NF-κB Transcriptional Activity Research Articles

NF-κB regulates brown adipocyte function through suppression of ANT2

The transcription factor nuclear factor of kappa-light-chain-enhancer of activated B cells (NF-κB) is expressed in brown adipocytes, but its role remains largely unknown in the cells. This issue was addressed in current study by examining NF-κB in brown adipocytes in vitro and in vivo. NF-κB activity was increased by differentiation of brown adipocytes through elevation of p65 (RelA) expression. The transcriptional activity of NF-κB was induced by the cold stimulation with an elevation in S276 phosphorylation of p65 protein. Inactivation of NF-κB in brown adipocytes made the knockout mice [uncoupling protein 1 (Ucp1)–CreER–p65f/f, U-p65-KO] intolerant to the cold environment. The brown adipocytes exhibited an increase in apoptosis, a decrease in cristae density and uncoupling activity in the interscapular brown adipose tissue (iBAT) of p65-KO mice. The alterations became severer after cold exposure of the KO mice. The brown adipocytes of mice with NF-κB activation (p65 overexpression, p65-OE) exhibited a set of opposite alterations with a reduction in apoptosis, an increase in cristae density and uncoupling activity. In mechanism, NF-κB inhibited expression of the adenine nucleotide translocase 2 (ANT2) in the control of apoptosis. Data suggest that NF-κB activity is increased in brown adipocytes by differentiation and cold stimulation to protect the cells from apoptosis through down-regulation of ANT2 expression.

SARS-CoV-2 Spike Glycoprotein S1 Induces Neuroinflammation in BV-2 Microglia

In addition to respiratory complications produced by SARS‐CoV‐2, accumulating evidence suggests that some neurological symptoms are associated with the disease caused by this coronavirus. In this study, we investigated the effects of the SARS‐CoV‐2 spike protein S1 stimulation on neuroinflammation in BV-2 microglia. Analyses of culture supernatants revealed an increase in the production of TNF-α, IL-6, IL-1β and iNOS/NO. S1 also increased protein levels of phospho-p65 and phospho-IκBα, as well as enhanced DNA binding and transcriptional activity of NF-κB. These effects of the protein were blocked in the presence of BAY11-7082 (1 µM). Exposure of S1 to BV-2 microglia also increased the protein levels of NLRP3 inflammasome and enhanced caspase-1 activity. Increased protein levels of p38 MAPK was observed in BV-2 microglia stimulated with the spike protein S1 (100 ng/ml), an action that was reduced in the presence of SKF 86,002 (1 µM). Results of immunofluorescence microscopy showed an increase in TLR4 protein expression in S1-stimulated BV-2 microglia. Furthermore, pharmacological inhibition with TAK 242 (1 µM) and transfection with TLR4 small interfering RNA resulted in significant reduction in TNF-α and IL-6 production in S1-stimulated BV-2 microglia. These results have provided the first evidence demonstrating S1-induced neuroinflammation in BV-2 microglia. We propose that induction of neuroinflammation by this protein in the microglia is mediated through activation of NF-κB and p38 MAPK, possibly as a result of TLR4 activation. These results contribute to our understanding of some of the mechanisms involved in CNS pathologies of SARS-CoV-2.

YEATS2 regulates the activation of TAK1/NF-κB pathway and is critical for pancreatic ductal adenocarcinoma cell survival.

The prognosis of pancreatic ductal adenocarcinoma (PDAC) is poor despite diagnostic progress and new chemotherapeutic regimens. Constitutive activation of NF-κB is frequently observed in PDAC. In this study, we found that YEATS2, a scaffolding protein of ATAC complex, was highly expressed in human PDAC. Depletion of YEATS2 reduced the growth, survival, and tumorigenesis of PDAC cells. The binding of YEATS2 is crucial for maintaining TAK1 activation and NF-κB transcriptional activity. Of importance, our results reveal that YEATS2 promotes NF-κB transcriptional activity through modulating TAK1 abundance and directly interacting with NF-κB as a co-transcriptional factor.

Expressing a truncated SARS-COV-2 spike protein in mouse heart induces cardiac hypertrophy

Abstract Cardiac injury is common in hospitalized and non-hospitalized COVID-19 patients, for which systemic inflammation stress is one of the causes (Topol, 2020). Although rare, COVID-19 cases that SARS-CoV-2 infecting cardiomyocytes (CMs) have been reported. In vitro, SARS-CoV-2 infection of human induced-pluripotent-cells derived CMs triggered innate immune responses and induced apoptosis (Bojkova et al., 2020; Chen et al., 2020). Therefore, the current literature indicates that the heart is attacked by SARS-CoV-2 directly or indirectly; however, the underlying mechanism remains largely unknown. Involved in the pathogenesis of heart diseases, Toll-like receptors (TLR) are a family of pattern recognition receptors that sense the pathogenic stimuli and signal the cardiac residential cells to cope with harsh conditions (Yu and Feng, 2018). Among the best characterized TLR signaling pathways is TLR4/NF-kB axis (Lu et al., 2008), in which TLR4 convey the danger signals through its down stream kinases, such as TAK1 and TBK1, to activate NF-kB. SARS-CoV-2 Spike protein is well known for its role of mediating virus entry into host cells, but its immunogenic role has not been clearly defined. Recently, we have found that SARS-CoV-2 Spike protein directly interacts with TLR4 and activates NF-kB transcriptional activity. Pharmaceutically blocking either TBK1 or TAK1 attenuates Spike protein's immunogenic activity. To pinpoint Spike protein's role in the heart, we generated an AAV to specifically express a truncated Spike protein (S1-TM) in the CMs. Our data show that expressing S1-TM in CMs induces cardiac hypertrophy and decreases heart systolic function in mice. On the molecular level, Spike protein increases RelA (p65 subunit of the NF-kB complex) and activates the expression of pro-inflammatory cytokine genes. In summary, our study suggests that Spike protein directly interacts with TLR4 to trigger innate immune signaling, and that Spike protein induced CM innate immune responses might be one of the underlying mechanisms of cardiac injury in COVID-19. Funding Acknowledgement Type of funding sources: Other. Main funding source(s): Masonic Medical Research Institute

MMP-9 reinforces radiation-induced delayed invasion and metastasis of neuroblastoma cells through second-signaling positive feedback with NFκB via both ERK and IKK activation.

Neuroblastoma (NB) progression is branded with hematogenous metastasis and frequent relapses. Despite intensive multimodal clinical therapy, outcomes for patients with progressive disease remain poor, with negligible long-term survival. Therefore, understanding the acquired molecular rearrangements in NB cells with therapy pressure and developing improved therapeutic strategies is a critical need to improve the outcomes for high-risk NB patients. We investigated the rearrangement of MMP9 in NB with therapy pressure, and unveiled the signaling that facilitates NB evolution. Radiation-treatment (RT) significantly increased MMP9 expression/activity, and the induced enzyme activity was persistently maintained across NB cell lines. Furthermore, RT-triggered NFκB transcriptional activity and this RT-induced NFκB were required/adequate for MMP9 maintenance. RT-triggered NFκB-dependent MMP9 actuated a second-signaling feedback to NFκB, facilitating a NFκB-MMP9-NFκB positive feedback cycle (PFC). Critically, MMP9-NFκB feedback is mediated by MMP9-dependent activation of IKKβ and ERK phosphotransferase activity. Beyond its tumor invasion/metastasis function, PFC-dependent MMP9 lessens RT-induced apoptosis and favors survival pathway through the activation of NFκB signaling. In addition, PFC-dependent MMP9 regulates 19 critical molecular determinants that play a pivotal role in tumor evolution. Interestingly, seven of 19 genes possess NFκB-binding sites, demonstrating that MMP9 regulates these molecules by activating NFκB. Collectively, these data suggest that RT-triggered NFκB-dependent MMP9 actuates feedback to NFκB though IKKβ- and ERK1/2-dependent IκBα phosphorylation. This RT-triggered PFC prompts MMP9-dependent survival advantage, tumor growth, and dissemination. Targeting therapy-pressure-driven PFC and/or selective inhibition of MMP9 maintenance could serve as promising therapeutic strategies for treatment of progressive NB.

GnRH pulse frequency and irregularity play a role in male aging.

Gonadotropin-releasing hormone (GnRH) has a role in hypothalamic control of aging, but the underlying patterns and relationship with downstream reproductive hormones are still unclear. Here we report that hypothalamic GnRH pulse frequency and irregularity increase before GnRH pulse amplitude slowly decreases during aging. GnRH is inhibited by nuclear factor (NF)-κB, and GnRH pulses were controlled by oscillations in the transcriptional activity of NF-κB. Exposure to testosterone under pro-inflammatory conditions stimulated both NF-κB oscillations and GnRH pulses. While castration of middle-aged mice induced short-term anti-aging effects, preventing elevation of luteinizing hormone (LH) levels after castration led to long-term anti-aging effects and lifespan extension, indicating that high-frequency GnRH pulses and high-magnitude LH levels coordinately mediate aging. Reprogramming the endogenous GnRH pulses of middle-aged male mice via an optogenetic approach revealed that increasing GnRH pulses frequency causes LH excess and aging acceleration, while lowering the frequency of and stabilizing GnRH pulses can slow down aging. In conclusion, GnRH pulses are important for aging in male mice.

Abstract P314: TGFβ2-Mediated Cardiac Fibroblast Activation Is Dependent On BET Bromodomain Proteins

Heart failure is a common and morbid disease frequently caused by dilated cardiomyopathy (DCM). Fibrotic remodeling is a key feature of DCM. Stress signaling pathways trigger transdifferentiation of quiescent cardiac fibroblasts into myofibroblasts (MF). Bromodomain and extra-terminal (BET) epigenetic reader proteins are stress-responsive distal signaling integrators that drive fibrosis. Here, we explored the role of BETs on distinct inflammatory pathways in ex vivo cultured cardiac fibroblasts. The Langendorff hanging heart procedure was used to isolate and culture primary non-myocytes from wild type hearts, which are almost exclusively fibroblasts. Cells were stimulated with TGFβ2 to trigger MF transdifferentiation. Prior to TGFβ2 treatment, fibroblasts were treated with 1 of the following: vehicle, the BET inhibitor JQ1, or siRNA targeting either Smad3 or Rela . MF-specific gene expression was assessed by Q-PCR. α-smooth muscle actin (αSMA) levels were assessed by immunocytochemistry (ICC) and immunoblotting. NFκB transcriptional activity was evaluated using a dual luciferase reporter assay. TGFβ2 activated MF-specific gene expression ( Acta2 , 9.0-fold; Col3a1 , 3.6-fold; Ctgf, 10.1-fold; Postn , 12.8-fold; p<0.05 for all vs. vehicle), and increased αSMA levels (1.5-fold by ICC and immunoblotting; p<0.05 vs. vehicle) in cultured primary cardiac fibroblasts. This was ablated by JQ1 and Smad3 siRNA. By contrast, Rela siRNA alone induced MF-specific gene expression ( Acta2 , 6.9-fold; Col3a1 , 7.0-fold; Ctgf, 3.7-fold; Postn , 11.2-fold; p<0.05 for all vs. vehicle), and increased αSMA staining by ICC (3.4-fold; p<0.05) but not protein (1.3-fold; p=NS). TGFβ2 further potentiated MF-specific gene expression ( Acta2 , 17.1-fold; Col3a1 , 7.1-fold; Ctgf, 13.2-fold; Postn , 42.5-fold; p<0.05 for all vs. vehicle) and αSMA protein (1.7-fold; p<0.05) in fibroblasts treated with Rela siRNA. NFκB transcriptional activity was reduced by TGFβ2 (-2.9-fold; p<0.05) and with JQ1 pre-treatment (-2.7-fold; p<0.05). These data define a critical role for BETs in TGFβ2-mediated gene transcription in cardiac fibroblasts. Surprisingly, NFκB does not appear to be a key activator of TGFβ2-stimulated fibroblast transdifferentiation.

CY-09 Inhibits NLRP3 Inflammasome Activation to Relieve Pain via TRPA1.

Peripheral tissue damage leads to inflammatory pain, and inflammatory cytokine releasing is the key factor for inducing the sensitization of nociceptors. As a calcium ion channel, TRPA1 plays an important role in pain and inflammation, thus becoming a new type of anti-inflammatory and analgesic target. However, there is no consensus on the role of this channel in mechanical hyperalgesia caused by inflammation. Here, we aim to explore the role and underlying mechanism of the inflammasome inhibitor CY-09 in two classic inflammatory pain models. We evaluated pain behavior on animal models, cytokine levels, intracellular Ca2+ levels, transient TRPA1 expression, NF-κB transcription, and NLPR3 inflammasome activation. Consistently, CY-09 reduced the production of inflammatory cytokines, intracellular Ca2+ levels, and the activation of TRPA1 by inhibiting the activation of inflammasomes, thereby reducing the proinflammatory polarization of macrophages and alleviating animal pain and injury. Importantly, AITC (TRPA1 agonist) significantly reversed the analgesic effect of CY-09, indicating that TRPA1 was involved in the analgesic effect of CY-09. Our findings indicate that CY-09 relieves inflammation and pain via inhibiting TRPA1-mediated activation of NLRP3 inflammasomes. Thus, NLRP3 inflammasome may be a potential therapeutic target for pain treatment and CY-09 may be a pharmacological agent to relieve inflammatory pain, which needs further research.

Chemical constituents and anti-inflammatory activity of the total alkaloid extract from Melodinus cochinchinensis (Lour.) Merr. and its inhibition of the NF-κB and MAPK signaling pathways

BackgroundMelodinus cochinchinensis (Lour.) Merr. is a medicinal plant, which is used as a folk medicine for treating meningitis and fractures. However, the anti-inflammatory activity of total alkaloid extract from M. cochinchinensis (MCTA) and its molecular mechanism are still not studied. PurposeThe aim of this study is to investigate the main chemical constituents of MCTA and explore its anti-inflammatory potential in both in vitro and in vivo assessments. MethodsUHPLC-ESI-HRMS/MS was applied to analyze the chemical profiling. The anti-inflammatory efficacy of MCTA was evaluated on lipopolysaccharide (LPS) induced RAW 264.7 cells and two common inflammation models in mice. The production of pro-inflammatory mediator and cytokine was tested using the ELISA method. The pathological change was analyzed by histological assessment. The expression of NF-κB, MAPKs and PPAR-γ proteins was evaluated using western blot analysis. ResultsA total of 21 monoterpenoid indole alkaloids (MIAs) were characterized by UHPLC-ESI-HRMS/MS. Aspidospermine- and quinolone-type alkaloids were found to be the major compounds. MCTA significantly decreased the production of NO, IL-1β, IL-6 and TNF-α in LPS-induced RAW 264.7 macrophages. MCTA significantly inhibited the phosphorylation of ERK1/2, JNK and p38 MAPK, suppressed the NF-κB transcriptional activation and improved the PPAR-γ expression. Moreover, the in vivo experiment exhibited that MCTA pretreatment markedly alleviated the xylene-induced ear edema and carrageenan-induced paw edema in mice and decreased the IL-1β, IL-6 and TNF-α expressions. ConclusionMCTA is rich in MIAs and exhibited a significant inhibitory effect on the production proinflammatory cytokines. The mechanism might be related to the inhibition of activation of NF-κB and MAPK pathways.

The protective effect of isoflurane pretreatment on liver IRI by suppressing noncanonical pyroptosis of liver macrophages

BackgroundLiver ischaemia–reperfusion injury (IRI) is a major complication in the perioperative period and often leads to liver failure and even systemic inflammation. Sufficient evidence has demonstrated that isoflurane has anti-inflammatory effects. We aimed to determine whether isoflurane pretreatment protects against liver IRI and to investigate the mechanisms involved in this protection. MethodsMale C57BL/6 mice were pretreated with or without isoflurane and subjected to 90 min of 70% liver ischaemia, followed by reperfusion for 6 h. Liver tissues and serum were analysed to assess liver IRI. To probe the mechanisms, liver macrophages isolated from C57BL/6 mice were pretreated with or without emulsified isoflurane for 30 min before incubation with 1 µg/ml lipopolysaccharide (LPS) for 24 h. Inflammatory cytokine production, intracellular Ca2+ levels, caspase-11 expression, NF-κB transcription, and NLRP3 inflammasome activation were assessed by ELISA, an intracellular Ca2+ concentration assay, immunohistochemistry, or Western blotting. ResultsIsoflurane preconditioning significantly relieved liver IRI in mice and LPS-induced inflammation in liver macrophages. Additionally, isoflurane pretreatment inhibited caspase-11 expression and noncanonical pyroptosis-related production of cytokines (IL-1β and IL-18). Interestingly, isoflurane preconditioning reduced intracellular Ca2+ levels, NF-κB translocation, and NLRP3 inflammasome activation in LPS-induced macrophages. Our results indicated that isoflurane preconditioning ameliorated liver IRI by suppressing noncanonical pyroptosis in liver macrophages. These findings suggest that isoflurane could be a pharmacological agent for liver IRI prevention and thus deserves more attention and further investigation.

CARMA1 is required for Notch1-induced NF-κB activation in SIL-TAL1-negative T cell acute lymphoblastic leukemia.

The NF-κB signaling pathway is an important downstream pathway of oncogenic Notch1 in T cell acute lymphoblastic leukemia (T-ALL) cells. However, the molecular mechanisms underlying the cascade activation of Notch1 in T-ALL cells are poorly understood. Here, we evaluated the role of CARMA1 in Notch1-induced NF-κB activation in T-ALL cells. CARMA1 was highly and specifically expressed in T-ALL cells and correlated with the prognosis of T-ALL patients. Interestingly, CARMA1 knockdown only inhibited the growth and proliferation of SIL-TAL1 fusion gene-negative T-ALL cells. In addition, CARMA1 knockdown arrested T-ALL cells at the G1 phase. Furthermore, CARMA1 knockdown significantly inhibited the proliferation of T-ALL cells in vivo and prolonged the survival of mice. Mechanistically, CARMA1 deficiency abolished Notch1-induced NF-κB transcriptional activation and significantly reduced expression levels of the NF-κB target genes c-Myc, Bcl-2, and CCR7. Taken together, these results of our study identify CARMA1 as one of the crucial mediators of Notch1-induced transformation of T-All cells, suggesting that CARMA1 is a promising therapeutic target for T-ALL due to its specific expression in lymphocytes. KEY MESSAGES: CARMA1 contributes to cell survival only in SIL-TAL1 negative T-ALL cells. CARMA1 is a crucial mediator of Notch1-induced activation of NF-κB pathway. CARMA1 is a promising therapeutic target for T-ALL.

Abstract 3040: Inhibition of Exportin-1 as part of combinatorial treatment for patients with high-risk neuroblastoma

Abstract Background: In many adult cancers, upregulation of the nuclear-to-cytoplasmic transport protein Exportin-1 (XPO1) correlates with poor outcome and responsiveness to selinexor, an FDA-approved XPO1 inhibitor. Similar data are emerging in childhood cancers, for which selinexor is being evaluated in early phase clinical studies. Our aims are to identify patients most likely to benefit from selinexor combination therapy and define the cellular mechanism responsible this effect. We hypothesize that selinexor and the proteasome inhibitor, bortezomib, act synergistically to decrease NF-kB-mediated proliferation by halting excessive exportation and proteasome degradation of IkB, an XPO1 cargo protein. Methods: We performed ion intensity-based label-free semiquantitative proteomic profiling on primary tumor material from fifty patients with uniformly treated high-risk NB, half of whom were long term survivors and half of whom died <18 months from diagnosis from disease progression. We cross-referenced this dataset with gene expression profiling data available through the R2 database. A panel of neuroblastoma cell lines was screened for XPO1 expression using Western blotting, sensitivity to selinexor and bortezomib as single agents, and in combination using cellular viability assays. Localization and quantification of IkB after vehicle or selinexor treatment was done by confocal imaging, using immunofluorescent labeling and Volocity software. Flow cytometry was used to define treatment effect on cell cycle arrest and apoptosis using BrdU and Annexin-V labeling, respectively. siRNA-mediated knockdown of IkB was used to investigate the relationship between presence of IkB and drug effectiveness. NF-kB activity was measured with a luciferase assay. Results: High tumor protein and gene expression of XPO1 correlates with poor outcome. Neuroblastoma cell lines demonstrate variably high XPO1 expression compared to normal tissue, as well as sensitivity to selinexor (IC50 range 4-300nM). IkB is retained in the nuclear compartment after treatment with selinexor. When selinexor is used in combination with bortezomib the effect on cell proliferation is synergistic. Flow cytometry demonstrates that selinexor alone contributes to G1-0 arrest while combination treatment results in significantly enhanced apoptosis. Measurement of NF-kB transcriptional activity shows decrease in activity with single agents and the least amount of activity in combination and siRNA against IkB diminishes the effect of single and combination treatment. Conclusions: This work demonstrates that XPO1 expression is a biomarker of highly aggressive neuroblastoma and may define patients most likely to be responsive to treatment with selinexor-based combinatorial therapies. Synergistic treatment effect with selinexor and bortezomib is mediated, in part, through the presence of IkB to regulate NF-kB signaling. Citation Format: Basia Galinski, Yossef Landesman, Daniel Weiser. Inhibition of Exportin-1 as part of combinatorial treatment for patients with high-risk neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3040.

Ivy leaves dry extract EA 575® mediates biased β2-adrenergic receptor signaling

Backgroundβ2-adrenergic receptor (β2-AR) stimulation activates the G protein/cAMP pathway, which is opposed by the GRK2/β-arrestin 2 pathway. The latter is undesirable in the treatment of respiratory diseases. Hypothesis/PurposeEA 575® is capable of mediating a biased β2-adrenergic signaling pathway. MethodsThe impact of the ivy leaves dry extract EA 575® on β2-adrenergic signaling was tested in a dynamic mass redistribution assay in HEK wild-type and in HEK β-arrestin knock-out cells. cAMP formation and recruitment of β-arrestin 2 were investigated using GloSensor™ and PathHunter® assays, respectively. NFκB transcriptional activity was determined in both HEK wild-type as well as HEK β-arrestin knock-out cells. ResultsEA 575® inhibits the recruitment of β-arrestin 2 and thereby enhances G protein/cAMP signaling under β2-stimulating conditions, as evidenced by a corresponding increase in cAMP formation. While β2-AR-mediated inhibition of NFκB transcriptional activity is β-arrestin-dependent, EA 575® leads to significant inhibition of NFκB transcriptional activity in β-arrestin knock-out cells and thus via a β-arrestin-independent signaling pathway. ConclusionEA 575® is the first active phytopharmaceutical ingredient for which biased β2-adrenergic activation has been described. This shift towards G protein/cAMP signaling provides the molecular basis for the clinically proven efficacy of EA 575® in the treatment of lower respiratory tract diseases. In this light, EA 575® could potentially reduce β-arrestin-mediated adverse effects in new combinatorial therapeutic approaches.

Discovery and biological evaluation of a small-molecule inhibitor of CRM1 that suppresses the growth of triple-negative breast cancer cells.

Dysregulation of the nuclear export machinery mediated by chromosomal maintenance 1 (CRM1, also known as exportin-1), is closely associated with various human disorders, such as breast cancer. Previously, we identified sulforaphene and its synthetic analogues as covalent inhibitors of CRM1. Herein, we describe the discovery and biological evaluation of another sulforaphene synthetic analogue, LFS-31, as a potential CRM1 inhibitor. In addition, we investigated the reversible binding mechanism of LFS-31 with CRM1 through molecular simulations coupled with bio-layer interferometry (BLI) and found relatively high binding affinity (KD=43.1 ± 35.3nM) between the LFS-31 and CRM1 groups. We found that LFS-31 exhibited a stronger growth suppression of triple-negative breast cancer (TNBC) cells than non-TNBC cells, and had minimal effect on normal breast cells. Pharmacological treatment of TNBC cells with LFS-31 at nanomolar concentrations led to the nuclear retention of IkBα resulting in strong suppression of NF-κB transcriptional activity and attenuated cell growth and proliferation, which collectively contributed to the antitumor responses. To the best of our knowledge, this is the first study to demonstrate the use of a sulforaphene analogue as a potent CRM1 inhibitor that targets the NF-κB signaling pathway for the targeted therapy of TNBC.

Efficacy of SPG-ODN 1826 Nanovehicles in Inducing M1 Phenotype through TLR-9 Activation in Murine Alveolar J774A.1 Cells: Plausible Nano-Immunotherapy for Lung Carcinoma.

Alveolar macrophages are the first line of defense against intruding pathogens and play a critical role in cancer immunology. The Toll-like receptor (TLR) family mediates an important role in recognizing and mounting an immune response against intruding microbes. TLR-9 is a member of the intracellular TLR family, which recognizes unmethylated CG motifs from the prokaryotic genome. Upon its activation, TLR-9 triggers downstream of the MyD-88-dependent transcriptional activation of NF-κB, and subsequently results in abundant inflammatory cytokines expression that induces a profound inflammatory milieu. The present exploratory investigation aimed at elucidating the potency of schizophyllan for entrapping ODN 1826 (SPG-ODN 1826)-mediated stimulation of TLR-9 in provoking an inflammatory-type response in murine alveolar macrophages. Schizophyllan (SPG), a representative of the β-glucan family, was used in the present study as a nanovehicle for endosomal trafficking of CpG ODN 1826. TEM analysis of SPG-ODN 1826 nanovehicles revealed that the prepared nanovehicles are spherical and have an average size of about 100 nm. Interestingly, SPG-ODN 1826 nanovehicles were competent in delivering their therapeutic payload within endosomes of murine alveolar macrophage (J774A.1) cells. Exposure of these nanovehicles within LPS stimulated J774A.1, resulted in a significant provocation of reactive oxygen species (ROS) (p < 0.01) in comparison to CpG ODN 1826 alone. Moreover, the formulated nanovehicles succeeded in generating a profound Th1-based cytokine profile constituted by enhanced expression of IFN-γ (p < 0.001) and IL-1β (p < 0.001) inflammatory cytokines. These findings clearly indicated the immunostimulatory potential of SPG-ODN 1826 nanovehicles for inducing the Th1-type phenotype, which would certainly assist in skewing M2 phenotype into the much-desired M1 type during lung cancer.

Inhibition of neuroinflammation by MIF inhibitor 3-({[4-(4-methoxyphenyl)-6-methyl-2-pyrimidinyl]thio}1methyl)benzoic acid (Z-312)

Microglial overactivation-mediated neuroinflammation contributes greatly to the pathogenesis of neurodegenerative diseases, such as Parkinson’s disease. Macrophage migration inhibitory factor (MIF) is a pleiotropic proinflammatory cytokine that is involved in the pathophysiology of various inflammatory diseases by inducing various proinflammatory cytokines. Compound 3-({[4-(4-methoxyphenyl)-6-methyl-2-pyrimidinyl]thio}methyl)benzoic acid (Z-312) is a novel small -molecule inhibitor of MIF tautomeric activity. In this study, we investigated the anti-inflammatory effects of Z-312 on liposaccharide (LPS)-induced neuroinflammation in vitro and in vivo. The results showed that Z-312 significantly decreased the production of nitric oxide (NO), interleukin (IL)-1β, tumor necrosis factor (TNF)-α and IL-6 in LPS-stimulated microglial cells. Mechanistically, nuclear translocation of the p65 subunit of nuclear factor (NF)-κB, degradation and phosphorylation of IκBα, NF-κB transcriptional activity and phosphorylation of p38 mitogen-activated protein kinase (MAPK) and JNK were markedly attenuated by pretreatment with Z-312 in BV-2 microglial cells. In addition, Z-312 suppressed the neurotoxic effects of cell culture medium of LPS-activated BV-2 microglia on cocultured mouse HT22 neuroblastoma cells. An in vivo study demonstrated that Z-312 markedly ameliorated microglial activation and subsequent DA neuron loss in an LPS-induced Parkinson’s disease (PD) mouse model. These results suggest that MIF inhibitor Z-312 may be a promising neuroprotective agent for the treatment of neuroinflammation-mediated neurological diseases.

Psiadin and plectranthone selectively inhibit colorectal carcinoma cells proliferation via modulating cyclins signaling and apoptotic pathways.

Three scarce terpenes, psiadin, plectranthone and saudinolide, were obtained after chromatographic isolation and purification from the aerial parts of the respective plants. Their identities were established based on their spectral data. Their anticancer effects against two human colorectal carcinoma cell lines, CCL233 and CCL235, along with the potential molecular mechanisms of action, were explored. Psiadin and plectranthone exhibited marked growth inhibition on both cell lines in a time- and dose-dependent manner with minimal cytotoxicity against normal breast cells (HB2). The terpenes even showed superior activities to the tested standards. Flow cytometry showed apoptosis induction and alteration in the cell cycle in colorectal cancer cells treated with both compounds. Nevertheless, it was also found that both compounds inhibited NF-κB transcriptional activity, induced mitochondrial membrane potential depolarization and increased the percentage of reactive oxygen species in the treated cancer cells in a dose-dependent manner as well. Since the anticancer effect of psiadin on cancer cells was higher than that produced by plectranthone, only psiadin was tested to determine its possible targets. The results suggested a high degree of specificity of action affecting particular cellular processes in both cancer cells. In conclusion, both terpenes, in particular psiadin, showed significant discriminative therapeutic potential between cancer and normal cells, a value that is missing in current chemotherapies.

Development and Biocompatibility Analysis of NBD Peptide Sustained- Release Microsphere Scaffold Nanoparticle SP-Sr-CaS/NBD.

In clinical treatment, it is difficult to carry out effective bone tissue transplantation and anti-inflammatory treatment at the same time due to bone defects and osteomyelitis where the tissue is contaminated or infected. As a downstream target of TNF-α, NF-κB has an inhibition effect on the proliferation and differentiation of cells surrounding the lesion. As a negative effect, it leads to a reduction in bone growth and development. In this study, the small molecule NBD polypeptide and bone conduction matrix Sr-CaS are microspheres, formed to prepare Sr-CaS, NBD drug-loaded sustained-release microspheres in order to achieve a Sr-CaS/NBD peptide drug-loaded sustained release microsphere scaffold material (SP-Sr-CaS/NBD). We prepared the microspheres and optimized the production process to obtain particles with stable morphological properties and sustained release properties. In vitro experiments demonstrated that SP-Sr-CaS/NBD could reduce TNF-α-induced cell growth inhibition, caspase-3 activity and NF-κB transcriptional activation as the function of continuous NBD peptide dosing regimen. Also, the introduction of the Sr-CaS matrix potentiates microspheres to promote cell proliferation and provides a basis to become a promising 3D bone scaffold material in the future.

Cigarette smoke extract-induced downregulation of p300 is responsible for the impaired inflammatory cytokine response of macrophages

Patients with chronic obstructive pulmonary disease (COPD) are susceptible to infection owing to the impaired immune function of alveolar macrophages. This is presumed to be caused, at least partially, by cigarette smoke (CS), which is a major risk factor for COPD. Although CS has been reported to inhibit Toll-like receptor (TLR) function and phagocytosis in macrophages, the molecular mechanism of CS-mediated impairment of macrophage immune function has not been completely elucidated. We investigated the effects of CS extracts (CSE) on macrophage immune function and its molecular mechanism. We assessed lipopolysaccharide (LPS, TLR4 ligand)-, Pam3CSK4 (TLR2 ligand)-, or CpG-oligodeoxynucleotide (TLR9 ligand)-induced IL-6, TNF-α, and IL-1β production in macrophages. Upregulation of IL-6, TNF-α, and IL-1β mRNA and protein by TLR ligands was suppressed on treatment with CSE. However, LPS-induced MAP kinase activation, IκBα degradation, and nuclear translocation of NF-κB were not impeded by CSE. In contrast, CSE significantly suppressed NF-κB transcriptional activity in the nucleus. We found that p300, which acetylates RelA/p65 at lysine 310, and acetyl-p65 (K310) were downregulated upon CSE treatment. Knock-down of p300 suppressed LPS-induced acetylation of NF-κB p65 and production of inflammatory cytokine. To summarize, these results suggest that CSE impair cytokine response by decreasing the expression levels of p300.

A Gender-Dependent Molecular Switch of Inflammation via MyD88/Estrogen Receptor-Alpha Interaction.

IntroductionMost Toll-like receptors and IL-1/IL-18 receptors activate a signaling cascade via the adaptor molecule MyD88, resulting in NF-κB activation and inflammatory cytokine and chemokine production. Females are less susceptible than males to inflammatory conditions, presumably due to protection by estrogen. The exact mechanism underlying this protection is unknown.MethodsMCF7 cells expressing wild-type or mutated LXXLL motif were used to determine MyD88/estrogen receptor (ER)-a interaction by immunoprecipitation and cell activation by ELISA and luciferase reporter assay. IL-1b and/or E2 were used to activate MCF7 cells expressing normal or knocked down levels of PRMT1. Finally, in situ proximity ligation assay with anti-MyD88 and anti-methylated ER-a (methER-a) antibodies was used to evaluate MyD88/methylated ER-a interaction in THP1 cells and histological sections.ResultsWe show that MyD88 interacts with a methylated, cytoplasmic form of estrogen receptor-alpha (methER-α). This interaction is required for NF-κB transcriptional activity and pro-inflammatory cytokine production, and is dissociated by estrogen. Importantly, we show a strong gender segregation in gametogenic reproductive organs, with MyD88/methER-α interactions found in testicular tissues and in ovarian tissues from menopausal women, but not in ovaries from women age 49 and less – suggesting a role for estrogen in disrupting this complex in situ.DiscussionCollectively, our results indicate that the formation of MyD88/methER-α complexes during inflammatory signaling and their disruption by estrogen may represent a mechanism that contributes to gender bias in inflammatory responses.