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

Abstract

The pro-inflammatory cytokines tumor necrosis factor-alpha (TNF-α) and interleukin (IL)-1β 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-1β, 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-1β. STAT1/3 activation synergistically increased TNF-α/IL-1β-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-β-induced Smad3 phosphorylation, resulting in decreased α-smooth muscle actin (SMA) expression. Moreover, fibrotic activities of LX-2 cells induced by TGF-β 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.

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

“…TNF-α and TGF-β have opposing roles in ECM remodeling and inflammatory responses. TNF-α has pro-inflammatory effects and increases TSG-6 expression in MSCs [ 30 ]. Therefore, we analyzed the effect of TGF-β on the regulation of TSG-6 expression in ASCs.…”

“…TSG-6 is a protein that exhibits tissue-protective and anti-inflammatory properties [ 27 ] and is expressed by cells including monocytes, neutrophils, macrophages, dendritic cells, and MSCs [ 1 , 28 , 29 , 30 ]. TSG-6 interacts with various extracellular matrix (ECM) proteins, including fibronectin, to stabilize and remodel the ECM [ 31 , 32 ] and can alleviate fibrosis by downregulating the phosphorylation of suppressor of mothers against decapentaplegic homolog (SMAD) [ 30 , 33 ]. In addition, TSG-6 plays an anti-inflammatory role by regulating the migration and proliferation of endothelial cells, neutrophils, mast cells, macrophage foam cells, vascular smooth muscle cells, and macrophages [ 34 , 35 ].…”

“…Since then, many studies have shown the immunomodulatory and reparative effects of TSG-6 in various diseases, such as atherosclerosis, myocardial infarction, corneal wounding, colitis, acute or chronic liver injury, acute lung injury, and rheumatoid arthritis [ 1 , 29 , 37 , 38 , 40 , 45 , 46 , 47 ]. Low concentrations (1 ng/mL) of TNF-α and IL-1β alone have been shown to slightly increase TSG-6 expression, but STAT-activating or interferon-γ (IFN-γ) + lipopolysaccharide (LPS) treatment conditions significantly increased TSG-6 expression in ASCs [ 30 ]. TSG-6 expression is also significantly increased in ASCs co-cultured with M1 macrophages [ 30 ].…”

“…Low concentrations (1 ng/mL) of TNF-α and IL-1β alone have been shown to slightly increase TSG-6 expression, but STAT-activating or interferon-γ (IFN-γ) + lipopolysaccharide (LPS) treatment conditions significantly increased TSG-6 expression in ASCs [ 30 ]. TSG-6 expression is also significantly increased in ASCs co-cultured with M1 macrophages [ 30 ]. These results suggest that IFN-γ + LPS-differentiated M1 macrophages increase the expression of TSG-6 in ASCs by increasing expression of TNF-α and IL-1β.…”

TGF-β1 induces formation of TSG-6-enriched extracellular vesicles in fibroblasts which can prevent myofibroblast transformation by modulating Erk1/2 phosphorylation

“…It exerts an anti-inflammatory function by counteracting the transcription of MMP-1 and MMP-3 and the activation of MMP-1 72 , can induce the expression of the anti-inflammatory cytokine IL-10 73 and downregulate the expression of the pro-inflammatory cytokine TNF-α 74 . Additionally, TSG-6 can help to attenuate the fibrotic process by inhibiting the activation and migration of neutrophils and macrophages 75 , 76 , which are involved in the inflammatory response associated with fibrosis. Finally, TSG-6 inhibits the activation of TGF-beta receptor I (TGFβRI) and downstream Smad signalling pathways 77 , thus blocking the activation of TGF-beta signalling.…”