MCAM,as a novel receptor for S100A8/A9, mediates progression of malignant melanoma through prominent activation of NF-κB and ROS formation upon ligand binding

The dynamic interaction between tumor cells and their microenvironment induces a proinflammatory milieu that drives cancer development and progression. The S100A8/A9 complex has been implicated in chronic inflammation, tumor development, and progression. The cancer microenvironment contributes to the up-regulation of this protein complex in many invasive tumors, which is associated with the formation of pre-metastatic niches and poor prognosis. Changing adhesive preference of cancer cells is at the core of the metastatic process that governs the reciprocal interactions of cancer cells with the extracellular matrices and neighboring stromal cells. Cell adhesion molecules (CAMs) have been confirmed to have high-level expression in various highly invasive tumors. The expression and function of CAMs are profoundly influenced by the extracellular milieu. S100A8/A9 mediates its effects by binding to cell surface receptors, such as heparan sulfate, TLR4 and RAGE on immune and tumor cells. RAGE has recently been identified as an adhesion molecule and has considerably high identity and similarity to ALCAM and MCAM, which are frequently over-expressed on metastatic malignant melanoma cells. In this study, we demonstrated that ALCAM and MCAM also function as S100A8/A9 receptors as does RAGE and induce malignant melanoma progression by NF-κB activation and ROS formation. Notably, MCAM not only activated NF-κB more prominently than ALCAM and RAGE did but also mediated intracellular signaling for the formation of lung metastasis. MCAM is known to be involved in malignant melanoma development and progression through several mechanisms. Therefore, MCAM is a potential effective target in malignant melanoma treatment.     

Neisseria meningitidis capsular polysaccharides induce inflammatory responses via TLR2 and TLR4-MD-2

CPS are major virulence factors in infections caused by Neisseria meningitidis and form the basis for meningococcal serogroup designation and protective meningococcal vaccines. CPS polymers are anchored in the meningococcal outer membrane through a 1,2-diacylglycerol moiety, but the innate immunostimulatory activity of CPS is largely unexplored. Well-established human and murine macrophage cell lines and HEK/TLR stably transfected cells were stimulated with CPS, purified from an endotoxin-deficient meningococcal serogroup B NMB-lpxA mutant. CPS induced inflammatory responses via TLR2- and TLR4-MD-2. Meningococcal CPS induced a dose-dependent release of cytokines (TNF-α, IL-6, IL-8, and CXCL10) and NO from human and murine macrophages, respectively. CPS induced IL-8 release from HEK cells stably transfected with TLR2/6, TLR2, TLR2/CD14, and TLR4/MD-2/CD14 but not HEK cells alone. mAb to TLR2 but not an isotype control antibody blocked CPS-induced IL-8 release from HEK-TLR2/6-transfected cells. A significant reduction in TNF-α and IL-8 release was seen when THP-1- and HEK-TLR4/MD-2-CD14- but not HEK-TLR2- or HEK-TLR2/6-transfected cells were stimulated with CPS in the presence of Eritoran (E5564), a lipid A antagonist that binds to MD-2, and a similar reduction in NO and TNF-α release was also seen in RAW 264.7 cells in the presence of Eritoran. CD14 and LBP enhanced CPS bioactivity, and NF-κB was, as anticipated, the major signaling pathway. Thus, these data suggest that innate immune recognition of meningococcal CPS by macrophages can occur via TLR2- and TLR4-MD-2 pathways.     

Magnolol Inhibits LPS-Induced Inflammatory Response in Uterine Epithelial Cells

Endometritis is an inflammation of the uterine lining that is commonly initiated at parturition. The uterine epithelial cells play an important role in defending against invading pathogens. Magnolol, a hydroxylated biphenyl compound isolated from Magnolia officinalis, has been shown to have anti-inflammatory effects. The aim of this study was to investigate the anti-inflammatory effect of magnolol in modifying lipopolysaccharide (LPS)-induced signal pathways in mouse uterine epithelial cells. We found that magnolol inhibited TNF-α and IL-6 production in LPS-stimulated mouse uterine epithelial cells. We also found that magnolol inhibited LPS-induced NF-κB activation, IκBα degradation, phosphorylation of ERK, JNK, and P38. Furthermore, magnolol could significantly inhibit the expression of TLR4 stimulating by LPS. These results suggest that magnolol exerts an anti-inflammatory property by downregulating the expression of TLR4 upregulated by LPS, thereby attenuating TLR4-mediated NF-κB and MAPK signaling and the release of pro-inflammatory cytokines. These findings suggest that magnolol may be a therapeutic agent against endometritis.     

Manganese superoxide dismutase induced by lipoteichoic acid isolated from Staphylococcus aureus regulates cytokine production in THP-1 cells

Lipoteichoic acid isolated from Staphylococcus aureus (aLTA) is known to regulate the production of pro-inflammatory cytokines through TLR2-mediated signaling pathways. In our previous study, we found that aLTA significantly increased manganese superoxide dismutase (MnSOD) in the THP-1 human monocyte-like cell line, but the role of MnSOD in the regulation of cytokine production was not elucidated. In the current study, we found that MnSOD was involved in aLTA-mediated cytokine production. The signaling pathways associated with aLTA-mediated MnSOD induction in THP-1 cells included TLR2-MyD88-IRAK2, JNK (c-Jun N-terminal kinases)1/2 and nuclear factor- κB (NF-κB). We also found MnSOD was involved in the regulation of IL-1β and TNF-α, which were induced by early signaling pathways, including JNK1/2, p38, and NF-κB p65. In addition, MnSOD was also involved in the production of IL-6 and CCL2 in aLTA-stimulated THP-1 cells through activation of late signaling pathways such as JAK2-STAT3. Taken together, our data suggest that aLTA-mediated MnSOD production involved in the regulation of cytokine production and it may be the cause of one of the excessive inflammatory reactions caused by S. aureus.     

NOD1 and NOD2 stimulation triggers innate immune responses of human periodontal ligament cells

Nod-like receptors (NLRs) are cytosolic sensors for microbial molecules. Νucleotide-binding oligomerization domain (NOD)1 and NOD2 recognize the peptidoglycan derivatives, meso-diaminopimelic acid (meso-DAP) and muramyl dipeptide (MDP), respectively, and trigger host innate immune responses. In the present study, we examined the function of NOD1 and NOD2 on innate immune responses in human periodontal ligament (PDL) cells. The gene expression of NOD1 and NOD2 was examined by RT-PCR. IL-6 and IL-8 production in culture supernatants was measured by ELISA. Western blot analysis was performed to determine the activation of NF-κB and MAPK in response to Tri-DAP and MDP. The genes of NOD1 and NOD2 appeared to be expressed in PDL cells. Although the levels of NOD2 expression were weak in intact cells, MDP stimulation increased the gene expression of NOD2 in PDL cells. Tri-DAP and MDP led to the production of IL-6 and IL-8 and the activation of NF-κB and MAPK in PDL cells. Toll-like receptor (TLR) stimulation led to increased gene expression of NOD1 and NOD2 in PDL cells. Pam3CSK4 (a TLR2 agonist) and IFN-γ synergized with Tri-DAP and MDP to produce IL-8 and IL-6 in PDL cells. Our results indicate that NOD1 and NOD2 are functionally expressed in human PDL cells and can trigger innate immune responses.     

Free heme is a danger signal inducing expression of proinflammatory proteins in cultured cells derived from normal rat hearts

Endogenous molecules from damaged tissue act as danger signals to trigger or amplify the immune/inflammatory response. In this study, we examined whether free heme induced pro-inflammatory proteins in cultured cells derived from normal hearts and investigated the cells targeted by heme, together with its mechanism of action in these cells. We cultured collagenase-isolated heart-derived cells from normal rats and examined whether free heme induced pro-inflammatory proteins, reactive oxygen species (ROS) production and NF-κB activation, by quantitative RT-PCR, ELISA and flow cytometry. Free heme increased mRNA of various pro-inflammatory proteins in cultured cardiac resident cells (CCRC) (at least 100-fold) and induced intracellular ROS formation. Approximately 85-90% of CCRC are fibroblast/smooth muscle cells and 10-15% are CD11bc-positive macrophages; therefore to examine individual target cells, macrophage-deleted (CD11bc-negative) CCRC, primary cultured cells (cardiac fibroblasts, arterial smooth muscle cells and cardiac microvascular endothelial cells) and macrophage cells lines (NR8383) were similarly treated. Free heme activated NF-κB and induced expression of some pro-inflammatory proteins, including IL-1 and TNF-α in NR8383. On the other hand, macrophage-deleted CCRC strongly increased expression of these proteins on treatment with IL-1 or TNF-α, but not free heme. Induction of expression of pro-inflammatory proteins by free heme was not inhibited by intracellular ROS reduction, but by protease and proteasome inhibitors capable of regulating NF-κB. These data suggest that free heme strongly induces various pro-inflammatory proteins in injured hearts through NF-κB activation in cardiac resident macrophages and through cross-talk between macrophages and fibroblast/smooth muscle cells mediated inter alia by IL-1, TNF-α.     

栀子苷抑制甲型流感病毒感染细胞中Toll样受体7/核因子-κB信号通路

目的 研究栀子苷对甲型流感病毒H3N2感染所致的Toll样受体7(Toll-like receptors7,TLR7)及其介导的细胞内转录因子核因子-κB(nuclear factor-kappa B,NF-κB)活性及前炎症细胞因子TNF-α和IL-6释放的影响,以探讨栀子苷干预甲型流感病毒作用的分子生物学机制.方法 甲型流感病毒H3N2作为刺激因素,利用双荧光素酶顺式报告系统和免疫荧光实验检测栀子苷对NF-κB转录活性及NF-κB核转位的影响.RT-PCR法进一步验证栀子苷对NF-κB上下游靶基因TLR7、TNF-α和IL-6 mRNA表达水平的影响.结果 通过NF-κB双萤光素酶报告系统检测发现,与正常对照组比较,病毒感染的细胞中NF-κB荧光素酶报告活性明显升高;与病毒损伤组比较,栀子苷治疗组NF-κB荧光素酶报告活性明显受到抑制.荧光倒置显微镜下观察NF-κB p65磷酸化水平及核转位变化结果显示,病毒感染的细胞中NF-κB磷酸化水平及人核率明显升高;与病毒损伤组比较,栀子苷治疗组NF-κB磷酸化水平及入核率明显受到抑制.RT-PCR结果显示栀子苷能明显抑制病毒诱导的TLR7、TNF-α和IL-6 mRNA的高表达.结论 栀子苷可拮抗甲型流感病毒H3N2感染细胞中TLR7介导的细胞内转录因子NF-κB信号转导通路的活化,并可抑制其下游靶基因TNF-α和IL-6 mRNA的高表达水平来发挥抗病毒感染的作用.     

Erythropoietin Protects Rat Brain Injury from Carbon Monoxide Poisoning by Inhibiting Toll-Like Receptor 4/NF-kappa B-Dependent Inflammatory Responses

Inflammatory responses play critical roles in carbon monoxide (CO) poisoning-induced cerebral injury. The present study investigated whether erythropoietin (EPO) modulates the toll-like receptor 4 (TLR4) and nuclear factor-kappa B (NF-κB) inflammatory signaling pathways in brain injury after acute CO poisoning. EPO (2500 and 5000 U/kg) was injected subcutaneously twice a day after acute CO poisoning for 2 days. At 48 h after treatment, the expression levels of TLR4 and NF-κB as well as the levels of inflammatory cytokines in the hippocampal tissues were measured. Our results showed that CO poisoning induced a significant upregulation of TLR4, NF-κB, and inflammatory cytokines in the injured rat hippocampal tissues. Treatment with EPO remarkably suppressed the gene and protein expression levels of TLR4 and NF-κB, as well as the concentrations of TNF-α, IL-1β, and IL-6 in the hippocampal tissues. EPO treatment ameliorated CO poisoning-induced histological edema and neuronal necrosis. These results suggested that EPO protected against CO poisoning-induced brain damage by inhibiting the TLR4–NF-κB inflammatory signaling pathway.     

MerTK negatively regulates Staphylococcus aureus induced inflammatory response via SOCS1/SOCS3 and Mal

ObjectiveStaphylococcus aureus (S. aureus), one of Gram-positive pathogen, is frequently associated with acute lung inflammation. The central feature of S. aureus acute lung inflammation are pulmonary dysfunctioning and impeded host defence response, which cause failure in inflammatory cytokines homeostasis and leads to serious tissue damage. However, the role of the Mer receptor tyrosine kinase (MerTK) in the lung following S. aureus infection remains elusive. Here, we investigate whether MerTK alleviates S. aureus induced uncontrolled inflammation through negatively regulating toll-like receptor 2 and 6 (TLR2/ TLR6) via suppressor of cytokine signalling 1, 3 (SOCS1/SOCS3).Methods and resultsWe found in mice lung tissues and RAW 264.7 macrophages upon S. aureus infection activates TLR2 and TLR6 driven mitogen-activated protein kinases (MAPKs) and nuclear factor kappa B (NF-κB) signalling pathways, resulting in production of inflammatory cytokines including tumour necrosis factor-α (TNF-α), interleukin 1β (IL-1β), interleukin 6 (IL-6). Furthermore, S. aureus-infection groups showed a significant up-regulation of MerTK which serves as mediator of SOCS1 and SOCS3. Subsequently, through feedback mechanism SOCS1/3 degrade Mal, resulting in inhibition of downstream TLR mediated inflammatory pathways. Moreover, MerTK^−/− mice lung tissues and silencing MerTK in RAW 264.7 inhibited the S. aureus-induced activation of MerTK, which significantly upregulated the phosphorylation of crucial protein in MAPKs (ERK, JNK, p38) and NF-κB (IĸBα, p65) signalling pathways, as well as the production of pro-inflammatory cytokines.ConclusionCollectively, these findings indicate the important role of MerTK in self-regulatory resolution of S. aureus-induced inflammatory pathways and cytokines through intrinsic SOCS1 and SOCS3 repressed feedback on TLR2, TLR6 both in vivo and in vitro.     

Celastrol attenuates impairments associated with lipopolysaccharide-induced acute respiratory distress syndrome (ARDS) in rats

Celastrol, a constituent from a traditional Chinese medicinal herb belonging to the family Celastraceae, has been shown to impart anti-inflammatory properties, in part, by inhibiting NF-κB activity and related induction of pro-inflammatory cytokine formation/release. The present study investigated the effects of celastrol in an animal model of acute respiratory distress syndrome (ARDS) induced by intratracheal administration of lipopolysaccharides (LPSs). Celastrol pre-treatment groups received celastrol by intraperitoneal injection on seven consecutive days before LPS treatment. In rats evaluated 24?h after LPS administration, oxygenation indices and lung injury were measured, as were levels of inflammatory cells and cytokines in isolated bronchoalveolar lavage fluid (BALF). Lung tissue expression of proteins involved in NF-κB and ERK/MAPK pathways were measured by Western blot analyses. Celastrol pre-treatments appeared to attenuate LPS-induced lung injury and inflammatory responses in the rats, including decreases in inducible aggregation\infiltration of inflammatory cells and production/release of pro-inflammatory cytokines into the lung airways. Celastrol appeared to also inhibit NF-κB activation, but had no effect on ERK/MAPK pathways in the LPS-induced ARDS. The results here thus indicated that celastrol pre-treatment could impart protective effects against LPS-induced ARDS, and that these effects may be occurring through an inhibition of induction of NF-κB signaling pathways.     

脂多糖预致敏的人骨髓间充质干细胞促炎机制研究

目的探索脂多糖（LPS）预致敏的人骨髓间充质干细胞（MSC）产生促炎功能的免疫调节机制。方法采用Real-time PCR和免疫荧光法检测MSC被预致敏前后TLR4信号通路相关分子（如TLR4、MyD88、TRAF6等）的表达水平,以及NF-κB的入核情况。通过Real-time PCR比较MSC被致敏前后促炎因子（IL-1β、IL-6、MIP-2、TNF-α）和Th1/Th2型细胞因子及其受体的表达差异。结果与未致敏的MSC相比,LPS预致敏的MSC中TLR4表达升高,NF-κB入核增加,促炎性因子IL-1β、IL-6、MIP-2、TNF-α表达升高,提示LPS预致敏可以激活MSC中的TLR4信号通路,并且诱导MSC中Th1型细胞因子及其受体表达升高,而Th2型细胞因子及其受体表达无变化或减少。结论MSC被LPS预致敏后TLR4信号通路激活,Th1型细胞因子及受体表达上调,从而诱导MSC分化成促炎表型。     

金黄葡萄球菌杀白细胞素(PVL)对THP-1巨噬细胞IL-8、IL-6表达的影响

目的 研究金黄葡萄球菌杀白细胞素( panton-valentine leucocidin,PVL)对THP-1巨噬细胞Toll样受体4(TLR4)/核因子κB(NF-κB)信号通路及IL-8、IL-6的表达影响,探讨PVL相关肺组织损伤的致病机制.方法 实验前用100nmol/L佛波酯(PMA)孵育THP-1细胞48...     

Luteoloside Protects the Uterus from <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>-Induced Inflammation,Apoptosis, and Injury

Luteoloside is a flavonoid extracted from several natural herbs that exhibits anti-microbial and anti-inflammation properties. Our study mainly identified the anti-inflammatory mechanism of action of luteoloside in Staphylococcus aureus-induced endometritis. Histopathological observations and myeloperoxidase (MPO) activity showed that luteoloside could protect the uterus from S. aureus-induced damage and ameliorate the infiltration of inflammatory cells. Quantitative PCR (qPCR) and ELISA analysis also revealed that luteoloside could decrease the expression of the pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 and increase the expression of the anti-inflammatory cytokine IL-10 both in vivo and in vitro. However, western blot analysis revealed that luteoloside inhibited the expression of TLR2 and IL-8 and inhibited the phosphorylation of IκBα and NF-κB p65. Moreover, luteoloside inhibited the apoptosis of endometrial epithelial cells (EECs), suppressed the phosphorylation of p53, and decreased the expression of caspase-3 induced by S. aureus. Furthermore, this study showed that luteoloside inhibited the expression of Bax but increased the expression of Bcl-2. These results indicate that luteoloside has anti-inflammatory properties by inhibiting the TLR2 and NF-κB signaling pathways and plays an anti-apoptotic role in S. aureus-induced endometritis, which may be valuable for the clinical treatment of S. aureus-induced inflammation.     

Receptor for advanced glycation end-products signals through Ras during tobacco smoke-induced pulmonary inflammation

We previously demonstrated up-regulation of the receptor for advanced glycation end-products (RAGE) and its ligands by cigarette smoke extract (CSE) in rat R3/1 cells, a type I-like alveolar epithelial cell line. However, RAGE-mediated intracellular signaling pathways that lead to pulmonary inflammation remained unclear. Using ELISAs, we demonstrate that alveolar epithelial cell lines exposed to 25% CSE for 2 hours induce the activation of Ras, a small GTPase that functions as a molecular switch in the control of several intracellular signaling networks. Conversely, cells treated with siRNA for RAGE (siRAGE) resulted in decreased Ras activation. Furthermore, Ras was significantly diminished in lungs from RAGE null mice exposed to chronic tobacco smoke when compared with smoke-exposed wild-type mice. The use of a luciferase reporter containing NF-κB binding sites also demonstrated elevated NF-κB activation in R3/1 cells after CSE stimulation and decreased NF-κB activation in cells transfected with siRAGE before CSE exposure. ELISA revealed an increase in the secretion of IL-1β and CCL5 by R3/1 cells, two cytokines induced by NF-κB and associated with leukocyte chemotaxis. Furthermore, real-time RT-PCR and ELISAs revealed decreased cytokine secretion in RAGE null mouse lung exposed to tobacco smoke compared with lungs from smoke-exposed wild-type animals. These results support the conclusion that CSE-induced RAGE expression functions in pathways that involve Ras-mediated NF-κB activation and cytokine elaboration. This RAGE-Ras-NF-κB axis likely contributes to inflammation associated with several smoking-related inflammatory lung diseases.     

Nuclear factor-κB mediates placental growth factor induced pro-labour mediators in human placenta

Prostaglandins, pro-inflammatory cytokines, extracellular matrix remodelling enzymes and nuclear factor-kappa B (NF-κB) are involved in the mechanisms of term and preterm parturition. Recent studies have reported an increase in angiogenesis-related genes during term and preterm labour, including placental growth factor (PLGF). In non-gestational tissues, PLGF induces inflammation via NF-κB. The aim of this study was to determine the effect of PLGF on the gene expression and release of pro-labour mediators in human placenta. Samples were obtained from normal pregnancies at the time of Caesarean section. Human placenta was incubated in the absence (basal control) or presence of a 10 ng/ml PLGF for 24 h. Inflammatory gene expression was analysed by quantitative RT-PCR, concentration of pro-inflammatory cytokines and prostaglandins was quantified by ELISA, and secretory matrix metalloproteinases (MMPs) activity by zymography. NF-κB DNA-binding activity and IκB-α (inhibitor of NF-κB) protein degradation were analysed by ELISA and Western blotting, respectively. PLGF significantly increased interleukin (IL)-6 and IL-8 gene expression and secretion, cyclooxygenase-2 expression and resultant prostaglandin (PG) E(2) and PGF(2α) release, and MMP-9 gene expression and enzyme production. PLGF induced the degradation of IκB-α whilst increasing NF-κB p65 DNA-binding activity. The PLGF-induced pro-labour responses were abrogated by co-treatment with the NF-κB inhibitor BAY 11-7082. In summary, the pro-inflammatory and pro-labour effects of PLGF in human placenta are mediated by NF-κB.     

S100A8, S100A9 and S100A12 activate airway epithelial cells to produce MUC5AC via extracellular signal‐regulated kinase and nuclear factor‐κB pathways

Airway mucus hyperproduction is a common feature of chronic airway diseases such as severe asthma, chronic obstructive pulmonary disease and cystic fibrosis, which are closely associated with neutrophilic airway inflammation. S100A8, S100A9 and S100A12 are highly abundant proteins released by neutrophils and have been identified as important biomarkers in many inflammatory diseases. Herein, we report a new role for S100A8, S100A9 and S100A12 for producing MUC5AC, a major mucin protein in the respiratory tract. All three S100 proteins induced MUC5AC mRNA and the protein in normal human bronchial epithelial cells as well as NCI‐H292 lung carcinoma cells in a dose‐dependent manner. A Toll‐like receptor 4 (TLR4) inhibitor almost completely abolished MUC5AC expression by all three S100 proteins, while neutralization of the receptor for advanced glycation end‐products (RAGE) inhibited only S100A12‐mediated production of MUC5AC. The S100 protein‐mediated production of MUC5AC was inhibited by the pharmacological agents that block prominent signalling molecules for MUC5AC expression, such as mitogen‐activated protein kinases, nuclear factor‐κB (NF‐κB) and epidermal growth factor receptor. S100A8, S100A9 and S100A12 equally elicited both phosphorylation of extracellular signal‐regulated kinase (ERK) and nuclear translocation of NF‐κB/degradation of cytosolic IκB with similar kinetics through TLR4. In contrast, S100A12 preferentially activated the ERK pathway rather than the NF‐κB pathway through RAGE. Collectively, these data reveal the capacity of these three S100 proteins to induce MUC5AC production in airway epithelial cells, suggesting that they all serve as key mediators linking neutrophil‐dominant airway inflammation to mucin hyperproduction.     

Receptor for advanced glycation endproducts (RAGE) deficiency protects against MPTP toxicity

Parkinson's disease (PD) is a common neurodegenerative disorder of unknown pathogenesis characterized by the loss of nigrostriatal dopaminergic neurons. Oxidative stress, microglial activation and inflammatory responses seem to contribute to the pathogenesis. The receptor for advanced glycation endproducts (RAGE) is a multiligand receptor of the immunoglobulin superfamily of cell surface molecules. The formation of advanced glycation end products (AGEs), the first ligand of RAGE identified, requires a complex series of reactions including nonenzymatic glycation and free radical reactions involving superoxide-radicals and hydrogen peroxide. Binding of RAGE ligands results in activation of nuclear factor-kappaB (NF-κB). We show that RAGE ablation protected nigral dopaminergic neurons against cell death induced by the neurotoxin MPTP that mimics most features of PD. In RAGE-deficient mice the translocation of the NF-κB subunit p65 to the nucleus, in dopaminergic neurons and glial cells was inhibited suggesting that RAGE involves the activation of NF-κB. The mRNA level of S100, one of the ligands of RAGE, was increased after MPTP treatment. The dopaminergic neurons treated with MPP(+) and S100 protein showed increased levels of apoptotic cell death, which was attenuated in RAGE-deficient mice. Our results suggest that activation of RAGE contributes to MPTP/MPP(+)-induced death of dopaminergic neurons that may be mediated by NF-κB activation.