Tim‐3 exacerbates kidney ischaemia/reperfusion injury through the TLR‐4/NF‐κB signalling pathway and an NLR‐C4 inflammasome activation

T cell immunoglobulin domain and mucin domain‐containing molecule‐3 (Tim‐3), a member of the immunoglobulin superfamily, has been shown to play a crucial role in host adaptive immunity and tolerance. However, its role in kidney ischaemia–reperfusion injury (IRI) remains unknown. In this study, we investigated the role and mechanism of Tim‐3 signalling after kidney IRI. In an established murine model of kidney IRI, we found that Tim‐3 expression is enhanced on monocytes/macrophages. Anti‐Tim‐3 antibody RMT3‐23 ameliorates biochemical and histological kidney injury, reduces apoptosis and decreases macrophage infiltration and cytokine production in ischaemic kidneys. Cell culture experiments also demonstrated that the role of Tim‐3 in IRI‐induced macrophage activation leads to the secretion of proinflammatory cytokines and chemokines. In addition, Toll‐like receptor (TLR)‐4 and Nod‐like receptor (NLR) family CARD domain‐containing protein 4 (NLR‐C4) expression were enhanced after kidney IRI and decreased significantly by RMT3‐23. Tim‐3 not only promotes TLR‐mediated nuclear factor kappa B (NF‐κB) activation and cytokine and chemokine release, but also participates in NLR‐C4 inflammasome activation. Taken together, our data confirm that Tim‐3 signalling enhances injury after kidney IRI and demonstrated that Tim‐3 is involved in regulating TLR‐4/NF‐κB signalling and NLR‐C4 inflammasome activation, which provide evidence that Tim‐3 signalling is critical for kidney IRI and may provide a new means to ameliorate kidney tissue immune responses in the clinics.     

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.     

Triggering of TLR‐3, ‐4, NOD2, and DC‐SIGN reduces viral replication and increases T‐cell activation capacity of HIV‐infected human dendritic cells

A variety of signals influence the capacity of dendritic cells (DCs) to mount potent antiviral cytotoxic T‐cell (CTL) responses. In particular, innate immune sensing by pathogen recognition receptors, such as TLR and C‐type lectines, influences DC biology and affects their susceptibility to HIV infection. Yet, whether the combined effects of PPRs triggering and HIV infection influence HIV‐specific (HS) CTL responses remain enigmatic. Here, we dissect the impact of innate immune sensing by pathogen recognition receptors on DC maturation, HIV infection, and on the quality of HS CTL activation. Remarkably, ligand‐driven triggering of TLR‐3, ‐4, NOD2, and DC‐SIGN, despite reducing viral replication, markedly increased the capacity of infected DCs to stimulate HS CTLs. This was exemplified by the diversity and the quantity of cytokines produced by HS CTLs primed by these DCs. Infecting DCs with viruses harboring members of the APOBEC family of antiviral factors enhanced the antigen‐presenting skills of infected DCs. Our results highlight the tight interplay between innate and adaptive immunity and may help develop innovative immunotherapies against viral infections.     

IRF‐5 and NF‐κB p50 co‐regulate IFN‐β and IL‐6 expression in TLR9‐stimulated human plasmacytoid dendritic cells

Synthetic oligonucleotides (ODN) expressing CpG motifs mimic the ability of bacterial DNA to trigger the innate immune system via TLR9. Plasmacytoid dendritic cells (pDCs) make a critical contribution to the ensuing immune response. This work examines the induction of antiviral (IFN‐β) and pro‐inflammatory (IL‐6) cytokines by CpG‐stimulated human pDCs and the human CAL‐1 pDC cell line. Results show that interferon regulatory factor‐5 (IRF‐5) and NF‐κB p50 are key co‐regulators of IFN‐β and IL‐6 expression following TLR9‐mediated activation of human pDCs. The nuclear accumulation of IRF‐1 was also observed, but this was a late event that was dependant on type 1 IFN and unrelated to the initiation of gene expression. IRF‐8 was identified as a novel negative regulator of gene activation in CpG‐stimulated pDCs. As variants of IRF‐5 and IRF‐8 were recently found to correlate with susceptibility to certain autoimmune diseases, these findings are relevant to our understanding of the pharmacologic effects of “K” ODN and the role of TLR9 ligation under physiologic, pathologic, and therapeutic conditions.     

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.     

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.     

Expression of classical NF‐κB pathway effectors in human ovarian carcinoma

Darb‐Esfahani S, Sinn B V, Weichert W, Budczies J, Lehmann A, Noske A, Buckendahl A‐C, Müller B M, Sehouli J, Koensgen D, Györffy B, Dietel M & Denkert C
(2010) Histopathology 56. 727–739
Expression of classical NF‐κB pathway effectors in human ovarian carcinoma Aims: Functional studies have demonstrated that nuclear factor (NF)‐κB promotes tumour progression in ovarian cancer cells. However, surprisingly little is known of the expression of effectors of the NF‐κB pathway in human ovarian cancer in vivo. Methods and results: Immunohistochemistry and in situ hybridization revealed that in a cohort of 85 primary ovarian carcinomas, total p65 expression was inversely correlated to nuclear and cytoplasmic phospho‐IκBα (P = 0.002 and P = 0.05, respectively), and IκBα mRNA expression (P = 0.032). In contrast, phospho‐p65 expression was paralleled by the expression of nuclear (P = 0.027) and cytoplasmic phospho‐IκBα (P = 0.01). Total p65 expression was an adverse prognostic factor for overall survival (P = 0.018). In contrast, total IκBα and phosphorylated nuclear and cytoplasmic IκBα expression were favourable prognostic markers (P = 0.001, P = 0.031, P = 0.001, respectively). Cytoplasmic phospho‐IκBα expression remained a significant prognostic factor on multivariate analysis (P = 0.010). In cultured, stimulated OVCAR‐3 ovarian cancer cells the cytoplasmic retranslocation of p65 was delayed by inhibition of the nuclear membrane transporter chromosomal region maintenance/exportin1 protein (CRM1). A positive association of p65 and CRM1 expression was demonstrated in ovarian cancer tissue (P < 0.0001). Conclusions: Total and phosphorylated IκBα protein expression might serve as markers for NF‐κB activation in human ovarian carcinoma. Cytoplasmic localization of p65 may be related to deregulated nucleocytoplasmic transport in carcinomas overexpressing CRM1.     

Olfactomedin 1 negatively regulates NF‐κB signalling and suppresses the growth and metastasis of colorectal cancer cells

Uncontrolled growth and distant metastasis are hallmarks of colorectal cancer (CRC), but the mechanisms are poorly understood. Olfactomedin 1 (OLFM1), a member of the olfactomedin domain‐containing protein family, plays an important role in the development of neurogenic tissues. Recently, OLFM1 deregulation was frequently observed in several cancers, and it was induced in colon cell lines after treatment with the demethylating agent 5‐aza‐2'‐deoxycytidine. However, the function of OLFM1 in CRC remains unknown. In this study, we reanalysed published microarray data and found that OLFM1 was significantly down‐regulated in primary CRC samples compared to adjacent non‐cancerous tissues. The results of immunohistochemistry indicated that decreased OLFM1 expression was significantly associated with lymph node status (p = 0.023), distant metastasis (p < 0.001), and AJCC/TNM stage (p = 0.013), and CRC patients with low OLFM1 expression had consistently poor overall survival (OS; p < 0.001) and progression‐free survival (PFS; p < 0.001). Further analysis demonstrated that OLFM1 was epigenetically silenced in CRC tissues and cell lines via promoter hypermethylation. Overexpression and knockdown of OLFM1 attenuated and increased, respectively, CRC cells' proliferation, migration, and invasion in vitro and metastasis to the lung and liver in vivo. Mechanistically, the promotion of growth and metastasis of CRC cells by silencing of OLFM1 was associated with the activation of the non‐canonical NF‐κB signalling pathway. OLFM1 interacted with NF‐κB‐inducing kinase (NIK; MAP3K14) and repressed the phosphorylation of its downstream substrate Ikappa B kinase alpha (IKKα). OLFM1 expression was negatively correlated with the phosphorylation level of IKKα in CRC tissue samples. Knockdown of NIK impaired the ability of OLFM1 to repress NF‐κB signalling, cell growth or migration. Thus, OLFM1 may be a valuable biomarker and therapeutic target for CRC patients. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Recombinant human brain natriuretic peptide attenuates trauma‐/haemorrhagic shock‐induced acute lung injury through inhibiting oxidative stress and the NF‐κB‐dependent inflammatory/MMP‐9 pathway

Acute lung injury (ALI) is one of the most serious complications in traumatic patients and is an important part of multiple organ dysfunction syndrome (MODS). Recombinant human brain natriuretic peptide (rhBNP) is a peptide with a wide range of biological activity. In this study, we investigated local changes in oxidative stress and the NF‐κB‐dependent matrix metalloproteinase‐9 (MMP‐9) pathway in rats with trauma/haemorrhagic shock (TH/S)‐induced ALI and evaluated the effects of pretreatment with rhBNP. Forty‐eight rats were randomly divided into four groups: sham operation group, model group, low‐dosage rhBNP group and high‐dosage rhBNP group (n = 12 for each group). Oxidative stress and MPO activity were measured by ELISA kits. MMP‐9 activity was detected by zymography analysis. NF‐κB activity was determined using Western blot assay. With rhBNP pretreatment, TH/S‐induced protein leakage, increased MPO activity, lipid peroxidation and metalloproteinase (MMP)‐9 activity were inhibited. Activation of antioxidative enzymes was reversed. The phosphorylation of NF‐κB and the degradation of its inhibitor IκB were suppressed. The results suggested that the protection mechanism of rhBNP is possibly mediated through upregulation of anti‐oxidative enzymes and inhibition of NF‐κB activation. More studies are needed to further evaluate whether rhBNP is a suitable candidate as an effective inhaling drug to reduce the incidence of TH/S‐induced ALI.     

Transcriptional regulation of pro‐apoptotic Par‐4 by NF‐κB/p65 and its function in controlling cell kinetics during early events in endometrial tumourigenesis

Prostatic apoptosis response‐4 (Par‐4) was first identified in prostatic cancer cells that were induced to undergo apoptosis. Recently, Par‐4 has been suggested to be a tumour suppressor gene that plays a role in the development of endometrial carcinomas (ECs), but the exact mechanism remains to be clarified. Here we examined gene activation signalling cascades and influence on cell kinetics during endometrial tumourigenesis. In normal endometrium, constitutively high levels of Par‐4 expression were observed in epithelial cells through the menstrual cycle, in contrast to the transient up‐regulation in stromal components in the menstrual stage, correlated positively with the phospho‐p65 (pp65) status and apoptosis. In contrast, most ECs exhibited significant down‐regulation as compared to normal endometrium, with positive links only to pp65 expression. In EC cell lines, transfection of the NF‐κB subunit p65 led to transactivation of Par‐4 through specific binding to its promoter region, in contrast to the suppression by active Akt, suggesting that the balance between the two signals may be important to determine Par‐4 expression levels. In addition, transient overexpression of Par‐4 resulted in the induction of not only apoptosis but also senescence, through changes in the expression of bcl‐2 and p21 , respectively. Together, these findings suggest that a signalling cascade involving sequential activation of NF‐κB/p65 and Par‐4 may participate in relatively early events of endometrial tumourigenesis, leading to modulation of cell kinetics including apoptosis and cell cycle progression. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

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.     

Anti‐Inflammatory and Neuroprotective Effects of Triptolide via the NF‐κB Signaling Pathway in a Rat MCAO Model

Stroke is the leading cause of neurological disability in humans. Middle cerebral artery occlusion (MCAO) followed by reperfusion is widely accepted to mimic stroke in basic medical research. Triptolide is one of the major active components of the traditional Chinese herb Tripterygium wilfordii Hook F, and has been reported to have potent anti‐inflammatory and immunosuppressive properties. Since its preclinical effects on stroke were still unclear, we decided to study the effects of Triptolide on focal cerebral ischemia/reperfusion injury in this study. The results showed that Triptolide treatment significantly attenuates brain infarction volume, water content, neurological deficits, and neuronal cell death rate, which were increased in the MCAO model rats. Immunohistochemistry was used to analyze the expression of glial fibrillary acidic protein (GFAP), Cyclooxygenase‐2 (COX‐2), inducible nitric oxide (iNOS), and NF‐κB in the ischemic brains. The administration of Triptolide showed down‐regulation of the iNOS, COX‐2, GFAP, and NF‐κB expression in MCAO rats. It also increased the expression of bcl‐2, and suppressed levels of bax and caspase‐3 compared with the MCAO group. Our findings revealed that Triptolide exerts its neuroprotective effects against inflammation with the involvement of inhibition of NF‐κB activation. Anat Rec, 299:256–266, 2016. © 2015 Wiley Periodicals, Inc.