.

The glycosaminoglycan hyaluronan (HA) is a ubiquitous, nonsulfated polysaccharide with diverse biological roles mediated through its interactions with HA-binding proteins (HABPs). Most HABPs belong to the Link module superfamily, including the major HA receptor, CD44, and secreted protein TSG-6, which catalyzes the covalent transfer of heavy chains from inter-α-inhibitor onto HA. The structures of the HA-binding domains (HABDs) of CD44 (HABD_CD44) and TSG-6 (Link_TSG6) have been determined and their interactions with HA extensively characterized. The mechanisms of binding are different, with Link_TSG6 interacting with HA primarily via ionic and CH-π interactions, whereas HABD_CD44 binds solely via hydrogen bonds and van der Waals forces. Here, we exploit these differences to generate HA oligosaccharides, chemically modified at their reducing ends, that bind specifically and differentially to these target HABPs. Hexasaccharides (HA6AN) modified with 2- or 3-aminobenzoic acid (HA6-2AA, HA6-3AA) or 2-amino-4-methoxybenzoic acid (HA6-2A4MBA), had increased affinities for Link_TSG6 compared to unmodified HA6AN. These modifications did not increase the affinity for CD44_HABD. A model of HA6-2AA (derived from the solution dynamic 3D structure of HA4-2AA) was docked into the Link_TSG6 structure, providing evidence that the 2AA-carboxyl forms a salt bridge with Arginine-81. These modeling results informed a second series of chemical modifications for HA oligosaccharides, which again showed differential binding to the two proteins. Several modifications to HA4 and HA6 were found to convert the oligosaccharide into substrates for heavy chain transfer, whereas unmodified HA4 and HA6 are not. This study has generated valuable research tools to further understand HA biology.

Human mesenchymal stromal cells ameliorate cisplatin-induced acute and chronic kidney injury via TSG-6.

Cisplatin is widely used in tumor chemotherapy, but nephrotoxicity is an unavoidable side effect of cisplatin. Several studies have demonstrated that mesenchymal stromal cells (MSCs) ameliorate cisplatin-induced kidney injury, but the underlying mechanisms are unknown. In this study, the cisplatin-induced kidney injury mouse model was established by subjecting a single intraperitoneal injection with cisplatin. One hour before cisplatin injection, the mice received human bone marrow MSCs (hBM-MSCs) with or without siRNA-transfection, recombinant human tumor necrosis factor-α-stimulated gene/protein 6 (rhTSG-6), or PBS through the tail vein. In addition, cisplatin-stimulated HK-2 cells were treated with hBM-MSCs or rhTSG-6. Human BM-MSCs treatment remarkably ameliorated cisplatin-induced acute and chronic kidney injury, as evidenced by significant reductions in serum creatinine (Scr), blood urea nitrogen, tubular injury, collagen deposition, α-smooth muscle actin accumulation, as well as inflammatory responses, and by remarkable increased anti-inflammatory factor expression and Treg cells infiltration in renal tissues. Furthermore, we found that only a few hBM-MSCs engrafted into damaged kidney and that the level of human TSG-6 in the serum of mice increased significantly following hBM-MSCs administration. Moreover, hBM-MSCs significantly increased the viability of damaged HK-2 cells and decreased the levels of inflammatory cytokines in the culture supernatant. However, the knockdown of the TSG-6 gene in hBM-MSCs significantly attenuated their beneficial effects in vivo and in vitro. On the contrary, treated with rhTSG-6 achieved similar beneficial effects of hBM-MSCs. Our results indicate that systemic administration of hBM-MSCs alleviates cisplatin-induced acute and chronic kidney injury in part by paracrine TSG-6 secretion.

Brain Ischemic Tolerance Triggered by Preconditioning Involves Modulation of Tumor Necrosis Factor-α-Stimulated Gene 6 (TSG-6) in Mice Subjected to Transient Middle Cerebral Artery Occlusion.

Ischemic preconditioning (PC) induced by a sub-lethal cerebral insult triggers brain tolerance against a subsequent severe injury through diverse mechanisms, including the modulation of the immune system. Tumor necrosis factor (TNF)-α-stimulated gene 6 (TSG-6), a hyaluronate (HA)-binding protein, has recently been involved in the regulation of the neuroimmune response following ischemic stroke. Thus, we aimed at assessing whether the neuroprotective effects of ischemic PC involve the modulation of TSG-6 in a murine model of transient middle cerebral artery occlusion (MCAo). The expression of TSG-6 was significantly elevated in the ischemic cortex of mice subjected to 1 h MCAo followed by 24 h reperfusion, while this effect was further potentiated (p < 0.05 vs. MCAo) by pre-exposure to ischemic PC (i.e., 15 min MCAo) 72 h before. By immunofluorescence analysis, we detected TSG-6 expression mainly in astrocytes and myeloid cells populating the lesioned cerebral cortex, with a more intense signal in tissue from mice pre-exposed to ischemic PC. By contrast, levels of TSG-6 were reduced after 24 h of reperfusion in plasma (p < 0.05 vs. SHAM), but were dramatically elevated when severe ischemia (1 h MCAo) was preceded by ischemic PC (p < 0.001 vs. MCAo) that also resulted in significant neuroprotection. In conclusion, our data demonstrate that neuroprotection exerted by ischemic PC is associated with the elevation of TSG-6 protein levels both in the brain and in plasma, further underscoring the beneficial effects of this endogenous modulator of the immune system.

The recombinant Link module of human TSG-6 suppresses cartilage damage in models of osteoarthritis: A potential disease-modifying OA drug.

OBJECTIVE: To investigate the role of endogenous TSG-6 in human osteoarthritis (OA) and assess the disease-modifying potential of a TSG-6-based biological treatment in cell, explant and animal models of OA. DESIGN: Knee articular cartilages from OA patients were analyzed for TSG-6 protein and mRNA expression using immunohistochemistry and RNAscope, respectively. The inhibitory activities of TSG-6 and its isolated Link module (Link_TSG6) on cytokine-induced degradation of OA cartilage explants were compared. Human mesenchymal stem/stromal cell-derived chondrocyte pellet cultures were used to determine the effects of Link_TSG6 and full-length TSG-6 on IL-1α-, IL-1ß-, or TNF-stimulated ADAMTS4, ADAMTS5, and MMP13 mRNA expression. Link_TSG6 was administered i.a. to the rat ACLTpMMx model; cartilage damage and tactile allodynia were assessed. RESULTS: TSG-6 is predominantly associated with chondrocytes in regions of cartilage damage where high TSG-6 expression aligns with low MMP13, the major collagenase implicated in OA progression. Link_TSG6 is more potent than full-length TSG-6 at inhibiting cytokine-mediated matrix breakdown in human OA cartilage explants;>50% of donor cartilages, from 59 tested, were responsive to Link_TSG6 treatment. Link_TSG6 also displayed more potent effects in 3D pellet cultures, suppressing ADAMTS4, ADAMTS5, and MMP13 gene expression, which was consistent with reduced aggrecanase and collagenase activities in explant cultures. Link_TSG6 treatment reduced touch-evoked pain behavior and dose-dependently inhibited cartilage damage in a rodent model of surgically-induced OA. CONCLUSIONS: Link_TSG6 has enhanced chondroprotective activity compared to the full-length TSG-6 protein and shows potential as a disease modifying OA drug via its inhibition of aggrecanase and collagenase activity.

Tumor Necrosis Factor (TNF)-α-Stimulated Gene 6 (TSG-6): A Promising Immunomodulatory Target in Acute Neurodegenerative Diseases.

Tumor necrosis factor (TNF)-α-stimulated gene 6 (TSG-6), the first soluble chemokine-binding protein to be identified in mammals, inhibits chemotaxis and transendothelial migration of neutrophils and attenuates the inflammatory response of dendritic cells, macrophages, monocytes, and T cells. This immunoregulatory protein is a pivotal mediator of the therapeutic efficacy of mesenchymal stem/stromal cells (MSC) in diverse pathological conditions, including neuroinflammation. However, TSG-6 is also constitutively expressed in some tissues, such as the brain and spinal cord, and is generally upregulated in response to inflammation in monocytes/macrophages, dendritic cells, astrocytes, vascular smooth muscle cells and fibroblasts. Due to its ability to modulate sterile inflammation, TSG-6 exerts protective effects in diverse degenerative and inflammatory diseases, including brain disorders. Emerging evidence provides insights into the potential use of TSG-6 as a peripheral diagnostic and/or prognostic biomarker, especially in the context of ischemic stroke, whereby the pathobiological relevance of this protein has also been demonstrated in patients. Thus, in this review, we will discuss the most recent data on the involvement of TSG-6 in neurodegenerative diseases, particularly focusing on relevant anti-inflammatory and immunomodulatory functions. Furthermore, we will examine evidence suggesting novel therapeutic opportunities that can be afforded by modulating TSG-6-related pathways in neuropathological contexts and, most notably, in stroke.

Characterization of the Involvement of Tumour Necrosis Factor (TNF)-α-Stimulated Gene 6 (TSG-6) in Ischemic Brain Injury Caused by Middle Cerebral Artery Occlusion in Mouse.

The identification of novel targets to modulate the immune response triggered by cerebral ischemia is crucial to promote the development of effective stroke therapeutics. Since tumour necrosis factor (TNF)-α-stimulated gene 6 (TSG-6), a hyaluronate (HA)-binding protein, is involved in the regulation of immune and stromal cell functions in acute neurodegeneration, we aimed to characterize its involvement in ischemic stroke. Transient middle cerebral artery occlusion (1 h MCAo, followed by 6 to 48 of reperfusion) in mice resulted in a significant elevation in cerebral TSG-6 protein levels, mainly localized in neurons and myeloid cells of the lesioned hemisphere. These myeloid cells were clearly infiltrating from the blood, strongly suggesting that brain ischemia also affects TSG-6 in the periphery. Accordingly, TSG-6 mRNA expression was elevated in peripheral blood mononuclear cells (PBMCs) from patients 48 h after ischemic stroke onset, and TSG-6 protein expression was higher in the plasma of mice subjected to 1 h MCAo followed by 48 h of reperfusion. Surprisingly, plasma TSG-6 levels were reduced in the acute phase (i.e., within 24 h of reperfusion) when compared to sham-operated mice, supporting the hypothesis of a detrimental role of TSG-6 in the early reperfusion stage. Accordingly, systemic acute administration of recombinant mouse TSG-6 increased brain levels of the M2 marker Ym1, providing a significant reduction in the brain infarct volume and general neurological deficits in mice subjected to transient MCAo. These findings suggest a pivotal role of TSG-6 in ischemic stroke pathobiology and underscore the clinical relevance of further investigating the mechanisms underlying its immunoregulatory role.

Role of TGF-ß and p38 MAPK in TSG-6 Expression in Adipose Tissue-Derived Stem Cells In Vitro and In Vivo.

Mesenchymal stem cells (MSCs) regulate immune cell activity by expressing tumor necrosis factor-α (TNF-α)-stimulated gene 6 (TSG-6) in inflammatory environments; however, whether anti-inflammatory responses affect TSG-6 expression in MSCs is not well understood. Therefore, we investigated whether transforming growth factor-ß (TGF-ß) regulates TSG-6 expression in adipose tissue-derived stem cells (ASCs) and whether effective immunosuppression can be achieved using ASCs and TGF-ß signaling inhibitor A83-01. TGF-ß significantly decreased TSG-6 expression in ASCs, but A83-01 and the p38 inhibitor SB202190 significantly increased it. However, in septic C57BL/6 mice, A83-01 further reduced the survival rate of the lipopolysaccharide (LPS)-treated group and ASC transplantation did not improve the severity induced by LPS. ASC transplantation alleviated the severity of sepsis induced by LPS+A83-01. In co-culture of macrophages and ASCs, A83-01 decreased TSG-6 expression whereas A83-01 and SB202190 reduced Cox-2 and IDO-2 expression in ASCs. These results suggest that TSG-6 expression in ASCs can be regulated by high concentrations of pro-inflammatory cytokines in vitro and in vivo, and that A83-01 and SB202190 can reduce the expression of immunomodulators in ASCs. Therefore, our data suggest that co-treatment of ASCs with TGF-ß or p38 inhibitors is not adequate to modulate inflammation.

TSG-6 inhibits hypertrophic scar fibroblast proliferation by regulating IRE1α/TRAF2/NF-κB signalling.

TNF-stimulated gene (TSG-6) was reported to suppress hypertrophic scar (HS) formation in a rabbit ear model, and the overexpression of TSG-6 in human HS fibroblasts (HSFs) was found to induce their apoptotic death. The molecular basis for these findings, however, remains to be clarified. HSFs were subjected to TSG-6 treatment. Treatment with TSG-6 significantly suppressed HSF proliferation and induced them to undergo apoptosis. Moreover, TSG-6 exposure led to reductions in collagen I, collagen III, and α-SMA mRNA and protein levels, with a corresponding drop in proliferating cell nuclear antigen (PCNA) expression indicative of impaired proliferative activity. Endoplasmic reticulum (ER) stress was also suppressed in these HSFs as demonstrated by decreases in Bip and p-IRE1α expression, downstream inositol requiring enzyme 1 alpha (IRE1α) -Tumor necrosis factor receptor associated factor 2 (TRAF2) pathway signalling was inhibited and treated cells failed to induce NF-κB, TNF-α, IL-1ß, and IL-6 expression. Overall, ER stress was found to trigger inflammatory activity in HSFs via the IRE1α-TRAF2 axis, as confirmed with the specific inhibitor of IRE1α STF083010. Additionally, the effects of TSG-6 on apoptosis, collagen I, collagen III, α-SMA, and PCNA of HSFs were reversed by the IRE1α activator thapsigargin (TG). These data suggest that TSG-6 administration can effectively suppress the proliferation of HSFs in part via the inhibition of IRE1α-mediated ER stress-induced inflammation (IRE1α/TRAF2/NF-κB signalling).

Mesenchymal stem cells protect against TBI-induced pyroptosis in vivo and in vitro through TSG-6.

BACKGROUND: Pyroptosis, especially microglial pyroptosis, may play an important role in central nervous system pathologies, including traumatic brain injury (TBI). Transplantation of mesenchymal stem cells (MSCs), such as human umbilical cord MSCs (hUMSCs), has been a focus of brain injury treatment. Recently, MSCs have been found to play a role in many diseases by regulating the pyroptosis pathway. However, the effect of MSC transplantation on pyroptosis following TBI remains unknown. Tumor necrosis factor α stimulated gene 6/protein (TSG-6), a potent anti-inflammatory factor expressed in many cell types including MSCs, plays an anti-inflammatory role in many diseases; however, the effect of TSG-6 secreted by MSCs on pyroptosis remains unclear. METHODS: Mice were subjected to controlled cortical impact injury in vivo. To assess the time course of pyroptosis after TBI, brains of TBI mice were collected at different time points. To study the effect of TSG-6 secreted by hUMSCs in regulating pyroptosis, normal hUMSCs, sh-TSG-6 hUMSCs, or different concentrations of rmTSG-6 were injected intracerebroventricularly into mice 4 h after TBI. Neurological deficits, double immunofluorescence staining, presence of inflammatory factors, cell apoptosis, and pyroptosis were assessed. In vitro, we investigated the anti-pyroptosis effects of hUMSCs and TSG-6 in a lipopolysaccharide/ATP-induced BV2 microglial pyroptosis model. RESULTS: In TBI mice, the co-localization of Iba-1 (marking microglia/macrophages) with NLRP3/Caspase-1 p20/GSDMD was distinctly observed at 48 h. In vivo, hUMSC transplantation or treatment with rmTSG-6 in TBI mice significantly improved neurological deficits, reduced inflammatory cytokine expression, and inhibited both NLRP3/Caspase-1 p20/GSDMD expression and microglial pyroptosis in the cerebral cortices of TBI mice. However, the therapeutic effect of hUMSCs on TBI mice was reduced by the inhibition of TSG-6 expression in hUMSCs. In vitro, lipopolysaccharide/ATP-induced BV2 microglial pyroptosis was inhibited by co-culture with hUMSCs or with rmTSG-6. However, the inhibitory effect of hUMSCs on BV2 microglial pyroptosis was significantly reduced by TSG-6-shRNA transfection. CONCLUSION: In TBI mice, microglial pyroptosis was observed. Both in vivo and in vitro, hUMSCs inhibited pyroptosis, particularly microglial pyroptosis, by regulating the NLRP3/Caspase-1/GSDMD signaling pathway via TSG-6. Video Abstract.

Antifibrotic TSG-6 Expression Is Synergistically Increased in Both Cells during Coculture of Mesenchymal Stem Cells and Macrophages via the JAK/STAT Signaling Pathway.

The pro-inflammatory cytokines tumor necrosis factor-alpha (TNF-α) and interleukin (IL)-1ß upregulate TNF-α-stimulated gene 6 (TSG-6); however, current knowledge about the optimal conditions for TSG-6 expression in mesenchymal stem cells (MSCs) is limited. Here, we investigated whether TSG-6 expression varies depending on the polarization state of macrophages co-cultured with adipose tissue-derived stem cells (ASCs) and analyzed the optimal conditions for TSG-6 expression in ASCs. TSG-6 expression increased in ASCs co-cultured with M0, M1, and M2 macrophages indirectly; among them, M1 macrophages resulted in the highest increase in TSG-6 expression in ASCs. TSG-6 expression in ASCs dramatically increased by combination (but not single) treatment of TNF-α, IL-1ß, interferon-gamma (IFN-γ), and lipopolysaccharide (LPS). In addition, phosphorylation of signal transducer and activator of transcription (STAT) 1/3 was observed in response to IFN-γ and LPS treatment but not TNF-α and/or IL-1ß. STAT1/3 activation synergistically increased TNF-α/IL-1ß-dependent TSG-6 expression, and JAK inhibitors suppressed TSG-6 expression both in ASCs and macrophages. In LX-2 hepatic stellate cells, TSG-6 inhibited TGF-ß-induced Smad3 phosphorylation, resulting in decreased α-smooth muscle actin (SMA) expression. Moreover, fibrotic activities of LX-2 cells induced by TGF-ß were dramatically decreased after indirect co-culture with ASCs and M1 macrophages. These results suggest that a comprehensive inflammatory microenvironment may play an important role in determining the therapeutic properties of ASCs by increasing TSG-6 expression through STAT1/3 activation.

Anti-inflammatory protein TNFα-stimulated gene-6 (TSG-6) reduces inflammatory response after brain injury in mice.

BACKGROUND: Current research suggests that the glial scar surrounding penetrating brain injuries is instrumental in preserving the surrounding uninjured tissue by limiting the inflammatory response to the injury site. We recently showed that tumor necrosis factor (TNF)-stimulated gene-6 (TSG-6), a well-established anti-inflammatory molecule, is present within the glial scar. In the present study we investigated the role of TSG-6 within the glial scar using TSG-6 null and littermate control mice subjected to penetrating brain injuries. RESULTS: Our findings show that mice lacking TSG-6 present a more severe inflammatory response after injury, which was correlated with an enlarged area of astrogliosis beyond the injury site. CONCLUSION: Our data provides evidence that TSG-6 has an anti-inflammatory role within the glial scar.

The intestinal injury caused by ischemia-reperfusion is attenuated by amniotic fluid stem cells via the release of tumor necrosis factor-stimulated gene 6 protein.

Ischemia/reperfusion (I/R) is implicated in the pathogenesis of various acute intestinal injuries. Amniotic fluid stem cells (AFSC) are beneficial in experimental intestinal diseases. Tumor necrosis factor-induced protein 6 (TSG-6) has been shown to exert anti-inflammatory effects. We aimed to investigate if AFSC secreted TSG-6 reduces inflammation and rescues intestinal I/R injury. The superior mesenteric artery of 3-week-old rats was occluded for 90 minutes and green fluorescent protein-labeled AFSC or recombinant TSG-6 was injected intravenously upon reperfusion. AFSC distribution was evaluated at 24, 48, and 72 hours after I/R. AFSC and TSG-6 effects on the intestine were assessed 48 hours postsurgery. Intestinal organoids were used to study the effects of TSG-6 after hypoxia-induced epithelial damage. After I/R-induced intestinal injury, AFSC migrated preferentially to the ileum, the primary site of injury, through blood circulation. Engrafted AFSC reduced ileum injury, inflammation, and oxidative stress. These AFSC-mediated beneficial effects were dependent on secretion of TSG-6. Administration of TSG-6 protected against hypoxia-induced epithelial damage in intestinal organoids. Finally, TSG-6 attenuated intestinal damage during I/R by suppressing genes involved in wound and injury pathways. This study indicates that AFSC or TSG-6 have the potential of rescuing the intestine from the damage caused by I/R.

Tumor Necrosis Factor-stimulated Gene-6 (TSG-6) Secreted by BMSCs Regulates Activated Astrocytes by Inhibiting NF-κB Signaling Pathway to Ameliorate Blood Brain Barrier Damage After Intracerebral Hemorrhage.

To investigate the influence of tumor necrosis factor-stimulated gene-6 (TSG-6) secreted by bone mesenchymal stem cells (BMSCs) on blood brain barrier (BBB) after intracerebral hemorrhage (ICH) and its related mechanisms. BMSCs and astrocytes were isolated and induced by TNF-α and LPS respectively. The effect of TSG-6 secreted by BMSCs on the proliferation and apoptosis of astrocytes and inflammatory response were assessed by CCK8, flow cytometry, and ELISA respectively. Then we studied the effects of TSG-6 secreted by BMSCs through the paracrine mechanism on the integrity of BBB after ICH via NF-κB signaling pathway in vitro and in vivo. We successfully isolated BMSCs and astrocytes. After LPS treatment of astrocytes, IL-1ß, IL-6, and TNF-α showed an upward trend. TSG-6 secreted by TNF-α-activated BMSCs could antagonize the inflammatory response in activated astrocytes. Through the co-culture of astrocytes and BMSCs and the ICH animal model, we found that TSG-6 regulates activated astrocytes by inhibiting the NF-κB signaling pathway and ameliorates BBB damage. Furthermore, we found that TNF-α-activated BMSCs secreted exosomes containing TSG-6 and played an anti-inflammatory effect. TSG-6 secreted by BMSCs regulates activated astrocytes by inhibiting the NF-κB signaling pathway, thereby ameliorating BBB damage.

HGF and TSG-6 Released by Mesenchymal Stem Cells Attenuate Colon Radiation-Induced Fibrosis.

Fibrosis is a leading cause of death in occidental states. The increasing number of patients with fibrosis requires innovative approaches. Despite the proven beneficial effects of mesenchymal stem cell (MSC) therapy on fibrosis, there is little evidence of their anti-fibrotic effects in colorectal fibrosis. The ability of MSCs to reduce radiation-induced colorectal fibrosis has been studied in vivo in Sprague-Dawley rats. After local radiation exposure, rats were injected with MSCs before an initiation of fibrosis. MSCs mediated a downregulation of fibrogenesis by a control of extra cellular matrix (ECM) turnover. For a better understanding of the mechanisms, we used an in vitro model of irradiated cocultured colorectal fibrosis in the presence of human MSCs. Pro-fibrotic cells in the colon are mainly intestinal fibroblasts and smooth muscle cells. Intestinal fibroblasts and smooth muscle cells were irradiated and cocultured in the presence of unirradiated MSCs. MSCs mediated a decrease in profibrotic gene expression and proteins secretion. Silencing hepatocyte growth factor (HGF) and tumor necrosis factor-stimulated gene 6 (TSG-6) in MSCs confirmed the complementary effects of these two genes. HGF and TSG-6 limited the progression of fibrosis by reducing activation of the smooth muscle cells and myofibroblast. To settle in vivo the contribution of HGF and TSG-6 in MSC-antifibrotic effects, rats were treated with MSCs silenced for HGF or TSG-6. HGF and TSG-6 silencing in transplanted MSCs resulted in a significant increase in ECM deposition in colon. These results emphasize the potential of MSCs to influence the pathophysiology of fibrosis-related diseases, which represent a challenging area for innovative treatments.

Characteristics of MSCs in Synovial Fluid and Mode of Action of Intra-Articular Injections of Synovial MSCs in Knee Osteoarthritis.

We have been studying mesenchymal stem cells (MSCs) in synovial fluid and the intra-articular injection of synovial MSCs in osteoarthritis (OA) knees. Here, mainly based on our own findings, we overview the characteristics of endogenous MSCs in the synovial fluid of OA knees and their mode of action when injected exogenously into OA knees. Many MSCs similar to synovial MSCs were detected in the synovial fluid of human OA knees, and their number correlated with the radiological OA grade. Our suspended synovium culture model demonstrated the release of MSCs from the synovium through a medium into a non-contacting culture dish. In OA knees, endogenous MSCs possibly mobilize in a similar manner from the synovium through the synovial fluid and act protectively. However, the number of mobilized MSCs is limited; therefore, OA progresses in its natural course. Synovial MSC injections inhibited the progression of cartilage degeneration in a rat OA model. Injected synovial MSCs migrated into the synovium, maintained their MSC properties, and increased the gene expressions of TSG-6, PRG-4, and BMP-2. Exogenous synovial MSCs can promote anti-inflammation, lubrication, and cartilage matrix synthesis in OA knees. Based on our findings, we have initiated a human clinical study of synovial MSC injections in OA knees.

Anti-inflammatory protein TSG-6 secreted by bone marrow mesenchymal stem cells attenuates neuropathic pain by inhibiting the TLR2/MyD88/NF-κB signaling pathway in spinal microglia.

BACKGROUND: Neuroinflammation plays a vital role in the development and maintenance of neuropathic pain. Recent evidence has proved that bone marrow mesenchymal stem cells (BMSCs) can inhibit neuropathic pain and possess potent immunomodulatory and immunosuppressive properties via secreting a variety of bioactive molecules, such as TNF-α-stimulated gene 6 protein (TSG-6). However, it is unknown whether BMSCs exert their analgesic effect against neuropathic pain by secreting TSG-6. Therefore, the present study aimed to evaluate the analgesic effects of TSG-6 released from BMSCs on neuropathic pain induced by chronic constriction injury (CCI) in rats and explored the possible underlying mechanisms in vitro and in vivo. METHODS: BMSCs were isolated from rat bone marrow and characterized by flow cytometry and functional differentiation. One day after CCI surgery, about 5 × 106 BMSCs were intrathecally injected into spinal cerebrospinal fluid. Behavioral tests, including mechanical allodynia, thermal hyperalgesia, and motor function, were carried out at 1, 3, 5, 7, 14 days after CCI surgery. Spinal cords were processed for immunohistochemical analysis of the microglial marker Iba-1. The mRNA and protein levels of pro-inflammatory cytokines (IL-1ß, TNFα, IL-6) were detected by real-time RT-PCR and ELISA. The activation of the TLR2/MyD88/NF-κB signaling pathway was evaluated by Western blot and immunofluorescence staining. The analgesic effect of exogenous recombinant TSG-6 on CCI-induced mechanical allodynia and heat hyperalgesia was observed by behavioral tests. In the in vitro experiments, primary cultured microglia were stimulated with the TLR2 agonist Pam3CSK4, and then co-cultured with BMSCs or recombinant TSG-6. The protein expression of TLR2, MyD88, p-p65 was evaluated by Western blot. The mRNA and protein levels of IL-1ß, TNFα, IL-6 were detected by real-time RT-PCR and ELISA. BMSCs were transfected with the TSG-6-specific shRNA and then intrathecally injected into spinal cerebrospinal fluid in vivo or co-cultured with Pam3CSK4-treated primary microglia in vitro to investigate whether TSG-6 participated in the therapeutic effect of BMSCs on CCI-induced neuropathic pain and neuroinflammation. RESULTS: We found that CCI-induced mechanical allodynia and heat hyperalgesia were ameliorated by intrathecal injection of BMSCs. Moreover, intrathecal administration of BMSCs inhibited CCI-induced neuroinflammation in spinal cord tissues. The analgesic effect and anti-inflammatory property of BMSCs were attenuated when TSG-6 expression was silenced. We also found that BMSCs inhibited the activation of the TLR2/MyD88/NF-κB pathway in the ipsilateral spinal cord dorsal horn by secreting TSG-6. Meanwhile, we proved that intrathecal injection of exogenous recombinant TSG-6 effectively attenuated CCI-induced neuropathic pain. Furthermore, in vitro experiments showed that BMSCs and TSG-6 downregulated the TLR2/MyD88/NF-κB signaling and reduced the production of pro-inflammatory cytokines, such as IL-1ß, IL-6, and TNF-α, in primary microglia treated with the specific TLR2 agonist Pam3CSK4. CONCLUSIONS: The present study demonstrated a paracrine mechanism by which intrathecal injection of BMSCs targets the TLR2/MyD88/NF-κB pathway in spinal cord dorsal horn microglia to elicit neuroprotection and sustained neuropathic pain relief via TSG-6 secretion.

TSG-6 Attenuates Oxidative Stress-Induced Early Brain Injury in Subarachnoid Hemorrhage Partly by the HO-1 and Nox2 Pathways.

BACKGROUND: Early brain injury (EBI) refers to acute brain injury during the first 72 h after subarachnoid hemorrhage (SAH), which is one of the major causes of poor prognosis after SAH. Here, we investigated the effect and the related mechanism of TSG-6 on EBI after SAH. MATERIALS AND METHODS: The Sprague-Dawley rat model of SAH was developed by the endovascular perforation method. TSG-6 (5µg) was administered by an intraventricular injection within 1.5 h after SAH. The effects of TSG-6 on EBI were assessed by neurological score, brain water content (BWC) and TUNEL staining. Immunofluorescence staining was used to assay NF-κB/p-NF-κB expression in microglia. Protein expression levels of heme oxygenase-1 (HO-1), NADPH oxidase 2 (Nox2), Bcl-2, Bax, and cleaved-caspase-3 were measured to investigate the potential mechanism. The enzyme activity of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) and the level of reactive oxygen species (ROS) were analyzed using commercially available kits. RESULTS: The results showed that TSG-6 treatment alleviated the neurobehavioral dysfunction and reduced BWC and the number of TUNEL-positive neurons in EBI after SAH. TSG-6 decreased the ROS level and enhanced the enzyme activity of SOD and GSH-Px after SAH. Furthermore TSG-6 inhibited the NF-κB activation, increased the protein expression levels of HO-1 and Bcl-2 and decreased the expression levels of Nox2, Bax, and cleaved-caspase-3. The administration of TSG-6 siRNA abolished the protective effects of TSG-6 on EBI after SAH. CONCLUSION: We found that TSG-6 attenuated oxidative stress and apoptosis in EBI after SAH partly by inhibiting NF-κB and activating HO-1 pathway in brain tissue.

TNF-stimulated gene 6 promotes formation of hyaluronan-inter-α-inhibitor heavy chain complexes necessary for ozone-induced airway hyperresponsiveness.

Exposure to pollutants, such as ozone, exacerbates airway inflammation and hyperresponsiveness (AHR). TNF-stimulated gene 6 (TSG-6) is required to transfer inter-α-inhibitor heavy chains (HC) to hyaluronan (HA), facilitating HA receptor binding. TSG-6 is necessary for AHR in allergic asthma, because it facilitates the development of a pathological HA-HC matrix. However, the role of TSG-6 in acute airway inflammation is not well understood. Here, we hypothesized that TSG-6 is essential for the development of HA- and ozone-induced AHR. TSG-6-/- and TSG-6+/+ mice were exposed to ozone or short-fragment HA (sHA), and AHR was assayed via flexiVent. The AHR response to sHA was evaluated in the isolated tracheal ring assay in tracheal rings from TSG-6-/- or TSG-6+/+, with or without the addition of exogenous TSG-6, and with or without inhibitors of Rho-associated, coiled-coil-containing protein kinase (ROCK), ERK, or PI3K. Smooth-muscle cells from mouse tracheas were assayed in vitro for signaling pathways. We found that TSG-6 deficiency protects against AHR after ozone (in vivo) or sHA (in vitro and in vivo) exposure. Moreover, TSG-6-/- tracheal ring non-responsiveness to sHA was reversed by exogenous TSG-6 addition. sHA rapidly activated RhoA, ERK, and Akt in airway smooth-muscle cells, but only in the presence of TSG-6. Inhibition of ROCK, ERK, or PI3K/Akt blocked sHA/TSG-6-mediated AHR. In conclusion, TSG-6 is necessary for AHR in response to ozone or sHA, in part because it facilitates rapid formation of HA-HC complexes. The sHA/TSG-6 effect is mediated by RhoA, ERK, and PI3K/Akt signaling.

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

BACKGROUND: An 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. METHODS: The 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. RESULTS: SAH 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. CONCLUSIONS: TSG-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.

TSG-6 - a double-edged sword for osteoarthritis (OA).

PURPOSE: To explore mechanisms underlying the association of TSG-6 with osteoarthritis (OA) progression. METHODS: TSG-6-mediated heavy chain (HC) transfer (TSG-6 activity) and its association with inflammatory mediators were quantified in knee OA (n=25) synovial fluids (SFs). Paired intact and damaged cartilages from the same individuals (20 tibial and 12 meniscal) were analyzed by qRT-PCR and immunohistochemistry (IHC) for gene and protein expression of TSG-6 and components of Inter-alpha-Inhibitor (IαI) and TSG-6 activity ± spiked in IαI. Primary chondrocyte cultures (n=5) ± IL1ß or TNFα were evaluated for gene expression. The effects of TSG-6 activity on cartilage extracellular matrix (ECM) assembly were explored using quantitative hyaluronan (HA)-aggrecan binding assays. RESULTS: TSG-6 activity was significantly associated (R > 0.683, P < 0.0002) with inflammatory mediators including TIMP-1, A2M, MMP3, VEGF, VCAM-1, ICAM-1 and IL-6. Although TSG-6 protein and mRNA were highly expressed in damaged articular and meniscal cartilage and cytokine-treated chondrocytes, there was little or no cartilage expression of components of the IαI complex (containing HC1). By IHC, TSG-6 was present throughout lesioned cartilage but HC1 only at lesioned surfaces. TSG-6 impaired HA-aggrecan assembly, but TSG-6 mediated HA-HC formation reduced this negative effect. CONCLUSIONS: TSG-6 activity is a global inflammatory biomarker in knee OA SF. IαI, supplied from outside cartilage, only penetrates the cartilage surface, restricting TSG-6 activity (HC transfer) to this region. Therefore, unopposed TSG-6 in intermediate and deep regions of OA cartilage could possibly block matrix assembly, leading to futile synthesis and account for increased risk of OA progression.