Role of NF-κB activation in LPS-induced endothelial barrier breakdown

Endothelial barrier breakdown contributes to organ failure in sepsis. The key mechanism by which the potent sepsis inductor lipopolysaccharide (LPS) disrupts the endothelial barrier is controversial. Here, we tested the hypothesis that NF-κB activation is critically involved in endothelial barrier breakdown. Application of LPS to monolayers of porcine pulmonary artery endothelial cells (PAEC) and human dermal microvascular endothelial cells (HDMEC) induced a rapid and sustained activation of NF-κB as revealed by translocation of its subunit p65 into the nuclei in nuclear extraction assays and by immunostaining. Measurements of transendothelial electrical resistance (TER) and intercellular gap formation demonstrated significant breakdown of endothelial barrier properties following LPS treatment for 3?h. Interestingly, monolayers recovered spontaneously beginning after 10?h. Increased cAMP prevented LPS-induced loss of endothelial barrier properties, but did not block NF-κB activation. Application of the cell-permeable NEMO-binding domain (NBD) synthetic peptide was effective to prevent NF-κB activation, but did neither block LPS-induced loss of TER nor intercellular gap formation. NBD peptide alone did not alter endothelial barrier properties, but enhanced the barrier-compromising effects when applied in combination with LPS. Similarly, siRNA-mediated knock-down of p65 in HDMECs did not prevent LPS-induced barrier breakdown. Known targets of NF-κB-derived protein expression of caveolin or vasodilator-stimulated phosphoprotein (VASP) remained unaltered by LPS treatment of endothelial cells. In summary, our data indicate that NF-κB activation by LPS is not critically involved in disruption of endothelial barrier properties. Rather, our data suggest that NF-κB activation acts as a part of a rescue mechanism.     

Salicortin suppresses lipopolysaccharide-stimulated inflammatory responses via blockade of NF-κB and JNK activation in RAW 264.7 macrophages

We isolated the phenolic glucoside salicortin from a Populus euramericana bark extract, and examined its ability to suppress inflammatory responses as well as the molecular mechanisms underlying these abilities, using lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Salicortin inhibited iNOS expression and the subsequent production of NO in a dose-dependent manner in the LPS-stimulated RAW 264.7 cells. Salicortin significantly suppressed LPS-induced signal cascades of NF-κB activation, such as IKK activation, IκBα phosphorylation and p65 phosphorylation in RAW 264.7 cells. In addition, salicortin inhibited the LPS-induced activation of JNK, but not ERK or p38 MAPK. Furthermore, salicortin significantly inhibited production of pro-inflammatory cytokines, such as TNF-α, IL-1β and IL-6 in the LPS-stimulated RAW 264.7 cells. These findings suggest that salicortin may show its anti-inflammatory activity by suppressing the LPS-induced expression of pro-inflammatory mediators through inhibition of NF-κB and JNK MAPK signaling cascades in macrophages. [BMB Reports 2014; 47(6): 318-323]     

Naringin lauroyl ester inhibits lipopolysaccharide-induced activation of nuclear factor κB signaling in macrophages

Naringin (Nar) has antioxidant and anti-inflammatory properties. It was recently reported that enzymatic modification of Nar enhanced its functions. Here, we acylated Nar with fatty acids of different sizes (C2–C18) using immobilized lipase from Rhizomucor miehei and investigated the anti-inflammatory effects of these molecules. Treatment of murine macrophage RAW264.7 cells with Nar alkyl esters inhibited lipopolysaccharide (LPS)-induced nitric oxide (NO) production, with Nar lauroyl ester (Nar-C12) showing the strongest effect. Furthermore, Nar-C12 suppressed the LPS-induced expression of inducible NO synthase by blocking the phosphorylation of inhibitor of nuclear factor (NF)-κB-α as well as the nuclear translocation of NF-κB subunit p65 in macrophage cells. Analysis of Nar-C12 uptake in macrophage cells revealed that Nar-C12 ester bond was partially degraded in the cell membrane and free Nar was translocated to the cytosol. These results indicate that Nar released from Nar-C12 exerts anti-inflammatory effects by suppressing NF-κB signaling pathway.     

Role of post-translational modifications of HTLV-1 Tax in NF-κB activation

Human T-cell leukemia virus type 1 (HTLV-1), the first human retrovirus discovered, is the etiological agent of adult-T-cell leukemia/lymphoma. The HTLV-1 encoded Tax protein is a potent oncoprotein that deregulates gene expression by constitutively activating nuclear factor-κB (NF-κB). Tax activation of NF-κB is critical for the immortalization and survival of HTLV-1-infected T cells. In this review, we summarize the present knowledge on mechanisms underlying Tax-mediated NF-κB activation, with an emphasis on post-translational modifications of Tax.     

Adriamycin induces myocardium apoptosis through activation of nuclear factor κB in rat

Adriamycin is one of the most effective and useful antineoplastic agents. Acute doxorubicin cardiotoxicity involved cardiomyocyte apoptosis. In this study, we investigated whether adriamycin induced myocardium apoptosis through activation of nuclear factor κB in rat. Forty male Wistar rats were randomly divided into five groups: control, ADR 5 mg/kg, ADR 10 mg/kg, ADR 15 mg/kg group and ADR + PDTC 200 mg/ml group. Myocardial apoptosis was detected by DNA fragmentation assay and TUNEL assay; Location and distribution of p-IκBα was observed by immunohistochemical assay; Myocardial expression of p-IκBα protein was assessed by Western blot analysis; Activity of NF-κB was evaluated by Electrophoretic Mobility Shift Assay. The myocardial apoptotic index, expression of p-IκBα, and binding activity of NF-κB increased significantly in ADR groups in dose-dependent manner. PDTC as a nonspecific inhibitor of NF-κB protected myocardium from apoptosis by inhibiting NF-κB activation. Adriamycin induces myocardium apoptosis through activation of nuclear factor κB in rat and NF-κB activation requires IκBα degradation.     

Chronic social isolation induces NF-κB activation and upregulation of iNOS protein expression in rat prefrontal cortex

Exposure of an organism to stress, results in oxidative stress and increased nitric oxide (NO) production in the brain. The role of the processes caused by chronic stress in the prefrontal cortex has not been fully investigated. Considering that chronic stress increases NO production by the enzyme nitric oxide synthase (NOS), we examined the cytosolic neuronal (nNOS) or inducible (iNOS) protein levels in the prefrontal cortex of rats exposed to 21 d of chronic social isolation stress, an animal model of depression, alone or in combination with 2 h of acute immobilization or cold (4 °C) stress (combined stress). Antioxidative status via cytosolic CuZnSOD and mitochondrial MnSOD activity, cytosolic redox status via reduced glutathione (GSH) concentration were determined. Furthermore, cytosolic inducible heat shock protein 70 (Hsp70i), cytosolic/nuclear distributions of NF-κB and serum corticosterone (CORT) were also investigated to elucidate the possible mechanism involved in the cellular NOS pathway. Our results showed that both acute stressors led to increases of CORT and nNOS protein while iNOS protein expression was unaffected. In contrast to the acute stress, chronic social isolation compromised hypothalamic–pituitary-adrenal axis functioning such that the normal stress response was impaired following subsequent acute stressors. Downregulated redox GSH status as well as decreased activity of CuZnSOD and MnSOD suggests the existence of oxidative stress which remained as such following combined stressors. Changes in redox-status associated with decreased Hsp70i protein expression enabled NF-κB translocation into the nucleus, causing increased cytosolic nNOS and iNOS protein expression. Results suggest that NOS signaling pathway plays a differential role between acute and chronic stress whereby state of oxidative/nitrosative stress after chronic social isolation is caused, at least in part, by NF-κB activation and increased iNOS protein expression.     

NF-κB signaling in fetal lung macrophages disrupts airway morphogenesis

Bronchopulmonary dysplasia is a common pulmonary complication of extreme prematurity. Arrested lung development leads to bronchopulmonary dysplasia, but the molecular pathways that cause this arrest are unclear. Lung injury and inflammation increase disease risk, but the cellular site of the inflammatory response and the potential role of localized inflammatory signaling in inhibiting lung morphogenesis are not known. In this study, we show that tissue macrophages present in the fetal mouse lung mediate the inflammatory response to LPS and that macrophage activation inhibits airway morphogenesis. Macrophage depletion or targeted inactivation of the NF-κB signaling pathway protected airway branching in cultured lung explants from the effects of LPS. Macrophages also appear to be the primary cellular site of IL-1β production following LPS exposure. Conversely, targeted NF-κB activation in transgenic macrophages was sufficient to inhibit airway morphogenesis. Macrophage activation in vivo inhibited expression of multiple genes critical for normal lung development, leading to thickened lung interstitium, reduced airway branching, and perinatal death. We propose that fetal lung macrophage activation contributes to bronchopulmonary dysplasia by generating a localized inflammatory response that disrupts developmental signals critical for lung formation.     

NF-κB1 inhibits TLR-induced IFN-β production in macrophages through TPL-2-dependent ERK activation

Although NF-κB1 p50/p105 has critical roles in immunity, the mechanism by which NF-κB1 regulates inflammatory responses is unclear. In this study, we analyzed the gene expression profile of LPS-stimulated Nfkb1(-/-) macrophages that lack both p50 and p105. Deficiency of p50/p105 selectively increased the expression of IFN-responsive genes, which correlated with increased IFN-β expression and STAT1 phosphorylation. IFN Ab-blocking experiments indicated that increased STAT1 phosphorylation and expression of IFN-responsive genes observed in the absence of p50/p105 depended upon autocrine IFN-β production. Markedly higher serum levels of IFN-β were observed in Nfkb1(-/-) mice than in wild-type mice following LPS injection, demonstrating that Nfkb1 inhibits IFN-β production under physiological conditions. TPL-2, a mitogen-activated protein kinase kinase kinase stabilized by association with the C-terminal ankyrin repeat domain of p105, negatively regulates LPS-induced IFN-β production by macrophages via activation of ERK MAPK. Retroviral expression of TPL-2 in Nfkb1(-/-) macrophages, which are deficient in endogenous TPL-2, reduced LPS-induced IFN-β secretion. Expression of the C-terminal ankyrin repeat domain of p105 in Nfkb1(-/-) macrophages, which rescued LPS activation of ERK, also inhibited IFN-β expression. These data indicate that p50/p105 negatively regulates LPS-induced IFN signaling in macrophages by stabilizing TPL-2, thereby facilitating activation of ERK.     

SIGNR1 ligation on murine peritoneal macrophages induces IL-12 production through NFκB activation

We have previously shown that murine resident peritoneal macrophages (PEMs) are activated in response to uptake of oligomannose-coated liposomes (OMLs), leading to production of interleukin (IL)-12. To understand the mechanism of activation of PEMs by OMLs, in the present study we investigated the role of a mannose-binding C-type lectin receptor, SIGNR1, in production of proinflammatory cytokines by PEMs, in which SIGNR1 acts as a physiological receptor for OMLs. Engagement of SIGNR1 on PEMs with an anti-SIGNR1-specific rat IgM antibody, ERTR9, induced production of IL-12 and tumor necrosis factor (TNF)-α from PEMs, while secretion of IL-6 and IL-1β was not detected with the same treatment. The level of phosphorylated IκB kinase in PEMs also increased in response to ERTR9 treatment of the cells. Treatment of PEMs with a specific nuclear factor kappa-B (NFκB) inhibitor, BAY11-7082, reduced ERTR9-dependent IL-12 production. Intraperitoneal treatment with BAY11-7082 also led to reduction of subsequent OML-induced IL-12 production from PEMs. These results indicate that SIGNR1-mediated intercellular signaling may induce production of cytokines such as IL-12 through NFκB activation.