Human mesenchymal stromal cells modulate T‐cell responses through TNF‐α‐mediated activation of NF‐κB

Although mesenchymal stromal cells (MSCs) possess the capacity to modulate immune responses, little is known about the mechanisms that underpin these processes. In this study, we show that immunosupression is mediated by activation of nuclear factor kappa B (NF‐κB) in human MSCs. This pathway is activated by TNF‐α that is generated following TCR stimulation of T cells. Inhibition of NF‐κB through silencing of IκB kinase β or the TNF‐α receptor abolishes the immunosuppressive capacity of MSCs. Our data also indicate that MSC‐associated NF‐κB activation primarily leads to inhibition of T‐cell proliferation with little effect on expression of the activation markers CD69 and CD25. Thus, our data support the hypothesis that the TNF‐α/NF‐κB signalling pathway is required for the initial priming of immunosuppressive function in human MSCs. Interestingly, drugs that interfere with NF‐κB activation significantly antagonise the immunoregulatory effect of MSCs, which could have important implications for immunosuppression regimens in the clinic.     

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.     

The atypical IκB protein IκBNS is important for Toll‐like receptor‐induced interleukin‐10 production in B cells

Although a major function of B cells is to mediate humoral immunity by producing antigen‐specific antibodies, a specific subset of B cells is important for immune suppression, which is mainly mediated by the secretion of the anti‐inflammatory cytokine interleukin‐10 (IL‐10). However, the mechanism by which IL‐10 is induced in B cells has not been fully elucidated. Here, we report that IκB_NS, an inducible nuclear IκB protein, is important for Toll‐like receptor (TLR)‐mediated IL‐10 production in B cells. Studies using IκB_NS knockout mice revealed that the number of IL‐10‐producing B cells is reduced in IκB_NS^?/? spleens and that the TLR‐mediated induction of cytoplasmic IL‐10‐positive cells and IL‐10 secretion in B cells are impaired in the absence of IκB_NS. The impairment of IL‐10 production by a lack of IκB_NS was not observed in TLR‐triggered macrophages or T‐cell‐receptor‐stimulated CD4⁺ CD25⁺ T cells. In addition, IκB_NS‐deficient B cells showed reduced expression of Prdm1 and Irf4 and failed to generate IL‐10⁺ CD138⁺ plasmablasts. These results suggest that IκB_NS is selectively required for IL‐10 production in B cells responding to TLR signals, so defining an additional role for IκB_NS in the control of the B‐cell‐mediated immune responses.     

Immunohistochemical analysis of NF‐κB signaling proteins IKKε, p50/p105, p52/p100 and RelA in prostate cancers

Seo SI, Song SY, Kang MR, Kim MS, Oh JE, Kim YR, Lee JY, Yoo NJ, Lee SH. Immunohistochemical analysis of NF‐κB signaling proteins IKKε, p50/p105, p52/p100 and RelA in prostate cancers. APMIS 2009; 117:623–8. Activation of nuclear factor‐kappa B (NF‐κB) signaling is considered an important mechanism in the development of prostate cancers. A recent study revealed that IκB kinase epsilon (IKKε), an activator of NF‐κB, was overexpressed in breast cancers and acted as an oncogene. Expression of NF‐κB members has been reported in prostate cancer tissues, but expression of IKKε has not yet been studied in prostate cancers. In this study, we attempted to explore as to whether expressions of IKKε and NF‐κB members p50/105, p52/p100 and RelA are altered in prostate cancers. We analyzed the expression of IKKε, p50/105, p52/p100 and RelA in 107 prostate adenocarcinoma tissues by immunohistochemistry using a tissue microarray (TMA) method. In the TMA, IKKε is expressed in basal cells, but not in alveolar cells in normal prostate glands. IKKε is expressed in 60.0% of prostate intraepithelial neoplasm (PIN) and 70.1% of the prostate cancers in the cytoplasm. Nuclear immunostainings of NF‐κB members p50/105, p52/p100 and RelA, which are considered activation of NF‐κB signaling, were observed respectively in 28.0%, 18.7% and 37.4% of the cancers. Nuclear staining was detected neither in normal alveolar cells nor in PIN. However, none of the expression of p50/105 nor p52/p100 nor RelA nor IKKε was associated with pathologic characteristics, including size of the cancers, age, Gleason score and stage. The increased cytoplasmic expression of IKKε as well as the increased nuclear expressions of p50/105, p52/p100 and RelA in the prostate cancers compared to normal alveolar cells suggested that overexpression of these proteins may be related to activation of the NF‐κB pathway and might play a role in tumorigenesis of prostate cancers.     

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.     

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.     

The Antiproliferation Effect of Berbamine on K562 Resistant Cells by Inhibiting NF‐κB Pathway

Imatinib mesylate is effective against Ph chromosome‐positive leukemia; however, resistance has been reported. High expression of bcr‐abl in mRNA and protein levels, and other alterations were found in patients who experienced imatinib treatment failures and thus it is important to design alternative treatment strategies. The aim of this study was to evaluate the in vitro effect of berbamine, on imatinib‐resistant chronic myelogenous leukemia (CML) K562 (K562‐r) cells, and explore the mechanisms. The growth of K562‐r cells was examined using the 3‐(4,5‐dimethylthiazol‐2yl)‐2,5‐diphenyl‐tetrazolium bromide (MTT) assay. Morphological analysis and DNA agarose electrophoresis were used to detect apoptosis in K562‐r cells, and the extent of the cells in the sub‐G1 cell cycle phase was measured using flow cytometry. The expression levels of BCR‐ABL, phospho‐BCR‐ABL, and nuclear factor κB (NF‐κB), IκBα, phospho‐IκBα, IκB kinases α(IKKα), and Survivin were determined by Western blot. bcr‐abl mRNA expression was determined by RT‐PCR. MTT assays indicated that berbamine significantly inhibited the proliferation of K562‐r cells. Cells with characteristics of apoptosis were confirmed by morphology examination and DNA agarose electrophoresis and percentage of apoptosis were increased after treatment with berbamine. The results also showed that berbamine was able to down‐regulate BCR‐ABL and phospho‐BCR‐ABL proteins by affecting bcr‐abl mRNA expression and decrease expression of nuclear NF‐κB, phospho‐IκBα, IKKα, and Survivin. Collectively, berbamine could inhibit the proliferation of K562‐r cells and induce apoptosis. The mechanisms may be related at least in part, to inhibit BCR‐ABL and its downstream NF‐κB signaling. Berbamine may provide an alternative candidate for the treatment of patients with CML resistant to imatinib therapy. Anat Rec, 292:945–950, 2009. © 2009 Wiley‐Liss, Inc.     

Signal regulatory protein α negatively regulates mast‐cell activation following FcεRI aggregation

Mast cells elicit allergic reaction through degranulation and release of proinflammatory mediators after aggregation of the IgE receptor FcεRI. Here we provide evidence to show that signal regulatory protein α (SIRPα), an ITIM‐containing receptor, is an endogenous regulator of IgE‐Ag induced mast‐cell activation. SIRPα expression is promptly reduced in mast cells in response to FcεRI aggregation. Impaired expression of SIRPα in mast cells facilitates FcεRI‐evoked degranulation and de novo synthesis of cytokines (IL‐4, IL‐13, IL‐6, and TNF‐α). We further demonstrate that SIRPα knockdown in mast cells accelerates calcium mobilization and affects cytoskeletal rearrangement (F‐actin disassembly and polymeric tubulin formation) after FcεRI aggregation. Mechanistic studies highlight the prolonged activation of NF‐κB and MAPKs as well as PLC‐γ after FcεRI stimulation as a consequence of the inhibition of SIRPα expression in mast cells. Immunoprecipitation analysis shows that SIRPα knockdown markedly increases IgE‐induced SHP2 interaction with PI3K regulatory subunit PI3Kp85 or IKK‐β in mast cells, indicating that SIRPα may accomplish this through its association and sequestration of SHP2. Collectively, our results strongly indicate that SIRPα is a biological important regulator of FcεRI signaling.