Peptide derived from SLAMF1 prevents TLR4-mediated inflammation in vitro and in vivo.

Inflammation plays a crucial role in the development and progression of many diseases, and is often caused by dysregulation of signalling from pattern recognition receptors, such as TLRs. Inhibition of key protein-protein interactions is an attractive target for treating inflammation. Recently, we demonstrated that the signalling lymphocyte activation molecule family 1 (SLAMF1) positively regulates signalling downstream of TLR4 and identified the interaction interface between SLAMF1 and the TLR4 adaptor protein TRIF-related adapter molecule (TRAM). Based on these findings, we developed a SLAMF1-derived peptide, P7, which is linked to a cell-penetrating peptide for intracellular delivery. We found that P7 peptide inhibits the expression and secretion of IFNß and pro-inflammatory cytokines (TNF, IL-1ß, IL-6) induced by TLR4, and prevents death in mice subjected to LPS shock. The mechanism of action of P7 peptide is based on interference with several intracellular protein-protein interactions, including TRAM-SLAMF1, TRAM-Rab11FIP2, and TIRAP-MyD88 interactions. Overall, P7 peptide has a unique mode of action and demonstrates high efficacy in inhibiting TLR4-mediated signalling in vitro and in vivo.

Benzyl-para-di-[5-methyl-4-(n-octylamino) pyrimidin-2(1H)one] as an interferon beta (IFN-ß) modulator.

IFN-ß is a cytokine that plays a significant role in the immune system. Inhibition of IFN-ß might be used as a therapeutic approach to treat septic shock. A peptidomimetic previously developed by our research team, 1-benzyl-5-methyl-4-(n-octylamino)pyrimidin-2(1H)-one (LT87), was used as an cardioprotective agent in a myocardial ischemia (MI) mouse model. We have developed new LT87 derivatives by synthetizing its dimers in an attempt to extend its structural variety and enhance its biological activity. A dimeric derivative, LT127, exhibited a dose-dependent inhibition of LPS-mediated IFN-ß and subsequent CXCL10 mRNA transcription. The effect was selective and transduced through TLR4- and TRAM/TRIF-mediated signaling, with no significant effect on MyD88-dependent signaling. However, this effect was not specific to TLR4, since a similar effect was observed both on TLR8- and MDA5/RIG-I-stimulated IFN-ß expression. Nevertheless, LT127 might serve as a drug candidate, specifically as an inhibitor for IFN-ß production in order to develop a novel therapeutic approach to prevent septic shock.

The TLR4 adaptor TRAM controls the phagocytosis of Gram-negative bacteria by interacting with the Rab11-family interacting protein 2.

Phagocytosis is a complex process that eliminates microbes and is performed by specialised cells such as macrophages. Toll-like receptor 4 (TLR4) is expressed on the surface of macrophages and recognizes Gram-negative bacteria. Moreover, TLR4 has been suggested to play a role in the phagocytosis of Gram-negative bacteria, but the mechanisms remain unclear. Here we have used primary human macrophages and engineered THP-1 monocytes to show that the TLR4 sorting adapter, TRAM, is instrumental for phagocytosis of Escherichia coli as well as Staphylococcus aureus. We find that TRAM forms a complex with Rab11 family interacting protein 2 (FIP2) that is recruited to the phagocytic cups of E. coli. This promotes activation of the actin-regulatory GTPases Rac1 and Cdc42. Our results show that FIP2 guided TRAM recruitment orchestrates actin remodelling and IRF3 activation, two events that are both required for phagocytosis of Gram-negative bacteria.

SLAMF1 is required for TLR4-mediated TRAM-TRIF-dependent signaling in human macrophages.

Signaling lymphocytic activation molecule family 1 (SLAMF1) is an Ig-like receptor and a costimulatory molecule that initiates signal transduction networks in a variety of immune cells. In this study, we report that SLAMF1 is required for Toll-like receptor 4 (TLR4)-mediated induction of interferon ß (IFNß) and for killing of Gram-negative bacteria by human macrophages. We found that SLAMF1 controls trafficking of the Toll receptor-associated molecule (TRAM) from the endocytic recycling compartment (ERC) to Escherichia coli phagosomes. In resting macrophages, SLAMF1 is localized to ERC, but upon addition of E. coli, it is trafficked together with TRAM from ERC to E. coli phagosomes in a Rab11-dependent manner. We found that endogenous SLAMF1 protein interacted with TRAM and defined key interaction domains as amino acids 68 to 95 of TRAM as well as 15 C-terminal amino acids of SLAMF1. Interestingly, the SLAMF1-TRAM interaction was observed for human but not mouse proteins. Overall, our observations suggest that SLAMF1 is a new target for modulation of TLR4-TRAM-TRIF inflammatory signaling in human cells.

Combined Inhibition of Complement and CD14 Attenuates Bacteria-Induced Inflammation in Human Whole Blood More Efficiently Than Antagonizing the Toll-like Receptor 4-MD2 Complex.

BACKGROUND: Single inhibition of the Toll-like receptor 4 (TLR4)-MD2 complex failed in treatment of sepsis. CD14 is a coreceptor for several TLRs, including TLR4 and TLR2. The aim of this study was to investigate the effect of single TLR4-MD2 inhibition by using eritoran, compared with the effect of CD14 inhibition alone and combined with the C3 complement inhibitor compstatin (Cp40), on the bacteria-induced inflammatory response in human whole blood. METHODS: Cytokines were measured by multiplex technology, and leukocyte activation markers CD11b and CD35 were measured by flow cytometry. RESULTS: Lipopolysaccharide (LPS)-induced inflammatory markers were efficiently abolished by both anti-CD14 and eritoran. Anti-CD14 was significantly more effective than eritoran in inhibiting LPS-binding to HEK-293E cells transfected with CD14 and Escherichia coli-induced upregulation of monocyte activation markers (P < .01). Combining Cp40 with anti-CD14 was significantly more effective than combining Cp40 with eritoran in reducing E. coli-induced interleukin 6 (P < .05) and monocyte activation markers induced by both E. coli (P < .001) and Staphylococcus aureus (P < .01). Combining CP40 with anti-CD14 was more efficient than eritoran alone for 18 of 20 bacteria-induced inflammatory responses (mean P < .0001). CONCLUSIONS: Whole bacteria-induced inflammation was inhibited more efficiently by anti-CD14 than by eritoran, particularly when combined with complement inhibition. Combined CD14 and complement inhibition may prove a promising treatment strategy for bacterial sepsis.

Using Confocal Microscopy to Investigate Intracellular Trafficking of Toll-Like Receptors.

Toll-like receptors (TLR) survey the extracellular space, cytoplasm, and endosomal compartments for signs of infection or tissue injury. Over the past decade, it has become evident that TLR activation and signal transduction can be regulated by subcellular compartmentalization of both the receptors and their downstream signaling components. Immunofluorescence and/or overexpression of fluorescently "tagged"' proteins teamed with confocal microscopy presents a powerful technique for studying the spatial organization of TLRs, their signaling mediators, and the dynamic processes they activate. This chapter details the common methods for determining the subcellular location of TLRs in both live and fixed cells.

CD14, TLR4 and TRAM Show Different Trafficking Dynamics During LPS Stimulation.

Toll-like receptor 4 (TLR4) is responsible for the immediate response to Gram-negative bacteria and signals via two main pathways by recruitment of distinct pairs of adaptor proteins. Mal-MyD88 [Mal (MyD88-adaptor-like) - MYD88 (Myeloid differentiation primary response gene (88))] is recruited to the plasma membrane to initiate the signaling cascade leading to production of pro-inflammatory cytokines while TRAM-TRIF [TRAM (TRIF-related adaptor molecule)-TRIF (TIR-domain-containing adapter-inducing interferon-ß)] is recruited to early endosomes to initiate the subsequent production of type I interferons. We have investigated the dynamics of TLR4 and TRAM during lipopolysaccharide (LPS) stimulation. We found that LPS induced a CD14-dependent immobile fraction of TLR4 in the plasma membrane. Total internal reflection fluorescence microscopy (TIRF) revealed that LPS stimulation induced clustering of TLR4 into small punctate structures in the plasma membrane containing CD14/LPS and clathrin, both in HEK293 cells and the macrophage model cell line U373-CD14. These results suggest that laterally immobilized TLR4 receptor complexes are being formed and prepared for endocytosis. RAB11A was found to be involved in localizing TRAM to the endocytic recycling compartment (ERC) and to early sorting endosomes. Moreover, CD14/LPS but not TRAM was immobilized on RAB11A-positive endosomes, which indicates that TRAM and CD14/LPS can independently be recruited to endosomes.

A role for the adaptor proteins TRAM and TRIF in toll-like receptor 2 signaling.

Toll-like receptors (TLRs) are involved in sensing invading microbes by host innate immunity. TLR2 recognizes bacterial lipoproteins/lipopeptides, and lipopolysaccharide activates TLR4. TLR2 and TLR4 signal via the Toll/interleukin-1 receptor adaptors MyD88 and MAL, leading to NF-κB activation. TLR4 also utilizes the adaptors TRAM and TRIF, resulting in activation of interferon regulatory factor (IRF) 3. Here, we report a new role for TRAM and TRIF in TLR2 regulation and signaling. Interestingly, we observed that TLR2-mediated induction of the chemokine Ccl5 was impaired in TRAM or TRIF deficient macrophages. Inhibition of endocytosis reduced Ccl5 release, and the data also suggested that TRAM and TLR2 co-localize in early endosomes, supporting the hypothesis that signaling may occur from an intracellular compartment. Ccl5 release following lipoprotein challenge additionally involved the kinase Tbk-1 and Irf3, as well as MyD88 and Irf1. Induction of Interferon-ß and Ccl4 by lipoproteins was also partially impaired in cells lacking TRIF cells. Our results show a novel function of TRAM and TRIF in TLR2-mediated signal transduction, and the findings broaden our understanding of how Toll/interleukin-1 receptor adaptor proteins may participate in signaling downstream from TLR2.

TLR3 mediates release of IL-1ß and cell death in keratinocytes in a caspase-4 dependent manner.

BACKGROUND: Inflammation and timely cell death are important elements in host defence and healing processes. Keratinocytes express high levels of Toll-like receptor 3 (TLR3), and stimulation of the receptor with its ligand polyinosinic-polycytidylic acid (polyI:C) is a powerful signal for release of a variety of proinflammatory cytokines. Caspase-4 is required for maturation of pro-IL-1ß through activation of caspase-1 in keratinocytes. METHODS: TLR3 in keratinocytes was stimulated with polyI:C. Induction of messenger RNA of pro-IL-1ß and inflammasomal components was measured using quantitative polymerase chain reaction methodology. Protein expression of IL-1ß was analysed with ELISA and Western blot techniques. Activation of apoptotic caspases was measured with flow cytometry, and cytotoxicity was determined. RESULTS: TLR3 induced release of substantial amounts of pro-IL-1ß in keratinocytes. NLRP3 or ASC dependent processing of IL-1ß into its cleaved bioactive form was found to be minimal. The release of IL-1ß was due to polyI:C induced cell death that occurred through a caspase-4 dependent manner. Caspase-1 did not seem to be involved in the polyI:C induced cytotoxicity despite that TLR3 stimulation induced activation of caspase-1. In addition, the apoptotic caspases -8, -9 and -3/7 were activated by polyI:C. CONCLUSION: TLR3 stimulation in keratinocytes induces a caspase-4 dependent release of pro-IL-1ß, but further processing to active IL-1ß is limited. Furthermore, TLR3 stimulation results in pyroptotic- and apoptotic cell death.

The GOLD domain-containing protein TMED7 inhibits TLR4 signalling from the endosome upon LPS stimulation.

Toll-like receptor 4 is an innate immune receptor responsible for the recognition of the Gram-negative cell wall component lipopolysaccharide. Here we show that transmembrane emp24 domain-containing protein 7 (TMED7) inhibits MyD88-independent toll-like receptor 4 signalling. TMED7 overexpression inhibits the ability of TRAM, an adaptor utilized by toll-like receptor 4, or lipopolysaccharide to activate the interferon regulatory factor 3-signalling pathway, whereas TMED7 knockdown enhances production of the cytokine, RANTES, following lipopolysaccharide stimulation. Upon lipopolysaccharide stimulation, TMED7 co-localizes with TRAM and toll-like receptor 4 in late endosomes where it encounters the negative regulator of TRAM, TAG. The TMED7 sequence is found in TAG because of a read-through from the tmed7 gene into the ticam2 gene. TMED7 is essential for TAG-mediated disruption of the TRAM/TRIF complex and the degradation of toll-like receptor 4. A TMED homologue, logjam, has a negative role in the Toll and IMD pathways in Drosophila melanogaster; therefore, TMEDs may have a conserved role in the regulation of innate immunity.

Toll-like receptor 4 facilitates binding of Shiga toxin to colon carcinoma and primary umbilical vein endothelial cells.

Infection with Shiga toxin (Stx)-producing, gram-negative bacteria can induce serious conditions such as dysentery and hemolytic uremic syndrome. In target cells, Stx is internalized by endocytosis, and travels through the Golgi apparatus and the endoplasmic reticulum to reach the cytosol, where it inhibits protein synthesis. Toll-like receptor 4 (TLR4) mediates the recognition of gram-negative bacteria. Here, we have investigated whether the cellular uptake and transport of Stx could involve TLR4. We found that upon small interfering RNA (siRNA)-mediated TLR4 depletion in epithelial colon carcinoma cells, Stx transport to the Golgi was strongly reduced, and this was primarily caused by diminished Stx cellular binding rather than by reduction in toxin uptake or endosome-to-Golgi transport. The reduced cellular binding of Stx upon siRNA-transfection was solely due to TLR4 depletion, because reconstitution of TLR4 expression by the introduction of an siRNA-resistant TLR4 gene completely abolished the TLR4-targeting siRNA-mediated effect. Importantly, the effect of TLR4 depletion was not restricted to cancer cells or epithelial cells, because primary human umbilical vein endothelial cells also displayed reduced Stx binding upon TLR4 depletion. These results indicate that although TLR4 is imperative in innate immunity against gram-negative bacteria, it may be exploited by bacterial toxins, for example Stx, to gain access and entry into cells.

The Rab11a GTPase controls Toll-like receptor 4-induced activation of interferon regulatory factor-3 on phagosomes.

Toll-like receptor 4 (TLR4) is indispensable for recognition of Gram-negative bacteria. We described a trafficking pathway for TLR4 from the endocytic recycling compartment (ERC) to E. coli phagosomes. We found a prominent colocalization between TLR4 and the small GTPase Rab11a in the ERC, and Rab11a was involved in the recruitment of TLR4 to phagosomes in a process requiring TLR4 signaling. Also, Toll-receptor-associated molecule (TRAM) and interferon regulatory factor-3 (IRF3) localized to E. coli phagosomes and internalization of E. coli was required for a robust interferon-ß induction. Suppression of Rab11a reduced TLR4 in the ERC and on phagosomes leading to inhibition of the IRF3 signaling pathway induced by E. coli, whereas activation of the transcription factor NF-κB was unaffected. Moreover, Rab11a silencing reduced the amount of TRAM on phagosomes. Thus, Rab11a is an important regulator of TLR4 and TRAM transport to E. coli phagosomes thereby controlling IRF3 activation from this compartment.

Intracellular Mycobacterium avium intersect transferrin in the Rab11(+) recycling endocytic pathway and avoid lipocalin 2 trafficking to the lysosomal pathway.

Iron is an essential nutrient for microbes, and many pathogenic bacteria depend on siderophores to obtain iron. The mammalian innate immunity protein lipocalin 2 (Lcn2; also known as neutrophil gelatinase-associated lipocalin, 24p3, or siderocalin) binds the siderophore carboxymycobactin, an essential component of the iron acquisition apparatus of mycobacteria. Here we show that Lcn2 suppressed growth of Mycobacterium avium in culture, and M. avium induced Lcn2 production from mouse macrophages. Lcn2 also had elevated levels and initially limited the growth of M. avium in the blood of infected mice but did not impede growth in tissues and during long-term infections. M. avium is an intracellular pathogen. Subcellular imaging of infected macrophages revealed that Lcn2 trafficked to lysosomes separate from M. avium, whereas transferrin was efficiently transported to the mycobacteria. Thus, mycobacteria seem to reside in the Rab11(+) endocytic recycling pathway, thereby retaining access to nutrition and avoiding endocytosed immunoproteins like Lcn2.

TAG, a splice variant of the adaptor TRAM, negatively regulates the adaptor MyD88-independent TLR4 pathway.

Toll-like receptor 4 (TLR4) signals the induction of transcription factor IRF3-dependent genes from the early endosome via the adaptor TRAM. Here we report a splice variant of TRAM, TAG ('TRAM adaptor with GOLD domain'), which has a Golgi dynamics domain coupled to TRAM's Toll-interleukin 1 receptor domain. After stimulation with lipopolysaccharide, TRAM and TAG localized to late endosomes positive for the GTPase Rab7a. TAG inhibited activation of IRF3 by lipopolysaccharide. Knockdown of TAG with small interfering RNA enhanced induction of the chemokine CCL5 (RANTES), but not of interleukin 8, by lipopolysaccharide in human peripheral blood mononuclear cells. TAG displaced the adaptor TRIF from TRAM. TAG is therefore an example of a specific inhibitor of the adaptor MyD88-independent pathway activated by TLR4. Targeting TAG could be useful in the effort to boost the immunostimulatory effect of TLR4 without causing unwanted inflammation.

IL-10 enhances MD-2 and CD14 expression in monocytes and the proteins are increased and correlated in HIV-infected patients.

Soluble proteins that bind LPS, like myeloid differentiation-2 (MD-2) and CD14, have essential roles in regulating LPS signaling through TLR4. During a gram-negative bacterial infection, the host may control the response by adjusting the levels of soluble MD-2 and CD14. To address the surface expression of MD-2 on human leukocytes, we developed a mAb, IIC1, that recognized MD-2 both free and when bound to TLR4. MD-2 was found on the surface of freshly isolated monocytes, on a subpopulation of CD19(+) B-cells and on CD15(+) neutrophils. LPS transiently reduced the MD-2 levels on monocytes, which is most likely due to endocytosis of the LPS receptor complex since MD-2 colocalized with TLR4 in early endosomes after LPS stimulation. In the absence of LPS, MD-2 partly colocalized with TLR4 in Golgi trans and medial compartments. Cultivating monocytes for 18-20 h resulted in loss of MD-2 expression on the surface, which was reversed either by LPS or IL-10. Furthermore, addition of IL-10, but not LPS, resulted in a considerable increase in mRNA for both MD-2 and CD14. Using ELISA, we demonstrated that IL-10 had a profound dose- and time-related effect on the release of soluble MD-2 and soluble CD14 from monocytes. In HIV-infected patients, the amounts of MD-2, CD14, and IL-10 increased significantly in the patient group with AIDS. Of interest, we found that IL-10, CD14, and MD-2 levels were positively correlated, suggesting that IL-10 may be a driving force for increased release of MD-2 and CD14 during systemic inflammation.

Cellular trafficking of lipoteichoic acid and Toll-like receptor 2 in relation to signaling: role of CD14 and CD36.

Lipoteichoic acid (LTA) is a central inducer of inflammatory responses caused by Gram-positive bacteria, such as Staphylococcus aureus, via activation of TLR2. Localization of TLR2 in relation to its coreceptors may be important for function. This study explores the signaling, uptake, and trafficking pattern of LTA in relation to expression of TLR2 and its coreceptors CD36 and CD14 in human monocytes. We found TLR2 expressed in early endosomes, late endosomes/lysosomes, and in Rab-11-positive compartments but not in the Golgi apparatus or endoplasmic reticulum (ER). Rapid internalization of fluorescently labeled LTA was observed in human monocytes, colocalizing with markers for early and late endosomes, lysosomes, ER, and Golgi network. Blocking CD14 and CD36 with antibodies inhibited LTA binding and LTA-induced TNF release from monocytes, emphasizing an important role for both molecules as coreceptors for TLR2. Importantly, blocking CD36 did not affect TNF release induced by N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2R,S)-propyl]-(R)-cysteinyl-seryl-(lysyl)3-lysine or LPS. Expression of CD14 markedly enhanced LTA binding to the plasma membrane and also enhanced NF-kappaB activation. LTA internalization, but not NF-kappaB activation, was inhibited in Dynamin-I K44A dominant-negative transfectants, suggesting that LTA is internalized by receptor-mediated endocytosis but that internalization is not required for signaling. In fact, immobilizing LTA and thereby inhibiting internalization resulted in enhanced TNF release from monocytes. Our results suggest that LTA signaling preferentially occurs at the plasma membrane, is independent of internalization, and is facilitated by CD36 and CD14 as coreceptors for TLR2.

Endocytic pathways regulate Toll-like receptor 4 signaling and link innate and adaptive immunity.

Immune responses are initiated when molecules of microbial origin are sensed by the Toll-like receptors (TLRs). We now report the identification of essential molecular components for the trafficking of the lipopolysaccharide (LPS) receptor complex. LPS was endocytosed by a receptor-mediated mechanism dependent on dynamin and clathrin and colocalized with TLR4 on early/sorting endosomes. TLR4 was ubiquitinated and associated with the ubiquitin-binding endosomal sorting protein hepatocyte growth factor-regulated tyrosine kinase substrate, Hrs. Inhibition of endocytosis and endosomal sorting increased LPS signaling. Finally, the LPS receptor complex was sorted to late endosomes/lysosomes for degradation and loading of associated antigens onto HLA class II molecules for presentation to CD4+ T cells. Our results show that endosomal trafficking of the LPS receptor complex is essential for signal termination and LPS-associated antigen presentation, thus controlling both innate and adaptive immunity through TLR4.

Crystal structure at 1.1 Angstroms resolution of an insect myrosinase from Brevicoryne brassicae shows its close relationship to beta-glucosidases.

The aphid Brevicoryne brassicae is a specialist feeding on Brassicaceae plants. The insect has an intricate defence system involving a beta-D-thioglucosidase (myrosinase) that hydrolyses glucosinolates sequestered from the host plant into volatile isothiocyanates. These isothiocyanates act synergistically with the pheromone E-beta-farnesene to form an alarm system when the aphid is predated. In order to investigate the enzymatic characteristics of the aphid myrosinase and its three-dimensional structure, milligram amounts of pure recombinant aphid myrosinase were obtained from Echerichia coli. The recombinant enzyme had similar physiochemical properties to the native enzyme. The global structure is very similar to Sinapis alba myrosinase and plant beta-O-glucosidases. Aphid myrosinase has two catalytic glutamic acid residues positioned as in plant beta-O-glucosidases, and it is not obvious why this unusual enzyme hydrolyses glucosinolates, the common substrates of plant myrosinases which are normally not hydrolyzed by plant beta-O-glucosidases. The only residue specific for aphid myrosinase in proximity of the glycosidic linkage is Tyr180 which may have a catalytic role. The aglycon binding site differs strongly from plant myrosinase, whereas due to the presence of Trp424 in the glucose binding site, this part of the active site is more similar to plant beta-O-glucosidases, as plant myrosinases carry a phenylalanine residue at this position.

Cell specific, cross-species expression of myrosinases in Brassica napus, Arabidopsis thaliana and Nicotiana tabacum.

A prototypical characteristic of the Brassicaceae is the presence of the myrosinase-glucosinolate system. Myrosinase, the only known S-glycosidase in plants, degrades glucosinolates, thereby initiating the formation of isothiocyanates, nitriles and other reactive products with biological activities. We have used myrosinase gene promoters from Brassica napus and Arabidopsis thaliana fused to the beta -glucuronidase (GUS) reporter gene and introduced into Arabidopsis thaliana, Brassica napus and/or Nicotiana tabacum plants to compare and determine the cell types expressing the myrosinase genes and the GUS expression regulated by these promoters. The A. thaliana TGG1 promoter directs expression to guard cells and phloem myrosin cell idioblasts of transgenic A. thaliana plants. Expression from the same promoter construct in transgenic tobacco plants lacking the myrosinase enzyme system also directs expression to guard cells. The B. napus Myr1.Bn1 promoter directs a cell specific expression to idioblast myrosin cells of immature and mature seeds and myrosin cells of phloem of B. napus. In A. thaliana the B. napus promoter directs expression to guard cells similar to the expression pattern of TGG1. The Myr1.Bn1 signal peptide targets the gene product to the reticular myrosin grains of myrosin cells. Our results indicate that myrosinase gene promoters from Brassicaceae direct cell, organ and developmental specific expression in B. napus, A. thaliana and N. tabacum.