Phosphorylation Of NF-κB P65 Subunit Research Articles

The NK Cell Receptor NKp46 Acts As an Essential Regulator of ILC1 Proliferation, Effector Function and Anti-AML Activity

Innate lymphoid cells (ILCs) are tissue-resident innate immune cells that respond rapidly to infection and secrete inflammatory mediators. Group 1 includes ILC1s and NK cells. Both cell types produce IFN-γ and require T-BET for their function; however, at least in mice, ILC1s do not express EOMES while NK cells do. The NKp46 receptor, encoded by the Ncr1 gene, plays an important role in NK cells. Non-NK immune cells, including ILC1s also express NKp46. However, the role of NKp46 in these non-NK immune cells remains largely unknown. We previously reported that NKp46 deficiency reduces both the percentages and numbers of ILC1s in mice but not the proportions of NK cells, ILC2s, and ILC3s (Wang et al., Plos Biology, 2018), suggesting that NKp46 may play a unique role in ILC1s. However, the precise mechanism by which NKp46 regulates ILC1s is yet to be determined. Using RNA-seq, we identified 35 upregulated genes and 66 downregulated genes in mutant ILC1s ( Ncr1 gfp/gfp) compared to wild-type ( Ncr1 +/+)ILC1s ( p < 0.05). Among the genes that were differentially expressed, we noted decreased expression of the cytokine receptors Il-18r1, Il-7rα, and Il-2rα in Ncr1 gfp/gfpILC1s compared to Ncr1 +/+ILC1s. Using gene set enrichment analysis of RNA-seq data from freshly sorted ILC1s from the livers of Ncr1 +/+and Ncr1 gfp/gfpmice, we identified the top 10 pathways associated with downregulated genes. Notably, NKp46 deletion significantly affected NF-κB. We validated this finding by treating ILC1s with NKp46 antibody and found that the phosphorylation of NF-κB p65 subunit was increased. The downregulation of IL-2Rα and IL-7Rα on Ncr1 gfp/gfpILC1s led us to investigate whether NKp46 also regulates ILC1 proliferation, survival, or persistence. We observed comparable levels of apoptosis in ILC1s freshly isolated from Ncr1 +/+and Ncr1 gfp/gfpmice. Thus, lack of NKp46 does not affect ILC1 apoptosis. However, expression of the proliferation marker Ki67 was significantly downregulated in freshly isolated Ncr1 gfp/gfpILC1s compared to Ncr1 +/+ILC1s. We also intravenously ( i.v.) co-transferred equal numbers of FACS-sorted Ncr1 +/+ and Ncr1 gfp/gfpILC1s labeled with CellTrace™ Violet dye (to distinguish donor cells from host cells) into immunodeficient recipient Rag2 −/− Il2γc −/− mice. After one week, the population of Ncr1 gfp/gfpILC1s was less than those of Ncr1 +/+ILC1s in the recipient Rag2 −/− Il2γc −/− mice ( p = 0.0002, n = 5), indicating that ILC1 proliferation requires NKp46 in vivo. To investigate the functional relevance ofNKp46, we i.v. injected C1498 AML cells into Ncr1 +/+or Ncr1 gfp/gfpmice. The Ncr1 gfp/gfpmice exhibited significantly more tumor growth than the Ncr1 +/+mice. Animal survival was significantly shorter in the Ncr1 gfp/gfpmice compared to Ncr1 +/+mice ( p = 0.0023, n > 9). Injection of additional Ncr1 +/+ILC1s into Ncr1 +/+mice increased survival compared to animals without additional Ncr1 +/+ILC1 administration ( p = 0.0183, n = 6). Further, this injection managed to rescue the dramatic decrease in the number of ILC1s observed in the Ncr1 gfp/gfpmice. This increased survival was in stark contrast to the Ncr1 gfp/gfpmice without Ncr1 +/+ILC1 injection, or those who received Ncr1 gfp/gfpILC1 injection. We also investigated the role of NKp46 in human ILC1s. Human ILC1s isolated from peripheral blood mononuclear cells (PBMCs) were treated with a human NKp46 antibody, increasing Ki67 expression. Next, we sorted NKp46 - and NKp46 + ILC1s from human PBMCs and co-cultured them with MOLM13 AML cells for 24 h and found the human NKp46 - ILC1s were significantly less cytotoxic compared to NKp46 + ILC1s. In conclusion, NKp46 is indispensable for ILC1 expression of IL-2Rα. Deficiency of NKp46 in mice reduces IL-2Rα expression on ILC1s by downregulating NF-κB signaling, thus impairing ILC1 proliferation. In a mouse model of AML, we see the functional consequences of cellular impairment as NKp46 deficiency eliminates the ability to control tumor growth and reduces survival. These changes can be reversed by injection of sufficient numbers of NKp46 + ILC1s into mice deficient for the receptor. Human NKp46 + ILC1s also exhibit stronger cytokine production and cytotoxicity than their NKp46 -counterparts, suggesting that NKp46 plays a similar role in both humans and mice. Our findings suggest a novel strategy for activating NKp46 signaling to promote ILC1 proliferation and anti-tumor activity.

Brain vitamin D3-auto/paracrine system in relation to structural, neurophysiological, and behavioral disturbances associated with glucocorticoid-induced neurotoxicity.

Vitamin D3 (VD3) is a potent para/autocrine regulator and neurosteroid that can strongly influence nerve cell function and counteract the negative effects of glucocorticoid (GC) therapy. The aim of the study was to reveal the relationship between VD3 status and behavioral, structural-functional and molecular changes associated with GC-induced neurotoxicity. Female Wistar rats received synthetic GC prednisolone (5 mg/kg b.w.) with or without VD3 (1000 IU/kg b.w.) for 30 days. Behavioral, histological, physiological, biochemical, molecular biological (RT-PCR, Western blotting) methods, and ELISA were used. There was no difference in open field test (OFT), while forced swim test (FST) showed an increase in immobility time and a decrease in active behavior in prednisolone-treated rats, indicative of depressive changes. GC increased the perikaryon area, enlarged the size of the nuclei, and caused a slight reduction of cell density in CA1-CA3 hippocampal sections. We established a GC-induced decrease in the long-term potentiation (LTP) in CA1-CA3 hippocampal synapses, the amplitude of high K+-stimulated exocytosis, and the rate of Ca2+-dependent fusion of synaptic vesicles with synaptic plasma membranes. These changes were accompanied by an increase in nitration and poly(ADP)-ribosylation of cerebral proteins, suggesting the development of oxidative-nitrosative stress. Prednisolone upregulated the expression and phosphorylation of NF-κB p65 subunit at Ser311, whereas downregulating IκB. GC loading depleted the circulating pool of 25OHD3 in serum and CSF, elevated VDR mRNA and protein levels but had an inhibitory effect on CYP24A1 and VDBP expression. Vitamin D3 supplementation had an antidepressant-like effect, decreasing the immobility time and stimulating active behavior. VD3 caused a decrease in the size of the perikaryon and nucleus in CA1 hippocampal area. We found a recovery in depolarization-induced fusion of synaptic vesicles and long-term synaptic plasticity after VD3 treatment. VD3 diminished the intensity of oxidative-nitrosative stress, and suppressed the NF-κB activation. Its ameliorative effect on GC-induced neuroanatomical and behavioral abnormalities was accompanied by the 25OHD3 repletion and partial restoration of the VD3-auto/paracrine system. GC-induced neurotoxicity and behavioral disturbances are associated with increased oxidative-nitrosative stress and impairments of VD3 metabolism. Thus, VD3 can be effective in preventing structural and functional abnormalities in the brain and behavior changes caused by long-term GC administration.

DOP34 Human MD2 deficiency - an inborn error of immunity predisposing to early onset Inflammatory Bowel Disease

Abstract Background Pattern recognition receptors (PRRs) serve as a hub of immune responses to microbes in the gut and play a critical role in maintaining intestinal homeostasis. Toll-like receptor 4 (TLR4) represents an important PRR that can recognize the gram-negative bacterial cell wall component lipopolysaccharide (LPS). Upon binding to LPS, TLR4 forms a multimeric complex with the indispensable co-receptor myeloid differentiation protein 2 (MD2) facilitating selection of TLR4 as cargo for endocytosis and TLR4-mediated activation of downstream signalling. Despite the critical role of TLR4 in innate immunity, no patients with TLR4 or MD2 deficiency have yet been reported. Methods To investigate potential genetic causes for two related patients presenting with very early onset Inflammatory Bowel Disease (VEO-IBD) and/or pneumonia, we have performed whole exome sequencing (WES). To assess the functional consequences of the identified LY96 (encoding for MD2) variant, we generated a CRISPR/Cas9-mediated knockout (KO) of MD2 or knockin (KI) of the patient mutation in induced pluripotent stem cells (iPSCs) and studied TLR4-mediated signalling, cytokine responses, and bacterial handling in iPSC-derived macrophages. Results Genetic analysis identified a 3 bp homozygous in-frame deletion in the LY96 gene (NM_015364.5, c.347_349delCAA; p.Thr116del) in our index patients following an autosomal recessive inheritance pattern. Immunoblotting revealed an altered protein expression of overexpressed Flag-tagged mutant MD2 protein due to impaired N-linked glycosylation. Correspondingly, iPSC-derived MD2-deficient macrophages showed an impaired LPS-induced TLR4 endocytosis. As a functional consequence, we could detect reduced NF-κB and MAPK signalling (phosphorylation of NF-κB p65 subunit, ERK1/2, and p38 MAPK) and dysregulated inflammasome activation (IL-1b production and secretion) upon challenge with LPS. Gentamycin protection assays and live cell imaging suggested that MD2-deficient macrophages exhibit an impaired phagocytosis of microbial pathogens. In addition, macrophages with mutant MD2 showed decreased cytokine expression (e.g., IFNB, IL6, IL10, and TNF) in response to LPS or gram-negative bacteria (E. coli and Salmonella typhimurium) but not gram-positive bacteria (Listeria monocytogenes). Conclusion Human MD2 deficiency is an inborn error of immunity that may predispose to VEO-IBD associated with altered TLR4-mediated signalling, cytokine responses, and bacterial handling. The first description of patients with MD2 deficiency provides critical insights on human TLR4/MD2 biology and warrants caution on therapeutics strategies targeting TLR4 signalling.

The Pseudomonas aeruginosa Secreted Protein PA3611 Promotes Bronchial Epithelial Cell Epithelial-Mesenchymal Transition via Integrin αvβ6-Mediated TGF-β1-Induced p38/NF-κB Pathway Activation.

Pseudomonas aeruginosa (PA) is an important pathogen that has been proven to colonize and cause infection in the respiratory tract of patients with structural lung diseases and to lead to bronchial fibrosis. The development of pulmonary fibrosis is a complication of PA colonization of the airway, resulting from repeated infection, damage and repair of the epithelium. Bronchial epithelial cell epithelial-mesenchymal transition (EMT) plays a vital role in bronchial fibrosis. To date, research on bronchial epithelial cell EMT caused by PA-secreted virulence factors has not been reported. Here, we found that PA3611 protein stimulation induced bronchial epithelial cell EMT with mesenchymal cell marker upregulation and epithelial cell marker downregulation. Moreover, integrin αvβ6 expression and TGF-β1 secretion were markedly increased, and p38 MAPK phosphorylation and NF-κB p65 subunit phosphorylation were markedly enhanced. Further research revealed that PA3611 promoted EMT via integrin αvβ6-mediated TGF-β1-induced p38/NF-κB pathway activation. The function of PA3611 was also verified in PA-infected rats, and the results showed that ΔPA3611 reduced lung inflammation and EMT. Overall, our results revealed that PA3611 promoted EMT via integrin αvβ6-mediated TGF-β1-induced p38/NF-κB pathway activation, suggesting that PA3611 acts as a crucial virulence factor in bronchial epithelial cell EMT and is a potential target for the clinical treatment of bronchial EMT and fibrosis caused by chronic PA infection.

Nipah virus W protein harnesses nuclear 14-3-3 to inhibit NF-\u03baB-induced proinflammatory response

Nipah virus (NiV) is a highly pathogenic emerging bat-borne Henipavirus that has caused numerous outbreaks with public health concerns. It is able to inhibit the host innate immune response. Since the NF-κB pathway plays a crucial role in the innate antiviral response as a major transcriptional regulator of inflammation, we postulated its implication in the still poorly understood NiV immunopathogenesis. We report here that NiV inhibits the canonical NF-κB pathway via its nonstructural W protein. Translocation of the W protein into the nucleus causes nuclear accumulation of the cellular scaffold protein 14-3-3 in both African green monkey and human cells infected by NiV. Excess of 14-3-3 in the nucleus was associated with a reduction of NF-κB p65 subunit phosphorylation and of its nuclear accumulation. Importantly, W-S449A substitution impairs the binding of the W protein to 14-3-3 and the subsequent suppression of NF-κB signaling, thus restoring the production of proinflammatory cytokines. Our data suggest that the W protein increases the steady-state level of 14-3-3 in the nucleus and consequently enhances 14-3-3-mediated negative feedback on the NF-κB pathway. These findings provide a mechanistic model of W-mediated disruption of the host inflammatory response, which could contribute to the high severity of NiV infection.

Cyclosporine A and Tacrolimus Induce Functional Impairment and Inflammatory Reactions in Endothelial Progenitor Cells.

Immunosuppressants are a mandatory therapy for transplant patients to avoid rejection of the transplanted organ by the immune system. However, there are several known side effects, including alterations of the vasculature, which involve a higher occurrence of cardiovascular events. While the effects of the commonly applied immunosuppressive drugs cyclosporine A (CsA) and tacrolimus (Tac) on mature endothelial cells have been addressed in several studies, we focused our research on the unexplored effects of CsA and Tac on endothelial colony-forming cells (ECFCs), a subgroup of endothelial progenitor cells, which play an important role in vascular repair and angiogenesis. We hypothesized that CsA and Tac induce functional defects and activate an inflammatory cascade via NF-κB signaling in ECFCs. ECFCs were incubated with different doses (0.01 µM–10 µM) of CsA or Tac. ECFC function was determined using in vitro models. The expression of inflammatory cytokines and adhesion molecules was explored by quantitative real-time PCR and flow cytometry. NF-κB subunit modification was assessed by immunoblot and immunofluorescence. CsA and Tac significantly impaired ECFC function, including proliferation, migration, and tube formation. TNF-α, IL-6, VCAM, and ICAM mRNA expression, as well as PECAM and VCAM surface expression, were enhanced. Furthermore, CsA and Tac led to NF-κB p65 subunit phosphorylation and nuclear translocation. Pharmacological inhibition of NF-κB by parthenolide diminished CsA- and Tac-mediated proinflammatory effects. The data of functional impairment and activation of inflammatory signals provide new insight into mechanisms associated with CsA and Tac and cardiovascular risk in transplant patients.

Astragaloside IV Attenuates Polymicrobial Sepsis-Induced Cardiac Dysfunction in Rats via IKK/NF-κB Pathway

To evaluate the protective effects of Astragaloside IV (AST) in a rat model of myocardial injury induced by cecal ligation and puncture (CLP). The model of sepsis-induced cardiac dysfunction was induced by CLP. Using a random number table, 50 specific pathogen free grade of Sprague Dawley rats were randomized into 5 groups: the sham group (sham), the model group (CLP, 18 h/72 h) and AST group (18 h/72 h). Except the sham group, the rats in other groups received CLP surgery to induce sepsis. CLP groups received intragastric administration with normal saline after CLP. AST groups received intragastric administration with AST solution (40 mg/kg) once a day. The levels of inflammatory mediators and oxidative stress markers in the serum of the septic rats were determined via enzyme-linked immunosorbent assay (ELISA) at different time point, such as interleukin 6 (IL-6), IL-10, high mobility group box-1 protein B1 (HMGB-1), superoxide dismutase (SOD), and malondialdehyde (MDA). Cardiac function was determined by echocardiography. Moreover, changes in myocardial pathology were evaluated using hematoxylin and eosin staining. The levels of lactate dehydrogenase (LDH) and creatine kinase-MB (CK-MB) were analysed to determine the status of CLP-induced myocardium. In addition, the apotosis of myocardial cells was analysed by terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL). The protein levels of B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X (Bax), IκB kinase α (IKKα), nuclear factor kappa B p65 (NF-κB p65) were detected by Western blot analysis. Moreover, survival rate was investigated. AST improved the survival rate of CLP-induced rats by up to 33.3% (P<0.05). The cardioprotective effect of AST was observed by increased ejection fraction, fractional shortening and left ventricular internal diameter in diastole respectively (P<0.01 or P<0.05). Subsequently, AST attenuated CLP-induced myocardial apoptosis and the ratio of Bcl-2/Bax in the myocardium, as well as the histological alterations of myocardium (P<0.01 or P<0.05); the generation of inflammatory cytokines (IL-6, IL-10, HMGB-1) and oxidative stress markers (SOD, MDA) in the serum was significantly alleviated (P<0.01 or P<0.05). On the other hand, AST markedly suppressed CLP-induced accumulation of IKK-α and NF-κB p65 subunit phosphorylation (P<0.01 or P<0.05). AST plays a significant protective role in sepsis-induced cardiac dysfunction and survival outcome. The possible mechanism of cardioprotection is dependent on the activation of the IKK/NF-κB pathway in cardiomyocytes.

Isorhapontigenin alleviates lipopolysaccharide-induced acute lung injury via modulating Nrf2 signaling

Nuclear factor erythroid-2 related factor 2 (Nrf2) is involved in mitigating various oxidative stress- and inflammation-induced diseases, including acute lung injury/acute respiratory distress syndrome (ALI/ARDS). Isorhapontigenin (ISO), from the Chinese herb Gnetum cleistostachyum, exhibits antioxidant and anti-inflammatory properties. In this study, we explored the protective effects of ISO in ALI and its underlying molecular mechanisms. ISO significantly mitigated ALI by reducing the lung wet/dry weight ratio, protein concentration in the bronchoalveolar lavage fluid (BALF), and the levels of myeloperoxidase and malondialdehyde. ISO also improved the superoxide dismutase and glutathione activity in vivo. Moreover, ISO effectively ameliorated the changes in IL-1β, IL-6, and TNF-α concentrations in BALF, prevented IκB degradation, and inhibited the phosphorylation of NF-κB p65 subunit in lung tissues; furthermore, it enhanced the nuclear translocation of Nrf2 and inhibited IL-1β, IL-6, TNF-α, iNOS, COX-2, and ROS production in lipopolysaccharide-treated RAW264.7 cells. The protective effects of ISO in ALI were significantly reversed in ML385-treated RAW264.7 cells and the mouse model, indicating its role in Nrf2-activation. In conclusion, ISO effectively ameliorated lipopolysaccharide-induced ALI by reducing inflammation and oxidative stress, primarily through activation of Nrf2 signaling.

Mesenchymal Stem Cell-derived Exosomes Rescue Oxygen-Glucose Deprivation-induced Injury in Endothelial Cells.

The effects of mesenchymal stem cell (MSC)-derived exosomes on brain microvascular endothelial cells under oxygen-glucose deprivation (OGD), which mimic cells in deep hypothermic circulatory arrest (DHCA) in vitro, are yet to be studied. MSCs were co-cultured with primary rat brain endothelial cells, which were then exposed to OGD. Cell viability, apoptosis, the inflammatory factors (IL-1β, IL-6, and TNF-α), and the activation of inflammation-associated TLR4-mediated pyroptosis and the NF-κB signaling pathway were determined. Furthermore, exosomes derived from MSCs were isolated and incubated with endothelial cells to investigate whether the effect of MSCs is associated with MSCderived exosomes. Apoptosis, cell viability, and the inflammatory response were also analyzed in OGD-induced endothelial cells incubated with MSC-derived exosomes. OGD treatment promoted endothelial cell apoptosis, induced the release of inflammatory factors IL-1β, IL-6, and TNF-α, and inhibited cell viability. Western blot analysis showed that OGD treatment-induced TLR4, and NF-κB p65 subunit phosphorylation and caspase-1 upregulation, while co-culture with MSCs could reduce the effect of OGD treatment on endothelial cells. As expected, the effect of MSC-derived exosomes on OGD-treated endothelial cells was similar to that of MSCs. MSC-derived exosomes alleviated the OGD-induced decrease in the viability of endothelial cells, and increased levels of apoptosis, inflammatory factors, and the activation of inflammatory and inflammatory focal pathways. Both MSCs and MSC-derived exosomes attenuated OGD-induced rat primary brain endothelial cell injury. These findings suggest that MSC-derived exosomes mediate at least some of the protective effects of MSCs on endothelial cells.

Tangeretin protects renal tubular epithelial cells against experimental cisplatin toxicity.

Objective(s):Cisplatin is an effective antineoplastic agent; its clinical utility, however, is limited by a few salient toxic side effects like nephrotoxicity. This study aimed to determine the potential protective effects of tangeretin, a citrus-derived flavonoid, against renal tubular cell injury in cisplatin-induced renal toxicity of rats.Materials and Methods:Tangeretin was injected intraperitoneally at 2.5 and 5 mg/kg doses for 10 days, and a single dose of cisplatin (8 mg/kg) was injected on the 7th day. Tests of kidney function and tubular injury in renal tissues and urine together with oxidative stress and inflammation markers were examined.Results:Tangeretin ameliorated cisplatin-induced elevations in serum creatinine, BUN, and histopathologic changes. It also attenuated kidney oxidative stress elicited by cisplatin as demonstrated by reduced MDA and increased GSH, CAT, and SOD activities, elevated Nrf2 expression and protein levels of its downstream effectors, HO-1 and NQO-1. Tangeretin further alleviated inflammation evoked by cisplatin as indicated by reduced NF-κB p65 subunit phosphorylation with a simultaneous decrement in its downstream effectors IL-1β and TNF-α expression and protein levels. Moreover, it declined caspase-3 protein levels and TUNEL positive cells in the kidneys, the markers of apoptosis and DNA fragmentation, thus improving renal endurance. Additionally, tangeretin mitigated renal levels of KIM-1 and NGAL, as well as urinary cystatin C and β2-microglobulin concentrations, the markers of renal tubular injury.Conclusion:Collectively, these data signify the binary profit of tangeretin: enhancement of renal protective mechanisms against cisplatin and attenuation of renal tubular cell injuries induced by the agent.

Urolithins Attenuate LPS-Induced Neuroinflammation in BV2Microglia via MAPK, Akt, and NF-κB Signaling Pathways.

Emerging data suggest that urolithins, gut microbiota metabolites of ellagitannins, contribute toward multiple health benefits attributed to ellagitannin-rich foods, including walnuts, red raspberry, strawberry, and pomegranate. However, there is limited data on whether the potential neuroprotective effects of these ellagitannin-rich foods are mediated by urolithins. Herein, we evaluated the potential mechanisms of antineuroinflammatory effects of urolithins (urolithins A, B, and C; 8-methyl-O-urolithin A; and 8,9-dimethyl-O-urolithin C) in BV2 murine microglia in vitro. Nitrite analysis and qRT-PCR suggested that urolithins A and B reduced NO levels and suppressed mRNA levels of pro-inflammatory genes of TNF-α, IL-6, IL-1β, iNOS, and COX-2 in LPS-treated microglia. Western blot revealed that urolithins A and B decreased phosphorylation levels of Erk1/2, p38 MAPK, and Akt, prevented IκB-α phosphorylation and degradation, and inhibited NF-κB p65 subunit phosphorylation and nuclear translocation in LPS-stimulated microglia. Our results indicated that urolithins A and B attenuated LPS-induced inflammation in BV2 microglia, which may be mediated by inhibiting NF-κB, MAPKs (p38 and Erk1/2), and Akt signaling pathway activation. The antineuroinflammatory activities of urolithins support their role in the potential neuroprotective effects reported for ellagitannin-rich foods warranting further in vivo studies on these ellagitannin gut microbial derived metabolites.

Triptolide suppresses growth and hormone secretion in murine pituitary corticotroph tumor cells via NF-kappaB signaling pathway

Triptolide is a principal diterpene triepoxide from the Chinese medical plant Tripterygium wilfordii Hook. f., whose extracts have been utilized in dealing with diverse diseases in traditional Chinese medicine for centuries. Recently, the antitumor effect of triptolide has been found in several pre-clinical neoplasm models, but its effect on pituitary corticotroph adenomas has not been investigated so far. In this study, we are aiming to figure out the antitumor effect of triptolide and address the underlying molecular mechanism in AtT20 murine corticotroph cell line. Our results demonstrated that triptolide inhibited cell viability and colony number of AtT20 cells in a dose- and time-dependent pattern. Triptolide also suppressed proopiomelanocortin (Pomc) mRNA expression and extracellular adrenocorticotropic hormone (ACTH) secretion in AtT20 cells. Flow cytometry prompted that triptolide leaded to G2/M phase arrest, apoptosis program and mitochondrial membrane depolarization in AtT20 cells. Moreover, dose-dependent activation of caspase-3 and decreased Bcl2/Bax proportion were observed after triptolide treatment. By western blot analysis we found that triptolide impeded phosphorylation of NF-κB p65 subunit and extracellular signal-regulated kinase (ERK), along with reduction of cyclin D1, without any impact on other NF-κB related protein expression like total p65, p50, IκB-α, p-IκB-α. Furthermore, the mouse xenograft model revealed the inhibition of tumor growth and hormone secretion after triptolide administration. Altogether this compound might be a potential pharmaceutical choice in managing Cushing’s disease.

Efficacy of Selinexor Is Dependent on IκB-α Expression and NF-Kb Deactivation in Multiple Myeloma Cells

Background: Selinexor is a Selective Inhibitor of Nuclear Export (SINE) compound that binds to and inhibits XPO1 mediated nuclear export, resulting in nuclear accumulation of tumor suppressor proteins (TSPs) including p53, pRB, and IκB-α. Selinexor has therapeutic benefit both pre-clinically and clinically (NCT01607892, NCT02336815) in multiple myeloma (MM). It has been previously demonstrated that the extent of NF-κB transcriptional inhibition is one of the critical mechanisms contributing to the efficacy and/or resistance to selinexor in cells. However, the mechanism leading to NF-κB inhibition after selinexor treatment is not fully understood. We hypothesized that the level of the cellular inhibitor of NF-κB, IκB-α, and its compartmental localization play an important role in NF-κB transactivation and response to selinexor. In this study, we investigate the effect of selinexor treatment on MM cells with low expression of IκB-α and high NF-kB activity in order to understand the mechanism of NF-κB inhibition by selinexor.Methods: IM9 and RPMI-8226cells were treated with selinexor in the presence or absence of 20 ng/mL tumor necrosis factor α (TNFα; inflammatory mimic) and whole protein lysates were analyzed by immunoblotting. Cytotoxic effects of selinexor were evaluated using standard viability assays. IκB-α knockdown was performed using transfection of specific siRNA duplexes. NF-κB transcriptional activity was analyzed using an ELISA assay.Results: Selinexor induces nuclear localization of IκB-α in MM cells. RNAi of IκB-α in MM cells reduced the cytotoxic effects of selinexor by 10-fold. In addition, knockdown of IκB-α reduces the synergy of the selinexor plus proteasome inhibitor (bortezomib or carfilzomib) combination. This data suggests that in MM, IκB-α plays a major role in cellular sensitivity to selinexor potentially through NF-κB activity. Selinexor inhibited NF-kB transcriptional activity in IM9 and RPMI-8226 cells with IC50 of 1079 nM and 591 nM respectively. Although the difference in NF-κB activity IC50 is only 2-fold between the two MM cell lines (MTT IC50s are ~100 nM), IM9 cells have a 100-fold higher basal NF-kB activity when compared to RPMI-8226 cells. Under TNFα stimulation NF-kB activity was induced by 1.5- and 35-fold in IM9 and RPMI-8226, respectively. We observed that selinexor treatment caused a dose dependent inhibition of IκB kinase (IKK)-mediated phosphorylation of serine 32/36 on IκB-α and serine 536 on the NF-κB p65 subunit (RelA) upon TNFα stimulation in both cell lines. In RPMI-8226 cells, selinexor reduced TNFα-induced IκB-α phosphorylation in a dose dependent manner and protected IκB-α from degradation. In IM9 cells that have high basal NF-κB activity, TNFα did not induce NF-kB activity or cause IκB-α degradation. However, selinexor treatment inhibited NF-kB activity below its basal level (70% reduction) which resulted in dose dependent reduction in the level of IκB-α protein perhaps through inhibition of NF-κB transcriptional control of IκB-α mRNA expression.Conclusions: IκB-α plays a major role in the cellular cytotoxicity of selinexor in cancer cells. Multiple myeloma cells lose sensitivity to selinexor treatment upon IκB-α silencing, which in turn reduces the cytotoxicity of selinexor. TNFα stimulation induces the phosphorylation of NF-κB p65 subunit and IκB-α through increased IKK activity resulting in IκB-α degradation and NF-κB activation. In RPMI-8226 cells, selinexor treatment blocked TNFα-induced degradation of IκB-α. However, in IM9 cells TNFα alone did not have any significant effect on IκB-α which might be due to the high basal NF-kB activity. Interestingly, IκB-α is also a transcriptional target of NF-kB. In IM9 cells, selinexor treatment reduces NF-kB activity below the high basal level in a dose dependent manner resulting in near complete inhibition of NF-kB-controlled IκB-α mRNA transcription and a loss of the IκB-α protein. Ultimately, selinexor treatment inhibits cell viability and NF-kB transcriptional activity regardless of basal NF-κB activity in MM cells. Because of this IκB-α/NF-kB transcriptional mechanism, selinexor treatment can inhibit both chronic (unresponsive to TNFα) and acute (TNFα-simulated) inflammatory signaling which makes selinexor an applicable therapy to cancer cells with a variety of aberrant signaling pathways. DisclosuresKashyap:Karyopharm Therapeutics: Employment, Equity Ownership. Klebanov:Karyopharm Therapeutics: Employment, Equity Ownership. Argueta:Karyopharm Therapeutics: Employment, Equity Ownership. Lee:Karyopharm Therapeutics: Employment, Equity Ownership. Shacham:Karyopharm Therapeutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Kauffman:Karyopharm Therapeutics Inc: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Landesman:Karyopharm Therapeutics Inc: Employment, Other: stockholder. Senapedis:Karyopharm Therapeutics: Employment, Equity Ownership.

Anti-rheumatic drug iguratimod (T-614) alleviates cancer-induced bone destruction via down-regulating interleukin-6 production in a nuclear factor-κB-dependent manner.

Cytokines are believed to be involved in a "vicious circle" of progressive interactions in bone metastasis. Iguratimod is a novel anti-rheumatic drug which is reported to have the capability of anti-cytokines. In this study, a rat model was constructed to investigate the effect of iguratimod on bone metastasis and it was found that iguratimod alleviated cancer-induced bone destruction. To further explore whether an anti-tumor activity of iguratimod contributes to the effect of bone resorption suppression, two human breast cancer cell lines MDA-MB-231 and MCF-7 were studied. The effect of iguratimod on tumor proliferation was detected by CCK-8 assay and flow cytometry. The effects of iguratimod on migration and invasion of cancer cells were determined by wound-healing and Transwell assays. Results showed that high dose (30 μg/mL) iguratimod slightly suppressed the proliferation of cancer cells but failed to inhibit their migration and invasion capacity. Interestingly, iguratimod decreased the transcription level of IL-6 in MDA-MB-231 cells in a concentration-dependent manner. Moreover, iguratimod partially impaired NF-κB signaling by suppressing the phosphorylation of NF-κB p65 subunit. Our findings indicated that iguratimod may alleviate bone destruction by partially decreasing the expression of IL-6 in an NF-κB-dependent manner, while it has little effect on the tumor proliferation and invasion.

Abstract 2899: Selinexor inhibits NF-κB activity by sequestering IkB-a in the nucleus and blocking IkB-a degradation

Abstract Background: Selinexor is a small-molecule therapeutic that inhibits XPO1 mediated nuclear export, resulting in nuclear accumulation of tumor suppressor proteins (TSPs) and subsequently cancer cell death while sparing normal cells. It has been previously demonstrated that the inhibitor of NFκB, IκB-, is localized to the cytoplasm by XPO1 in several cancer cell lines and that treatment of cancer cells with selinexor reduces NF-κB transcriptional activity. The mechanism of NF-κB inhibition by selinexor, however, is not fully understood. We hypothesized that nuclear retention of IκB- and down-regulation of IκB- kinase (IKK) in response to selinexor treatment would inhibit NF-κB transcriptional activity. Methods: U2OS (Osteosarcoma) cells were treated with selinexor in the presence or absence of TNF stimulation and whole protein lysates were analyzed by Western blotting. IκB- localization was evaluated by immunofluorescence microscopy and NF-κB transcriptional activity by ELISA (Thermo Scientific). Results: TNFα induced the phosphorylation of NF-κB p65 subunit on serine 536 and IκB- on serine 32/36 through IKK. This resulted in the dissociation of IκB-α from NF-κB and led to IκB-α degradation via the 26S proteasome. Free NF-κB could now migrate into the nucleus and initiate transcriptional activation supporting tumorigenicity and inflammation. The IKK kinase is a complex made of two kinases (IKKα and IKKβ) and one regulatory subunit, NEMO/IKKγ. We found that selinexor treatment blocked IKK activity through the down regulation of the IKK (IKKγ) gamma subunit protein levels. This inhibition was dose dependent and prevented IκB- phosphorylation, thereby protecting IκB- from degradation. The protection of intact IκB- from degradation and its forced nuclear accumulation through XPO1 inhibition enabled inhibition of NF-κB transcriptional activity even in the presence of TNFα. Selinexor did not alter the protein levels of IKK or IKK. Conclusions: Selinexor blocks TNFα induced degradation of IκB- by reducing the levels of IKK. In addition, selinexor increased nuclear IκB- levels through the inhibition of nuclear export. This blocked NF-κB activity and enhanced cancer cell death. We are currently investigating the beneficial effects of combining selinexor with proteasome inhibitors, which are known to prevent IκB- degradation. Citation Format: Trinayan Kashyap, Christian Argueta, Boris Klebanov, TJ Unger, Benjamin Link, Maxwell Werman, Margaret Lee, Sharon Shacham, Yosef Landesman. Selinexor inhibits NF-κB activity by sequestering IkB-a in the nucleus and blocking IkB-a degradation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2899.

LncRNA-HOTAIR promotes TNF-α production in cardiomyocytes of LPS-induced sepsis mice by activating NF-κB pathway

BackgroundMounting studies have illustrated an important role of HOTAIR in cancer progress, but few studies have reported its function in cardiac disease, including cardiac-associated sepsis. This study aimed to investigate the function of HOTAIR in sepsis, involving its association with the level of tumor necrosis factor-alpha (TNF-α), an important inducer of myocardial dysfunction during LPS-induced sepsis. MethodsSepsis mice model was established by LPS administration, and myocardial dysfunction was evaluated with hemodynamic parameters. HOTAIR expression in isolated cardiomyocytes and TNF-α production in the circulation were detected, as well as the protein levels of phosphorylated p65. HL-1 cells were subjected to LPS treatment in vitro for functional studies, including luciferase report assays for NF-κB activity. ResultsHOTAIR expression was significantly upregulated in cardiomyocytes from sepsis mice, in line with increased TNF-α production and p65 phosphorylation, while similar results were also observed in LPS treated HL-1 cells, which was then reversed by HOTAIR interference. Functional studies demonstrated that HOTAIR showed positive regulation on p65 phosphorylation and NF-κB activation, while HOTAIR-induced TNF-α production was repressed by NF-κB inhibitor. Further in vivo studies confirmed that HOTAIR silence can improve cardiac function of sepsis mice, and markedly decreased TNF-α production in the circulation. ConclusionHOTAIR upregulation in cardiomyocytes of LPS-induced sepsis mice promoted TNF-α production in the circulation by activating NF-κB, involving the phosphorylation of NF-κB p65 subunit. Moreover, HOTAIR silence preserved cardiac function of sepsis mice during LPS-induced sepsis.

Isoliensinine, a Bioactive Alkaloid Derived from Embryos of Nelumbo nucifera, Induces Hepatocellular Carcinoma Cell Apoptosis through Suppression of NF-κB Signaling.

Isoliensinine (isolie) is an alkaloid produced by the edible plant Nelumbo nucifera. Here, we unveiled that isolie was able to provoke HepG2, Huh-7, and H22 hepatocellular carcinoma (HCC) cell apoptosis. Isolie decreased NF-κB activity and constitutive phosphorylation of NF-κB p65 subunit at Ser536 in HCC cells. Overexpression of p65 Ser536 phosphorylation mimics abrogated isolie-mediated HCC cell apoptosis. Furthermore, intraperitoneal injection of isolie inhibited the growth of Huh-7 xenografts in nude mice. Additionally, isolie given by both intraperitoneal injection and gavage diminished the proliferation of transplanted H22 cells in Kunming mice. Reduced tumor growth in vivo was associated with inhibited p65 phosphorylation at Ser536 and declined NF-κB activity in tumor tissues. Finally, we revealed that isolie was bioavailable in the blood of mice and exhibited no detectable toxic effects on tumor-bearing mice. Our data provided strong evidence for the anti-HCC effect of isolie.

Asiaticoside attenuates lipopolysaccharide-induced acute lung injury via down-regulation of NF-κB signaling pathway.

Asiaticoside (AS), a triterpene glycoside isolated from Centella asiatica, has been shown to possess potent anti-inflammatory activity. However, the detailed molecular mechanisms of AS on lipopolysaccharide (LPS)-induced acute lung injury (ALI) model in mice are scanty. The purpose of this study was to evaluate the effect of AS on LPS-induced mouse ALI via down-regulation of NF-κB signaling pathway. We investigated the efficacy of AS on cytokine levels induced by LPS in bronchoalveolar lavage fluid (BALF) and RAW 264.7 cells. The production of cytokine (TNF-α and IL-6) was measured by enzyme-linked immunosorbent assay (ELISA). The lung wet-to-dry weight ratios were measured in LPS-challenged mice, and lung histopathologic changes observed via paraffin section were assessed. To further study the mechanism of AS protective effects on ALI, the activation of NF-κB p65 subunit and the degradation of IκBα were tested by western blot assay. We found that AS treatment at 15, 30 or 45mg/kg dose-dependently attenuated LPS-induced pulmonary inflammation by reducing inflammatory infiltration, histopathological changes, descended cytokine production, and pulmonary edema initiated by LPS. Furthermore, our results suggested that AS suppressed inflammatory responses in LPS-induced ALI through inhibition of the phosphorylation of NF-κB p65 subunit and the degradation of its inhibitor IκBα, and might be a new preventive agent of ALI in the clinical setting.

Cepharanthine Attenuates Lipopolysaccharide-Induced Mice Mastitis by Suppressing the NF-κB Signaling Pathway

Cepharanthine (CEP), a biscoclaurine alkaloid isolated from Stephania cepharantha Hayata, has been reported to have potent anti-inflammatory properties. However, the anti-inflammatory effects of CEP on a mouse model of lipopolysaccharide (LPS)-induced mastitis and its underlying molecular mechanisms remain to be elucidated. The purpose of the present study was to investigate the effects of CEP on LPS-induced mouse mastitis. The mouse model of mastitis was induced by inoculation of LPS through the canals of the mammary gland. CEP was administered intraperitoneally at 1 h before and 12 h after induction of LPS. The results show that CEP significantly attenuates the infiltration of neutrophils, suppresses myeloperoxidase activity, and reduces the levels of TNF-α, IL-1β, and IL-6 in LPS-induced mouse mastitis. Furthermore, CEP inhibited the phosphorylation of NF-κB p65 subunit and the degradation of its inhibitor IκBα. All the results suggest that CEP exerts potent anti-inflammatory effects on LPS-induced mouse mastitis. Accordingly, CEP might be a potential therapeutic agent for mastitis.

AGGF1 is a novel anti-inflammatory factor associated with TNF-α-induced endothelial activation

Endothelial activation contributes to the development of vascular inflammation and subsequent vascular diseases, particularly atherosclerosis. AGGF1, a new member of angiogenic factors with a FHA and a G-patch domain, has been shown critical for the regulation of vascular differentiation and angiogenesis. In this study, we found that various inflammatory cytokines strongly induced the expression of AGGF1 in endothelial cells (ECs) and identified AGGF1 as a novel anti-inflammatory factor both in vivo and in vitro. Overexpression of AGGF1 significantly repressed the expression of pro-inflammatory molecules such as E-Selectin, ICAM-1, and IL-8 and the adhesion of monocytes onto ECs activated by TNF-α. Conversely, the knockdown of AGGF1 resulted in the increased expressions of these pro-inflammatory molecules and the enhanced monocyte-EC interaction. We further demonstrated that AGGF1 potently attenuated TNF-α triggered NF-κB pathway, as indicated by the decreased promoter activity, nuclear distribution and phosphorylation of NF-κB p65 subunit as well as the increased protein level of IκBα. This inhibitory effect of AGGF1 was further proved through blocking the phosphorylation of ERK induced by TNF-α. Finally, we showed that the FHA domain of AGGF1 was required for its anti-inflammatory effect. Thus, our findings for the first time demonstrate that AGGF1 suppresses endothelial activation responses to TNF-α by antagonizing the ERK/NF-κB pathway, which makes AGGF1 a promising therapeutic candidate for the prevention and treatment of inflammatory diseases.