Unveiling the role of TNF‐α in mesenchymal stromal cell‐mediated immunosuppression

Mesenchymal stromal cells (MSCs) are multipotent progenitors of mesodermal origin that not only differentiate into osteoblasts, chondrocytes, connective stromal cells, and adipocytes, but also exert immunoregulatory activities, usually induced by soluble molecules released during the cross‐talk between MSCs and their target immune cell populations. In this issue of the European Journal of Immunology, Dorronsoro et al. [Eur. J. Immunol. 2014. 44: 480–488] demonstrate for the first time that TNF‐α released by activated T cells confers immunosuppressive properties upon MSCs by binding to TNF‐R1 and activating the NF‐kB pathway. Such findings may improve our knowledge of the mechanisms underlying the reported efficacy of human MSCs administered locally or systemically to patients with autoimmune/inflammatory disorders, such as Crohn's disease and graft versus host disease, as discussed in this commentary.     

5,7‐dihydroxy‐3,4,6‐trimethoxyflavone inhibits the inflammatory effects induced by Bacteroides fragilis enterotoxin via dissociating the complex of heat shock protein 90 and IκBα and IκB kinase‐γ in intestinal epithelial cell culture

Enterotoxin produced by enterotoxigenic Bacteroides fragilis (BFT) has been associated with mucosal inflammation and diarrhoeal diseases. In this study, the anti‐inflammatory molecular mechanism of 5,7‐dihydroxy‐3,4,6‐trimethoxyflavone (eupatilin) was characterized in an HT‐29 intestinal epithelial cell line stimulated with BFT. Pre‐treatment of HT‐29 cells with eupatilin decreased the production significantly of both interleukin (IL)‐8 and prostaglandin E₂ induced by BFT in a dose‐dependent manner. BFT‐activated nuclear factor‐kappaB (NF‐κB) signals in HT‐29 cells and pretreatment with eupatilin suppressed NF‐κB activation that resulted in the significant inhibition of IL‐8 and cyclo‐oxygenase‐2 expression. BFT‐induced phosphorylation of both IκBα and IκB kinase (IKK) signals was prevented in eupatilin‐pretreated HT‐29 cells. Transfection of siRNA for IKK‐α and IKK‐β decreased the production of IL‐8 and prostaglandin E₂; however, the transfection of IKK‐β siRNA showed a more significant reduction of BFT‐induced IκBα phosphorylation compared with that of IKK‐α siRNA. In addition, herbimycin A, a specific inhibitor of heat shock protein 90 (Hsp90), decreased the BFT‐induced activation of IKK and NF‐κB, suggesting that Hsp90 is associated with a pathway of IKK‐NF‐κB‐IL‐8/cyclo‐oxygenase‐2 gene signalling. Furthermore, eupatilin dissociated the complex between Hsp90 and IKK‐γ in BFT‐stimulated HT‐29 cells. These results suggest that eupatilin can suppress the NF‐κB signalling pathway by targeting the Hsp90‐IKK‐γ complex in intestinal epithelial cells and may attenuate BFT‐induced inflammatory responses.     

Vitamin K3 attenuates lipopolysaccharide‐induced acute lung injury through inhibition of nuclear factor‐κB activation

Vitamin K is a family of fat‐soluble compounds including phylloquinone (vitamin K₁), menaquinone (vitamin K₂) and menadione (vitamin K₃). Recently, it was reported that vitamin K, especially vitamins K₁ and K₂, exerts a variety of biological effects, and these compounds are expected to be candidates for therapeutic agents against various diseases. In this study, we investigated the anti‐inflammatory effects of vitamin K₃ in in vitro cultured cell experiments and in vivo animal experiments. In human embryonic kidney (HEK)293 cells, vitamin K₃ inhibited the tumour necrosis factor (TNF)‐α‐evoked translocation of nuclear factor (NF)‐κB into the nucleus, although vitamins K₁ and K₂ did not. Vitamin K₃ also suppressed the lipopolysaccharide (LPS)‐induced nuclear translocation of NF‐κB and production of TNF‐α in mouse macrophage RAW264·7 cells. Moreover, the addition of vitamin K₃ before and after LPS administration attenuated the severity of lung injury in an animal model of acute lung injury/acute respiratory distress syndrome (ARDS), which occurs in the setting of acute severe illness complicated by systemic inflammation. In the ARDS model, vitamin K₃ also suppressed the LPS‐induced increase in the serum TNF‐α level and inhibited the LPS‐evoked nuclear translocation of NF‐κB in lung tissue. Despite marked efforts, little therapeutic progress has been made, and the mortality rate of ARDS remains high. Vitamin K₃ may be an effective therapeutic strategy against acute lung injury including ARDS.     

Synergistic effect of interleukin‐17 and tumour necrosis factor‐α on inflammatory response in hepatocytes through interleukin‐6‐dependent and independent pathways

The proinflammatory cytokines interleukin (IL)‐17 and tumour necrosis factor (TNF)‐α are targets for treatment in many chronic inflammatory diseases. Here, we examined their role in liver inflammatory response compared to that of IL‐6. Human hepatoma cells (HepaRG, Huh7.5 and HepG2 cells) and primary human hepatocytes (PHH) were cultured with IL‐6, IL‐17 and/or TNF‐α. To determine the contribution of the IL‐6 pathway in the IL‐17/TNF‐α‐mediated effect, an anti‐IL‐6 receptor antibody was used. IL‐17 and TNF‐α increased in synergy IL‐6 secretion by HepaRG cells and PHH but not by Huh7.5 and HepG2 cells. This IL‐17/TNF‐α synergistic cooperation enhanced the levels of C‐reactive protein (CRP) and aspartate aminotransferase (ASAT) in HepaRG cell and PHH cultures through the induction of IL‐6. IL‐17/TNF‐α also up‐regulated IL‐8, monocyte chemoattractant protein (MCP)‐1 and chemokine (C‐C motif) ligand 20 (CCL20) chemokines in synergy through an IL‐6‐independent pathway. Interestingly, first exposure to IL‐17, but not to TNF‐α, was crucial for the initiation of the IL‐17/TNF‐α synergistic effect on IL‐6 and IL‐8 production. In HepaRG cells, IL‐17 enhanced IL‐6 mRNA stability resulting in increased IL‐6 protein levels. The IL‐17A/TNF‐α synergistic effect on IL‐6 and IL‐8 induction was mediated through the activation of extracellular signal‐regulated kinase (ERK)‐mitogen‐activated protein kinase, nuclear factor‐κB and/or protein kinase B (Akt)–phosphatidylinositol 3‐kinase signalling pathways. Therefore, the IL‐17/TNF‐α synergistic interaction mediates systemic inflammation and cell damage in hepatocytes mainly through IL‐6 for CRP and ASAT induction. Independently of IL‐6, the IL‐17A/TNF‐α combination may also induce immune cell recruitment by chemokine up‐regulation. IL‐17 and/or TNF‐α neutralization can be a promising therapeutic strategy to control both systemic inflammation and liver cell attraction.     

Suppression of oxLDL‐Induced MMP‐9 and EMMPRIN Expression by Berberine via Inhibition of NF‐κB Activation in Human THP‐1 Macrophages

Upregulation of matrix metalloproteinases (MMPs) and extracellular matrix metalloproteinase inducer (EMMPRIN) by macrophages leads to atherosclerotic plaque rupture by degradation of the extracellular matrix. NF‐κB activation regulates many key inflammatory genes linked to atherosclerosis. In the present study, the function of berberine, a natural extract from Rhizoma coptidis, on MMP‐9 and EMMPRIN expression, the role of NF‐κB activation in oxLDL‐stimulated macrophages, and the possible mechanism in which NF‐κB activation is involved were investigated. Berberine inhibited the expression of MMP‐9 and EMMPRIN at both mRNA and protein levels. The phosphorylation of IκB‐α and nuclear translocation of p65 protein were reduced by berberine, suggesting that NF‐κB activation was inhibited by berberine in oxLDL‐stimulated macrophages. Overall, berberine suppressed the expression of MMP‐9 and EMMPRIN by at least reducing partly the activity of NF‐κB in oxLDL‐induced macrophages. Anat Rec, 2012. © 2011 Wiley Periodicals, Inc.     

Hepatocyte‐specific activation of NF‐κB does not aggravate chemical hepatocarcinogenesis in transgenic mice

The NF‐κB signalling pathway plays important roles in liver organogenesis and carcinogenesis. Mouse embryos deficient in IKKβ die in mid‐gestation, due to excessive apoptosis of hepatoblasts. Although activation of the NF‐κB signalling pathway has been demonstrated in human hepatocellular carcinoma, the role of NF‐κB is controversial. Here, we have generated transgenic mice in which a constitutively active form of IKKβ was expressed in a hepatocyte‐specific manner. Using electrophoretic mobility shift assay, we documented increased NF‐κB activities and up‐regulated levels of NF‐κB downstream target genes, Bcl‐xL and STAT5, in the transgenic mouse livers. These results confirmed that the NF‐κB pathway was activated in the livers of the transgenic mice. However, there was no significant difference in tumour formation between transgenic and wild‐type mice up to an age of 50 weeks. When we treated the transgenic mice with the chemical carcinogen diethylnitrosamine (DEN), we observed no significant differences in the incidence and size of liver tumours formed in these mice with and without DEN treatment at 35 weeks of age, suggesting that the activated NF‐κB pathway in the livers of the transgenic mice did not enhance hepatocarcinogenesis. Interestingly, some of the transient transgenic embryos (E12.5) had abnormal excessive accumulation of nucleated red blood cells in their developing livers. In summary, NF‐κB activation in hepatocytes did not significantly affect chemical hepatocarcinogenesis. In addition, the TTR/IKKCA transgenic mice may serve as a useful model for studying the role of NF‐κB activation in hepatocarcinogenesis as well as inflammatory and metabolic diseases. Copyright © 2008 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Mycoplasma pneumoniae lipids license TLR‐4 for activation of NLRP3 inflammasome and autophagy to evoke a proinflammatory response

Mycoplasma pneumoniae is an obligate pathogen that causes pneumonia, tracheobronchitis, pharyngitis and asthma in humans. It is well recognized that membrane lipoproteins are immunostimulants exerting as lipopolysaccharides (LPS) and play a crucial role in the pathogenesis of inflammatory responses upon M. pneumoniae infection. Here, we report that the M. pneumoniae‐derived lipids are another proinflammatory agents. Using an antibody‐neutralizing assay, RNA interference or specific inhibitors, we found that Toll‐like receptor 4 (TLR‐4) is essential for M. pneumoniae lipid‐induced tumour necrosis factor (TNF)‐α and interleukin (IL)‐1β production. We also demonstrate that NLR family pyrin domain containing 3 inflammasome (NLRP3) inflammasome, autophagy and nuclear factor kappa B (NF‐κB)‐dependent pathways are critical for the secretion of proinflammatory cytokines, while inhibition of TLR‐4 significantly abrogates these events. Further characterization revealed that autophagy‐mediated inflammatory responses involved the activation of NF‐κB. In addition, the activation of NF‐κB promoted lipid‐induced autophagosome formation, as revealed by assays using pharmacological inhibitors, 3‐methyladenine (3‐MA) and Bay 11‐7082, or silencing of atg5 and beclin‐1. These findings suggest that, unlike the response to lipoprotein stimulation, the inflammation in response to M. pneumoniae lipids is mediated by the TLR‐4 pathway, which subsequently initiates the activation of NLRP3 inflammasome and formation of a positive feedback loop between autophagy and NF‐κB signalling cascade, ultimately promoting TNF‐α and Il‐1β production in macrophages.     

Golgi phosphoprotein 3 (GOLPH3) promotes hepatocellular carcinoma cell aggressiveness by activating the NF‐κB pathway

Hepatocellular carcinoma (HCC) is one of the most lethal malignancies, in which the NF‐κB pathway plays an important role and is constitutively activated. Better understanding of the molecular pathogenesis of HCC and the NF‐κB pathway are needed to improve patient outcomes. Herein, we identified an unappreciated protein involved in NF‐κB‐induced activation, Golgi phosphoprotein 3 (GOLPH3). The mRNA and protein expression levels of GOLPH3 were frequently up‐regulated in HCC and GOLPH3 expression correlated closely with clinical stage and survival in both the testing and validation cohorts. Ectopic over‐expression of GOLPH3 in PLC/PRF/5 (PLC) and Huh7 HCC cells protected against cisplatin‐induced apoptosis, promoted angiogenesis and proliferation and increased the aggressiveness of HCC cells in vitro and in vivo, whereas inhibition of GOLPH3 led to decreased aggressiveness. Through analysis of two published HCC patient profiles, GOLPH3 expression significantly correlated with NF‐κB signalling. Furthermore, we demonstrated that GOLPH3 promoted K63‐linked polyubiquitination of tumour necrosis factor receptor‐associated factor 2 (TRAF2), receptor interacting protein (RIP) and NF‐κB essential modulator (NEMO) and substantially sustained the activation of NF‐κB in HCC cells. Taken together, our findings provided evidence that GOLPH3 is a prognostic and/or potential therapeutic biomarker for HCC patients and plays an important role in activation of the NF‐κB pathway during HCC progression. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

NF‐κB‐inducing kinase is a key regulator of inflammation‐induced and tumour‐associated angiogenesis

Angiogenesis is essential during development and in pathological conditions such as chronic inflammation and cancer progression. Inhibition of angiogenesis by targeting vascular endothelial growth factor (VEGF) blocks disease progression, but most patients eventually develop resistance which may result from compensatory signalling pathways. In endothelial cells (ECs), expression of the pro‐angiogenic chemokine CXCL12 is regulated by non‐canonical nuclear factor (NF)‐κB signalling. Here, we report that NF‐κB‐inducing kinase (NIK) and subsequent non‐canonical NF‐κB signalling regulate both inflammation‐induced and tumour‐associated angiogenesis. NIK is highly expressed in endothelial cells (ECs) in tumour tissues and inflamed rheumatoid arthritis synovial tissue. Furthermore, non‐canonical NF‐κB signalling in human microvascular ECs significantly enhanced vascular tube formation, which was completely blocked by siRNA targeting NIK. Interestingly, Nik^?/? mice exhibited normal angiogenesis during development and unaltered TNFα‐ or VEGF‐induced angiogenic responses, whereas angiogenesis induced by non‐canonical NF‐κB stimuli was significantly reduced. In addition, angiogenesis in experimental arthritis and a murine tumour model was severely impaired in these mice. These studies provide evidence for a role of non‐canonical NF‐κB signalling in pathological angiogenesis, and identify NIK as a potential therapeutic target in chronic inflammatory diseases and tumour neoangiogenesis. © 2014 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.