Lipopolysaccharide Signaling Research Articles

Investigation of inflammatory responses of alveolar epithelial cells induced by lipopolysaccharide and mechanism

Lipopolysaccharide (LPS) can activate alveolar epithelial cells (AECs) and induce inflammatory injury. Toll-like receptor-4 (TLR-4) is integrally involved in LPS signaling and has a requisite role in the activation of NF-κB. NF-κB is a key intercellular signaling event that mediates cell inflammatory responses. The aim of the study is to investigate in an in vitro model the inflammatory responses of AECs induced by LPS and the probable mechanism underlined the observed inflammatory responses. So cytokines of ICAM-1, TNF-α and IL-8 secreted by LPS-activated AECs were observed. And the initial signal molecule (the expression of TLR-4 mRNA) and the key intracellular steps (the activation of NF-κB) were studied in detail. The study was performed on A549 cells (Human lung adenocarcinoma cell line). A549 cells were divided into two groups: control, and LPS interference group. Proinflammatory cytokines ICAM-1, TNF-α and IL-8 were detected by ELISA or radioimmunological methods. The expression of TLR-4 mRNA was detected by real time PCR. The activation of NF-κB was detected by Western blot (proteins of I-κBα and NF-κB p65). Compared with control group, ICAM-1 and TNF-α of LPS-stimulated group were significantly higher and peaked after 2h before gradually declining at 6 and 12 h; IL-8 was higher after 2 h, which continued up to 12 h. The expression of TLR-4 mRNA of LPS group was significantly higher and peaked after 2 h and gradually declining at 6 and 12 h. Meanwhile, NF-κB was activated after 0.5, 2, 6 and 12 h indicated by the significant degradation of IκB-α and the significant release of NF-κB P65 and its subsequent translocation into the nucleus approximately synchronized. Taken together, the results demonstrate that LPS can induce AECs inflammatory injury via activating TLR-4 and subsequently activating NF-κB.

Role of CLIC4 in the host innate responses to bacterial lipopolysaccharide

Chloride intracellular channel (CLIC) 4 has diverse functions in membrane trafficking, apoptosis, angiogenesis and cell differentiation. CLIC4 is abundantly expressed in macrophages, but its role in innate immune functions is unclear. Here, we show that primary murine macrophages express increased amounts of CLIC4 after exposure to bacterial lipopolysaccharide (LPS). Endogenous CLIC4 level was significantly elevated in the brain, heart, lung, kidney, liver and spleen after LPS injection of mice. Stable macrophage lines overexpressing CLIC4 produced more TNF, IL-6, IL-12 and CCL5 than mock transfectants when exposed to LPS. To explore the role of CLIC4 in vivo, we generated CLIC4-null mice. These mice were protected from LPS-induced death, and had reduced serum levels of inflammatory cytokines. Upon infection with Listeria monocytogenes, CLIC4-deficient mice were impaired in their ability to clear infection, and their macrophages responded to Listeria by producing less inflammatory cytokines and chemokines than the WT controls. When challenged with LPS in vitro, deletion of clic4 gene had little effect on MAPK and NF-κB activation, but led to a reduced accumulation of phosphorylated interferon response factor 3 (IRF3) within macrophages. Conversely, overexpression of CLIC4 enhanced LPS-mediated IRF3. Thus, these findings suggest that CLIC4 is an LPS-induced product that can serve as a positive regulator of LPS signaling.

Surfactant blocks lipopolysaccharide signaling by inhibiting both NFκB and PARP activation in experimental ARDS

Surfactant plays an important role in lung homeostasis and is also involved in maintaining innate immunity within the lung. Lipopolysaccharide (LPS) is known to elicit acute proinflammatory responses in lung diseases such as acute respiratory distress syndrome and is responsible for the expression of the inducible isoform of nitric oxide synthase (iNOS). Because cells are exposed to low pH within the microenvironment of inflammatory lesions, the potential role of low environmental pH on iNOS expression was investigated. Low environmental pH-induced iNOS gene transcription involved the activation of nuclear factor-κB (NF-κB) transcription factor and IκB kinases. Here, we find that exposure of cells to low environmental pH increased both nitrite accumulation and activation of NF-κB-signaling pathway by Western blot and immunohistochemistry. In addition, treatment with surfactant prevents NF-κB translocation to the nucleus by preventing phosphorylation of IκBα, and its subsequent degradation by IKKα, and canceling low pH-induced nitrite accumulation. Surfactant also inhibited the LPS-induced PARP activation. Therefore, surfactant may regulate lung homeostasis by neutralizing acidic microenvironment in inflammatory lesions that leads to the upregulation of iNOS activity through the activation of NF-κB pathway and by PARP activation.

Antioxidant signaling via Nrf2 counteracts lipopolysaccharide-mediated inflammatory responses in foam cell macrophages

Inflammatory conditions and oxidative stress contribute to the development of atherosclerosis. Nuclear factor E2-related factor 2 (Nrf2) is a redox-sensitive transcription factor known for its antioxidant, anti-inflammatory, and, thus, cell-protective properties. Its role in effecting a deactivated state of oxidized low-density lipoprotein (oxLDL)-generated foam cell macrophages (FCMs), a prevailing cellular phenotype of atherosclerotic lesions, has not been investigated yet. In this study RAW264.7- or mouse peritoneal macrophage-derived FCMs showed reduced mRNA expression of proinflammatory cytokines such as IL-1β and IL-6 and an attenuated production of reactive oxygen species (ROS), as analyzed by hydroethidine in response to lipopolysaccharide (LPS) and compared to LPS-treated control macrophages. In peritoneal FCMs from Nrf2−/− mice (C57BL/6J), the LPS-induced proinflammatory response was restored. OxLDL induced heme oxygenase (HO)-1, which was Nrf2-dependent, and inhibition of HO-1 activity in FCMs using zinc protoporphyrin-IX allowed the cells to regain a proinflammatory phenotype. Mechanistically, oxLDL attenuated ROS-dependent activation of CCAAT/enhancer binding protein (C/EBP) family members in FCMs, thereby reducing cytokine expression. Thus, in FCMs the Nrf2/HO-1 axis intervenes in LPS signaling. ROS production is impaired, C/EBP transactivation is reduced, and consequently the expression of proinflammatory mediators is attenuated, thereby shaping a desensitized FCM phenotype. This macrophage phenotype may be important for the progression of atherosclerosis.

Downregulation effects of beta-elemene on the levels of plasma endotoxin, serum TNF-alpha, and hepatic CD14 expression in rats with liver fibrosis

It has been demonstrated that β-elemene could protect against carbon tetrachloride (CCl(4))-induced liver fibrosis in our laboratory work, and the aim of this paper is to reveal the protective mechanisms of β-elemene. The hepatic fibrosis experimental model was induced by the hypodermical injection of CCl(4) in Wistar male rats. β-elemene was intraperitoneally administered into rats for 8 weeks (0.1 mL/100 g bodyweight per day), and plasma endotoxin content was assayed by biochemistry. The serum TNF-α level was detected using radioactive immunity. CD14 expression in rat livers was measured by immunohistochemistry and Western blot. The results showed that β-elemene can downregulate the levels of plasma endotoxins, serum TNF-α, and hepatic CD14 expression in rats with liver fibrosis. β-elemene plays an important role in downregulating the lipopolysaccharide signal transduction pathway, a significant pathway in hepatic fibrosis development.

Role of p115RhoGEF in lipopolysaccharide-induced mouse brain microvascular endothelial barrier dysfunction

Background In endothelial cells, exposure to lipopolysaccharide (LPS) results in barrier dysfunction through a complex signaling mechanism. The RhoA/Rho-kinase pathway plays a significant role in endothelial cell permeability. p115RhoGEF, a specific guanine nucleotide exchange factors (GEFs) activates RhoA, triggering RhoA-dependent cytoskeletal remodeling. However, little is known about the role of p115RhoGEF in LPS-induced brain endothelial barrier breakdown. We hypothesized that suppression of p115RhoGEF may inhibit activation of RhoA and prevent LPS-induced brain microvascular endothelial cell hyperpermeability. Methods The cultured monolayer of bEnd.3 cells, an immortalized mouse brain endothelial cell line, was used in this study. bEnd.3 cells were pretreated with specific siRNA to knockdown p115RhoGEF or C3 transferase to inhibit RhoA activity, and then incubated with LPS (5 μg/ml). The degree of RhoA activation was determined by a Rhotekin-based pull-down assay, and expression of p115RhoGEF, zonula occludens-1 (ZO-1), occludin and claudin-5 proteins were detected by Western blot analysis. The barrier function was measured by transendothelial electrical resistance (TEER). F-actin cytoskeleton was visualized by Rhodamine-phalloidin staining. Results The expression level of p115RhoGEF protein was significantly increased in LPS-treated bEnd.3 cells. The activity of RhoA was enhanced after LPS stimulation and pretreatment with p115RhoGEF siRNA or exoenzyme C3 transferase reduced RhoA activation significantly as shown by the pull-down assay. Furthermore, depletion of p115RhoGEF partially prevented the LPS-induced decrease in TEER, stress fiber formation and tight junction proteins degradation. Conclusions These results suggest that p115RhoGEF is important for LPS signaling to RhoA and LPS-induced endothelial barrier dysfunction, providing new insight into the function of RhoGEFs in inflammation.

Betulinic acid inhibits endotoxin-stimulated phosphorylation cascade and pro-inflammatory prostaglandin E(2) production in human peripheral blood mononuclear cells.

Betulinic acid (BA) is a naturally occurring triterpenoid widely distributed throughout the plant kingdom. We previously reported that BA inhibits lipopolysaccharide (LPS)-induced interleukin-6 production through modulation of nuclear factor κB (NF-κB) in human peripheral blood mononuclear cells (hPBMCs). This study attempted to identify other mechanisms through which BA modulates LPS signalling in mononuclear cells. The effects of BA on signalling pathways downstream were focused on in this study. We determined the ability of BA to interfere with p38 and extracellular regulated kinase (ERK) phosphorylation as well as Akt phosphorylation and nuclear factor-κB activation using LPS-activated hPBMCs as an in vitro model. LPS-induced endotoxin shock in mice was the in vivo model employed. BA inhibited LPS-induced COX-2 protein expression and prostaglandin E(2) production and also attenuated LPS-induced ERK and Akt phosphorylation, but not p38 in hPBMCs. BA abolished LPS-induced IκBα phosphorylation and thus normalized the levels of IκBα in cytosol. BA also inhibited LPS-induced reactive oxygen species formation and lactate dehydrogenase release. Interestingly, BA improved the life span of mice in endotoxin shock and also inhibited PGE(2) production and myeloperoxidase activity in vivo. BA modulates LPS-induced COX-2 expression in hPBMCs by inhibiting ERK and Akt pathways as well as by modulating IκBα phosphorylation. At the same time, no cell toxicity was observed. The effect of the drug was confirmed through in vivo experiments. The study gives an insight into the molecular mechanisms of BA.

ESeroS-GS modulates lipopolysaccharide-induced macrophage activation by impairing the assembly of TLR-4 complexes in lipid rafts

The binding of lipopolysaccharides (LPS) to macrophages results in inflammatory responses. In extreme cases it can lead to endotoxic shock, often resulting in death. A broad range of antioxidants, including tocopherols, can reduce LPS activity in vitro and in vivo. To elucidate the underlying mechanisms of their action, we investigated the effect of the sodium salt of γ-L-glutamyl-S-[2-[[[3,4-dihydro-2,5,7,8-tetramethyl-2-(4,8,12-trimethyltridecyl)-2H-1-benzopyran-6-yl]oxy]carbonyl]-3-[[2-(1H-indol-3-yl)ethyl]amino]-3-oxopropyl]-L-cysteinylglycine (ESeroS-GS), a novel α-tocopherol derivative, on LPS-induced inflammation in vitro and in vivo. ESeroS-GS reduced the transcription of TNF-α, IL-1β, IL-6 and iNOS genes in a dose-dependent manner in RAW264.7 macrophages, and inhibited the release of these inflammatory factors. In addition, ESeroS-GS inhibited LPS-induced mortality in a mouse sepsis model. Electrophoretic mobility shift assays (EMSA) and reporter gene assays revealed that ESeroS-GS down-regulated the transcriptional activity of NF-κB. By analyzing the partitioning of CD14 and Toll-like receptor 4 (TLR-4) in cell membrane microdomains, we found that ESeroS-GS attenuates the binding of LPS to RAW264.7 cells via interfering with the relocation of CD14 and TLR-4 to lipid rafts, blocking the activation of interleukin-1 receptor-associated kinase 1 (IRAK-1), and inhibiting the consequent phosphorylation of TAK1 and IKKα/β, which together account for the suppression of NF-κB activation. Taken together, our data suggest that ESeroS-GS can modulate LPS signaling in macrophages by impairing TLR-4 complex assembly via a lipid raft dependent mechanism.

Cloning and characterization of a shrimp ML superfamily protein

ML superfamily proteins, including MD-1, MD-2, Niemann-Pick type C2 (Npc2) protein, GM2 activator protein, phosphatidylinositol/phosphatidylglycerol transfer protein (PG/PI-TP) and mite allergen Der p 2, bind to specific lipids and play important roles in lipid-recognition and metabolism. Among these ML (MD-2-related lipid-recognition) proteins, MD-2 is essential for lipopolysaccharide (LPS) signaling and the following secretion of proinflammatory factors. In this report, we identified the cDNA and gene of an ML protein from an important white shrimp Litopenaeus vannamei and named it LvML. The gene consists of four exons and three introns. The putative LvML contains 6 cysteines which may form 3 disulfide bonds that are conserved in ML proteins. Reverse transcription PCR analysis showed that in the examined tissues LvML mRNA is only expressed in the hepatopancreas, while not in hemocytes, eyestalk, gill, heart, stomach, intestine, nerve core, muscle or pyloric caecum. Its expression is positively regulated after injection of LPS. Then enzyme-linked immunosorbent assay showed that the recombinant LvML possessed activity of binding to LPS, and that the binding was inhibited by pre-incubation with LPS. We suggested that the LvML may play roles in the shrimp innate immunity.

LPS-induced Cytokine Production in Human Monocytes and Macrophages

Lipopolysaccharide (LPS) from Gram-negative bacteria is one of the most potent innate immune-activating stimuli known. Here we review the current understanding of LPS effects on human monocyte and macrophage function. We provide an overview of LPS signal transduction with attention given to receptor cooperativity and species differences in LPS responses, as well as the role of tyrosine phosphorylation and lysine acetylation in signalling. We also review LPS-regulated transcription, with emphasis on chromatin remodeling and primary versus secondary transcriptional control mechanisms. Finally, we review the regulation and function of LPS-inducible cytokines produced by human monocytes and macrophages including TNFα, the IL-1 family, IL-6, IL-8, the IL-10 family, the IL-12 family, IL-15 and TGFβ.

Lipopolysaccharide signaling in the carotid chemoreceptor pathway of rats with sepsis syndrome

In addition to their role in cardiorespiratory regulation, carotid body (CB) chemoreceptors serve as sensors for inflammatory status and as a protective factor during sepsis. However, lipopolysaccharide-induced sepsis (LPS) reduces CB responsiveness to excitatory or depressant stimuli. We tested whether LPS exerts a direct effect on the carotid chemoreceptor pathway, the CB and its sensory ganglion. We determined that the rat CB and nodose-petrosal-jugular ganglion complex (NPJgc) express TLR4, TNF-α and its receptors (TNF-R1 and TNF-R2). LPS administration (15mg/kg intraperitoneally) evoked MyD88-mechanism pathway activation in CB and NPJgc, with NF-κB p65, p38 MAPK, and ERK activation. Consistently, LPS increased TNF-α and TNF-R2. Double-labeling studies showed that the aforementioned pathway occurs in TH-containing glomus cells and NPJgc neurons, components of the chemosensitive neural pathway. Thus, our results suggest that LPS acting directly through TLR4/MyD88-mechanism pathways increases TNF-α and TNF-R2 expression in the carotid chemoreceptor pathway. These results show a novel afferent pathway to the central nervous system during endotoxemia, and could be relevant in understanding sepsis pathophysiology and therapy.

Inhibition of p38 MAPK during cellular activation modulate gene expression of head kidney leukocytes isolated from Atlantic salmon ( Salmo salar) fed soy bean oil or fish oil based diets

Head kidney leukocytes isolated from Atlantic salmon fed either a diet based on fish oil (FO) or soy bean oil (VO) were used in order to evaluate if different lipid sources could contribute to cellular activation of the salmon innate immune system. A specific inhibitor of p38 MAPK, SB202190, was used to investigate the effect of lipopolysaccharide (LPS) signalling in the head kidney leukocytes. The results show that LPS up regulate IL- 1β, TNF- α, Cox2 expression in leukocytes isolated from fish fed either diet. The p38 MAPK inhibitor, SB202190, reduced the LPS induced expression of these genes in both dietary groups. In LPS stimulated leukocytes isolated from VO fed fish, SB202190 showed a clear dose dependent inhibitory effect on IL- 1β, TNF- α and Cox2 expression. This effect was also observed for Cox2 in leukocytes isolated from FO fed fish. Furthermore, there was a stronger mean induction of Cox2 in LPS stimulated leucocytes isolated from the VO-group compared to LPS stimulated leukocytes isolated from the FO-group. In both dietary groups, LPS stimulation of salmon head kidney leukocytes increased the induction of CD83, a dendrite cell marker, while the inhibitor reduced CD83 expression in the VO fed fish only. The inhibitor also clearly reduced hsp27 expression in VO fed fish. Indicating a p38 MAPK feedback loop, LPS significantly inhibited the expression of p38MAPK itself in both diets, while SB202190 increased p38MAPK expression especially in the VO diet group. hsp70 expression was not affected by any treatment or feed composition. There were also differences in p38MAPK protein phosphorylation comparing treatment groups but no obvious difference comparing the two dietary groups. The results indicate that dietary fatty acids have the ability to modify signalling through p38 MAPK which may have consequences for the fish’s ability to handle infections and stress. Signalling through p38MAPK is ligand dependent and affects gene and protein expression differently.

Severe gunshot injuries in a porcine model: impact on central markers of innate immunity

the mechanisms behind lipopolysaccharide (LPS) tolerance remain obscure. LPS signals through Toll-like receptor 4 (TLR4) and severe trauma/haemorrhage may influence binding and signalling through this receptor, e.g. by changing membrane expression or by releasing endogenous ligands like High Mobility Group Box 1 (HMGB1). The aim of this study was to examine these relations further in a porcine model with standardized trauma. nine anaesthetized pigs sustained one gunshot through the femur and one pistol shot through the upper abdomen. Blood was sampled before and 90 min after shooting. The samples were stimulated for 4 h with LPS 10 ng/ml or an equivalent amount of normal saline. The leucocyte response was evaluated by measuring the tumour necrosis factor-α (TNF-α) and CXC ligand 8 (CXCL8) in the supernatant. Flow cytometry was used to measure the surface expression of TLR4 on CD14+ monocytes. HMGB1 concentrations were measured in the plasma. trauma and treatment caused a significant decline in the LPS-stimulated concentrations of TNF-α [4.53 ± 0.24 pg/ml (ln) at 0 min, 3.54 ± 0.35 pg/ml (ln) at 90 min, P=0.026], but did not modify the release of CXCL8. Monocyte TLR4 expression was unchanged. Plasma HMGB1 increased significantly [<0.92 vs. 3.02 ± 0.19 ng/ml (ln), P<0.001]. The concentrations of TNF-α and CXCL8 did not correlate with TLR4 expression or HMGB1 concentrations. the results suggest that trauma-induced LPS tolerance is not primarily regulated by TLR4 expression on circulating CD14+ monocytes or by the release of HMGB1 from damaged tissues.

Emodin suppresses lipopolysaccharide-induced pro-inflammatory responses and NF-κB activation by disrupting lipid rafts in CD14-negative endothelial cells.

Emodin [1,3,8-trihydroxy-6-methylanthraquinone] has been reported to exhibit vascular anti-inflammatory properties. However, the corresponding mechanisms are not well understood. The present study was designed to explore the molecular target(s) of emodin in modifying lipopolysaccharide (LPS)-associated signal transduction pathways in endothelial cells. Cultured primary human umbilical vein endothelial cells (HUVECs; passages 3-5) were pre-incubated with emodin (1-50 µg·mL(-1) ). LPS-induced expression of pro-inflammatory cytokines [interleukin (IL)-1β, IL-6] and chemokines (IL-8; CCL2/MCP-1) were determined by reverse transcription-PCR and elisa. Nuclear factor-κB (NF-κB) activation, inhibitor of κB (IκB)α degradation and Toll-like receptor-4 (TLR-4) were detected by immunocytochemistry and Western blotting. Cholesterol depletion [by methyl β-cyclodextrin (MBCD), a specific cholesterol binding agent] and cholesterol replenishment were further used to investigate the roles of lipid rafts in activation of HUVECs. Emodin inhibited, concentration-dependently, the expression of LPS-induced pro-inflammatory cytokines (IL-1β, IL-6) and chemokines (IL-8, CCL2) and, in parallel, inhibited NF-κB activation and IκBα degradation in HUVECs. However, emodin did not inhibit the NF-κB activation and IκBα degradation induced by IL-1β. The cholesterol binding agent, MBCD, inhibited LPS-induced NF-κB activation in passaged HUVECs [which also lack the LPS receptor, membrane CD14 (mCD14)], showing that lipid rafts played a key role in LPS signalling in mCD14-negative HUVECs. Moreover, emodin disrupted the formation of lipid rafts in cell membranes by depleting cholesterol. Lipid rafts were crucial in facilitating inflammatory responses of mCD14-negative HUVECs to LPS. Emodin disrupted lipid rafts through depleting cholesterol and, consequently, inhibited inflammatory responses in endothelial cells.

Apolipoprotein(a) inhibits lipopolysaccharide-induced IL-6 secretion in human astrocytoma cell line by interfering with lipopolysaccharide signaling

To examine the role of lipoprotein(a) [Lp(a)] on the inflammatory response of cells in the nervous system by investigating its effect on lipopolysaccharide (LPS)-induced interleukin-6 (IL-6) secretion. Human astrocytoma U373 cells were treated with recombinant apolipoprotein(a) [r-apo(a)] A10K (175-11 nM), alone or in combination with LPS (100 and 10 ng/ml). IL-6 levels were evaluated by immunoblotting. Statistical analysis was performed by one-way ANOVA. r-apo(a) caused dose-dependent inhibition of LPS-induced IL-6 secretion (100 ng/ml LPS, p = 0.0205; 10 ng/ml LPS, p = 0.0005). Pre-treatment of cells with 88 nM r-apo(a), rinsing, and activation with 10 ng/ml LPS did not reverse the inhibition (p = 0.0048), which could be reversed by supplementation with excess serum (5-20%) (p = 0.0454) or recombinant CD14 (2.0-0.05 μg/ml) (p = 0.0230). Our data indicate that apo(a) plays a natural anti-endotoxin role which relies on its interference with cell-associated and serum components of LPS signaling.

Lung Disease Associated with 1-Antitrypsin Deficiency

α(1)-Antitrypsin (A1AT) is a polyvalent, acute-phase reactant with an extensive range of biological functions that go beyond those usually linked to its antiprotease (serpin) activities. Genetic mutations cause a systemic deficiency of A1AT, leading to liver and pulmonary diseases, including emphysema and chronic bronchitis. The pathogenesis of emphysema, which involves the destruction of small airway structures and alveolar units, is triggered by cigarette smoke and pollutants. The tissue damage caused by these agents is further potentiated by the mutual interactions between apoptosis, oxidative stress, and protease/antiprotease imbalance. These processes lead to the activation of endogenous mediators of tissue destruction, including the lipid ceramide, extracellular matrix proteins, and abnormal inflammatory cell signaling. In this review, we propose that A1AT has a range of actions that are not restricted to protease inhibition but rather extend to mitigate a range of these pathological processes involved in the development of emphysema. We discuss the evidence indicating that A1AT blocks apoptosis by binding and inhibiting active caspase-3 and modulates a broad range of inflammatory responses induced by neutrophils and by lipopolysaccharide and tumor necrosis factor-α signaling.

Lipopolysaccharide stress induces cell-type specific production of murine leukemia virus type-endogenous retroviral virions in primary lymphoid cells

Some murine-endogenous retroviruses, making up ∼10 % of the mouse genome, are induced during the course of experimental sepsis in which lipopolysaccharide (LPS), a pathogenic component of Gram-negative bacteria, often plays a critical role. In this study, we investigated whether LPS stress induces the production of murine leukemia virus type-endogenous retrovirus (MuLV-ERV) virions in primary lymphoid cells. LPS treatment of cells (single-cell suspensions and sorted B- and T-cells) isolated from seven lymphoid organs of C57BL/6J mice resulted in a differential increase in the production of MuLV-ERV virions in most cells examined. Interestingly, among the 34 unique MuLV-ERV U3 sequences cloned from the viral genomic RNAs, the nuclear respiratory factor 1 (transcription factor) element was present only in the 20 U3 sequences that were derived from the LPS-induced MuLV-ERV U3 bands. Using the U3 sequences as a probe, 55 putative MuLV-ERV loci were mapped onto the C57BL/6J mouse genome and 15 of them retained full coding potential. Furthermore, one full-length recombinant MuLV-ERV originating from a locus on chromosome 13 was determined to be responsive to LPS stress. The findings from this study suggest that LPS stress differentially activates MuLV-ERV virion production in lymphoid organs in a cell type- and MuLV-ERV-specific manner. Further investigation is needed to define the role of MuLV-ERVs in the LPS signalling pathway(s) in general, as well as in the pathogenesis of sepsis.

Exploring the LPS/TLR4 signal pathway with small molecules

The identification of the bacterial endotoxin receptors for innate immunity, most notably TLR4 (Toll-like receptor 4), has sparked great interest in therapeutic manipulation of the innate immune system. In the present mini-review, several natural and synthetic molecules that modulate the TLR4-mediated LPS (lipopolysaccharide) signalling in animals and humans are considered, and their mechanisms of action are discussed. The process of LPS sensing and signal amplification in humans is based on the sequential action of specific receptors situated in the extracellular side of the innate immunity cells, which bind and transfer LPS to TLR4: LBP (LPS-binding protein), CD14, MD-2 (myeloid differentiation protein 2). We classified the compounds active on TLR4 pathway depending on the specific molecular targets (LPS, LBP, CD14, MD-2 or TLR4). Small molecules developed by our group are described that inhibit LPS-stimulated TLR4 activation by selectively targeting the LPS-CD14 interaction. These compounds have an interesting antiseptic shock, anti-inflammatory and anti-neuropathic pain activity in vivo.

In this issue

PROTEOMICSVolume 10, Issue 15 In this issue In this issue First published: 27 July 2010 https://doi.org/10.1002/pmic.201090067AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Abstract Monocytes (mis)lead charge against malicious lipo-polysaccharide Monocytes play a critical role in the functioning of the innate immune system (sometimes). After exposure to low levels of lipopolysaccharide (LPS), monocytes exhibit a refractory period of no response to subsequent exposure to LPS. Zhang et al. took proteomic quantitation tools (iTRAQ) in hand and went exploring. After identifying over 1600 proteins, half of which were membrane proteins and 250 of those tightly regulated by LPS, they found that more than 90% of mitochondrial proteins were downregulated, as were several proteins from the LPS signal pathway. Upregulated were proteins from a number of organelles (ER, Golgi, etc.). Mitochondrial DNA copy number declined, as expected for a target of an invader. Induction of TNF-α leads to enhanced production of reactive oxygen and nitrogen species. Given proper control of monocyte LPS response levels, sepsis could become a threat of the past. pp. 2780–2789 Help wanted: Astronomers needed for study of multimagnitude astrocytomas Candidate must be familiar with 2-D star patterns/2-D gel patterns. No black holes involved (yet)… Actually this paper is published by a group including molecular biological, cell biological, radiological and neurological “astronomers”. Astrocytomas, one of the more frequent and lethal brain tumors, often show up as graded types: (II) diffuse infiltrative, (III) anaplastic, and (IV) glioblastoma multiforme. Gimenez et al. explored the differences between the various grades with proteomic tools including 2-DE, MALDI-TOF and ESI-MS/MS. Two proteins of particular interest were found: nucleophosmin (NPM) and raf kinase inhibitor protein (RKIP). Further studies of these two will clarify their potential as markers and/or targets for diagnosis. pp. 2812–2821 “Gee, I could have used a V8” Context matters: the title of this paragraph could be referring to an eight-cylinder internal combustion engine with cylinders arranged in a v format with respect to each other, to a brand of vegetable juice mixture, or to a protease derived from the bacterium Staphylococcus aureus V8. Given that this is a journal devoted to proteomics, chances are very high that the title refers to a protease. Gupta et al. found that establishing such specificity was critical to determining the recognition sequence of proteinases for use in label-free proteolysis/mass spec. sequencing. The authors have developed a software tool for identifying potential sites using digestion by trypsin and three other enzymes to establish the cutting rules for a new protease. The method works on a proteome-wide scale. pp. 2833–2844 Volume10, Issue15No. 15 August 2010 RelatedInformation

Children with atopic histories exhibit impaired lipopolysaccharide‐induced Toll‐like receptor‐4 signalling in peripheral monocytes

The hygiene hypothesis states that early exposure to bacterial products such as lipopolysaccharide (LPS) may be protective against the development of allergic diseases. Whether atopic disease affects the ability of immune cells to respond to LPS is unclear. Our laboratory has demonstrated previously that children express high levels of Toll-like receptor (TLR)-4 on CD4(+) cells in nasal mucosa. To determine if children with a history of allergic disease have impaired responses to LPS on circulating CD4(+) leucocytes. Peripheral blood mononuclear cells from children (aged 2-18) and adults with or without a history of atopic conditions were cultured with/without IL-4 or LPS for up to 24 h. Expression of surface TLR-4, CD14, CD4, CD3, as well as of intracellular phosphorylated (p42/p44) ERK and p38 mitogen-activated protein kinase (MAPK) were assessed by flow cytometry. A history of atopy in children was associated with impaired LPS-induced TLR-4-dependent phosphorylation of (p42/44) ERK and p38 MAPK by CD4(+) monocytes. Decreased LPS signalling was reproduced by pre-incubation of control cells with recombinant IL-4. LPS stimulation also decreased TLR-4 expression on monocytes from children without atopic histories but not from atopic subjects. CD4(+) T lymphocytes showed limited LPS responsiveness, regardless of atopic status. In contrast with non-atopic children, TLR-4 expression on monocytes of children with atopic histories decreased as a function of age. This study provides evidence for defective LPS recognition on circulating CD4(+) leucocytes of subjects with atopic histories compared with those from non-atopic children. CD4(+) TLR4(+) monocytes from children with atopic histories failed to phosphorylate MAPKs. Our results suggest that a history of atopic disease is associated with impaired TLR-4-mediated innate immune function compared with non-atopic children.