Recombinant pyrin domain protein attenuates allergic inflammation by suppressing NF‐κB pathway in asthmatic mice

Pyrin domain (PYD), a subclass of protein motif known as the death fold, is frequently involved in inflammation and immune responses. PYD modulates nuclear factor‐kappa B (NF‐κB) signalling pathway upon various stimuli. Herein, a novel recombinant pyrin domain protein (RPYD) was generated. Its role and mechanism in inflammatory response in an ovalbumin (OVA) induced asthma model was investigated. After OVA challenge, there was inflammatory cell infiltration in the lung, as well as airway hyper‐responsiveness (AHR) to inhaled methacholine. In addition, eosinophils increased in the bronchoalveolar lavage fluids, alone with the elevated levels of Th‐2 type cytokines [interleukin (IL)‐4, IL‐5 and IL‐13], eotaxin, and adhesion molecules. However, the transnasal administration of RPYD before the OVA challenge significantly inhibited these asthmatic reactions. Moreover, RPYD markedly suppressed NF‐κB translocation, reduced phosphorylation of p38 MAPK, and thus attenuated the expression of intercellular adhesion molecule 1 and IL‐6 in the BEAS‐2B cells stimulated by proinflammatory cytokines in vitro. These findings indicate that RPYD can protect asthma host from OVA‐induced airway inflammation and AHR via down‐regulation of NF‐κB and p38 MAPK activities. RPYD may be used as a potential medicine for the treatment of asthma in clinic.     

Selective blockade of endothelial NF‐κB pathway differentially affects systemic inflammation and multiple organ dysfunction and injury in septic mice

Endothelium has long been considered both a source and a target of systemic inflammation. However, to what extent endothelial activation contributes to systemic inflammation remains unclear. This study addresses the relative contribution of endothelial activation to systemic inflammation and multiple organ dysfunction and injury (MOD/I) in an E. coli peritonitis model of sepsis. We prevented endothelial activation using transgenic (TG) mice that conditionally overexpress a mutant I‐κBα, a NF‐κB inhibitor, selectively on endothelium. TG mice and their transgene negative littermates (WT) were injected with saline or E. coli (10⁸ CFU per mouse). At 7 h after E. coli infection, markers of systemic inflammation, endothelial activation, and MOD/I were assessed. WT‐E. coli mice showed significantly increased serum levels of TNF‐α, IL‐1β, IFN‐γ, IL‐6, KC, and MCP‐1; tissue levels of TNF‐α, IL‐6, KC, MCP‐1, ICAM‐1, and VCAM‐1; endothelial leakage index in heart, lungs, liver, and kidney; significantly increased serum levels of AST, ALT, BUN, and creatinine; and increased mortality. Blockade of NF‐κB‐mediated endothelial activation in TG mice had no effects on serum levels of TNF‐α, IL‐1β, IFN‐γ, IL‐6, KC, and MCP‐1 (markers of systemic inflammation), and tissue levels of TNF‐α, IL‐6, KC, and MCP‐1, but significantly reduced tissue levels of ICAM‐1 and VCAM‐1 (markers of endothelial inflammation and activation) in those four organs. TG‐E. coli mice displayed reversed endothelial leakage index; reduced serum levels of AST, ALT, BUN, and creatinine; and improved survival. Our data demonstrate that endothelial NF‐κB‐driven inflammatory response contributes minimally to systemic inflammation, but plays a pivotal role in septic MOD/I, suggesting that endothelium is mainly a target rather than a source of systemic inflammation. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

NF‐κB signaling regulates the generation of intermediate progenitors in the developing neocortex

In addition to its well‐established role during immune system function, NF‐κB regulates cell survival and synaptic plasticity in the mature nervous system. Here, we show that during mouse brain development, NF‐κB activity is present in the neocortical ventricular and subventricular zones (VZ and SVZ), where it regulates proliferative pool maintenance. Activation of NF‐κB signaling, by expression of p65 or an activated form of the IκB kinase complex subunit IKK2, inhibited neuronal differentiation and promoted retention of progenitors in the VZ and SVZ. In contrast, blockade of the pathway with dominant negative forms of IKK2 and IκBα promoted neuronal differentiation both in vivo and in vitro. Furthermore, by modulating both the NF‐κB and Notch pathways, we show that in the absence of canonical Notch activity, after knockdown of the pathway effector CBF1, NF‐κB signaling promoted Tbr2 expression and intermediate neural progenitor fate. Interestingly, however, activation of NF‐κB in vivo, with canonical Notch signaling intact, promoted expression of the radial glial marker Pax6. This work identifies NF‐κB signaling as a regulator of neocortical neurogenesis and suggests that the pathway plays roles in both the VZ and SVZ.     

Regulation of Inflammation by the NF‐κB Pathway in Ovarian Cancer Stem Cells

Citation Leizer AL, Alvero AB, Fu HH, Holmberg JC, Cheng Y‐C, Silasi D‐A, Rutherford T, Mor G. Regulation of inflammation by the NF‐κB pathway in ovarian cancer stem cells. Am J Reprod Immunol 2011; 65: 438–447 Problem The NFκB pathway is a major source of pro‐inflammatory cytokines, which may contribute to cancer chemoresistance. We showed that constitutive NFκB activity is characteristic of the ovarian cancer stem cells (OCSCs). The aim of this study is to determine whether the inhibition of NFκB by Eriocalyxin B (EriB) in the OCSCs may induce cell death in otherwise chemoresistant cells. Methods OCSCs and mature ovarian cancer cells (mOCCs) were treated with increasing concentrations of EriB. Cell viability was measured using the Celltiter 96 assay, and caspase activity was quantified using Caspase‐Glo? assay. Cytokine levels were quantified using xMAP technology. Results EriB decreased the percent of viable cells in all cultures tested with GI₅₀ of 0.5–1 μm after 48 hrs of treatment. The intracellular changes associated with EriB‐induced cell death are: (i) inhibition of NF‐κB activity; (ii) decreased cytokine production; (iii) activation of caspases; and (iv) down‐regulation of XIAP. In addition, EriB is able to sensitize OCSCs to TNFα and FasL‐mediated cell death. Conclusion Inhibition of the NFκB pathway induces cell death in the OCSCs. Because the OCSCs may represent the source of recurrence and chemoresistance, the use of NFκB inhibitors like EriB may prevent recurrence in patients with ovarian cancer.     

Gx‐50 reduces β‐amyloid‐induced TNF‐α, IL‐1β, NO,and PGE2 expression and inhibits NF‐κB signaling in a mouse model of Alzheimer's disease

Chronic inflammation, which is regulated by overactivated microglia in the brain, accelerates the occurrence and development of Alzheimer's disease (AD). Gx‐50 has been investigated as a novel drug for the treatment of AD in our previous studies. Here, we investigated whether gx‐50 possesses anti‐inflammatory effects in primary rat microglia and a mouse model of AD, amyloid precursor protein (APP) Tg mice. The expression of TNF‐α, IL‐1β, NO, prostaglandin E2, and the expression of iNOS and COX2 were inhibited by gx‐50 in amyloid β (Aβ) treated rat microglia; additionally, microglial activation and the expression of IL‐1β, iNOS, and COX2 were also significantly suppressed by gx‐50 in APP⁺ transgenic mice. Furthermore, gx‐50 inhibited the activation of NF‐κB and MAPK cascades in vitro and in vivo in APP‐Tg mice. Moreover, the expression of TLR4 and its downstream signaling proteins MyD88 and tumor necrosis factor receptor associated factor 6 (TRAF6) was reduced by gx‐50 in vitro and in vivo. Interestingly, silencing of TLR4 reduced Aβ‐induced upregulation of IL‐1β and TRAF6 to levels similar to gx‐50 inhibition; moreover, overexpression of TLR4 increased the expression of MyD88 and TRAF6, which was significantly reduced by gx‐50. These findings provide strong evidence that gx‐50 has anti‐inflammatory effects against Aβ‐triggered microglial overactivation via a mechanism that involves the TLR4‐mediated NF‐κBB/MAPK signaling cascade.     

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.     

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.     

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.     

Alternative NF‐κB signaling regulates mTEC differentiation from podoplanin‐expressing precursors in the cortico‐medullary junction

The thymic epithelium forms specialized niches to enable thymocyte differentiation. While the common epithelial progenitor of medullary and cortical thymic epithelial cells (mTECs and cTECs) is well defined, early stages of mTEC lineage specification have remained elusive. Here, we utilized in vivo targeting of mTECs to resolve their differentiation pathways and to determine whether mTEC progenitors participate in thymocyte education. We found that mTECs descend from a lineage committed, podoplanin (PDPN)‐expressing progenitor located at the cortico‐medullary junction. PDPN⁺ junctional TECs (jTECs) represent a distinct TEC population that builds the thymic medulla, but only partially supports negative selection and thymocyte differentiation. Moreover, conditional gene targeting revealed that abrogation of alternative NF‐κB pathway signaling in the jTEC stage completely blocked mTEC development. Taken together, this study identifies jTECs as lineage‐committed mTEC progenitors and shows that NF‐κB‐dependent progression of jTECs to mTECs is critical to secure central tolerance.