Lipopolysaccharide Internalization Research Articles

Brainendothelial GSDMD activation mediates inflammatory BBB breakdown.

The blood-brain barrier (BBB) protects the central nervous system from infections or harmful substances1; its impairment can lead to or exacerbate various diseases of the central nervous system2-4. However, the mechanisms of BBB disruption during infection and inflammatory conditions5,6 remain poorly defined. Here we find that activation of the pore-forming protein GSDMD by the cytosolic lipopolysaccharide (LPS) sensor caspase-11 (refs. 7-9), but not by TLR4-induced cytokines, mediates BBB breakdown in response to circulating LPS or during LPS-induced sepsis. Mice deficient in the LBP-CD14 LPS transfer and internalization pathway10-12 resist BBB disruption. Single-cell RNA-sequencing analysis reveals that brain endothelial cells (bECs), which express high levels of GSDMD, have a prominent response to circulating LPS. LPS acting on bECs primes Casp11 and Cd14 expression and induces GSDMD-mediated plasma membrane permeabilization and pyroptosis in vitro and in mice. Electron microscopy shows that this features ultrastructural changes in the disrupted BBB, including pyroptotic endothelia, abnormal appearance of tight junctions and vasculature detachment from thebasement membrane. Comprehensive mouse genetic analyses, combined with a bEC-targeting adeno-associated virus system, establish that GSDMD activation in bECs underlies BBB disruption by LPS. Delivery of active GSDMD into bECs bypasses LPS stimulation and opens the BBB. In CASP4-humanized mice, Gram-negative Klebsiella pneumoniae infection disrupts the BBB; this is blocked by expression of a GSDMD-neutralizing nanobody in bECs. Our findings outline a mechanism for inflammatory BBB breakdown, and suggest potential therapies for diseases of the central nervous system associated with BBB impairment.

Galectin-3 Mediates Endotoxin Internalization and Caspase-4/11 Activation in Tubular Epithelials and Macrophages During Sepsis and Sepsis-Associated Acute Kidney Injury.

Besides being recognized by membrane receptor TLR4, lipopolysaccharide (LPS) can also be internalized into the cytosol and activate Caspase-4/11 pyroptotic pathways to further amplify inflammation in sepsis. The objective of this study was to investigate whether Galectin-3 (Gal3) could promote the uptake of LPS by governing RAGE or administering endocytosis, consequently activating Caspase 4/11 and mediating pyroptosis in sepsis-associated acute kidney injury (SA-AKI). By pinpointing Gal3, LPS, and EEA1 (endosome-marker) or LAMP1 (lysosome-marker) respectively, immunofluorescence discovered that Gal3 and LPS were mainly aggregated in early endosomes initially and translocated into lysosomes afterwards. In cells and animal models, Gal3 and the Caspase-4/11 pathways were simultaneously activated, and the overexpression of Gal3 could exacerbate pyroptosis, whereas inhibition of Gal3 or the knockdown of its expression could ameliorate pyroptosis, reduce the pathological changes of SA-AKI and improve the survival of the animals with SA-AKI. Silencing RAGE reduced pyroptosis in primary tubular epithelial cells (PTCs) activated by Gal3 and LPS but not in cells activated by Gal3 and outer membrane vesicles (with LPS inside), whereas pyroptosis in both was reduced by blockade of Gal3, indicating Gal3 promoted pyroptosis through both RAGE-dependent and RAGE-independent pathways. Our investigation further revealed a positive correlation between serum Gal3 and pyroptotic biomarkers IL-1 beta and IL-18 in patients with sepsis, and that serum Gal3 was an independent risk factor for mortality. Through our collective exploration, we unraveled the significant role of Gal3 in the internalization of LPS and the provocation of more intense pyroptosis, thus making it a vital pathogenic factor in SA-AKI and a possible therapeutic target.Gal3 enabled the internalization of endotoxin into endosomes and lysosomes via both RAGE-dependent (A) and RAGE-independent (B) pathways, leading to pyroptosis. The suppression of Gal3 curbed Caspase4/11 noncanonical inflammasomes and diminished sepsis and SA-AKI.

Colchicine overdose impairs the capacity of Kupffer cells to clear foreign particles and endotoxins

Colchicine is an anti-inflammatory drug with a narrow therapeutic index. Its binding to tubulin prevents microtubule polymerization; however, little is known about how depolymerization of microtubules interferes with the phagocytosis function of Kupffer cells (KC). Here, we applied functional intravital imaging techniques to investigate the influence of microtubule disruption by colchicine on KC morphology, as well as its capacity to clear foreign particles and bacterial lipopolysaccharide (LPS) in anesthetized mice. Intravital imaging of KC in healthy mice showed the typical elongated morphology, localization at the luminal side of the sinusoidal endothelial cells, and moving cell protrusions. In contrast, at colchicine doses of 1 mg/kg and higher (intraperitoneal), KC appeared roundish with strongly reduced protrusions and motility. To study the functional consequences of these alterations, we analyzed the capacity of KC to phagocytose fluorescent nanospheres (100 nm-size) and LPS. After tail vein injection, the nanospheres formed aggregates of up to ~ 5 µm moving along the sinusoidal bloodstream. In controls, the nanosphere aggregates were rapidly captured by the Kupffer cell protrusions, followed by an internalization process that lasted up to 10 min. Similar capture events and internalization processes were observed after the administration of fluorescently labeled LPS. In contrast, capture and internalization of both nanospheres and LPS by KC were strongly reduced in colchicine-treated mice. Reduced phagocytosis of LPS was accompanied by aggravated production of inflammatory cytokines. Since 0.4 mg/kg colchicine in mice has been reported to be bio-equivalent to human therapeutic doses, the here-observed adverse effects on KC occurred at doses only slightly above those used clinically, and may be critical for patients with endotoxemia due to a leaky gut–blood barrier.

Cigarette smoke promotes inflammasome-independent activation of caspase-1 and -4 leading to gasdermin D cleavage in human macrophages.

Mechanisms and consequences of gasdermin D (GSDMD) activation in cigarette smoke (CS)-associated inflammation and lung disease are unknown. GSDMD is a downstream effector of caspase-1, -8, and -4. Upon cleavage, GSDMD generates pores into cell membranes. Different degrees of GSDMD activation are associated with a range of physiological outputs ranging from cell hyperactivation to pyroptosis. We have previously reported that in human monocyte-derived macrophages CS extract (CSE) inhibits the NLRP3 inflammasome and shifts the response to lipopolysaccharide (LPS) towards the TLR4-TRIF axis leading to activation of caspase-8, which, in turn, activates caspase-1. In the present work, we investigated whether other ASC-dependent inflammasomes could be involved in caspase activation by CSE and whether caspase activation led to GSDMD cleavage and other downstream effects. Presented results demonstrate that CSE promoted ASC-independent activation of caspase-1 leading to GSDMD cleavage and increased cell permeability, in the absence of cell death. GSDMD cleavage was strongly enhanced upon stimulation with LPS+CSE, suggesting a synergistic effect between the two stimuli. Noteworthy, CSE promoted LPS internalization leading to caspase-4 activation, thus contributing to increased GSDMD cleavage. Caspase-dependent GSDMD cleavage was associated with mitochondrial superoxide generation. Increased cleaved GSDMD was found in lung macrophages of smokers compared to ex-smokers and non-smoking controls. Our findings revealed that ASC-independent activation of caspase-1, -4, and -8 and GSDMD cleavage upon exposure to CS may contribute to macrophage dysfunction and feed the chronic inflammation observed in the smokers' lung.

The Mechanism of Lipopolysaccharide Escaping the Intestinal Barrier in Megalobrama amblycephala Fed a High-Fat Diet.

With the popularity of western food characterized by excessive fat and sugars, obesity has currently been a public health issue. Low-grade chronic inflammation accompanied by obesity increases the risk of multiple epidemics such as diabetes, cancer and cardiovascular diseases. Here, we show that feeding Megalobrama amblycephala with a high-fat diet (HFD) drives obesity-related chronic inflammation and the penetration of lipopolysaccharide (LPS). Interference with antibiotics inhibits the produce of LPS and this alleviates the sustained release of pro-inflammatory factors induced by HFD. LPS penetration is attributed to weakened intestinal mucus barrier after high-fat exposure. Mechanically, the consumption of HFD inhibits the secretion of mucin 2 (MUC2) due to the induction of endoplasmic reticulum stress mediated by the inositol-requiring enzyme 1 (IRE1) /X box-binding protein 1 (XBP1) pathway in goblet cells. Furthermore, excessive lipid exacerbates the leakage of LPS across the intestinal epithelial cell barrier via the transcellular pathway. Mechanically, lipid increases the internalization of LPS in intestinal epithelial cells depending on the activation of fatty acid translocase (FAT/CD36). These results demonstrate that HFD causes the penetration of LPS due to the weakened intestinal mucosal barrier and the assistance of CD36.

A Novel Lipopolysaccharide Recognition Mechanism Mediated by Internalization in Teleost Macrophages.

Macrophages in teleosts are less sensitive to lipopolysaccharide (LPS) compared to mammals. The functional equivalent of the mammalian LPS surface receptor in teleost macrophages for the pro-inflammatory response is either non-existent or replaced by negative regulation. LPS signaling in teleost macrophages remains unclear. Here, we found a scavenger receptor class B 2a (PaSRB2a) that played a crucial role in LPS signaling in teleost macrophages. The internalization of LPS and subsequent pro-inflammatory responses in macrophages were mediated by PaSRB2a, which is a novel isoform of the mammalian SRB2 gene. LPS internalization by PaSRB2a is dependent on its C-terminal intracellular domain. Following LPS internalization, it interacts with the ayu intracellular receptors nucleotide-binding oligomerization domain protein 1 (PaNOD1) and PaNOD2. Moreover, LPS pre-stimulation with sub-threshold concentrations reduced the effect of secondary LPS treatment on pro-inflammatory responses that were mediated by PaSRB2a. The pro-inflammatory responses in LPS-treated ayu were down-regulated upon PaSRB2a knockdown by lentivirus siRNA delivery. In grass carp and spotted green pufferfish, SRB2a also mediated LPS internalization and pro-inflammatory responses. Our work identifies a novel LPS signaling pathway in teleosts that differs from those in mammals, and contributes to our understanding of the evolution of pathogen recognition in vertebrates.

An MD2-derived peptide promotes LPS aggregation, facilitates its internalization in THP-1 cells, and inhibits LPS-induced pro-inflammatory responses.

MD2, a 160-residue accessory glycoprotein, is responsible for the recognition and binding of Gram-negative bacterial membrane component, lipopolysaccharide (LPS). Internalization of pathogen inside the mononuclear phagocytes has also been attributed to MD2 which leads to the clearance of pathogens from the host. However, not much is known about the segments in MD2 that are responsible for LPS interaction or internalization of pathogen inside the defense cells. A 16-residue stretch (MD54) from MD2 protein has been identified that possesses a short heptad repeat sequence and four cationic residues enabling it to participate in both hydrophobic and electrostatic interactions with LPS. An MD54 analog of the same size was also designed in which a leucine residue at a heptadic position was replaced with an alanine residue. MD54 but not its analog, MMD54 induced aggregation of LPS and aided in its internalization within THP-1 monocytes. Furthermore, MD54 inhibited LPS-induced nuclear translocation of NF-κB in PMA-treated THP-1 and TLR4/MD2/CD14-transfected HEK-293Tcells and the production of pro-inflammatory cytokines. In addition, in in vivo experiments, MD54 showed marked protection and survival of mice against LPS-induced inflammation and death. Overall, we have identified a short peptide with heptad repeat sequence from MD2 that can cause aggregation of LPS and abet in its internalization within THP-1 cells, resulting in attenuation of LPS-induced pro-inflammatory responses in vitro and in vivo.

Iron oxide nanoparticles modulate lipopolysaccharide-induced inflammatory responses in primary human monocytes.

Co-stimulation of the immune system to more than one agent concomitantly is very common in real life, and considering the increasing use of engineered nanoparticles and nanomaterials, it is highly relevant to assess the ability of these materials to modulate key innate immune responses, which has not yet been studied in detail. We investigated the immunomodulatory effects of 10 nm and 30 nm iron oxide nanoparticles (IONPs) on primary human monocytes in the presence and absence of Toll-like receptor 4 agonist lipopolysaccharide (LPS). Prior to the cell studies, we characterized the physicochemical properties of the nanoparticles in cell culture medium and ensured that the nanoparticles were free from biological contamination. Cellular uptake of the IONPs in monocytes was assessed using transmission electron microscopy. Using enzyme-linked immunosorbent assay, we found that the IONPs per se did not induce the production of proinflammatory cytokines tumor necrosis factor-α, interleukin-6, and interleukin-1β. However, the IONPs had the ability to suppress LPS-induced nuclear factor kappa B activation and production of proinflammatory cytokines in primary human monocytes in an LPS and a particle dose-dependent manner. Using confocal microscopy and fluorescently labeled LPS, we showed that the effects correlated with impaired LPS internalization by monocytes in the presence of IONPs, which could be partly explained by LPS adsorption onto the nanoparticle surface. Additionally, the results from particle pretreatment experiments indicate that other cellular mechanisms might also play a role in the observed effects, which warrants further studies to elucidate the additional mechanisms underlying the capacity of IONPs to alter the reactivity of monocytes to LPS and to mount an appropriate cellular response.

Kukoamine B promotes TLR4-independent lipopolysaccharide uptake in murine hepatocytes.

Free bacterial lipopolysaccharide (LPS) is generally removed from the bloodstream through hepatic uptake via TLR4, the LPS pattern recognition receptor, but mechanisms for internalization and clearance of conjugated LPS are less clear. Kukoamine B (KB) is a novel cationic alkaloid that interferes with LPS binding to TLR4. In this study, KB accelerated blood clearance of LPS. KB also enhanced LPS distribution in the hepatic tissues of C57 BL/6 mice, along with LPS uptake in primary hepatocytes and HepG2 cells. By contrast, KB inhibited LPS internalization in Kupffer and RAW 264.7 cells. Loss of TLR4 did not affect LPS uptake into KB-treated hepatocytes. We also detected selective upregulation of the asialoglycoprotein receptor (ASGPR) upon KB treatment, and ASGPR colocalized with KB in cultured hepatocytes. Molecular docking showed that KB bound to ASGPR in a manner similar to GalNAc, a known ASGPR agonist. GalNAc dose-dependently reduced KB internalization, suggesting it competes with KB for ASGPR binding, and ASGPR knockdown also impaired LPS uptake into hepatocytes. Finally, while KB enhanced LPS uptake, it was protective against LPS-induced inflammation and hepatocyte injury. Our study provides a new mechanism for conjugated LPS hepatic uptake induced by the LPS neutralizer KB and mediated by membrane ASGPR binding.

Human caspase-4 and caspase-5 regulate the one-step non-canonical inflammasome activation in monocytes.

Monocytes promote the early host response to infection releasing key pro-inflammatory cytokines, such as IL-1β. The biologically inactive IL-1β precursor is processed to active form by inflammasomes, multi-protein complexes activating caspase-1. Human monocytes exhibit an unconventional one-step pathway of inflammasome activation in response to lipopolysaccharide (LPS) alone. Although this lineage-restricted mechanism is likely to contribute to the pathology of endotoxin shock, signalling pathways regulating this mechanism are currently unknown. Here we report that caspase-4 and caspase-5 mediate IL-1α and IL-1β release from human monocytes after LPS stimulation. Although caspase-4 remains uncleaved, caspase-5 undergoes rapid processing upon LPS treatment. We also identify an additional caspase-5 cleavage product in LPS-stimulated monocytes, which correlates with IL-1 secretion. This one-step pathway requires Syk activity and Ca2+ flux instigated by CD14/TLR4-mediated LPS internalization. Identification of caspase-4/5 as the key determinants of one-step inflammasome activation in human monocytes provides potential targets for therapeutic intervention in endotoxin shock.

Liposomal Lipopolysaccharide Initiates TRIF-Dependent Signaling Pathway Independent of CD14

Lipopolysaccharide (LPS) is recognized by CD14 with Toll-like receptor 4 (TLR4), and initiates 2 major pathways of TLR4 signaling, the MyD88-dependent and TRIF-dependent signaling pathways. The MyD88-dependent pathway induces inflammatory responses such as the production of TNF-α, IL-6, and IL-12 via the activation of NFκB and MAPK. The TRIF-dependent pathway induces the production of type-I IFN, and RANTES via the activation of IRF-3 and NFκB, and is also important for the induction of adaptive immune responses. CD14 plays a critical role in initiating the TRIF-dependent signaling pathway response to LPS, to support the internalization of LPS via endocytosis. Here, we clearly demonstrate that intracellular delivery of LPS by LPS-formulated liposomes (LPS-liposomes) initiate only TRIF-dependent signaling via clathrin-mediated endocytosis, independent of CD14. In fact, LPS-liposomes do not induce the production of TNF-α and IL-6 but induce RANTES production in peritoneal macrophages. Additionally, LPS-liposomes could induce adaptive immune responses effectively in CD14-deficient mice. Collectively, our results strongly suggest that LPS-liposomes are useful as a TRIF-dependent signaling-based immune adjuvant without inducing unnecessary inflammation.

Inhibition of clathrin/dynamin-dependent internalization interferes with LPS-mediated TRAM–TRIF-dependent signaling pathway

Recognition of lipopolysaccharide (LPS) by Toll-like receptor 4 (TLR4) activates two district proinflammatory signaling pathway and initiates LPS internalization. To investigate roles of LPS internalization, a traditionally regarded metabolic pathway for LPS, in regulation of these two pathways, three internalization inhibitors, monodansylcadaverine (MDC, a clathrin inhibitor), dynasore (DS, a dynamin inhibitor) and chloroquine (CQ, an endosome acidifying maturation inhibitor) were applied to induce internalization dysfunction in macrophages. Results showed MDC and DS affected LPS internalization but did not interfere with their colocalization. Additionally, they decreased cytokines and chemokines release and inhibited signaling molecules activation mediated by TRAM–TRIF-dependent pathway as determined by protein array. In contrast, CQ did not inhibit LPS internalization but affected the colocalization. It also suppressed macrophage activation mediated by both MyD88-dependent and TRAM–TRIF-dependent pathways. The above data indicated that LPS internalization was clathrin/dynamin dependent and it was essential for activation of TRAM–TRIF-dependent signaling pathway.

Cell‐surface nucleolin is involved in lipopolysaccharide internalization and signalling in alveolar macrophages

C23 (nucleolin) shuttling between the nucleus, cytoplasm and cell surface has been implicated in controlling regulatory processes and may play a role in pathogen infection and autoimmune diseases. It has been reported that cell surface-expressed C23 on THP-1 monocytes is involved in the inflammatory response induced by LPS (lipopolysaccharide). This study investigates whether C23 is a membrane receptor for LPS during LPS-induced AMs (alveolar macrophages) activation. First, using immunofluorescence and microscopy, we detected the expression of C23 on the surface of AMs. Second, using LPS affinity columns, we demonstrated that C23 directly binds to LPS. Third, we found that LPS colocalized with C23 on both the cell surface and in the cytoplasm. Finally, knockdown of C23 expression on the cell surface using siRNA (small interfering RNA) led to significant reductions in the internalization of LPS, in LPS-induced NF-κB (nuclear factor κB)-DNA binding and in the protein expression of TNF (tumour necrosis factor)-α and IL-6 (interleukin-6). These findings provide evidence that cell-surface C23 on AMs may serve as a receptor for LPS and are essential for internalization and transport of LPS. Furthermore, C23 participates in the regulation of LPS-induced inflammation of AMs, which indicates that cell-surface C23 is a new and promising therapeutic target for the treatment of bacterial infections.

Loss of Heat Shock Protein gp96 Does Not Impair Toll Like Receptor Signaling In Vitro

Background: The heat shock protein gp96 is an endoplasmic reticulum chaperone for multiple protein substrates. We showed that a lack of gp96 in intestinal macrophages of Crohn's Disease (CD) patients is correlated with loss of tolerance against the host gut flora, leading to chronic inflammation. A recent manuscript suggested gp96 to be the major chaperone for TLRs in murine macrophages. Therefore, we studied the impact of gp96 knockdown on TLR function in the monocytic cell line Mono Mac 6 (MM6). Methods: For stable gp96 knockdown a lentiviral system was used. Stimulations were performed with lipopolysaccharide (LPS), a ligand of TLR 4. Internalization was shown using FITC-labeled LPS. TLR folding and functionality was investigated by Western blotting and flow cytometric analysis. IL-8 secretion upon stimulation with LPS was determined by ELISA. Results: Flow cytometric analysis of TLR 2 and 4 showed similar patterns on the cell surface of gp96knockdown cells as well as on control MM6 cells. Internalization of LPS by gp96-knockdown cells was shown using a FITC-labeled derivative. No reduction by gp96 knockdown was observed. Western blot analysis of phospho-IκB/IκB and phospho-NFκB/NFκB ratios did not reveal a significant difference in TLR mediated NFκB signaling between gp96 knockdown and control cells. Induction of IL-8 secretion upon stimulation with LPS was comparable between knockdown and control cells. Conclusions: In contrast to recent reports loss of gp96 does not have pivotal effects on functionality of TLRs in human MM6 cells. These findings indicate that the loss of tolerance against commensal gut flora is caused by different mechanisms yet to be identified.

The antimicrobial peptide cathelicidin enhances activation of lung epithelial cells by LPS

Epithelial cells (ECs) are usually hyporesponsive to various microbial products. Detection of lipopolysaccharide (LPS), the major component of gram-negative bacteria, is impeded, at least in part, by intracellular sequestration of its receptor, Toll-like receptor-4 (TLR4). In this study, using human bronchial ECs (hBECs) as a model of mucosal epithelium, we tested the hypothesis that the human LPS-binding, membrane-active cationic host defense peptide cathelicidin LL-37 augments epithelial response to LPS by facilitating its delivery to TLR4-containing intracellular compartments. We found that LL-37 significantly increases uptake of LPS by ECs with subsequent targeting to cholera toxin subunit B-labeled structures and lysosomes. This uptake is peptide specific, dose and time dependent, and involves the endocytotic machinery, functional lipid rafts, and epidermal growth factor receptor signaling. Cathelicidin-dependent LPS internalization resulted in significant increased release of the inflammatory cytokines IL-6 and IL-8. This indicates that, in ECs, this peptide may replace LPS-binding protein functions. In polarized ECs, the effect of LL-37 was restricted to the basolateral compartment of the epithelial membrane, suggesting that LL-37-mediated activation of ECs by LPS may be relevant to disease conditions associated with damage to the epithelial barrier. In summary, our study identified a novel role of LL-37 in host-microbe interactions as a host factor that licenses mucosal ECs to respond to LPS.

O-Antigen Delays Lipopolysaccharide Recognition and Impairs Antibacterial Host Defense in Murine Intestinal Epithelial Cells

Although Toll-like receptor (TLR) 4 signals from the cell surface of myeloid cells, it is restricted to an intracellular compartment and requires ligand internalization in intestinal epithelial cells (IECs). Yet, the functional consequence of cell-type specific receptor localization and uptake-dependent lipopolysaccharide (LPS) recognition is unknown. Here, we demonstrate a strikingly delayed activation of IECs but not macrophages by wildtype Salmonella enterica subsp. enterica sv. (S.) Typhimurium as compared to isogenic O-antigen deficient mutants. Delayed epithelial activation is associated with impaired LPS internalization and retarded TLR4-mediated immune recognition. The O-antigen-mediated evasion from early epithelial innate immune activation significantly enhances intraepithelial bacterial survival in vitro and in vivo following oral challenge. These data identify O-antigen expression as an innate immune evasion mechanism during apical intestinal epithelial invasion and illustrate the importance of early innate immune recognition for efficient host defense against invading Salmonella.

Endotoxin Tolerance of RAW264.7 Correlates with p38-dependent Up-regulation of Scavenger Receptor-A

Pre-exposure to lipopolysaccharide (LPS) leads to hyposensitivity to secondary LPS stimulation, known as endotoxin tolerance. The role of macrophage scavenger receptor-A (SR-A) in endotoxin tolerance is unknown. In this study, LPS was shown to induce SR-A expression in the mouse macrophage cell line, RAW264.7, in dose- and time-dependent manners, which correlated with inflammatory cytokine suppression in RAW264.7 on secondary LPS stimulation. Over-expression of SR-A in RAW264.7 suppressed tumour necrosis factor (TNF)-alpha release and nuclear factor (NF)-kappa B activation, demonstrating the involvement of SR-A in endotoxin tolerance. LPS-pre-treated RAW264.7 cells could bind and internalize more fluorescein isothiocyanate (FITC)-LPS than untreated cells and both the SR-A ligand, fucoidan, and anti-SR-A 2F8 antibodies completely suppressed LPS-induced binding and internalization of FITC-LPS by RAW264.7. LPS-induced SR-A expression on RAW264.7 was completely suppressed by the p38-specific inhibitor, SB203580, but not by inhibition of toll-like receptor 4 (TLR4) signalling with MTS510, demonstrating that p38- but not TLR4-dependent up-regulation of SR-A was involved in endotoxin tolerance through binding and internalization of LPS.

β2-Integrin-induced p38 MAPK Activation Is a Key Mediator in the CD14/TLR4/MD2-dependent Uptake of Lipopolysaccharide by Hepatocytes

The liver is the main organ that clears circulating lipopolysaccharide (LPS), and hepatocytes are a major cell type involved in LPS uptake. Little is known about the mechanisms for LPS internalization in hepatocytes and what signaling pathways are involved. We show here that LPS uptake is initiated after formation of a multi-receptor complex within lipid rafts. We find that essential components for LPS uptake are CD14, TLR4, MD2, and the beta2-integrin CD11b/CD18. Activation of p38 MAPK is also essential for the initiation of LPS uptake, and interestingly, we show that this activation is not through TLR4 signaling by MyD88 but through activation of TIRAP via CD11b/CD18. However, TLR4/MD2 remain essential components at the cell surface as part of the LPS receptor complex. We therefore suggest novel roles for TLR4/MD2, CD11b/CD18, TIRAP, and p38 MAPK in LPS uptake by hepatocytes.

PGJ2 Inhibition of LPS-induced Inflammatory Mediator Expression from Rat Alveolar Macrophages

Studies suggested that 15-deoxy-Δ-12,14-prostaglandin J2 (PGJ2) may exert anti-inflammatory effects, including in the lung. Thus, in vitro studies were conducted to (1) investigate whether PGJ2 inhibited the production of inflammatory mediators from lipopolysaccharide (LPS)-exposed primary rat alveolar macrophages (AM), and (2) investigate possible mechanisms underlying PGJ2-mediated inhibition of inflammatory mediator production. These studies determined that PGJ2 inhibited LPS-induced nitric oxide (NO) production in a concentration- and time-dependent manner. PGJ2-mediated inhibition of NO, as well as of tumor necrosis factor-α (TNF-α) and macrophage inflammatory protein-2 (MIP-2), was also determined to be dependent on the time of addition of PGJ2 relative to LPS, and suggested the PGJ2 inhibitory mechanism is an early event. PGJ2 was shown not to interfere with binding or internalization of LPS by AM, indicating this was not responsible for PGJ2 inhibitory effects. Another possible mechanism underlying PGJ2-mediated inhibition was via peroxisome proliferator-activated receptor-γ (PPAR-γ). However, biochemical studies suggested that PGJ2-mediated inhibition was not occurring through PPAR-γ dependent mechanism, and molecular studies further established that both LPS and PGJ2 decrease PPAR-γ mRNA expression. A third possible mechanism underlying PGJ2-mediated inhibition was by alteration of nuclear factor (NF)-κB. Molecular studies confirmed that LPS stimulated NF-κB mRNA expression, and PGJ2 reduced this stimulation, which is consistent with PGJ2 effect on LPS-induced production of NO, TNF-α and MIP-2. Thus, data in this study established that PGJ2 inhibited LPS-induced inflammatory mediator production in rat AM, and this inhibition is mediated, at least in part, by reducing the expression of NF-κB mRNA.

MAC1 mediates LPS‐induced production of superoxide by microglia: The role of pattern recognition receptors in dopaminergic neurotoxicity

Microglia-derived superoxide is critical for the inflammation-induced selective loss of dopaminergic (DA) neurons, but the underlying mechanisms of microglial activation remain poorly defined. Using neuron-glia and microglia-enriched cultures from mice deficient in the MAC1 receptor (MAC1-/-), we demonstrate that lipopolysaccharide (LPS) treatment results in lower TNFalpha response, attenuated loss of DA neurons, and absence of extracellular superoxide production in MAC1-/- cultures. Microglia accumulated fluorescently labeled LPS in punctate compartments associated with the plasma membrane, intracellular vesicles, and the Golgi apparatus. Cytochalasin D (CD), an inhibitor of phagocytosis, blocked LPS internalization. However, microglia derived from Toll-like receptor 4 deficient mice and MAC1-/- mice failed to show a significant decrease in intracellular accumulation of labeled LPS, when compared with controls. Pretreatment with the scavenger receptor inhibitor, fucoidan, inhibited 79% of LPS accumulation in microglia without affecting superoxide, indicating that LPS internalization and superoxide production are mediated by separate phagocytosis receptors. Together, these data demonstrate that MAC1 is essential for LPS-induced superoxide from microglia, implicating MAC1 as a critical trigger of microglial-derived oxidative stress during inflammation-mediated neurodegeneration.