Taxanes inhibit human TLR4 signaling by binding to MD-2

LPS is the primary ligand of Toll-like receptor 4, activating it through binding to its accessory protein MD-2. Murine but not human cells expressing MD-2/TLR4 are also activated by paclitaxel. Paclitaxel binds to human MD-2. The binding site of paclitaxel overlaps with the binding site of bis-ANS and LPS, which results in the ability of taxanes to inhibit LPS signaling in the system with human receptors. Circular dichroic spectra of human MD-2 indicated differences in the chemical environment in the presence of paclitaxel and docetaxel. Molecular docking identified the interacting residues of MD-2 and suggests that hydrophobic interactions govern the binding, while the C-3′N group where the paclitaxel and docetaxel differ is exposed on the surface of MD-2.     

MD-2 is required for the full responsiveness of mast cells to LPS but not to PGN

To address the role played by MD-2 in mast cell recognition of LPS, we examined bone marrow-derived mast cells (BMMCs) from MD-2 gene-targeted mice. BMMCs from MD-2-/- mice showed impaired cytokine production (TNF-alpha, IL-6, IL-13, and IL-1beta) in response to LPS from Escherichia coli, but not to peptidoglycan (PGN) from Staphylococcus aureus. In a mast cell-dependent acute septic model, MD-2 deficiency of mast cell resulted in significantly higher mortality due to defective neutrophil recruitment and the production of cytokines in the peritoneal cavity, which was similar to mice with TLR4-deficient mast cells. The TLR2-dependent activation of skin mast cells by PGN was not altered by the absence of MD-2 in vivo. Collectively, MD-2 is essential for the recognition of LPS by TLR4 but not for that of PGN by TLR2 of mast cells.     

Penta-acylated lipopolisaccharide binds to murine MD-2 but does not induce the oligomerization of TLR4 required for signal transduction

A mutant lipopolysaccharide (LPS) lacking a myristate chain in lipid A was shown to be non-pathogenic both in humans and mice. The mutant penta-acylated LPS from the lpxM-strain did not induce TNF-alpha production in murine peritoneal macrophages, or activation of NF-kappaB in transfected cells expressing murine TLR4/MD-2. We prepared a recombinant murine MD-2 in Escherichia coli (E. coli), and examined the binding function. Unexpectedly, specific binding was detected to both wild type and mutant LPS. However, the mutant LPS did not induce conformation changes or oligomerization of TLR4, which have been shown to be required for signal transduction. Mutant LPS appears to fail to induce appropriate conformational changes, resulting in oligomerization of the murine complex for triggering cell responses.     

The Toll-like receptor 4 region Glu24-Pro34 is critical for interaction with MD-2

Toll-like receptor 4 (TLR4) is a signaling receptor for lipopolysaccharide (LPS) but requires MD-2, a molecule associated with the extracellular TLR4 domain, to respond efficiently to LPS. The purpose of this study was to determine the critical stretch of primary sequence in the TLR4 region involved in MD-2 recognition. TLR4 and TLR4/2a chimera consisting of the TLR4 region Met(1)-Phe(54) and the TLR2 region Ala(53)-Ser(784) were coprecipitated with MD-2, but the deletion mutant TLR4(Delta E24-P34) in which the TLR4 region Glu(24)-Pro(34) was deleted failed to coprecipitate. In agreement with the MD-2 binding, LPS-conjugated beads sedimented TLR4 and TLR4/2a chimera but not TLR2 with MD-2. TLR4(Delta E24-P34) barely coprecipitated with LPS-beads. The cells that had been cotransfected with TLR4(Delta E24-P34) and MD-2 did not induce NF-kappa B activation in response to LPS. These results clearly demonstrate that the amino-terminal TLR4 region of Glu(24)-Pro(34) is critical for MD-2 binding and LPS signaling.     

The CD14 region spanning amino acids 57-64 is critical for interaction with the extracellular Toll-like receptor 2 domain

CD14 has been shown to enhance Toll-like receptor 2 (TLR2)-mediated signaling in response to peptidoglycan. Anti-CD14 monoclonal antibody MEM-18, whose epitope was located at the amino acid residues 57-64, blocked the binding of sCD14 to the recombinant soluble form of the extracellular TLR2 domain (sTLR2). The deletion mutant sCD14Delta57-64 lacking the amino acid residues 57-64 failed to bind to sTLR2. Cotransfection of wild type mCD14 but not mCD14Delta57-64 with TLR2 enhanced NF-kappaB activation in response to peptidoglycan. These results indicate that the CD14 region spanning amino acids 57-64 is critical for interacting with TLR2 and enhancing TLR2-mediated peptidoglycan signaling.     

Therapeutic targeting of innate immunity with Toll-like receptor 4 (TLR4) antagonists

Early recognition of invading bacteria by the innate immune system has a crucial function in antibacterial defense by triggering inflammatory responses that prevent the spread of infection and suppress bacterial growth. Toll-like receptor 4 (TLR4), the innate immunity receptor of bacterial endotoxins, plays a pivotal role in the induction of inflammatory responses. TLR4 activation by bacterial lipopolysaccharide (LPS) is achieved by the coordinate and sequential action of three other proteins, LBP, CD14 and MD-2 receptors, that bind lipopolysaccharide (LPS) and present it to TLR4 by forming the activated (TLR4-MD-2-LPS)(2) complex. Small molecules active in modulating the TLR4 activation process have great pharmacological interest as vaccine adjuvants, immunotherapeutics or antisepsis and anti-inflammatory agents. In this review we present natural and synthetic molecules active in inhibiting TLR4-mediated LPS signalling in humans and their therapeutic potential. New pharmacological applications of TLR4 antagonists will be also presented related to the recently discovered role of TLR4 in the insurgence and progression of neuropathic pain and sterile inflammations.     

The lipopolysaccharide-recognition mechanism in cells expressing TLR4 and CD14 but lacking MD-2

We analysed the lipopolysaccharide (LPS)-recognition mechanism in cells expressing TLR4 and CD14 but lacking MD-2. When TLR4 and CD14 were transiently expressed in HEK293 cells, cell-surface expression of TLR4 was observed, although the expression level was lower than that in cells coexpressing MD-2. We found that membrane CD14-TLR4 complexes were formed in these cells in response to LPS stimulation even in the absence of MD-2 expression, although NF-kappaB-dependent reporter activity was not induced. A strong activation of NF-kappaB was observed when these cells were stimulated with LPS followed by soluble MD-2 in this order, even when excess LPS was removed after formation of the CD14-TLR4 complex by washing cells prior to sMD-2 addition. From these results, we propose an additional LPS-recognition mechanism. In cells expressing TLR4 and CD14 but lacking MD-2, LPS is first transferred to membrane CD14 with the aid of LPS binding protein, which leads to the formation of the TLR4-CD14 complex. Then, the binding of soluble MD-2 to this complex triggers the transmembrane signal transduction. Cells expressing TLR4 and CD14 but lacking MD-2, such as airway epithelial cells, may be activated in response to LPS by this mechanism.     

Microglial Heparan Sulfate Proteoglycans Facilitate the Cluster-of-Differentiation 14 (CD14)/Toll-like Receptor 4 (TLR4)-Dependent Inflammatory Response

Microglia rapidly mount an inflammatory response to pathogens in the central nervous system (CNS). Heparan sulfate proteoglycans (HSPGs) have been attributed various roles in inflammation. To elucidate the relevance of microglial HSPGs in a pro-inflammatory response we isolated microglia from mice overexpressing heparanase (Hpa-tg), the HS-degrading endoglucuronidase, and challenged them with lipopolysaccharide (LPS), a bacterial endotoxin. Prior to LPS-stimulation, the LPS-receptor cluster-of-differentiation 14 (CD14) and Toll-like receptor 4 (TLR4; essential for the LPS response) were similarly expressed in Ctrl and Hpa-tg microglia. However, compared with Ctrl microglia, Hpa-tg cells released significantly less tumor necrosis factor-α (TNFα), essentially failed to up-regulate interleukin-1β (IL1β) and did not initiate synthesis of proCD14. Isolated primary astroyctes expressed TLR4, but notably lacked CD14 and in contrast to microglia, LPS challenge induced a similar TNFα response in Ctrl and Hpa-tg astrocytes, while neither released IL1β. The astrocyte TNFα-induction was thus attributed to CD14-independent TLR4 activation and was unaffected by the cells HS status. Equally, the suppressed LPS-response in Hpa-tg microglia indicated a loss of CD14-dependent TLR4 activation, suggesting that microglial HSPGs facilitate this process. Indeed, confocal microscopy confirmed interactions between microglial HS and CD14 in LPS-stimulated microglia and a potential HS-binding motif in CD14 was identified. We conclude that microglial HSPGs facilitate CD14-dependent TLR4 activation and that heparanase can modulate this mechanism.     

Mannose binding lectin and lung collectins interact with Toll-like receptor 4 and MD-2 by different mechanisms

Background

We have previously shown that lung collectins, surfactant protein A (SP-A) and surfactant protein D, interact with Toll-like receptor (TLR) 2, TLR4, or MD-2. Bindings of lung collectins to TLR2 and TLR4/MD-2 result in the alterations of signaling through these receptors, suggesting the immunomodulatory functions of lung collectins. Mannose binding lectin (MBL) is another collectin molecule which has structural homology to SP-A. The interaction between MBL and TLRs has not yet been determined.

Methods

We prepared recombinant MBL, and analyzed its bindings to recombinant soluble forms of TLR4 (sTLR4) and MD-2.

Results

MBL bound to sTLR4 and MD-2. The interactions were Ca²⁺-dependent and inhibited by mannose or monoclonal antibody against the carbohydrate-recognition domain of MBL. Treatment of sTLR4 or MD-2 by peptide N-glycosidase F significantly decreased the binding of MBL. SP-A bound to deglycosylated sTLR4, and this property did not change in chimeric molecules of SP-A/MBL in which Glu¹⁹⁵–Phe²²⁸ or Thr¹⁷⁴–Gly¹⁹⁴ of SP-A were replaced with the corresponding MBL sequences.

General Significance

These results suggested that MBL binds to TLR4 and MD-2 through the carbohydrate-recognition domain, and that oligosaccharide moieties of TLR4 and MD-2 are important for recognition by MBL. Since our previous studies indicated that lung collectins bind to the peptide portions of TLRs, MBL and lung collectins interact with TLRs by different mechanisms. These direct interactions between MBL and TLR4 or MD-2 suggest that MBL may modulate cellular responses by altering signals through TLRs.     

MD-2-dependent human Toll-like receptor 4 monoclonal antibodies detect extracellular association of Toll-like receptor 4 with extrinsic soluble MD-2 on the cell surface

MD-2 is essential for lipopolysaccharide (LPS) recognition of Toll-like receptor 4 (TLR4) but not for cell surface expression. The TLR4/MD-2 complex is formed intracellularly through co-expression. Extracellular complex formation remains a matter for debate because of the aggregative nature of secreted MD-2 in the absence of TLR4 co-expression. We demonstrated extracellular complex formation using three independent monoclonal antibodies (mAbs), all of which are specific for complexed TLR4 but unreactive with free TLR4 and MD-2. These mAbs bound to TLR4-expressing Ba/F3 cells only when co-cultured with MD-2-secreting Chinese hamster ovary cells or incubated with conditioned medium from these cells. All three mAbs bound the extracellularly formed complex indistinguishably from the intracellularly formed complex in titration studies. In addition, we demonstrated that two mAbs lost their affinity for TLR4/MD-2 on LPS stimulation, suggesting that these mAbs bound to conformation-sensitive epitopes. This was also found when the extracellularly formed complex was stimulated with LPS. Additionally, we showed that cell surface TLR4 and extrinsically secreted MD-2 are capable of forming the functional complex extracellularly, indicating an additional or alternative pathway for the complex formation.     

Kir3.1 channel is functionally involved in TLR4-mediated signaling

We aimed to study the involvement of Kir3.1 channel in TLR4-mediated signaling. LPS stimulation induced the recruitment of TLR4 and Kir3.1 into the lipid raft in THP-1 cells. Treatment with Tertiapin-Q, an inhibitor of Kir3.1, markedly abolished the recruitment of TLR4 into the lipid raft and inhibited the LPS-induced NF-κB activation, resulting in decreased production of TNF-α, IL-1β, and IL-6. To verify the specific role of the Kir3.1 channel, we generated Kir3.1-knockdown THP-1 cells. The Kir3.1^KD THP-1 cells exhibited inhibition of NF-κB activation and production of these pro-inflammatory cytokines in response to TLR4 stimulation. Taken together, our results demonstrate that the Kir3.1 channel is involved in the TLR4-mediated signal at an early event by facilitating the recruitment of TLR4 into lipid raft.     

Expression and purification of recombinant human angiopoietin-2 produced in Chinese hamster ovary cells

Angiopoietin-2 (Ang2) is a complex regulator of vascular remodeling that plays a role in both blood vessel sprouting and blood vessel regression through its receptor Tie2. Recombinant Chinese hamster ovary (rCHO) cell lines expressing a high level (20 microg/mL) of recombinant human Ang2 protein (rhAng2) with an amino-terminal FLAG-tag was constructed by transfecting the expression vectors into dihydrofolate reductase (dhfr)-deficient CHO cells and the subsequent gene amplification in medium containing stepwise increments in methotrexate level such as 0.02, 0.08, and 0.32 microM. The rhAng2 secreted from rCHO cells was purified at a purification yield of 53.6% from the cultured medium using an anti-FLAG M2 agarose affinity gel. SDS-PAGE and Western blot analyses showed that rCHO cells secret rhAng2 as a homodimeric glycoprotein form. Furthermore, rhAng2 binds to the Tie2 receptor and phosphorylates Tie2 in a concentration-dependent manner. Therefore, our rhAng2 could be useful for clarifying biological effect of exogenous Ang2 in the future.     

利用荧光共振能量转移技术研究活细胞TLR4与MD-2作用结构域

脂多糖(LPS)的识别和信号转导是宿主发生防御反应的关键,Toll样受体4(TLR4)与髓样分化蛋白-2(MD-2)形成复合物在LPS的识别及其信号转导中发挥了重要作用．研究TLR4与MD-2结合的功能结构域,对于深入了解LPS信号转导机制及其内毒素休克的防治具有重要意义．运用基于强度的三通道荧光共振能量转移技术(FRET)及基因突变和转染技术,研究了活细胞TLR4与MD-2作用的结构域．结果表明：N端Glu²⁴～Met⁴¹缺失使TLR4与MD-2结合能力明显下降;LPS刺激后TLR4聚合迅速增加,而缺失Glu²⁴～Met⁴¹的TLR4不能聚合．上述结果提示,TLR4的Glu²⁴～Met⁴¹不仅是结合MD-2的区域,并且还参与了LPS刺激后TLR4的聚合作用．     

Assessment of cell engineering strategies for improved therapeutic protein production in CHO cells

Recombinant glycoprotein therapeutics have proven to be invaluable pharmaceuticals for the treatment of various diseases. Chinese hamster ovary (CHO) cells are widely used in industry for the production of these proteins. Several strategies for engineering CHO cells for improved protein production have been tried with considerable results. The focus has mainly been to increase the specific productivity and to extend the culture longevity by preventing programmed cell death. These CHO cell engineering strategies, particularly those developed in Korea, are reviewed here.     

TLR4 signaling induced TLR2 expression in the process of mimic cerebral ischemia/reperfusion <Emphasis Type="Italic">in vitro</Emphasis>

Both TLR4 and TLR2 participated in the mediation of the inflammatory injury in the process of partial cerebral ischemia/reperfusion. However, it still remains unclear whether a crosstalk exists between TLR2 and TLR4 in ischemic cerebral damage. In the present study, we investigated the effect of TLR4 signaling on TLR2 expression during mimic cerebral I/R in vitro. BV-2 cells were cultured and treated with ischemia/reperfusion, then transfected with the plasmid pEGFP-H1/TLR4-siRNA, the plasmid pEGFP-H1/control sequence-siRNA and the blank plasmid, respectively. Interestingly, the expression of TLR2 and TLR4 mRNA and protein, NF-κB p65 mRNA and supernatant TNF-α level were significantly higher in ischemia/reperfusion treated cells than those lack of ischemia/reperfusion treatment, and as compared with those in ischemia/reperfusion treated cells without transfection, no significant differences about the above mentioned gene and protein expression were found in the blank plasmid tranfected cells and the plasmid pEGFP-H1/control sequence-siRNA transfected cells respectively, while the expression levels in the plasmid pEGFP-H1/TLR4-siRNA transfected cells were significantly lower. Additionally, in order to determine the effects of pyrrolidinediethyldithiocarbamate (PDTC), an NF-κB inhibitor, on the TLR4-induced TLR2 expression in BV-2 cells treated with ischemia/reperfusion, it was found that TLR4 and TLR2 mRNA expressions in PDTC pretreated cells were significantly lower in comparison with normal saline pretreated cells and non-pretreated cells. The data suggested that TLR2 activation, signaled by TLR4 and regulated by NF-κB, might be directly involved play an important role in ischemia/reperfusion induced brain damage.     

Effect of herbal melanin on IL-8: a possible role of Toll-like receptor 4 (TLR4)

The production of IL-8 can be induced by LPS via TLR4 signaling pathway. In this study, we tested the effect of a herbal melanin (HM) extract, from black cumin seeds (Nigella sativa L.), on IL-8 production. We used HM and LPS in parallel to induce IL-8 production by THP-I, PBMCs, and TLR4-transfected HEK293 cells. Both HM and LPS induced IL-8 mRNA expression and protein production in THP-1 and PBMCs. On applying similar treatment to HEK293 cells that express TLR4, MD2, and CD14, both HM and LPS significantly induced IL-8 protein production. We have also demonstrated that HM and LPS had identical effects in terms of IL-8 stimulation by HEK293 transfected with either TLR4 or MD2-CD14. Melanin extracted from N. sativa L. mimics the action of LPS in the induction of IL-8 by PBMC and the other used cell lines. Our results suggest that HM may share a signaling pathway with LPS that involves TLR4.     

Lipopolysaccharide induces proliferation and osteogenic differentiation of adipose-derived mesenchymal stromal cells in vitro via TLR4 activation

Multipotent mesenchymal stromal cells (MSC) are capable of multi-lineage differentiation and support regenerative processes. In bacterial infections, resident MSC can come intocontact with and need to react to bacterial components. Lipopolysaccharide (LPS), a typical structure of Gram-negative bacteria, increases the proliferation and osteogenic differentiation of MSC. LPS is usually recognized by the toll-like receptor (TLR) 4 and induces pro-inflammatory reactions in numerous cell types. In this study, we quantified the protein expression of TLR4 and CD14 on adipose-derived MSC (adMSC) in osteogenic differentiation and investigated the effect of TLR4 activation by LPS on NF-κB activation, proliferation and osteogenic differentiation of adMSC. We found that TLR4 is expressed on adMSC whereas CD14 is not, and that osteogenic differentiation induced an increase of the amount of TLR4 protein whereas LPS stimulation did not. Moreover, we could show that NF-κB activation via TLR4 occurs upon LPS treatment. Furthermore, we were able to show that competitive inhibition of TLR4 completely abolished the stimulatory effect of LPS on the proliferation and osteogenic differentiation of adMSC. In addition, the inhibition of TLR4 leads to the complete absence of osteogenic differentiation of adMSC, even when osteogenically stimulated. Thus, we conclude that LPS induces proliferation and osteogenic differentiation of adMSC in vitro through the activation of TLR4 and that the TLR4 receptor seems to play a role during osteogenic differentiation of adMSC.     

Immunohistochemical localization of TLR2 and CD14 in gingival tissue of healthy individuals and patients with chronic periodontitis

We used immunohistochemistry to quantify and compare the expression of Toll-like receptor 2 (TLR2) and cluster of differentiation 14 (CD14) in gingival tissues of both healthy individuals and patients with chronic periodontitis. We also correlated the expression of TLR2 and CD14 with the histological grades of chronic periodontitis. We examined 30 gingival specimens from chronic periodontitis patients and 10 from healthy individuals. Tissues from both groups were immunostained with antibodies against TLR2 and CD14. TLR2 and CD14 were expressed by endothelial cells, fibroblasts, lymphocytes and plasma cells. The immunohistochemical expression of TLR2 and CD14 was significantly greater in inflammatory cells of the chronic periodontitis group than in healthy individuals. Expression of these molecules was greater in the inflammatory cells of connective tissue adjacent to pocket epithelium in both groups. The expression of TLR2 and CD14 was greatest in the periodontitis group that was classified as severe grade, followed by moderate and mild grades, which suggests a role of TLR2 and CD14 in the pathogenesis of chronic periodontitis. The positive correlation of TLR2 and CD14 expression levels with the severity grades of chronic periodontitis suggests that they are correlated also with disease severity; therefore, they may be useful for predicting disease progression. Our findings are consistent with the possibility that CD14 acts as a co-receptor for TLR2.