Lipopolysaccharide-induced Protein Research Articles

A common edible insect (Antheraea assamensis) protein hydrolysate regulates LPS-induced oxidative stress and inflammation by modulating the TLR4/NF- κ β Signaling Pathway

Abstract Dietary intake of antioxidant and anti-inflammatory-rich foods is a growing approach for fighting inflammation and its associated disorders. Edible insects are gaining popularity as a food source; however, their therapeutic properties, anti-inflammatory activity and their mechanism of action remain largely unexplored. Herein, we evaluate the effectiveness of Antheraea assamensis pupae hydrolysates against oxidative stress and inflammation. The proteins isolated from the pupae were digested with pepsin or pancreatin, or pepsin + pancreatin and passed through a 30 kDa molecular cut-off membrane. The low molecular weight protein hydrolysates were characterised using RP-HPLC, HRMS, XRD, and FTIR-ATR. Hydrolysate from pepsin + pancreatin digestion showed significantly higher DPPH (77.13±2.57%), superoxide (72.08±1.15%) and hydroxyl (56.32±0.90%) radical scavenging activity in a cell-free system. Further, it reduced intercellular ROS production and suppressed the lipopolysaccharide-induced protein expression of TLR4, p-IKKβ, p-NF-, IL-1β, and COX2 in RAW 264.7 macrophages. Furthermore, it inhibited the secretion of pro-inflammatory mediators, including IL-6, IL-1β, and MIP-2. These findings suggest that A. assamensis pupae hydrolysates can be a potential source of bioactive peptides for managing oxidative stress and inflammation.

S-1-propenyl-L-cysteine suppresses lipopolysaccharide-induced expression of matrix metalloproteinase-1 through inhibition of tumor necrosis factor-α converting enzyme-epidermal growth factor receptor axis in human gingival fibroblasts.

Periodontal disease is the most common dental health problem characterized by the destruction of connective tissue and the resorption of alveolar bone resulting from a chronic infection associated with pathogenic bacteria in the gingiva. Aged garlic extract has been reported to improve gingival bleeding index and probing pocket depth score in patients with mild to moderate periodontitis. Although our previous study found that aged garlic extract and its constituents suppressed the tumor necrosis factor-α-induced inflammatory responses in a human gingival epithelial cell line, the mechanism underlying the effect of aged garlic extract on the destruction of the gingiva remains unclear. The present study investigated the effect of S-1-propenyl-L-cysteine, one of the major sulfur bioactive compounds in aged garlic extract, on the lipopolysaccharide-induced expression of matrix metalloproteinases in human gingival fibroblasts HGF-1 cells. Matrix metalloproteinases are well known to be closely related to the destruction of the gingiva. We found that S-1-propenyl-L-cysteine suppressed the lipopolysaccharide-induced expression and secretion of matrix metalloproteinase-1 in HGF-1 cells. In addition, S-1-propenyl-L-cysteine inhibited the lipopolysaccharide-induced phosphorylation of epidermal growth factor receptor and expression of the active form of tumor necrosis factor-α converting enzyme. Furthermore, the inhibitors of epidermal growth factor receptor tyrosine kinase and tumor necrosis factor-α converting enzyme, AG-1478 and TAPI-1, respectively, reduced the lipopolysaccharide-induced protein level of matrix metalloproteinase-1, as did S-1-propenyl-L-cysteine. Taken together, these results suggested that S-1-propenyl-L-cysteine suppresses the lipopolysaccharide-induced expression of matrix metalloproteinase-1 through the blockade of the tumor necrosis factor-α converting enzyme-epidermal growth factor receptor axis in gingival fibroblasts.

Research progress on Mincle as a multifunctional receptor.

Macrophage-induced C-type lectin (Mincle), a lipopolysaccharide-induced protein, is widely expressed on antigen-presenting cells. Mincle acts as a pattern recognition receptor that recognizes pathogen-associated molecular patterns of pathogens such as bacteria and fungi, mainly glycolipids, which induces an acquired immune response against microbial infection. Interestingly, Mincle can also identify patterns of lipid damage-associated molecule patterns released by injured cells, such as Sin3-associated protein 130 and β-glucosylceramides, which induces sterile inflammation and ultimately accelerates the progression of stroke, obesity, hepatitis, kidney injury, autoimmune diseases and tumors by promoting tissue inflammation. This article will review the various functions of Mincle, such as mediating sterile inflammation of tissues to accelerate disease progression, initiating immune responses to fight infection and promoting tumor progression.

Mincle: 20 years of a versatile sensor of insults.

Macrophage-inducible C-type lectin, better known as Mincle, is a member of the C-type lectin receptor family and is encoded by Clec4e. Mincle was an orphan receptor for a long time after having been discovered as a lipopolysaccharide-induced protein, yet later an adjuvant glycolipid in mycobacteria-trehalose dimycolate-was identified as a ligand. Ligands for Mincle were also found existing in bacteria, fungi and even mammals. When confronted with foreign elements, Mincle can recognize characteristic pathogen-associated molecular patterns, mostly glycolipids, from Mycobacterium tuberculosis and other pathogens, and thus induce immune responses against infection. To maintain self-homeostasis, Mincle can recognize lipid-based damage-associated molecular patterns, thereby monitoring the internal environment. The mechanism by which Mincle functions in the immune system is also becoming more clear along with the identification of its ligands. Being expressed widely on antigen-presenting cells, Mincle activation leads to the production of cytokines and chemokines, neutrophil infiltration and other inflammatory responses. Besides, Mincle can induce acquired immunity such as antigen-specific T-cell responses and antibody production as an adjuvant receptor. In this review, we will retrospectively sketch the discovery and study of Mincle, and outline some current work on this receptor.

Lipopolysaccharide-induced protein kinase D activation mediated by interleukin-1β and protein kinase C

Protein kinase D (PKD), a newly described serine/threonine kinase, has been implicated in many signal transduction pathways. The present study was designed to determine whether and how PKD is activated in inflammation. The results demonstrated that lipopolysaccharide (LPS, 30 μg/ml) stimulated PKD and protein kinase C (PKC) phosphorylation in spinal neurons within 0.5 h, and the activation reached a maximum at 3 or 8 h and declined at 12 h. The phosphorylation could be inhibited by the selective inhibitors for PKC (100 nM), mainly for PKCα and PKCβ, suggesting the involvement of the PKC pathway. Particularly, PKCα might be critical for LPS-induced PKD activation since the PKCβ inhibitor (100 nM) observed no effect on the phosphorylation of PKD. Furthermore, the expression of interleukin-1β (IL-1β) was significantly induced by LPS within 0.5 h, and reached a maximum at 8 h. IL-1 receptor antagonist inhibited PKD and PKCs activation induced by LPS at a concentration of 50 nM and achieved maximum at 1000 nM. These results demonstrated for the first time that PKD could be activated by LPS in spinal neurons, might via the IL-1β/PKCα pathway. Additionally, immunostaining showed an increase in number of phosphorylated PKD-immunoreactive cells of adult spinal dorsal horn induced by intraplantar injected carrageenan (2 μg/100 μl), and antisense oligodeoxynucleotide to IL-1 receptor type I (50 μg/10 μl, intrathecal injected) inhibited the PKD activation, suggesting an involvement of IL-1β/PKD pathway in inflammation in adult spinal cord.

Effects of Cytokine Antagonists on the Hepatic Acute-Phase Response

Endotoxin-induced hepatic acute-phase protein synthesis has been thought to be primarily regulated through cytokines such as interleukin-1 (IL-1) and interleukin-6 (IL-6). Previously, it was found that a 23-kDa murine acute-phase protein, the lipopolysaccharide (LPS)-induced protein (LIP), was synthesized following treatment of hepatocytesin vitrowith LPS. Since this protein was also induced by IL-1 and IL-6, the present studies were undertaken to determine if the effect of endotoxin was mediated through these cytokines. Primary cultures of murine hepatocytes were treated with LPS, IL-1, IL-6, or an LPS-stimulated macrophage supernatant in the presence or absence of the IL-1 receptor antagonist (IL-1 RA) and/or an anti-IL-6 antibody. The cells were then radiolabeled with [35S]methionine. LIP was detected by electrophoresis and autoradiography of the secreted proteins.In vitro,IL-1 RA completely inhibited the stimulation of LIP synthesis elicited by IL-1 and the macrophage supernatant, but did not affect LPS-stimulated synthesis of this protein. The anti-IL-6 antibody inhibited IL-6-triggered synthesis of LIP, but had no effect on LPS-stimulated synthesis. Hepatocytes isolated from mice treatedin vivowith both IL-1 RA and LPS synthesized LIP to the same degree as hepatocytes isolated from mice treated with LPS alone. LPS-stimulated synthesis of LIPin vitrodoes not require IL-1 or IL-6 as an obligatory intermediate. These results are consistent with the hypothesis that endotoxin can directly stimulate hepatocyte acute-phase protein synthesis in the absence of cytokines.

A role for MARCKS, the alpha isozyme of protein kinase C and myosin I in zymosan phagocytosis by macrophages.

Myristoylated, alanine-rich C-kinase substrate (MARCKS) is a lipopolysaccharide-induced protein kinase C (PKC) substrate that has been proposed to regulate actin-membrane interactions, as well as actin structure at the membrane. We studied the distribution of MARCKS, the alpha isozyme of PKC (PKC alpha), and myosin I in lipopolysaccharide-treated peritoneal macrophages ingesting zymosan particles. MARCKS, PKC alpha, and myosin I colocalized with F-actin and talin in the cortical cytoplasm adjacent to forming phagocytic cups. After particle ingestion was completed, myosin I, F-actin, and talin were no longer enriched in the vicinity of the phagosome. By contrast, MARCKS and PKC alpha remained associated with the phagosome membrane until after acquisition of the lysosomal marker Lamp-1. Vinculin was not detected on phagosomes at any time point examined. Phagocytosis of zymosan was accompanied by rapid and sustained phosphorylation of MARCKS. Inhibitors of PKC reduced zymosan binding to the macrophage surface and blocked the focal accumulation of F-actin, talin, phosphotyrosine-containing proteins, MARCKS, and PKC alpha beneath attached particles. We propose that PKC-dependent phosphorylation is an early signal required for zymosan phagocytosis and that MARCKS and PKC alpha have a role in phagosome maturation. The colocalization of F-actin and MARCKS at the cytoplasmic face of the nascent phagosome reinforces the hypothesis that MARCKS regulates actin structure at the membrane. Our data also suggest that myosin I functions as a mechanical motor during particle uptake.

Lipopolysaccharide-induced protein tyrosine phosphorylation in human macrophages is mediated by CD14.

Induced protein tyrosine phosphorylation is an early intracellular event in LPS-stimulated murine macrophages that appears to play a role in signal transduction. We have now demonstrated that LPS also increases protein tyrosine phosphorylation in human monocyte-derived macrophages and in the human monocytic cell line, THP-1. This response was rapidly elicited by biologically active forms of LPS or lipid A, at concentrations of these bacterial components that stimulate anti-bacterial responses by human macrophages. Inhibition of the LPS-induced tyrosine phosphorylation response with the tyrosine kinase inhibitor, herbimycin A, was accompanied by the inhibition of the secretion of TNF-alpha by human macrophages. These results extend previous work with murine macrophages and provide further support for the hypothesis that induced protein tyrosine phosphorylation is an important signaling reaction in macrophages after LPS exposure. In addition, CD14, which is thought to be a receptor for LPS, appeared to mediate the induced phosphorylation response in human macrophages and THP-1 cells at low LPS concentrations. Two antibodies against CD14, 3C10 and 60b, which have been shown to prevent LPS binding to CD14, specifically inhibited the protein tyrosine phosphorylation induced by nanogram per milliliter concentrations of LPS in these cells. The antibody-mediated inhibition did not appear to involve engagement of surface FcR because a preparation of F(ab')2/Fab fragments of the 60b antibody also prevented LPS-induced tyrosine phosphorylation. At higher concentrations of LPS (> or = 10 ng/ml), however, anti-CD14 antibodies did not prevent the induction of protein tyrosine phosphorylation, suggesting that a lower affinity CD14-independent pathway also mediates the tyrosine phosphorylation response. Because CD14-dependent and CD14-independent recognition of LPS appear to lead to the same functional responses by macrophages, induced protein tyrosine phosphorylation may be part of a shared intracellular signaling pathway.

Comparison of Heat-Shock-Induced and Lipopolysaccharide-Induced Protein Changes and Tumoricidal Activity in a Chicken Mononuclear Cell Line

The functions of a chicken mononuclear phagocytic cell line MQ-NCSU were examined after exposure to nonthermal (lipopolysaccharide, LPS) and thermal (heat shock, HS) treatments. The protein profiles and tumoricidal factor activity of MQ-NCSU cells exposed to 15 μg LPS under control (41 C) temperatures expressed enhanced synthesis of classical 23-, 70-, and 90-kDa HS proteins (HSP), a heat-inducible 32-kDa protein (P32), and a novel LPS-induced 120-kDa protein (P120). In comparison to LPS treatment, MQ-NCSU cells exposed to 45 C (HS) expressed HSP23, HSP70, HSP90, and P32 but not P120. Combined exposure of MQ-NCSU cells to HS (45 C) and LPS (15 μg) induced an alteration in the initial and optimal expression and duration of synthesis of the HSP and the LPS-induced P120. The tumoricidal activity of supernatants from LPS-treated and untreated MQ-NCSU cells cultured at 45 C was significantly depressed as compared with the controls (cultured at 41 C). The supernatants collected from LPS-treated and untreated MQ-NCSU cultures maintained at 41 C were exposed to 45 C temperature for up to 48 h. The tumoricidal potential of these supernatants was not affected. The present study demonstrates that LPS exposure induces several “stress” proteins in macrophages, some of which have molecular similarity with the classical HSP. In addition, LPS induces a unique 120-kDa protein not produced following HS alone, which may serve as a differential protein associated with activated or tumoricidal phenotype of macrophages. Heat shock suppresses the tumoricidal potential of LPS-treated MQ-NCSU cells in a regulatory manner that does not appear to be a result of thermal denaturation of the tumoricidal factor secreted in the culture supernatant.

A role for protein kinase C-mediated phosphorylation in the mobilization of arachidonic acid in mouse macrophages

Mouse peritoneal macrophages respond to activators of protein kinase C and to zymosan particles and calcium ionophore by rapid enhancement of a phospholipase A pathway and mobilization of arachidonic acid. The pattern of protein phosphorylation induced in these cells by 4 beta-phorbol 12-myristate 13-acetate (PMA), 1,2-dioctanoyl-sn-glycerol, exogenous phospholipase C and by zymosan and ionophore A23187 was found to be virtually identical. The time course of phosphorylation differed among the phosphoprotein bands and in only some of those identified (i.e., those of 45 and 65 kDa) was the phosphorylation sufficiently rapid to be involved in the activation of the phospholipase A pathway. Phosphorylation of lipocortin I or II could not be detected. Down-regulation of kinase C by a 24-h pretreatment with PMA resulted in extensive inhibition of both protein phosphorylation and the mobilization of arachidonic acid in response to PMA or dioctanoylglycerol. The phosphorylation of the 45 kDa protein in response to zymosan and A23187 was also inhibited by pretreatment with PMA, while only arachidonic acid release induced by zymosan was inhibited by this pretreatment. Depletion of intracellular calcium had little effect on kinase C-dependent phosphorylation, although arachidonic acid mobilization is severely inhibited under these conditions. Bacterial lipopolysaccharide and lipid A induced a phosphorylation pattern different from that induced by PMA, and down-regulation of protein kinase C did not affect lipopolysaccharide-induced protein phosphorylation. The results indicate (i) that protein kinase C plays a critical role also in zymosan-induced activation of the phospholipase A pathway mobilizing arachidonic acid; (ii) that such activation requires calcium at some step distal to kinase C-mediated phosphorylation and (iii) that phosphorylation of lipocortins does not explain the kinase C-dependent activation.